Documentation:PATH417archive2020W2/Case 2

Path 417 Cases
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Nimah Kelly
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	PATH4172020W2/Case_1 PATH417:2020W2/Case_2 Course:PATH4172020W2/Case_3 Course:PATH4172020W2/Case_4

1. Case 2

A Stiff Neck

18-year-old Mary has just moved into the dormitory at her university. One day, her roommate finds her lying in bed under her sheets. She is complaining of fever, chills, bad headache and a stiff neck. She is staying under the covers because the light is hurting her eyes. Her roommate calls 911 and an ambulance takes Mary to the local hospital.

The emergency room physician asks Mary about her recent vaccinations and she reports that she has not had any since she was in elementary school. The physician documents a fever of 39.2°C and low blood pressure. He sends blood and cerebral spinal fluid to the Microbiology Laboratory. She is started immediately on intravenous antibiotics. Mary’s blood and cerebral spinal fluid grow Neisseria meningitidis and she is diagnosed with meningococcal meningitis.

1. The Body System

(i) Describe the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). Also make note of any key history findings, what samples are taken (and why) and the meaning of any laboratory results reported. (There is no need to describe the laboratory testing as this is the basis of another question).

(ii) Which body system is affected? In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.

(iii) What antibiotics might have been in the intravenous drip i.e. what is the antibacterial treatment of choice and how do the(se) antibiotic(s) work to rid the body of the organism?

(iv) Why did the doctor ask about Mary’s vaccination history? Is this a reportable communicable disease?

2. The Microbiology Laboratory

(i) Other than the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.

(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?

(iii) Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.

(iv) What are the results expected from these tests allowing for the identification of the bacteria named in this case.

3. Bacterial Pathogenesis

Using the following pathogenic steps outline the pathogenesis of the bacteria named as being responsible for this infection.

i. Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria

ii. Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.

iii. Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.

iv. Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case?

4. The Immune Response

i. Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

ii. Host damage: what damage ensues to the host from the immune response?

iii. Bacterial evasion: how does the bacteria attempt to evade these host response elements.

iv. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria ?

Q.1 The Body System

i. Describe the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). Also make note of any key history findings, what samples are taken (and why) and the meaning of any laboratory results reported. (There is no need to describe the laboratory testing as this is the basis of another question).

Meningococcal meningitis is an infection caused by the bacterium Neisseria meningitidis and is characterized by inflammation of the meningeal membrane which serves as a protective barrier of the central nervous system (1). Signs of infection include fever and low blood pressure which are detected by the emergency room physician (2). Infected individuals may experience symptoms of chills,  headache, stiff neck and the possibility of aching muscles, all of which align with Mary’s expressed symptoms (1). Mary has also mentioned that she is unable to tolerate bright light which is a common symptom of meningococcal meningitis known as photophobia (3). Photophobia is characterized by sensitivity or feeling of uncomfortableness to light (3). Additional symptoms that may also be observed with this bacterial infection are nausea and vomiting (1). However, approximately 10-20% of acute cases may progress into the systemic form of the disease known as meningococcemia which results in vascular injury and development of skin rashes (1). It is also important to note that objective characteristics observed in infants and young children are harder to detect and therefore vary in comparison to adults where symptoms are clearer (1). Young children may experience irritability, lethargy, and/or poor feeding (4). Symptoms in older patients also slightly differ since they are less likely to experience headache and neck stiffness and are more likely to have altered mental status(4). However, these characteristics do not apply to Mary as she is neither an infant or considered an older individual.

Stiffness of the hamstrings and inability to fully extend the leg when the hip is flexed at 90° indicate a positive Kernig’s sign. Involuntary flexion of the hip and knees indicate a positive Brudzinski sign.

In the hospital, Mary is examined by the emergency room physician where he has documented a high fever of 39.2°C and low blood pressure. High fever may be triggered by swelling and inflammation occurring in the meningeal membranes surrounding the brain and spinal cord (1). Low blood pressure may also indicate the development of shock with the possibility of organ failure (5). When meningitis is suspected, three physical signs will be assessed through physically maneuvering the patient in an attempt to stretch the meninges to elicit meningeal irritation (2). These clinical maneuvers include assessing nuchal rigidity by passively flexing the neck of the patient which, if the test yields a positive result, will be met with palpable resistance (2). Kernig’s sign will also be examined which involves positioning the patient supine whilst placing their hips at a 90 degree angle (2). A positive test result will be achieved if there is either pain or passive extension of the patient’s knee (2). Additionally, Brudzinki’s sign will be tested in which a patient is positioned supine and is asked to passively flex their neck (2). This test will yield a positive result if the performance of the manoeuvre results in reflexive flexion of the hip and knee (2). All three of these physical signs suggest, but do not confirm a potential diagnosis of meningococcal meningitis (2). In addition to this, the emergency room physician has asked Mary whether or not her vaccinations were up to date. Although Mary had been vaccinated with the monovalent conjugate C meningococcal (Men-C) vaccine in elementary school, healthy young adults like Mary should have received a second Men-C or meningococcal quadrivalent vaccination which protects against a wide array of Neisseria meningitidis serotypes (6,7). Moreover, meningitis outbreaks are quite common in college dormitories where individuals are living in close proximity with one another (8). Therefore, Mary’s lack of vaccination and living situation has made her more susceptible to this particular bacterial infection.

Collection of cerebrospinal fluid is performed through a lumbar puncture. Needle is placed between two lumbar vertebrae bones.

To confirm Mary’s diagnosis, samples of her blood and cerebrospinal fluid (CSF) were sent to the laboratory where Neisseria meningitidis was detected. Based on the key symptoms that Mary experienced such as neck stiffness, which can be identified through Brudzinski’s and Kernig’s sign, the emergency room physician was able to determine that both blood and cerebrospinal fluid are the samples to be collected (3). The blood sample collected can be used for the growth and isolation of N.meningitidis through the blood agar plate (BAP) and chocolate agar plate mediums (CAP) (9). Moreover, the collection of cerebrospinal fluid occurs due to suspected infection of the meningeal layers where CSF is housed in the subarachnoid space. A lumbar puncture is performed in the lower back where a needle is inserted between two lumbar vertebrae bones for the collection of CSF (10). From this sample, white blood cells, blood, protein and glucose can be analyzed to determine whether the infection is bacterial, viral or fungal (11). If the sample is cloudy, has an increased white blood cell count, increased protein amount and decreased glucose levels, then diagnosis of meningococcal meningitis can be confirmed (11). Immediately after the samples were taken, Mary was put on antibiotics. Prompt treatment with antibiotics for individuals with suspected meningococcal meningitis occurs to initiate the process of recovery and reduce the risk of complications that may arise (5). Moreover, sample collection must occur before the administration of any antimicrobial agent as these agents may reduce success of isolation and diagnosis in the samples (10).

References:

1. Meningococcal meningitis - NORD (national organization for rare disorders) [Internet]. Rarediseases.org. 2015 [cited 2021 Feb 11]. Available from: https://rarediseases.org/rare- diseases/meningococcal-meningitis/

1. Tracy A, Waterfield T. How to use clinical signs of meningitis. Arch Dis Child Educ Pract Ed. 2020;105(1):46–9.
2. Pace D, Pollard AJ. Meningococcal disease: clinical presentation and sequelae. Vaccine. 2012;30 Suppl 2:B3-9.
3. Mount HR, Boyle SD. Aseptic and bacterial meningitis: Evaluation, treatment, and prevention. Am Fam Physician. 2017;96(5):314–22.
4. Signs of bacterial meningitis [Internet]. Ada.com. [cited 2021 Feb 11]. Available from: https://ada.com/conditions/bacterial-meningitis/
5. Public Health Agency of Canada. Meningococcal vaccine: Canadian Immunization Guide [Internet]. Canada.ca. 2007 [cited 2021 Feb 24]. Available from: https://www.canada.ca/en/public-health/services/publications/healthy-living/canadian-immunization-guide-part-4-active-vaccines/page-13-meningococcal-vaccine.html
6. Vaccines in BC [Internet]. Bccdc.ca. [cited 2021 Feb 24]. Available from: http://www.bccdc.ca/health-professionals/clinical-resources/vaccines-in-bc
7. Why your child needs the meningitis B vaccine before college - johns Hopkins all children’s hospital [Internet]. Hopkinsallchildrens.org. [cited 2021 Feb 11]. Available from: https://www.hopkinsallchildrens.org/ACH-News/General-News/Why-Your-Child-Needs-the- Meningitis-B-Vaccine-Befo
8. Laboratory Methods for the Diagnosis of Meningitis. Center for Disease Control and Prevention. Available at: https://www.cdc.gov/meningitis/lab-manual/chpt06-culture-id.pdf.
9. Morse SA. Neisseria, Moraxella, Kingella and Eikenella. In: Baron S, editor. Medical Microbiology Galveston, Texas: University of Texas Medical Branch at Galveston; 1996.
10. Mace SE. Acute bacterial meningitis. Emerg Med Clin North Am. 2008;26(2):281–317, viii.

ii. Which body system is affected? In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.

A schematic depicting the pathogenesis and pathophysiology of bacterial meningitis.

Neisseria meningitidis, the causative pathogen of Mary’s meningococcal meningitis, is a gram-negative diplococcal pathogenic bacterial species that commonly causes septicemia and meningitis in humans (1). Meningococcal meningitis is a disease that is characterized by the inflammation of the meninges (2). The meninges are composed of an innermost pia mater, subarachnoid space, arachnoid mater and outermost dura mater (3).

N. meningitidis has been found to colonize the nasopharynx of a sizable proportion of the population as approximately 3-30% of individuals contain N. meningitidis as part of their residential flora (1). However, infection can be initiated by the pathogenic species when the bacteria adhere to non-ciliated epithelial cells and pass through the nasopharyngeal membrane to enter into the blood circulation where they are able to avoid immune responses and proliferate (1, 4).

Once within the circulation, the bacteria must avoid immune responses by various mechanisms, one which includes the composition of its cell wall which is non-immunogenic (4). For example, in group B meningococci, their peptidoglycan displays structural similarity to human neural cell adhesion molecules which allows for immune evasion (4). Additionally, N. meningitidis releases an endotoxin which is an important part of the pathophysiology of disease as it binds receptors such as CD14 and TLRs in host inflammatory cells and macrophages which results in the production of inflammatory cytokines such as TNFα, IL-6, IL-8, etc. (4). In addition, neutrophils can be activated by both binding of these endotoxins or through complement mediated mechanisms, which results in the promotion of their antimicrobial responses such as production of reactive oxygen species and respiratory burst which can in turn cause endothelial cell damage and capillary disruption and leakage (4). This inflammatory process is responsible for irritation of nerve roots that run throughout the meninges, which can result in her symptoms of neck stiffness and headache (5).

Additionally, it has been demonstrated that bacterial as well as host-derived reactive oxygen and nitrogen species which both serve as highly reactive intermediates that can cause neuronal tissue damage (2). Furthermore, patients with bacterial meningitis can experience neuronal loss in the hippocampus due to exposure to different factors including bacterial toxins and cytotoxic products from host immune cells (2).

This image shows meningococcal penetration of the blood brain barrier. It highlights how bacteria interaction with the meninges triggers TNF-α, IL-6 and IL-8 cytokine release that causes inflammation of the meninges

The body system involved and implicated in meningococcal meningitis is the central nervous system, consisting of the brain and spinal cord, which is protected from external pathogens and other harmful substances that may be in the blood circulation by the blood brain barrier (BBB) as well as the blood-cerebrospinal (B-CSF) barrier (6). To cause meningitis, the bacteria must then pass through the BBB and the B-CSF barrier, which is formed by the choroid plexus of the meninges within the cerebral ventricles (6). To cross this barrier and also initially adhere to and colonize the submucosa of the nasopharynx, N. meningitidis tightly adheres to non-ciliated epithelial host cells and endothelial cells in the choroid plexus and vasculature, through type IV pili (1). The main component of these pili is the pilin which is encoded by the bacterial pilE gene (1). It has been demonstrated that CD145, which is a surface glycoprotein that is a member of the immunoglobulin superfamily, acts as a receptor for the type IV pili on cerebral endothelial cells allowing for adherence of the bacteria to the cerebral vasculature (7). Then, N. meningitidis has been suggested to invade choroid plexus epithelial cells through the basolateral side of these cells and be transported through an intracellular mechanism and then colonizes on the apical surface of these cells (6).

Once within the subarachnoid space, N. meningitidis endotoxin within the CSF largely results in the production of pro-inflammatory cytokines and chemokines (TNFα and IL-1/6) which increase protein concentrations and granulocyte accumulation (8). The normal function of the choroid plexus of the meninges is to produce approximately 50% of all cerebrospinal fluid, which is important for delivering nutrients, removing metabolic wastes and cushioning the nervous tissue of both the brain and the spinal cord, in addition to also forming the B-CSF barrier (6). However, during bacterial meningitis, outflow resistance to the movement of CSF in the subarachnoid space can result due to increased intracranial pressure and CSF fluid accumulation resulting from the development of cerebral edema (9). This condition can be due to either vasogenic, cytotoxic or interstitial cerebral edema and can be fatal as it can result in cerebral herniation or hydrocephalus (9). Additionally, normal cerebral blood flow can be altered first by demonstrating an increase and then decrease which indicates a loss of normal cerebrovascular autoregulation (indicating that cerebral blood flow, contrary to normal, fluctuates in tangent with mean arterial pressure) (10). These alterations in blood flow can have clinical implications as decreased cerebral blood flow can result in reduced nutrient delivery to brain tissue (including glucose and oxygen) (10). If not treated promptly, within a few days this can result in a wide array of neurological symptoms such as vision loss, memory loss, inability to walk, loss of sensation, confusion, altered consciousness and difficulty speaking (10).

References

1. Meningococcal meningitis - NORD (national organization for rare disorders) [Internet]. Rarediseases.org. 2015 [cited 2021 Feb 11]. Available from: https://rarediseases.org/rare- diseases/meningococcal-meningitis/
2. Tracy A, Waterfield T. How to use clinical signs of meningitis. Arch Dis Child Educ Pract Ed. 2020;105(1):46–9.
3. Pace D, Pollard AJ. Meningococcal disease: clinical presentation and sequelae. Vaccine. 2012;30 Suppl 2:B3-9.
4. Mount HR, Boyle SD. Aseptic and bacterial meningitis: Evaluation, treatment, and prevention. Am Fam Physician. 2017;96(5):314–22.
5. Signs of bacterial meningitis [Internet]. Ada.com. [cited 2021 Feb 11]. Available from: https://ada.com/conditions/bacterial-meningitis/
6. Public Health Agency of Canada. Meningococcal vaccine: Canadian Immunization Guide [Internet]. Canada.ca. 2007 [cited 2021 Feb 24]. Available from: https://www.canada.ca/en/public-health/services/publications/healthy-living/canadian-immunization-guide-part-4-active-vaccines/page-13-meningococcal-vaccine.html
7. Vaccines in BC [Internet]. Bccdc.ca. [cited 2021 Feb 24]. Available from: http://www.bccdc.ca/health-professionals/clinical-resources/vaccines-in-bc
8. Why your child needs the meningitis B vaccine before college - johns Hopkins all children’s hospital [Internet]. Hopkinsallchildrens.org. [cited 2021 Feb 11]. Available from: https://www.hopkinsallchildrens.org/ACH-News/General-News/Why-Your-Child-Needs-the- Meningitis-B-Vaccine-Befo
9. Laboratory Methods for the Diagnosis of Meningitis. Center for Disease Control and Prevention. Available at: https://www.cdc.gov/meningitis/lab-manual/chpt06-culture-id.pdf .
10. Morse SA. Neisseria, Moraxella, Kingella and Eikenella. In: Baron S, editor. Medical Microbiology Galveston, Texas: University of Texas Medical Branch at Galveston; 1996.
11. Mace SE. Acute bacterial meningitis. Emerg Med Clin North Am. 2008;26(2):281–317, viii.

iii. What antibiotics might have been in the intravenous drip i.e. what is the antibacterial treatment of choice and how do the(se) antibiotic(s) work to rid the body of the organism?

Before Diagnosis and Identification of Causative Agent of Meningitis: Broad Spectrum Antibiotics

Mary is started on intravenous antibiotics immediately after signs and symptoms are reported and before the laboratory tests identify the causative agent of infection. The signs and symptoms of meningococcal meningitis are similar to those of acute meningitis caused by H. influenzae, S. pneumoniae and other bacterial pathogens, therefore, initial antibiotic treatment must account for these other potential causes (1).

The current approach to meningitis treatment is to use broad-spectrum antibiotics to cover most possible causative organisms until diagnosis is confirmed (2). When acute bacterial meningitis is suspected, intravenous antibiotic therapy is started immediately and consists of vancomycin and a third-generation cephalosporin (eg. ceftriaxone or cefotaxime) (3).

Vancomycin (pink) binds and blocks transglucosidase (yellow) and transpeptidase (green) from binding to the peptidoglycan precursor to inhibit cell wall synthesis. Retrieved from: https://pharmaxchange.info/2011/04/mechanism-of-action-of-vancomycin/

Vancomycin Mechanism of Action

Vancomycin binds the D-Ala-D-Ala terminal of the peptidoglycan (PG) precursor to inhibit bacterial cell wall synthesis (4). Vancomycin is bulky and prevents binding of PG precursors to penicillin binding protein enzymes that catalyze transglycosylation and transpeptidation (cross-linking between PG precursors) for bacterial cell wall synthesis, and effectively inhibit bacterial cell wall synthesis (4). Inhibition of cell wall synthesis leads to bacteria cell death (4).

Beta lactams bind penicillin binding protein (PBP) and inhibit the enzyme to block transpeptidation and cross linking and bacterial cell wall synthesis. Retrieved from: https://tmedweb.tulane.edu/pharmwiki/doku.php/betalactam_pharm

Cephalosporins Mechanism of Action

Cephalosporins are beta-lactams, which contain a beta-lactam ring, also known as a four-membered cyclic amide, to mimic the PG precursor structure (5). Cephalosporins bind and inhibit bacterial penicillin-binding protein enzymes by binding to and blocking the penicillin binding protein active site such that the PG precursor cannot bind; this inhibits transpeptidation and formation of the bacterial cell wall (4, 5). In British Columbia, ceftriaxone is administered intravenously or intramuscularly. Cephalosporins are able to penetrate into the majority of body fluids and extracellular fluids of body tissues, especially during inflammation due to increased diffusion (6). Therefore, ceftriaxone is often chosen for treatment of meningitis due to its ability to access the CSF (6). Therefore, the third-generation cephalosporins penetrate the central nervous system very well and are effective against N. meningitidis, S. pneumoniae, H. influenzae, E.coli, and Streptococcus agalactiae, but not against L. monocytogenes which are other bacteria that can cause infections with similar signs and symptoms (2).

Adjunctive Steroids

Adjunctive corticosteroids can also reduce the severity of neurological complications in bacterial meningitis, and are used before diagnosis when the causative agent is unknown (2). These steroids have been found to decrease cytokine release and have anti-inflammatory effects (7).  Dexamethasone is recommended for both adults and children with suspected meningitis, in particular if S. pneumoniae is the causative microbe (2). Treatment should start before initiation of antibiotic therapy and the recommended duration of dexamethasone treatment is 4 days in adults (2). Dexamethasone has been found to reduce brain edema, intracranial pressure and overall inflammation of the meninges (8). There is only evidence of the use of steroids in Pneumococcal meningitis caused by S. pneumoniae, so when N. meningitidis is identified, the steroid treatment will stop (2).

Before Diagnosis and Identification of Causative Agent of Meningitis: Broad Spectrum Antibiotics

Therefore, because Mary starts immediately on the antibiotic treatment before receiving laboratory test results, the intravenous antibiotics of choice likely included vancomycin and a cephalosporin, which both function to inhibit bacterial cell wall synthesis and kill the bacteria, with adjunctive steroids to reduce inflammation (3, 4). These antibiotics are broad-spectrum and are effective against the multitude of bacteria that could be responsible for the initial signs and symptoms of infection.

After Diagnosis and Identification of N. meningitidis as the Causative Agent

Once N. meningitidis is confirmed, the antibiotic therapy can be narrowed down to a specific antibiotic treatment (2).

Past Treatment of Choice: Penicillin and Chloramphenicol

Previously before third generation cephalosporins were discovered and used, a combination of penicillin and chloramphenicol were used after confirmation of N. meningitidis diagnosis (2). Penicillin is a bactericidal beta-lactam that inhibits bacterial cell wall synthesis by binding and inhibiting bacterial penicillin binding proteins (4). Penicillin has poor penetration to the CNS in the absence of an inflamed blood-CSF barrier, but high and frequent doses can achieve a good therapeutic level (2). Penicillin is highly effective against penicillin-sensitive strains (2). However, meningococcal strains with reduced sensitivity to penicillin have emerged, demonstrating reduced affinity for penicillin for penicillin-binding protein 2 due to changes in the penA gene sequence (2, 9). Penicillin can still be used as an effective treatment, provided there is evidence of susceptibility on antimicrobial susceptibility testing (2, 4). When penicillin is used for treatment, an additional antibiotic such as ceftriaxone, ciprofloxacin or rifampin is necessary in order to clear N. meningitidis colonies from the nasopharynx (10).

Chloramphenicol is bactericidal to meningococci and can achieve high concentration in the CSF (2). Chloramphenicol is a broad-spectrum antibiotic with primarily bacteriostatic activity that is mediated through diffusion through the bacterial cell wall and prevents bacterial peptide bond and protein synthesis by binding to the 50S ribosomal subunit (11).

Current Treatment of Choice

Currently, the standard treatment following N. meningitidis diagnosis is a third-generation cephalosporin (mechanism described above), which is effective against both penicillin-sensitive and penicillin-resistant strains (2, 3). In British Columbia, ceftriaxone is the third-generation cephalosporin used and is administered intravenously. Ceftriaxone has the advantage of more convenient IV dosing, only being given every 12 hours while penicillin requires a dose every 6 hours (12). The recommended duration of antibiotic therapy for N. meningitidis 7 days, but will need to be modified to the individual based on clinical response (2, 13).

References

1. Communicable Disease Control. BC Centre for Disease Control. 2017; Available at: http://www.bccdc.ca/health-professionals/clinical-resources/communicable-disease-control-manual/communicable-disease-control. Accessed February 10, 2021.
2. Ala'Aldeen DAA, Turner DPJ. Neisseria meningitidis. In: Gillespie SH, Hawkey PM, editors. Principles and Practice of Clinical Bacteriology: John Wiley & Sons; 2006. p. 205-220.
3. Schmitz JE, Stratton CW. Chapter 98 - Neisseria meningitidis. In: Tang Y, Sussman M, Liu D, Poxton I, Schwartzman J, editors. Molecular Medical Microbiology 2nd Ed. Boston: Academic Press; 2015. p. 1729-1750.
4. Fernandez, R. Antibiotics that affect the cell wall: Mechanisms of resistance [unpublished lecture notes]. MICB308: Paradigms in bacterial pathogenesis, Vancouver: University of British Columbia; lecture given 2021 Jan 15.
5. Lemke TL, Williams DA, eds. (2013). Foye's Principles of Medicinal Chemistry (Seventh ed.). Philadelphia, PA: Lippincott Williams & Wilkins. pp. 1093–1094, 1099–1100. ISBN 9781609133450.
6. Arumugham VB, Cascella M. Third Generation Cephalosporins. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2020.
7.  H EB, M L, R B. Diagnosis and treatment of bacterial meningitis. Arch Dis Child. 1136;2003;88(7):615-620:88 7 615.
8. Hoffman O, Weber RJ. Pathophysiology and treatment of bacterial meningitis. Ther Adv Neurol Disord. 2009;2(6):1–7.
9. Manchanda V, Gupta S, Bhalla P. Meningococcal disease: History, epidemiology, pathogenesis, clinical manifestations, diagnosis, antimicrobial susceptibility and prevention. Indian J Med Microbiol 2006;24(1):7-19.
10. Nadel S. Treatment of Meningococcal Disease. Journal of Adolescent Health. 2016 Aug 1;59(2, Supplement):S21–8.
11. PubChem. Chloramphenicol [Internet]. [cited 2021 Feb 12]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/5959
12. American Academy of Pediatrics. Meningococcal infections. In: Red Book: 2018 Report of the Committee on Infectious Diseases, 31st ed, Kimberlin DW, Brady MT, Jackson MA, Long SS (Eds) (Eds), American Academy of Pediatrics, Itasca, IL 2018. P.550.
13. Tunkel AR, Hartman BJ, Kaplan SL, Kaufman BA, Roos KL, Scheld WM, et al. Practice Guidelines for the Management of Bacterial Meningitis. Clin Infect Dis 2004;39(9):1267-1284

iv. Why did the doctor ask about Mary’s vaccination history? Is this a reportable communicable disease?

Several vaccines are administered to young children and adolescents to prevent meningococcal meningitis with different countries using different vaccine types. N. meningitidis has twelve serogroups, six of which are capable of causing epidemics (serogroups A, B, C, W, X, and Y) and different vaccine types protect against different combinations of serogroups (1). In the United States, the meningococcal conjugate vaccine (Men-ACWY) protects against serogroups A, B, C, W, and Y and the meningococcal B vaccine (Men-B) protects against serogroup B. Each vaccine is administered to a different age group; young children and adolescents aged 11-16 have developing immune systems and are given the Men-ACWY vaccine with one dose at 11 years and a booster at age 16. Teenagers age 16-18 are given the Men-B vaccine (2). In Canada, infants are given the meningococcal C vaccine (Men-C) between the age of 1 and 2. Young children may receive a dose of the Men-C vaccine in elementary school, but many provinces in Canada like British Columbia will administer the quadrivalent Men-ACWY vaccine to students in grade 9 (3).

If we assume that Mary was raised in Canada, her medical history states that her last reported series of vaccinations was in elementary school; as a result, we can deduce from the Canadian childhood vaccination schedule that she received at least the Men-C vaccine but has most likely missed her Men-ACWY vaccine. As a result, this makes her highly susceptible to infection from every serogroup of N. meningitidis.

Meningitis caused by N. meningitidis infection is incredibly contagious. Meningococcal meningitis is listed by the BC Centre for Disease Control as a reportable communicable disease (4) . This classification means that there is a reporting requirement for cases and contact tracing of the patient must be performed to identify individuals at-risk for infection. Bacterial meningitis can spread respiratory droplets, contact with respiratory fluids such as, kissing, sharing food and water, sharing a toothbrush, etc (1,5–7). High-density residential spaces, such as college dorms, dramatically increase the risk of infection in unprotected individuals (WHO). While the bacteria can occupy the nasopharynx of an infected individual, spreading the disease through sneezing or coughing is more difficult (8). Once a positive CSF or blood sample of N. meningitidis is confirmed, the serotype in question is recorded and public health agencies are notified of a case and work can be done to mitigate the spread of the disease (5,7). Close contacts of an infected individual who have interacted with the patient seven days prior to infection are notified and are required to undergo prophylactic antibiotic treatment for up to ten days. The current series of prophylactic antibiotics include penicillin, ceftriaxone, ciprofloxacin, and rifampin (5). Prophylactic treatment is intended to prevent infection regardless of a close contact’s immunization status.  

A roadmap from the identification of a positive sample of N. meningitidis to deploying prophylactic treatment to close contacts

In Mary’s context, her living situation in her college dorm is one with a high density of young adults who may share the same washroom and shower facilities. This makes it very likely that an outbreak could occur in Mary’s dorm and the surrounding student housing community. After Mary has been diagnosed, close contacts such as Mary’s roommate, Mary’s Residence Advisor, and anyone who she had met or interacted with during the Move-In and settling period. As Mary is moving into a new dorm, it is very likely that she has interacted with a lot of her floormates and other people living in the building as a normal university community building activity. Thus, it is imperative that public health agencies notify Mary’s university so they can take early action in mitigating the spread of N. meningitidis through methods such as quarantining the entire dorm, closing the dorm and relocating any students not at-risk, and prescribing the 10-day prophylactic antibiotic treatment to everyone residing in the dorm building.

References

1. WHO | Meningococcal meningitis [Internet]. WHO. World Health Organization; [cited 2021 Feb 26]. Available from: http://www.who.int/immunization/diseases/meningitis/en/
2. Meningococcal Vaccination | CDC [Internet]. Centers for Disease Control and Prevention. 2021 [cited 2021 Feb 26]. Available from: https://www.cdc.gov/vaccines/vpd/mening/index.html
3. Canada PHA of. Meningococcal vaccine: Canadian Immunization Guide [Internet]. Government of Canada. 2007 [cited 2021 Feb 26]. Available from: https://www.canada.ca/en/public-health/services/publications/healthy-living/canadian-immunization-guide-part-4-active-vaccines/page-13-meningococcal-vaccine.html
4. British Columbia G of. List of Reportable Communicable Diseases in BC [Internet]. BC Centre for Disease Control; 2021. Available from: http://www.bccdc.ca/Documents/BC%20Reportable%20Disease%20List.pdf
5. Meningitis | About Bacterial Meningitis Infection | CDC [Internet]. 2019 [cited 2021 Feb 26]. Available from: https://www.cdc.gov/meningitis/bacterial.html
6. Meningitis - Symptoms and causes [Internet]. Mayo Clinic. [cited 2021 Feb 26]. Available from: https://www.mayoclinic.org/diseases-conditions/meningitis/symptoms-causes/syc-20350508
7. Canada PHA of. Invasive Meningococcal Disease [Internet]. Government of Canada. 2014 [cited 2021 Feb 26]. Available from: https://www.canada.ca/en/public-health/services/immunization/vaccine-preventable-diseases/invasive-meningococcal-disease/health-professionals.html
8. Exley RM, Sim R, Goodwin L, Winterbotham M, Schneider MC, Read RC, et al. Identification of Meningococcal Genes Necessary for Colonization of Human Upper Airway Tissue. Infect Immun. 2009 Jan 1;77(1):45–51.

Q2: The Microbiological Laboratory

i. Other than the stated bacterial cause, what are the most common bacterial pathogens associated with this infectious scenario.

Meningitis is the inflammation of the meninges, specifically the arachnoid and the pia mater, which are the fluid membranes that envelope the brain and spinal cord (1, 2). Meningitis can be caused by various pathogens, such as viruses, parasites, fungi and bacteria (2). Many types of bacteria can cause bacterial meningitis, the most common are Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae and Listeria monocytogenes (1, 2). The current hypothesis is that the bacteria enter the central nervous system (CNS) to cause meningitis using two different methods (1). The first method is that the infection causes bacteremia, which allows the bacteria to spread to the CNS from the bloodstream (1). The second method is direct access to the CNS through injuries to the dura mater or local infections, such as sinus or middle ear infections (1, 3). Once the bacteria is within the CNS and spinal cord, they multiply and, depending on the bacterium, they induce inflammation using various strategies such as toxins and the activation of the immune system (4).

Probable Pathogens Causing Bacterial Meningitis (Hoffman & Weber, 2009)

The inflammation of the CNS and spinal cord as a result of meningitis may cause symptoms such as headaches, fever, a stiff neck, photophobia (sensitivity to light), nausea, joint pain, seizures and symptoms of shock (ex. low blood pressure, rapid heartbeat, difficulty breathing and low urine output) (2). Mary was immediately treated with antibiotics because she was showing signs and symptoms of meningitis, such as fever, chills, headache, stiff neck, low blood pressure and photophobia (4). Bacterial infections causing meningitis can be fatal or cause severe complications, therefore, early treatment with antibiotics is critical (1, 4). Mary was diagnosed with meningococcal meningitis after laboratory techniques confirmed the presence of Neisseria meningitidis within her blood and cerebrospinal fluid (CSF).

Neisseria meningitidis (meningococcus) is a gram-negative diplococcus with an antiphagocytic polysaccharide capsule that causes both sporadic and epidemic disease in humans (5). N. menigitidis is the leading worldwide cause of bacterial meningitis and sepsis in otherwise healthy individuals (5). N. meningitidis is transmitted by direct contact with nasal or oral secretions or by inhalation of large droplet nuclei (5). In most individuals, N. meningitidis asymptomatically colonizes the upper respiratory tract (5). However, in some individuals, meningococci colonize the upper respiratory mucosal surfaces and may spread to nearby mucosal surfaces, such as the lower respiratory tract, and cross the mucosal barrier to enter the bloodstream (5). If the bacteria is not cleared, the infection may spread to the CNS via the bloodstream to cause meningitis (5). The induction of the local inflammatory response in the CNS and the release of the N. meningitidis endotoxin lipooligosaccharide (LOS) results in the inflammation of the CNS, causing meningitis (5, 6).

Meningococci are typically classified by their capsular polysaccharides, into 12 serogroups (7). Capsular serogroups A, B, V, C, Y and W-135 are the most common serogroups of N. meningitidis that cause invasive meningococcal disease resulting in meningitis, therefore, it is likely that Mary is infected with one of these serogroups (5). N. meningitidis causes a highly contagious infection that mostly affects teenagers and young adults; it can cause epidemics within university dorms, boarding schools and military bases (2). The Centre for Disease Control (CDC) recommends a meningococcal conjugate vaccine protecting against serogroups A, C, W and Y for all preteen and teens 11 to 12 years old with a booster dose at 16 years old and children and adults at increased risk for meningococcal disease (ex. immunocompromised) (8). In the case study, the patient states that she has not had any vaccinations since she was in elementary school, therefore, it is plausible that she did not receive her meningococcal booster vaccine when she was 16 years old, resulting in her having a higher chance of contracting meningococcal disease (2). In addition, Mary has just moved into her dormitory which is a common location for local N. meningitidis epidemics (2). Mary’s signs and symptoms, key history of skipped vaccinations and living situation supports her diagnosis of meningococcal meningitis.

Before Mary was diagnosed with meningococcal meningitis from laboratory diagnostic techniques, the physicians may have predicted that she was infected with a variety of other bacteria that most commonly cause meningitis such as Streptococcus pneumoniae, Haemophilus influenzae and Listeria monocytogenes (1). S. pneumoniae is a gram-positive diplococcus with an anti-phagocytic polysaccharide capsule that can cause pneumonia, bacteremia, meningitis, sinusitis, otitis media and bacterial respiratory infections (3). S. pneumoniae is a typical member of the respiratory tract flora; the invasion of the respiratory mucosal surfaces results in pneumonia (9). Meningitis caused by S. pneumoniae may result from bacteremia or by direct access to the CNS from infection in the sinuses or middle ear (3). Once S. pneumoniae is in the CNS, its ability to escape phagocytosis and induce inflammation results in the condition of meningitis (3). Pneumococcal disease is most common in young children, therefore, the CDC recommends the Pneumococcal conjugate vaccine (PCV13) for children younger than 2 years old which protects against the thirteen types of pneumococcal bacteria (10). Based on the information that Mary was last vaccinated in elementary school, it is likely that she has been immunized against S. pneumoniae.

Haemophilus influenzae is a small, gram-negative, pleomorphic cocci bacilli and a pathogen that is usually found within the upper respiratory tract (11). The Type B strain of H. influenzae can cause meningitis, epiglottitis and other invasive infections in infants and children (11). Type B spreads from one individual to another by direct contact or via secretions and/or aerosol (12). H. influenzae type B colonizes the nasopharynx and, with the help of the polysaccharide capsule composed of polyribosylribitol phosphate (PRP), the bacterium may disrupt the epithelium and capillary endothelium to cause bacteremia (11, 12). Meningitis caused by H. influenzae may be a result of bacteria in the bloodstream reaching the CNS or directly from lymphatic drainage from the upper respiratory tract (12). However, most cases of H. influenzae meningitis in adults are a result of non typable strains of H. influenzae (12). Non-typable strains are unencapsulated and cannot invade the capillaries, therefore, they enter the CNS by direct access from infection of the sinuses or middle ear or trauma to the dura (12). The CDC recommends a H. influenzae (Hib) vaccine for all children younger than 5 years old (13). H. influenzae is most common in young children, therefore, it is not recommended that older children and adults receive the vaccine unless they are immunocompromised (13). It is likely that Mary received her Hib vaccine when she was a young child.

Listeria monocytogenes is a gram-positive bacillus and a zoonotic foodborne pathogen (14). The bacteria can be found in food such as unpasteurized cheeses and lunch meats (2). L. monocytogenes can cause self-limited gastroenteritis or more severe, invasive disease such as meningitis that most commonly affects pregnant women, newborns and individuals with cell-mediated immunodeficiencies (14). Unlike other bacteria that cause meningitis, L. monocytogenes has tropism for the brain itself as well as the meninges, therefore, infection of these areas by L. monocytogenes causes inflammation and meningitis (14). The patient from the case study, Mary, is an otherwise healthy 18 year old girl that does not show any of the risk factors for contracting meningitis from L. monocytogenes.

References:

1.     Hoffman O, Weber J. Review: Pathophysiology and treatment of bacterial meningitis. Ther Adv Neurol Disord. 2009;2(6):401-412.

2.     Meningitis - Symptoms and causes [Internet]. Mayo Clinic. 2020 [cited 13 February 2021]. Available from: https://www.mayoclinic.org/diseases-conditions/meningitis/symptoms-causes/syc-20350508#:~:text=Meningitis%20is%20an%20inflammation%20of,fever%20and%20a%20stiff%20neck

3.     Janoff EN, Musher DM. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 9th ed. Philadelphia, PA: Elsevier; 2020. Streptococcus pneumoniae.

4.     Signs of bacterial meningitis | Ada [Internet]. Ada. 2020 [cited 13 February 2021]. Available from: https://ada.com/conditions/bacterial-meningitis/

5.     Tzeng Y, Stephens D. Epidemiology and pathogenesis of Neisseria meningitidis. Microb Infect. 2000;2(6):687-700.

6.     Stephens DS. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 9th ed. Philadelphia, PA: Elsevier; 2020. Neisseria menigitidis.

7.     Kenneth Todar M. Pathogenic neisseriae [Internet]. Online Textbook of Bacteriology. [cited 12 February 2021]. Available from: http://textbookofbacteriology.net

8.     Meningococcal Vaccination | CDC [Internet]. Cdc.gov. 2019 [cited 13 February 2021]. Available from: https://www.cdc.gov/vaccines/vpd/mening/index.html#:~:text=CDC%20recommends%20routine%20meningococcal%20conjugate,increased%20risk%20for%20meningococcal%20disease

9.     Patterson MJ. Medical microbiology 4^th edition. Texas: University of Texas Medical Branch at Galveston; 1996. 21, Streptococcus.

10.  Pneumococcal Vaccination | CDC [Internet]. Cdc.gov. 2019 [cited 13 February 2021]. Available from: https://www.cdc.gov/vaccines/vpd/pneumo/index.html

11.  Murphy TF. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 9th ed. Philadelphia, PA: Elsevier; 2020. Haemophilus species, including H. influenzae and H. ducreyi.

12.  Musher DM. Medical microbiology 4^th edition. Texas: University of Texas Medical Branch at Galveston; 1996. 21, Haemophilus species.

13.  Hib Vaccination | Haemophilus Influenzae Type b | CDC [Internet]. Cdc.gov. 2018 [cited 13 February 2021]. Available from: https://www.cdc.gov/vaccines/vpd/hib/index.html

14.  Johnson JE, Mylonakis E. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 9th ed. Philadelphia, PA: Elsevier; 2020. Listeria monocytogenes.

ii. What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?

The Microbiology Lab plays a central role in the presumptive identification and surveillance of bacterial meningitis. It has been specified that Mary’s blood and cerebral spinal fluid (CSF) were collected and sent to the Microbiology Laboratory.  Ideally, these are to be collected prior to administering antimicrobial therapy to ensure the causative bacteria remains viable. The bacteria that most-often cause meningitis (N. meningitidis, S. pneumoniae, and H. influenzae) are fastidious and fragile organisms, and the proper collection and transport of specimens is required for their positive identification (1, 2).

Blood collection

Figure 1. Collection of CSF by lumbar puncture. (US CDC)

A blood sample is collected if there is suspected bacteriemia. The volume of blood collected is 5-10mL for adults and 1-3mL from children. The blood sample is collected using a glass tube, with no anti-coagulant component present. For this reason, the collected blood is immediately added to blood culture broth for microbiological culture. Suitable media for blood culturing are trypticase soy broth (TSB) or brain heart infusion (BHI) broth, although these require specific supplementation to support the growth of the fastidious organisms. A chemical inhibitor such as 0.025% sodium polyanetholesulfonate should also be added to neutralize the antimicrobial components present in the patient’s blood. The inoculated blood broth should be transported directly to the microbiology laboratory or incubated at 36 ± 1^oC with 5% CO₂ until this is possible.

CSF collection

The collection of cerebral spinal fluid (CSF) is an invasive procedure involving puncture of the patient’s lumbar spine and should be performed by trained individuals in a sterile environment. Ideally 3mL is collected and split into three different 1mL tubes for chemical, microbiological, and cytological analysis. If only 1mL can be obtained from the patient it should be prioritized for the microbiology lab. Although, blood contamination present in the first tube may potentially interfere with the downstream microbiological culture; if possible, the second or third collection tube should be given to the microbiology lab. Once the CSF has been collected, the sample must be delivered to the microbiology lab as soon as possible, optimally within one hour. If is not possible, the CSF sample may be inoculated into Trans-Isolate (T-I) medium and stored at 36 ± 1^oC with 5% CO₂. T-I medium is the preferred medium for culturing the bacterial causes of meningitis. Once received by the microbiology laboratory, the sample is examined by plating onto chocolate or blood agar media and through additional biochemical methods.

Importance of the Microbiology Laboratory

Figure 2. Flow chart of CSF specimen processing (US CDC)

Rapid diagnosis and treatment

By rapidly identifying the infectious agent, the microbiology lab assists in promptly decerning and administering the appropriate antibiotics to treat Mary’s disease. Through the development of rapid diagnostic tests, such as the PCR-based BioFire ME Panel, the microbiology lab is able to identify N. meningitidis as the causative agent within approximately one hour (3). By quickly diagnosing and treating Mary’s illness, the laboratory can now also play a role in providing swift contract tracing, as any intimate contacts of Mary’s will require antibiotic prophylactic treatment. Bacterial meningitis can be treated immediately with intravenous antibiotics and sometimes corticosteroids, depending on the type of bacteria. Drainage of infected sinuses or mastoids can also be done. Aseptic meningitis can be treated with bed rest, fluids, and pain medication as it typically improves in several weeks. Meningitis due to autoimmune disease may be treated with corticosteroids. There are other types of meningitis with their own respective treatments and therefore, identifying the underlying cause is important. In Mary’s case, she had bacterial meningitis and will receive treatment and antibiotics specific to N.meningitidis. This will include penicillin, ampicillin, or a combination of penicillin and chloramphenicol (4,5).

Surveillance

The microbiology lab collects species and serotype information to inform public health decisions. Importantly, by tracking serotype or serogroup circulation in the population, this can help inform public health responses through the implementation of specific vaccine programs. This is the case with the successful men-C and men-ACYW vaccine programs in Canada (6). Alternatively, to advise which antibiotics to administer depending on current trends in antimicrobial resistance, working to prevent the emergence of resistant infection on both individual and community levels (2).

References:

1. Popovic T, Ajello G, Facklam R. Laboratory manual for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. [Geneva]: World Health Organization; 1999.
2. Centers for Disease Control and Prevention. Laboratory Methods for the Diagnosis of Meningitis Caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. 2^nd edition.
3. Leber AL. et al. Multicenter Evaluation of BioFire FilmArray Meningitis/Encephalitis Panel for Detection of Bacteria, Viruses, and Yeast in Cerebrospinal Fluid Specimens. Journal of Clinical Microbiology Aug 2016, 54 (9) 2251-2261; DOI: 10.1128/JCM.00730-16
4. Negi S, Grover S, Rautela S, Rawat D, Gupta S, Khare S, et al. (2010). Direct detection and serogroup characterization of Neisseria meningitidis from outbreak of meningococcal meningitis in Delhi. Iran J Microbiol. 2(2):73–9.
5. Andrew J. Pollard, Gary Probe, Colleen Trombley, Annette Castell, Sue Whitehead, J. Mark Bigham, Sylvie Champagne, Judy Isaac-Renton, Rusung Tan, Malcolm Guiver, Ray Borrow, David P. Speert, Eva Thomas. (2002). Evaluation of a Diagnostic Polymerase Chain Reaction Assay for Neisseria meningitidis in North America and Field Experience During an Outbreak. Arch Pathol Lab Med. 126 (10): 1209–1215.
6. Public Health Agency of Canada. 2015. Meningococcal vaccine. Canadian Immunization Guide. http://www.phac-aspc.gc.ca/publicat/cig-gci/p04-meni-eng.php.

iii. Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.

There are several different tests that can be used to identify and differentiate the bacteria from other serotypes.

Spinal Tap Procedure

A cytological examination of the CSF, which is the analysis of cells using a microscope to determine the structure and function of cells, may first be performed for a general diagnosis of bacterial meningitis (1, 2). Cellular irregularities correlated with bacterial meningitis include turbidity, increased opening pressure (>180 mm water), pleocytosis; WBC counts >10 cells/mm^3, decreased glucose concentration (<45 mg/dl) and increased protein concentration (>45 mg/dl) (1).

The analysis of the CSF for white blood cell, color, glucose levels, and protein levels can be performed to detect infection. Blood cells can be counted manually or using an instrument such as GloCyte or hemocytometer (3).  To measure glucose and total protein concentrations, immunochemistry analysis can be done using machines such as the Cobas 6000 c501 biochemistry analyzer with reagents GLUC3 and TPUC3 for glucose and protein, respectively (4). Changes in glucose and protein concentrations can be observed after infection. Therefore, this is a quick and efficient way to detect the presence of infection.

Microbiological Cultures

Proper streaking and growth of N. meningitidis on a BAP.

In clinics, primary culture is the gold standard for case confirmation of the bacterial pathogen causing disease within the patient (1). Primary culture is the process in which samples of bacteria that have been isolated from a patient's fluids, which in this case is CSF, are plated on various media (1). Common primary culture media is a blood agar plate (BAP) and a chocolate agar plate (CAP) (1). The growth of the isolated bacteria on certain media can give a clue to which bacteria is present, for example, N .meningitidis grows on both blood agar plate and chocolate agar plate while H. influenzae only grows on chocolate agar plate (1). In addition, primary culture provides valuable information about the structure and morphology of the isolated bacterium which can help with the diagnosis (1).

Bacterial isolation from the CSF can be performed.  First, the CSF should be concentrated by centrifugation (5), a process that separates particles based on their density. A portion of the sediment should then be cultured on chocolate or blood agar plate (5). Chocolate agar contains blood that has been heated and lysed to release factors that aid in the growth of bacteria (6). Blood agar is an enriched medium that is made using peptone, beef extract, agar, salt, and distilled water (7). Both plates serve to increase the amount of N. meningitidis while suppressing growth of others. The plates should be incubated in a candle jar or CO2 incubator (5). Medium to large, blue-gray mucoid convex colonies form in 48 hours at 35-37 degrees Celsius (8). After isolation is complete, other experiments to select for specific bacteria, or increase population size, should be performed

Selective media can be used to improve the isolation of the desired bacteria. For primary isolation of N. meningitidis, a chocolate agar base containing vancomycin, colistin, nystatin and trimethoprim can be used (8). Vancomycin, colistin, and trimethoprim are antibiotics, while nystatin is an antifungal medication that purifies the isolation. Other drugs such as penicillin, amikacin, ampicillin, nalidixic acid, chloramphenicol, cefotaxime, ceftriaxone, imipenem, ofloxacin, moxifloxacin, cefuroxime and ciprofloxacin can be used to select for N.meningitidis (9).

After a purified sample is obtained, N.meningitidis can be identified using Gram-staining, oxidase test and carbohydrate utilization reactions.

Flow Chart for Identification and Characterization of a N.meningitidis Isolate (Popovic T, Ajello G & Facklam R, 1999)

Gram-staining Test

Gram-staining differentiates gram-positive bacteria from gram-negative bacteria. A gram-positive bacterium has thicker cell walls while a gram-negative bacterium has a thinner cell wall with an outer membrane (10). The Gram stain differentiates between gram-positive and gram-negative bacteria through colors which are purple and pink respectively. First, a crystal violet is used which dyes everything purple using the gram’s iodine mixture which allows it to adhere to gram-positive cells (10). Then, an acetone-alcohol mixture is applied that washes away the stain from everything except the gram-positive organisms. The safranin is the counterstain that smears all successfully washed cells pink (10).

Kovac’s Oxidase Test

The oxidase test detects the presence of a cytochrome oxidase system. If positive, the reagent N, N, N’, N’-tetramethyl-p-phenylenediamine dihydrochloride turns blue due to oxidation indicating the presence of a cytochrome c system (11). If negative, the reagent remains colorless (11). The process can be done by soaking a strip of filter paper with 1% reagent, rubbing a speck of the purified bacterial culture on it, and noting the changes (11). This test helps with the identification of N. meningitidis, however, other members of the Neisseria genus may give a similar reaction (1).

Carbohydrate Utilization Test

Carbohydrate utilization, the cystine trypticase agar (CTA) method is used to confirm that the isolate is N.meningitidis, and not another member of the Neisseria genus (1). A carbohydrate utilization test is used to determine whether a bacterium can utilize a certain carbohydrate by testing for the presence of gas and/or acid produced from fermentation (12). The production of acid changes the pH and can be detected by a pH indicator. Typically, phenol red carbohydrate broths are used for carbohydrate fermentation tests where the carbohydrate itself can vary (12). In the case of phenol red, as the broth gets more acidic, the indicator turns red (12). Depending on the pH indicator used though different colors can be observed. While testing for the presence of N. meningitidis, glucose, maltose, lactose and sucrose should all be used in separate broths.

Slide Agglutination Serogrouping (SASG) Test

If the carbohydrate utilization test demonstrates that the isolate from the patient’s sample is N. meningitidis, serological tests to identify the serogroup should be completed (1). As stated previously, N. meningitidis strains are separated into 12 serogroups based on their capsular polysaccharide, however, the serogroups A, B, C, W135 and Y are the most common causes of meningitis (1). Depending on the resources and time available, it may not be efficient to test for all serogroups, therefore, the choice of serogroups tested may be based on knowledge of the prevalence of a certain serogroups within a particular region (). For example, in North America, Europe and Australia, serogroups B and C are the most common, while serogroup A is prevalent in Africa and Asia (13).

Slide agglutination serogrouping (SASG) tests are used to identify the serogroup of N. meningitidis samples (1). In SASG tests, the isolate is sectioned on the slide with as many sections required for each serogroup and the serogroup-specific antisera is added to the glass slide (14). After 2 minutes, the SASG are examined and rated based on the intensity of agglutination (1). A positive result is indicated by a level 3 or 4+, meaning strong agglutination, except for serogroup B that is determined to be positive with a rating of 2 or higher (1).

Classification of N. meningitidis into Serotypes and Subtypes: Monoclonal Antibodies

Further classification of N. meningitidis into serotypes and serosubtypes can be determined via monoclonal antibodies (Mabs) (1). It may be important to identify serotypes of N. meningitidis if there is a prevalent spread of a specific, highly pathogenic serotype (14). Serotypes of N. meningitidis are classified based on two major outer membrane proteins PorB and PorA (14). In this method, whole cell suspensions of the isolate are added to strips and then mixed with Mabs specific for the outer-membrane antigen in a well (14). The strips are then graded for the staining intensity of each dot (positive, weak or negative) correlated to each suspected serotype (14).

Latex Agglutination Test

The Latex agglutination test is a method to check for certain antibodies or antigens in body fluids (15). CSF or blood is mixed with latex beads coated with a specific antibody or antigen and if the pathogen is present, the beads will agglutinate (15). Commercially available kits could be used where one drop of CSF is suspended in the specific latex antisera for serogroups A, B, C, D, W135, Y and mixed and tested for agglutination (15).

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a technique that uses nucleic acid primers to bind to specific nucleic acid sequences unique to the bacteria, in this case N. meningitidis (16). By amplifying small segments of DNA, one can visualize the chain length which is otherwise difficult. To complete a PCR, the sample is first heated to allow the DNA to denature or separate into two single strands of DNA (16). Next, a Taq Polymerase enzyme builds two new strands of DNA using the original strands, and the process continues (16). The entire process takes a few hours to complete (16).

There are many possible PCR primers one can use to amplify certain genomes specific to N. meningitidis. One such set of primers included the ctrA forward and reverse primers were used (17). The ctrA gene encodes a conserved outer membrane protein involved in capsular polysaccharide transport (17). The siaD gene was also amplified using the forward and reverse primers siaD B, siaD C and siaDY/W135 (17). The siaD gene contains sequences that can be amplified to allow serogroup discrimination which is important for treatment (17).

Gel electrophoresis is then used to identify the serotype based on the location of the band on the gel where smaller bands of DNA move faster than larger bands. In the case of the above genes, the ctraA gene had a product size of 111, the siaD B gene had a product size of 457, the siaD C gene had a product size of 442 and the siaD/Y/W135 had a product size of 698 (17). Orf-2of myn B gene can be used for Serogroup A, and it has a base pair of 400 (18).

By understanding the proteins on the bacteria, and creating the proper primers, one can use PCR to determine the identity of the bacterium.

Rapid Meningococcal Meningitis Diagnostic Tests

Although presumptive identification of a bacterium, such as N. meningitidis, can be made based on the diagnostic information provided by a gram stain and primary culture, other tests such as rapid meningococcal meningitis diagnostic tests may be performed (1). Rapid meningococcal meningitis diagnostic tests are inexpensive and easily produced tests that can relatively accurately determine the serogroup (commonly invasive serogroups A, C, W135 and Y) of the suspected sample of N. meningitidis (1). The test uses the principle of flow immunochromatography in which gold particles and nitrocellulose are covered with monoclonal antibodies that bind to serogroup-specific polysaccharide antigens in the sample of CSF (1). Two dipsticks (RDT1 test for serogroups A and W135/Y and RDT2 tests for serogroups C and Y) are placed within 2 separate tubes of the CSF (1). Red lines on the dipstick will demonstrate whether one of the four main invasive N. meningitidis serogroups are present in the CSF samples (1).

MALDI-TOF

Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid, sensitive, and economical procedure used to identify bacteria using either intact cells or cell extracts (19). The sample is placed in a UV-absorbing matrix pad and exposed to a laser pulse where Desorption and ionization with a laser beam generates protonated ions from the sample (19). The protons are accelerated off the matrix pad and move towards a detector where they are detected and measured using different types of mass analyzers like quadrupole mass analyzers, ion trap analyzers and, time of flight (TOF) analyzers (19). Individual signals are separated by their respective mass to charge ratio (19). Based on the TOF information, a peptide mass fingerprint (PMF) is generated for the sample (19). Identification of microbes by MALDI-TOF MS is done by either comparing the PMF of an unknown organism with the PMFs contained in the database, or by matching the masses of biomarkers of unknown organism with the proteome database (19).

To perform MALDI-TOF analysis, proteins should be extracted according to manufacturer instructions which will be different for each machine. But an example protocol is discussed below. After centrifugation of the CSF, blood cells should be removed through aspiration (20). The sample pellets should be washed with deionized water  and ethanol (20). The pellets should be treated with 70% formic acid and 100% acetonitrile to extract bacterial cell components (20). The extract should be centrifuged and taken for analysis by MALDI-TOF (20). A microliter of the supernatant was deposited on the target chip with a matrix solution, which included alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2% trifluoroacetic acid (20). The peak lists which were generated using the MALDI-TOF spectra should be compared with a reference library (20). Reliability scores ranged from 0-3 where scores below 1.7 were unreliable, 1.7-2.0 scores were regarded as genus level identification and scores greater than 2.0 were regarded as species level identification. (20)

References:

1. Popovic T, Ajello G, Facklam R. Laboratory manual for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. [Geneva]: World Health Organization; 1999.
2. Cytologic evaluation: MedlinePlus Medical Encyclopedia [Internet]. Medlineplus.gov. 2021 [cited 13 February 2021]. Available from: https://medlineplus.gov/ency/article/002323.htm
3. Hod EA, Brugnara C, Pilichowska M, Sandhaus LM, Luu HS, Forest SK, et al. (2018). Automated cell counts on CSF samples: A multicenter performance evaluation of the GloCyte system. Int J Lab Hematol. 40(1):56–65.
4. Mlinarić A, Vogrinc Ž, Drenšek Z. (2018). Effect of sample processing and time delay on cell count and chemistry tests in cerebrospinal fluid collected from drainage systems. Biochem Med (Zagreb). 15;28(3):030705.
5. Morse SA. (1996). Neisseria, Moraxella, Kingella and Eikenella. In: Baron S, editor. Medical Microbiology. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 14. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7650/
6. Murray TS, Haycocks N, Fink SL. (2016). Introduction to the Laboratory Diagnosis of Infectious Diseases. In: Head, Neck, and Orofacial Infections. p. 94–102. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780323289450000053
7. Aryal S. (2018) Blood Agar-Compisition, Preparation, Uses and Pictures. Retrieved from: https://microbiologyinfo.com/blood-agar-composition-preparation-uses-and-pictures/
8. Kaiser, G. (2020). Isolation and Identification of Neisseriae, Mycobacteria, and Obligate Anaerobes. Retrieved from: https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_II/Lab_16%3A_Isolation_and_Identification_of_Neisseriae%2C_Mycobacteria%2C_and_Obligate_Anaerobes
9. Negi S, Grover S, Rautela S, Rawat D, Gupta S, Khare S, et al. (2010). Direct detection and serogroup characterization of Neisseria meningitidis from outbreak of meningococcal meningitis in Delhi. Iran J Microbiol. 2(2):73–9.
10. Microscope. (2020). How to do a Gram’s stain test. Retrieved from: https://www.microscope.com/education-center/how-to-guides/grams-stain/
11. Aryal, S. (2018). Oxidase Test-Principle, Uses, Procedure, Types, Result, Interpretation, Examples and Limitations. Retrieved from: https://microbiologyinfo.com/oxidase-test-principle-uses-procedure-types-result-interpretation-examples-and-limitations
12. Tankeshwar, A. (2016). Carbohydrate Fermentation Test: Uses, Principle, Procedure and Results. Retrieved from: https://microbeonline.com/carbohydrate-fermentation-test-uses-principle-procedure-results/
13. Meningitis | Lab Manual | Epidemiology | CDC [Internet]. Cdc.gov. 2016 [cited 13 February 2021]. Available from: https://www.cdc.gov/meningitis/lab-manual/chpt02-epi.html
14. Meningitis | Lab Manual | Id and Characterization of Neisseria | CDC [Internet]. Cdc.gov. 2016 [cited 13 February 2021]. Available from: https://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-nm.html
15. 1.     Negi S, Grover S, Rautela S, Rawat D, Gupta S, Khare S, et al. (2010). Direct detection and serogroup characterization of Neisseria meningitidis from outbreak of meningococcal meningitis in Delhi. Iran J Microbiol. 2(2):73–9.
16. National Human Genome Research Institute (2020). Polymerase Chain Reaction (PCR) Fact Sheet. Retrieved from: https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet
17. Andrew J. Pollard, Gary Probe, Colleen Trombley, Annette Castell, Sue Whitehead, J. Mark Bigham, Sylvie Champagne, Judy Isaac-Renton, Rusung Tan, Malcolm Guiver, Ray Borrow, David P. Speert, Eva Thomas. (2002). Evaluation of a Diagnostic Polymerase Chain Reaction Assay for Neisseria meningitidis in North America and Field Experience During an Outbreak. Arch Pathol Lab Med. 126 (10): 1209–1215.
18. Hod EA, Brugnara C, Pilichowska M, Sandhaus LM, Luu HS, Forest SK, et al. (2018). Automated cell counts on CSF samples: A multicenter performance evaluation of the GloCyte system. Int J Lab Hematol. 40(1):56–65.
19. Singhal N, Kumar M, Kanaujia PK, Virdi JS. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front Microbiol. 2015;6:791.
20. Segawa S, Sawai S, Murata S, Nishimura M, Beppu M, Sogawa K, et al. Direct application of MALDI-TOF mass spectrometry to cerebrospinal fluid for rapid pathogen identification in a patient with bacterial meningitis. Clinica Chimica Acta. 2014 Aug;435:59–61.

iv. What are the results expected from these tests allowing for the identification of the bacteria named in this case.

Spinal Tap

From the spinal tap, CSF appears clear and colorless but during collection, the initial pressure of the sample may have increased, and CSF could be cloudy due to presence of microorganisms (1). As few as 200 white blood cells per mm³ could make the CSF appear cloudy (1). Normally CSF has about 5 WBC per mm³ in adults (1). But in individuals with bacterial meningitis, a WBC count higher than 1000 per mm³ can be seen. Having a white blood cell count less than 100 per mm3 is more common in viral meningitis (2). CSF in bacterial meningitis typically is cloudy due to polymorphonuclear leukocytes (3). Normal protein levels in CSF in adults is typically 18-58 mg/dL and is reached between 6 and 12 months of age (4). In bacterial meningitis an average of 418mg/dL is noted. RBC can contaminate the CSF in a traumatic tap situation, but this can be corrected by subtracting 1mg/dL of protein (5). Generally, CSF glucose is about two thirds of serum glucose measured during the preceding two or four hours in a normal adult but in individuals with bacterial meningitis, these decrease (5). Some studies show that glucose levels do not change despite infection, so glucose should not be used in isolation for diagnosis (5). Therefore, Mary’s sample of CSF are expected to demonstrate the indicated abnormalities associated with meningitis because she is infected with N. meningitidis.

Gram stain of N. meningitidis in CSF with associated PMNs.

N. meningitidis colonies on a CAP.

Microbiological Culture

In order to identify N. meningitidis, the bacteria must be able to grow on BAP and CAP at 35-37°C with ~5% CO₂. Colonies from BAP will be round and grey, and on CAP will appear to be colorless-to-grey opaque colonies, which distinguishes the bacteria from the other potential bacterium (6).

Gram-staining test

The presence of oxidase-positive colonies and Gram-negative diplococci provides a presumptive identification of N meningitidis (5). N. meningitidis are gram-negative bacteria, therefore, the bacteria isolated from Mary’s CSF should not be able to retain the primary stain and instead be stained with the counterstain, which is red or pink (17, 19).

Table 1. Carbohydrate utilization by some Neisseria and Moraxella spp.

Kovac’s and carbohydrate utilization

Kovac's oxidase test: a negative and positive reaction on filter paper.

Kovac’s oxidase test determines the presence of cytochrome oxidase which will be present in bacteria that use cytochrome c as part of their respiratory chain (17). As N. meningitidis uses cytochrome c as part of their respiratory chain, N. meningitidis will cause the oxidase reagent to turn purple. However, other members of the Neisseria family may give a positive reaction, so it is important to follow up a positive test with carbohydrate utilization which can validate the identity of the strain. 4 tubes with different carbohydrates (glucose, maltose, lactose and sucrose) are used with a phenol red indicator that turns yellow in the presence of acid at pH 6.8 or less. Since N. meningitidis can oxidize glucose and maltose, but not lactose and sucrose, it will produce acid from oxidizing those specific carbohydrates. There should only be reactions with glucose and maltose, confirming the identity of N. meningitidis (6).

Serotyping

Testing CSF samples with latex agglutination, we would detect ABCY or W 135 antigens for N. meningitidis (7). The results from the ELISA would identify IgG and IgM antibodies that are specific for N. meningitidis cell surface markers (8). For latex agglutination, a positive result, meaning that the latex antibody binds to an antigen in the isolate, is indicated by agglutination of the latex particles and slight clearing of the suspension (9).  Mary is showing signs and symptoms of meningitis, therefore, her sample will have antigens from invasive serogroups of N. meningitidis that will agglutinate with the latex antibodies.

A slide agglutination serogrouping test (SASG) is used to determine which invasive serogroup (A, B, C, W135 or Y) the patient is infected with (9). I am not sure which serogroup of N. meningitidis Mary is infected with, however, one of the SASG test should come back positive which is indicated by an agglutination intensity of 3+ or 4+, except for group B which is considered positive with a rating of 2+ or higher (9).

CTA sugar reactions for N. meningitidis with utilization of glucose (dextrose) and maltose, indicated by acid production (color change to yellow) and no utilization of lactose or sucrose.

Further classification of N. meningitidis into serotypes and serosubtypes can be determined via monoclonal antibodies (Mabs) (9). Similar to the serogroup, I am uncertain which serotype of N. meningitidis Mary is infected with. Since N. meningitidis is present within Mary’s CSF, one of the monoclonal antibody tests should come back positive which is indicated by the dot on the testing strip to be graded as intense relative to the reference strain (9).

PCR

For the RT-PCR assay, we would expect to see 2 genes targeted which are ctrA and sodC. By targeting and identifying these genes in RT-PCR, it can be used to detect N. meningitidis. (6).  The ctrA PCR test has a sensitivity of 93.15% (23) meaning that it can correctly identify the bacteria 93% of the time.

MALDI-TOF

MALDI-TOF is a form of mass spectrometry that can generate a unique protein mass spectrum for the given microorganism, then comparing it to a reference database for rapid identification (15). Using MALDI-TOF for N. meningitidis identification in the laboratory can be unreliable because this species is highly variable, which can be problematic as rapid implementation of treatment is extremely important (15). Shown in figure D is the mass spectra of whole-cell extract for N. meningitidis (16).

Diagnostic Tool/ Test	Purpose	N. meningitidis Results
Gram Stain	Used to distinguish between bacteria into two large groups based on their cell wall morphology.	Gram-negative diplococci; bacteria from sample stain red or pink.
Primary Culture: Blood Agar Plate (BAP)	Primary culture is used for case confirmation and to visualize bacteria morphology.	Grows on BAP. Bacteria appear grey, unpigmented and look round, smooth, moist and convex with a clearly defined edge.
Primary Culture: Chocolate Agar Plate (CAP)	Primary culture is used for case confirmation and to visualize bacteria morphology.	Grows on CAP. Bacteria appear lage, colorless/grey opaque colonies.
Kovac’s Oxidase Test	Determines the presence of cytochrome oxidase which will be present in bacteria that use cytochrome c as their respiratory chain.	Isolated bacteria turns filter paper purple; the bacteria uses cytochrome c as part of their respiratory chain.
Carbohydrate Utilization Test	Used to confirm that the isolate is N. meningitidis, and not another member of the Neisseria genus. Four different carbohydrates (glucose, maltose, lactose and sucrose) are added to test tubes to determine if the isolated bacterium causes their oxidation.	Test tubes containing glucose and maltose turn yellow, indicating the production of acid and oxidation.
Slide Agglutination Serogrouping	Identify the serogroup of N. meningitidis sample.	A positive result is indicated by a level 3 or 4+, meaning strong agglutination, except for serogroup B that is determined to be positive with a rating of 2 or higher.
Monoclonal Antibodies	Classification of N. meningitidis into serotypes and serosubtypes.	High intensity of two major outer membrane proteins PorB and PorA.
Latex Agglutination Test	Check for certain antibodies or antigens in body fluids that may be unique for specific bacteria.	ABCY or W 135 antigens for N. meningitidis.
PCR and Gel Electrophoresis	Uses nucleic acid primers to bind to specific nucleic acid sequences unique to the bacteria to amplify, visualize and identify bacteria type.	Dependent on the primers used. But generally, would expect to see 2 genes targeted which are ctrA and sodC. Serotype grouping can also be done.
Rapid Meningococcal Meningitis Diagnostic Tests	Inexpensive and easily produced tests that can relatively accurately determine the serogroup (commonly invasive serogroups A, C, W135 and Y) of N. meningitidis.	Red lines on the dipstick will demonstrate whether one of the four main invasive N. meningitidis serogroups are present in the CSF samples.
MALDI-TOF	Rapid, sensitive, and economical procedure used to identify bacteria using either intact cells or cell extracts.	Dependant on the reference spectrum used.

References:

1. Seehusen DA, Reeves MM, Fomin DA. Cerebrospinal fluid analysis. Am Fam Physician. 2003 Sep 15;68(6):1103–8.
2. Fishman RA. Cerebrospinal fluid in diseases of the nervous system. 2d ed. Philadelphia: Saunders, 1992.
3. Arevalo CE, Barnes PF, Duda M, Leedom JM. (1989) Cerebrospinal fluid cell counts and chemistries in bacterial meningitis. South Med J. 82:1122–7.
4. Conly JM, Ronald AR. Cerebrospinal fluid as a diagnostic body fluid. (1983). Am J Med. 751B:102–8.
5. Dougherty JM, Roth RM (1986). Cerebral spinal fluid. Emerg Med Clin North Am. 4:281–97.
6. Meningitis | Lab Manual | Id and Characterization of Neisseria | CDC [Internet]. 2019 [cited 2021 Feb 12]. Available from: https://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-nm.html`
7. Mohammadi SF, Patil AB, Nadagir SD, Nandihal N, Lakshminarayana SA. Diagnostic value of latex agglutination test in diagnosis of acute bacterial meningitis. Ann Indian Acad Neurol. 2013;16(4):645–9.
8. Clarke SC, Reid J, Thom L, Denham BC, Edwards GFS. Laboratory confirmation of meningococcal disease in Scotland, 1993–9. J Clin Pathol. 2002 Jan;55(1):32–6.
9. Popovic T, Ajello G, Facklam R. Laboratory manual for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. [Geneva]: World Health Organization; 1999.
10. Negi S, Grover S, Rautela S, Rawat D, Gupta S, Khare S, et al. (2010). Direct detection and serogroup characterization of Neisseria meningitidis from outbreak of meningococcal meningitis in Delhi. Iran J Microbiol. 2(2):73–9.
11. MS may not be reliable. Clin Microbiol Infect. 2019 Jun;25(6):717–22.
12. Ilina EN, Borovskaya AD, Malakhova MM, Vereshchagin VA, Kubanova AA, Kruglov AN, et al. Direct Bacterial Profiling by Matrix-Assisted Laser Desorption−Ionization Time-of-Flight Mass Spectrometry for Identification of Pathogenic Neisseria. J Mol Diagn. 2009 Jan;11(1):75–86.

Q3: Bacterial Pathogenesis

i. Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria

Figure 1. Neisseria meningitidis, a Gram-negative diplococcal bacteria

Neisseria meningitidis, also known as meningococcus, is the leading cause of meningococcal meningitis worldwide [1]. It is part of the genera Neisseria of the Neisseriaceae family of Gram-negative diplococcus bacteria, along with Neisseria gonorrhoeae. The structure and morphology of N. meningitidis is similar to N. gonorrhoeae; however, certain strains also have a polysaccharide capsule [2], which N. gonorrhoeae do not possess. The polysaccharide capsule is an important endotoxin and virulence factor for invasive N. meningitidis strains [2,3]. Invasive N. meningitidis can cause meningococcal disease, which can involve meningococcal meningitis, lethal shock, sepsis, and fever [4]. These types of infections are associated with high rates of morbidity and mortality [5]. Other manifestations of disease include septic arthritis, pneumonia, pericarditis, conjunctivitis, otitis, sinusitis and urethritis [1].

There are some structural differences between the Nesseria meningitidis capsular polysaccharides, but this is as a result of the different serogroups [6]. N. meningitidis includes 12 different serogroups (A, B, C, E, H, I, K, L, W, X, Y, Z), also called “clonal complexes,” that are categorized by genotypes [5]. Meningococcal infection is a global, but not uniform problem (2). The disease patterns vary widely over time, between geographical regions, age groups and bacterial serotypes [7]. Most of the cases worldwide are primarily caused by the six serogroups associated with N. meningitidis: A, B, C, W, X, and Y [8].

Geographical Locations

Approximately 1.2 million cases of N. meningitidis infection occur worldwide each year, with roughly 500,000 of these cases involving invasive meningococci and 135,000 resulting in mortality [5,7]. Although meningococcal disease occurs worldwide, the incidence varies depending on geographic region and climate, as well as the prevalent serogroup in each region. Serogroup A meningococci are the most common cause of epidemics, while serogroups B and C are most likely to cause sporadic infection in developed countries [3].

Figure  2. Areas  with  frequent  epidemics  of  meningococcal  meningitis.

The highest meningococcal meningitis attack rates occur in the “meningitis belt” of sub Saharan Africa, where Serogroups A and X are commonly seen [9]. This belt extends from Ethiopia in the east to Senegal in the west, and includes 18 countries with more than 270 million individuals [7]. The major epidemics of meningococcal disease within this belt occur every 5-12 years, and attack rates can reach 1000 cases per 100 000 people [10]. Serogroup A has been responsible for most of the meningococcal outbreaks within the meningitis belt [10]. However, after the mass vaccinations program for Serogroup A in 2010, the countries within the meningitis belt were able to eliminate the majority of epidemics attributed to this serogroup [10]. Recently, the epidemics in this area have been primarily due to Serogroups C and W [10]. The African meningitis belt has high rates of meningococcal meningitis because of the environmental conditions in this area, which include dry seasons, dust and wind, overcrowding of individuals, and higher rates of underlying conditions. Underlying conditions that can increase the risk of infection include immunosuppressive disorders, such as HIV and AIDs [5]. Outbreaks can occur as endemics, epidemics, or hyperendemics; however, endemics are most common in developed nations [11].

Hot weather climates with low humidity, wind and dusty conditions are known to increase the risk of invasive disease [9] because low humidity and dust causes chronic irritation or damage, which can harm the integrity of the pharyngeal and respiratory epithelium and leave the host vulnerable to infection and disease [12]. As a result, N. meningitidis is able to attach to these mucosal cells using pili and other surface proteins and more easily cross the potentially damaged mucosal barrier to enter the bloodstream to cause disease [9]. It is suggested that encapsulation can also enhance survival of N. meningitidis strains outside of the host [5]. The dry seasons facilitate enhanced transmission of respiratory droplets containing N. meningitidis, as respiratory droplets are at a higher density in drier environments. Consequently, infection rates are lower in rainy seasons, during the summer and fall. Population factors can also increase transmission, including crowded populations, increased travel, and smoking [5]. College campuses are a reported environment where outbreaks have occurred due to close proximity of individuals; for example, serogroup B outbreaks have been reported on college campuses [5].

Figure  3.  Worldwide  serogroup  distribution  of  invasive  meningococcal  disease. 

Epidemics of N. meningitidis occur in other regions of the world as well; however, the outbreaks and overall rates of disease in these areas are much lower in comparison to the meningitis belt in sub-Saharan Africa [10]. Serogroups B, C and Y of N. meningitidis are seen in areas of Europe, North America, and Australia [10]. Serogroup C has occasionally caused large epidemics in developed countries, accounting for 30% of disease in the USA and Europe [7]. Outbreaks of meningitis have been higher in Europe than in the USA, and were mainly due to Serogroup C, before the introduction of their vaccination campaign in 1999. Since then, most of the cases of N. meningitidis in Europe are due to Serogroup B (about 80%) [7]. Currently in the USA, meningococcal disease caused by Serogroup A is very rare, and is mainly due to Serogroups B and C [7]. Recently, Serogroup Y has emerged in the USA and has caused more than 25% of meningococcal disease in the last decade [7]. Epidemic outbreaks in Brazil have been linked to the transition from rainy to dry season and similarly, in the United States and Europe, disease is most common in late winter and early spring [1]. Within Asia, large outbreaks of Serogroup A have occurred in China, Nepal, India, and Russia, but diseases caused by Serogroup B and C are now increasing [7]. The annual Hajj pilgrimage in Saudi Arabia has also been associated with outbreaks of meningitis due to Serogroup W and A [7,10], due to the large amount of individuals in close contact with each other, crowded living conditions, and the large amount of travel associated with this event [9].

The incidence of cases in children and adolescence is rising, which may due to the fact that young adults have an increased number of bacteria in their bodies that produce more propionic acid, and N. meningitidis is known to use this acid to grow. However, advances in prevention and treatment are enhancing control of disease outbreaks. This includes the increased distribution and use of meningococcal conjugate vaccines (MCVs) for serogroups A, C, W, X, and Y, new vaccines being developed for serogroup B, the increased use of targeted antibiotics to reduce the amount of carriers, and a decrease in population risk factors, such as smoking and overcrowding [5]. Challenges still remain, however, in improving accessibility to MCVs in developing countries and preventing a rise in antibiotic resistance in N. meningitidis. Additionally, there is the risk of new invasive strains that can arise; for example, new invasive cases of serogroups C have reportedly emerged in both Africa and China [5].

N. meningitidis remains a worldwide threat, which can be demonstrated by the increase in prevalence of infection by serogroups W, X, and Y, the persistence of serogroup A causing disease in the African meningitis belt, and the continued prevalence of serogroups B and W causing disease in Canada [4,5]. Higher mortality rates are seen in developing countries at approximately 20% of cases, compared to 10 to 15% of cases in industrialized nations. Internationally, morbidity occurs in an additional 11 to 19% of infected individuals, and can include visual and learning impairments, seizures, developmental delays, motor and cognitive dysfunction, scarring, limb and digit loss, and behavioural problems [5]. The prevalence of different serogroups in geographical areas is dynamic and changing over time. The affected age population also varies by serogroup and location; for example, serogroup W strains are becoming more prominent in Canada, mostly affecting elderly populations and adolescents [5]. The specific serogroup of N. meningitidis that is causing Mary’s infection is likely to be influenced by her geographic location, among other factors that will be discussed in this journal.

Host Location

Figure 4. Nasopharynx, the primary location of N. meningitidis reservoirs and initial attachment

Humans are the only known host of N. meningitidis, and the bacteria normally reside in the posterior nasopharynx of the human host [13]. In particular, N. meningitidis attaches to and colonizes the mucosal membranes within the upper respiratory tract. Meningococci can also be a part of the normal non-pathogenic flora in the respiratory tract of healthy individuals [13]. Specifically, N. meningitidis attach to non-ciliated columnar epithelial cells in the nasopharynx through adherence by outer membrane pili and other proteins [2]. Type IV pili (Tfp) are proteinaceous filaments that extend from the bacterial surface and bind host epithelial cell surfaces [9]. Opacity associated adhesion proteins (Opa and Opc) in the outer membrane interact with host receptors to cause tight secondary binding [9]. After this attachment, the bacteria are able to persist in the mucosa by forming meningococcal microcolonies encased in a secreted biofilm matrix [9]. N. meningitidis also has several minor adhesin proteins, such as meningococcal surface fibril (Msf) and meningococcal adhesion and penetration protein (App) that promote binding to epithelial cells in the nasopharyngeal mucosa [9]. Therefore, N. meningitidis is well adapted to colonize human mucosal surfaces. Furthermore, the human nasopharynx is a suitable reservoir for N. meningitidis because it provides optimal conditions for growth, including a CO₂ content of 3 to 4%, high humidity of 75 to 80%, and a temperature of approximately 34°C [5]. Additionally, iron is a mineral found in human hosts that is necessary for the survival, colonization, and disease-causing infection of N. meningitidis. The Fur gene regulatory system of N. meningitidis controls approximately 80 genes involved in adhesion, invasion, and endotoxin production, and the presence of iron is needed to upregulate expression of these genes [4]. Once the bacteria have colonized the nasopharynx of the host, they can also spread into the bloodstream and eventually go and affect the central nervous system, particularly the meningeal layers covering the brain and spinal cord.

Host and Bacteria Contact

The human host will likely come into contact with N. meningitidis bacteria through direct interaction with carriers. Carriers are individuals that have a nasopharynx colonized with these meningococci [14]. Carriers of N. meningitidis can facilitate transmission to other individuals through direct contact with or inhalation of respiratory droplets [5]. It can also be transmitted sexually; however this is less common. It has been estimated that the total number of N. meningitidis carriers worldwide ranges from 230 million to over 1 billion individuals, comprising 3 to 25% of the total human population [2]. Carriers are a major source of transmission and are a necessary factor in any disease outbreak. The carrier usually spreads the meningococcal bacteria through respiratory and throat secretions, such as air droplets or saliva. This often occurs during actions such as kissing, coughing, and hugging, and such contact usually occurs over a lengthy period of time for the bacteria to effectively spread [14]. N. meningitidis enters the host primarily via the inhalation of large respiratory droplets, as there is widespread belief that meningococci are fastidious bacteria that can only survive a few hours on inanimate surfaces [7], even when they are encapsulated [15]. The bacteria often undergo desiccation on surfaces, which is why entry usually occurs via air-borne droplets [15]. Therefore, when an individual breathes in air near someone who is infected, the respiratory droplets containing N.meningitidis can deposit into the nasopharynx region of the body. Infection occurs upon the aspiration of infective particles that attach to epithelial cells in the nasopharyngeal and oropharyngeal mucosa [12]. The bacteria can then cross the musical barrier and can enter the bloodstream, causing meningococcemia [12]. If this systemic infection is not cleared, blood-borne bacteria may enter the central nervous system and cause meningitis [12].

N. meningitidis are not as contagious as pathogens that cause the common cold, and infection with this bacteria likely occurs when individuals live in close proximity to each other [14]. It is plausible that Mary came into close contact with an infected individual on her campus or in her dormitory, allowing transmission via direct contact or inhalation of respiratory droplets. This is because college dormitories have crowded living conditions, as students live in close proximity to each other and often have to share bathrooms [16]. As mentioned before, close contact and crowding of individuals, especially young adults who are known to be common carriers of N. meningitidis, can increase the transmission of this bacteria and the rate of invasive disease [1]. It is likely that an individual living in the same dormitory as Mary was harboring these bacteria and upon close contact with this individual, she would have contracted the pathogen. Since Neisseria meningitis is transmissible through respiratory droplets, close face-to-face contact with an infected individual could easily lead to an infection. Perhaps Mary was exposed to the respiratory, nasal or oral droplets of an asymptomatic carrier of the bacteria via a sneeze, kiss or even a shared utensil [9]. Since she has not had any vaccines since elementary school, it is likely that she does not have a meningococcal vaccine and thus, did not have sufficient bactericidal antibodies to protect her from this infection and disease. It is also possible that she may have a deficient complement system, which would also make her vulnerable to infection [9].

References:

1. Tzeng, Y, Stephens, DS. 2000. Epidemiology and pathogenesis of Neisseria meningitidis. Microbes and Infections 2:687-700. https://doi.org/10.1016/S1286-4579(00)00356-7
2. Pathogenic Neisseriae: Gonorrhea, Neonatal Ophthalmia and Meningococcal Meningitis (Internet). Textbook of Bacteriology. 2021(cited 13 February 2021). Available from: http://textbookofbacteriology.net/neisseria_5.html
3. Baron S. Neisseria – Medical Microbiology. University of Texas Medical Branch at Galveston;1996.
4. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis meningococcemia, and Neisseria meningitidis. 2007. The Lancet 369(9580):2196-210.
5. Bennett JE, Dolin R, Blaser MJ. Mandell Douglas, and Bennett’s Principles and Practice of Infectious Diseases: 2-volume set. Elsevier Health Sciences; 2014 Aug 28.
6. Stephens DS. Neisseria meningitidis. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases [Internet]. 9th ed. Philadelphia, PA: Elsevier; 2020 [cited 2021 Feb 12]. p. 2585-2607.e7. Available from: https://www.clinicalkey.com/?fbclid=IwAR0C9KCpxA2S6Olh7vMNVB91fBsI_8-B0KOV4M5Y6h5VkBQgP3zAfAafkzE#!/content/book/3-s2.0-B9780323482554002113
7. Rouphael, NG, Stephens, DS. 2012. Neisseria meningitidis: biology, microbiology, and epidemiology. Methods Mol Biol. 799:1-20. doi:10.1007/978-1-61779-346-2_1
8. Meningococcal Disease Chapter 8. 2019.Centre for Disease Control and Prevention. https://www.cdc.gov/vaccines/pubs/surv-manual/chpt08-mening.html
9. Scmitz, JE, Stratton, CW. 2015 Chapter 98-Nesseiria meningitidis. In D. Liu, I. Poxton, J. Schwartzman, M. Sussman, Y. Tang. Molecular Medical Microbiology. Elsevier Ltd Nashville, Tennessee https://doi.org/10.1016/B978-0-12-397169-2.00098-6
10. Meningococcal Disease (Meningococcal Disease in Other countries). 2019. Centre for Disease Control and Prevention. https://www.cdc.gov/meningococcal/global.html
11. Feldman C, Anderson R. Meningococcal pneumonia: a review. 2019. Pneumonia 11(1):1-3
12. Morse, SA. 1996. Neisseria, Moraxella, Kingella and Eikenella. In S. Baron (ed.), Medical Microbiology. University of Texas Medical Branch at Galveston, Galveston, Texas.
13. Hill DJ, Griffiths NJ, Borodina E, Virji M. 2010. Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease. Clinical science 118(9):547–564. https://doi.org/10.1042/CS20090513
14. Meningococcal Disease (Causes and Spread to Others). 2019.Centre for Disease Control and Prevention. https://www.cdc.gov/meningococcal/about/causes-transmission.html
15. Tzeng, YL, Martin, LE, Stephens, DS. 2014. Environmental survival of Neisseria meningitidis. Epidemiology and infection, 142(1): 187–190. https://doi.org/10.1017/S095026881300085X
16. Meningococcal (Global in other Countries). 2019. Centre for Disease Control and Prevention. Available from: https://www.cdc.gov/meningococcal/global.html

ii. Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.

N. meningitidis is an obligate commensal of the human nasopharyngeal mucosa (1). About 10% of normal individuals are carriers during endemic disease, but very few ever develop meningococcal disease (2). Carriers are individuals, who are infected and can transmit N. meningitidis to others, but may not show any symptoms (2). This carriage rate increases during epidemics (2). Meningococci is spread through respiratory droplets and oral and nasal secretions (3,4). Person to person transmission can also occur from contact with upper respiratory secretions through activities like kissing and sharing eating utensils (4). Infection occurs upon the aspiration of infective particles that attach to epithelial cells in the nasopharyngeal and oropharyngeal mucosa (3). These infections usually follow an asymptomatic or mildly symptomatic nasopharyngeal carrier state (3). The bacteria then crosses the musical barrier and can enter the bloodstream, causing meningococcemia (3). If this systemic infection is not cleared, blood-borne bacteria may enter the central nervous system and cause meningitis (3).

Entry and colonization is the first step of the disease process and is necessary for bacterial propagation and survival (1). First, N. meningitidis enters the human host through the aspiration of infective particles or close contact with respiratory secretions (2,3). Then, N. meningitidis use surface adhesive proteins on their outer membrane that enable the initial attachment of the bacteria onto the respiratory non ciliated epithelial cells of the nasopharynx (5). These adhesive proteins can be divided into major and minor groups (5). The major adhesins include pili and opacity proteins, and are expressed on the bacterial surface (1).

Type IV Pili (Tfp)

N.meningitidis possess Type IV pili (Tfp), which are surface-exposed fibres on the bacteria that are 6nm in diameter and can aggregate to form bundles (5). Type IV pili is the major adhesin involved in initial attachment to host cells (2). Type IV pili are proteinaceous filaments that extend from the bacterial surface and bind host non ciliated epithelial cell surfaces (2). At the molecular level, different proteins of this multiprotein complex play important roles in entry and adherence of N. meningitidis (1). PilC proteins, PilC1 and PilC2, are required for the formation and assembly of pili (1). Additionally, PilC1 modulates adhesiveness and is necessary for association with host cells (1). Thus, at the molecular level, PilC proteins are key factors in initial adherence (1). Type IV pili bind the host protein CD46, also known as membrane cofactor protein (MCP), on non ciliated columnar epithelial cells (2). After initial attachment, PilT, an active molecular motor molecule, promotes pilus retraction, which mediates twitching motility (1). This force generated by pili retraction is proposed to be the motive force responsible for bringing the bacteria in close association with the host cell (1).This twitching motility is also important for passage through the epithelial mucus layer, movement over epithelial surfaces and microcolony formation (6). The twitching motility allows for close contact between the bacterial surface proteins and host receptors on the epithelial surface (2). Then, the initial attachment by Type IV pili is further facilitated by opacity associated adhesion proteins (Opa and Opc),  lipooligosaccharide (LOS), and glycolipid adhesins (1).

Opacity Associated Adhesion Proteins (Opa and Opc)

Opacity associated adhesion proteins in the outer membrane interact with host receptors to cause tight secondary binding to support adhesion and invasion into host cells (2) .Opa and Opc are the two outer membrane opacity proteins that contribute to N. meningitidis adhesion (6). Opa proteins are encoded by multiple genes and are composed of eight transmembrane domains arranged in a beta-barrel formation (5). The Opc protein is also a beta-barrel protein with five transmembrane domains, and mediates adhesion and invasion of cells via a trimolecular complex (5,6).  Both Opa and Opc interact with host CD66 protein and other antigen related cell adhesion molecule (CECAM) receptors (2).CECAM receptors are expressed in high levels during inflammatory reactions caused by cytokines, which helps facilitate Opa interaction, cellular attachment, and invasion during meningococcal meningitis (5). Additionally, both Opa and Opc also interact with cell-surface associated heparan sulfate proteoglycans (HSPG) found on most epithelial cells for adhesion and invasion (6). It is important to note that Opa proteins and CD66-related proteins are variably expressed in bacterial and host cells, respectively (1). So, depending on the particular CD66 variant and Opa proteins expressed, different host cell responses can occur, such as binding, uptake or activation of signal transduction pathways (1). In general, in the nasopharynx mucosa, Opa and Opc bind host CD66, which results in tight secondary binding (2).

Lipooligosaccharide (LOS) and Minor Adhesins

LOS is an endotoxin found on the outer membrane, which plays a role in the adherence of meningococci to host cells (7). LOS lacks the repeating O side chain of lipopolysaccharide (LPS) found in enteric gram negative bacteria (2). Instead, LOS consists of three parts: lipid A, a core oligosaccharide and highly variable short oligosaccharides of α-, β-, γ- chains (2). These α-chain structures mimic human I and i antigens to escape host immune mechanisms (2). Additionally, LOS binds to receptors of the innate immune system cells, which can trigger the secretion of cytokines and increase the inflammatory response (7). The inflammatory response can then upregulate the expression of CECAM and HSPG receptors, which can aid bacterial binding and adhesion (6,7).

N. meningitidis also has several minor adhesin proteins, such as meningococcal surface fibril (Msf), N. meningitidis adhesin A (NadA) and meningococcal adhesion and penetration protein (App) that promote adhesion and binding to epithelial cells in the nasopharyngeal mucosa (2, 8). These multiple adhesins have different receptor specificities, which suggests they may interact cooperatively with different receptors on the same target cell or may act at different stages during infection and mediate adhesion to different cell types at different sites (8).

Following attachment to host cells in the nasopharynx, meningococcal microcolonies are encased in a secreted biofilm matrix form and persist in the mucosa (2). Biofilm formation allows the bacterial cells to be resistant to antimicrobial agents and allow them to persist in and colonize the nasopharyngeal mucosa (2).

Nutrient Acquisition

It is important to note N. meningitidis needs nutrients, cofactors and carbon sources for this sustained colonization (2). So, N. meningitidis has developed mechanisms to acquire these resources from the human host (2). Some proposed mechanisms involve iron scavenging and lactate acquisition pathways (2). Iron binding proteins, such as transferrin binding protein (TbpA and TbpB) lactoferrin binding protein (LbpA and LbpB), hemoglobin receptor (HmbR), haptoglobin-hemoglobin (HpuA and HpuB) allow meningococci to acquire iron (6, 9). TbpA and TbpB are outer membrane proteins that together function as the N. meningitidis transferrin receptor complex with a high affinity binding domain to bind and uptake iron from the host cell (9). Similarly, LbpA and LbpB form the lactoferrin receptor in N. meningitidis (9). Interestingly, lactoferrin predominates as the main source of iron in the nasopharynx (9). Thus, acquisition of iron from lactoferrin is a key factor of meningococci in the entrance and persistence in the nasopharynx (9). The specific interactions between TbpA and transferrin and LbpA and lactoferrin from the host is a current area of ongoing research (9). However, the process is known to involve high-affinity binding of transferrin or lactoferrin to the receptor complex, the stripping of iron from the respective molecule, and the transport of iron through the outer membrane into the periplasm (9). In a similar sense, the binding and interaction between hemoglobin and HmbR and haptoglobin-hemoglobin and HpuA and HpuB is also an ongoing study of research; however it is predicted that the process of iron acquisition involves the stripping of heme from the respective complex and subsequent transport into the periplasm (9). Because iron is an essential growth factor, iron binding proteins are key cellular factors in N. meningitidis colonization (6).

Immune System Evasion

N. meningitidis has also developed different mechanisms to evade the immune system in order to colonize and persist in the nasopharynx (2). Firstly, meningococci is known to secrete an immunoglobulin protease that cleaves IgA antibodies to interfere with host recognition, opsonization, phagocytosis and complement mediated killing (2). Additionally, N. meningitidis incorporates N-acetylneuraminic acid (NANA), the most common sialic acid found on host cells, into their bacterial envelope as a form of molecular mimicry to decrease recognition by the host immune system (2). Also, as mentioned above, LOS α-chain structures mimic human I and i antigens to escape host immune mechanisms (2). Finally, N. meningitidis also has shown resistance mechanisms against host antimicrobial peptides, such as lactoferrin, lysozyme and LL-37 cathelicidin (2). Together, these processes allow for protection of N. meningitidis from the host immune response, which can facilitate their persistence and colonization (6).

References

1. Bourdoulous, S, Nassif, X. 2006. Mechanisms of attachment and invasion. In M. Frosch, M.C.J. Maiden (eds.), Weinheim, FRG: Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. https://doi.org/10.1002/3527608508.ch13
2. Schmitz, JE, Stratton, CW. 2015. Chapter 98 - Neisseria meningitidis. In D. Liu, I. Poxton, J. Schwartzman, M. Sussman, Y. Tang. Molecular Medical Microbiology. Elsevier Ltd, Nashville, Tennessee https://doi.org/10.1016/B978-0-12-397169-2.00098-6
3. Morse, SA. 1996. Neisseria, Moraxella, Kingella and Eikenella. In S. Baron (ed.), Medical Microbiology. University of Texas Medical Branch at Galveston, Galveston, Texas.
4. Tzeng, Y, Stephens, DS. 2000. Epidemiology and pathogenesis of neisseria meningitidis. Microbes and Infection. 2. 687-700. https://doi.org/10.1016/S1286-4579(00)00356-7
5. Hill, DJ, Griffiths, NJ, Borodina, E, Virji, M. 2010. Cellular and molecular biology of  Neisseria meningitidis colonization and invasive disease. Clin science. 118:547–564. https://doi.org/10.1042/CS20090513
6. Rouphael, NG, Stephens, DS. 2012. Neisseria meningitidis: biology, microbiology, and epidemiology. Methods Mol Biol. 799:1-20. doi:10.1007/978-1-61779-346-2_1
7. Zarantonelli, ML, Huerre, M, Taha, MK, Alonso, JM. 2006. Infection and Immunity. Differential Role of Lipooligosaccharide of Neisseria meningitidis in Virulence  and Inflammatory Response during Respiratory Infection in Mice. Infect Immun. 74: 5506-5512. https://doi.org/10.1128/IAI.00655-06
8. Pizza, M, Rappuoli, R. 2014. Neisseria meningitidis: pathogenesis and immunity. Curr opin in microbiol. 23:68-72.
9. Perkins-Balding, D, Ratliff-Griffin, M, Stojiljkovic, I. 2004. Iron Transport Systems in Neisseria meningitidis. Microbiol and Molecular Biol Rev. 68:154-171.

iii. Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.

After initial adherence to the host nasopharynx epithelial cells is complete, N. meningitidis will undergo proliferation and dissemination. During the growth of N. meningitidis colonies, some bacteria will undergo autolysis. This results in the release of the bacterial cell wall in a soluble form into the local environment, which contains endotoxins such as LOS and lipopolysaccharide (LPS) (1). LOS and LPS are analogous to each other in terms of structures, aside from the O-antigen which lacks exclusively in the structure of LOS. While LPS is present in all Gram-negative bacteria, LOS is mainly present on mucosal and enteric bacterial species, such as N. meningitidis (2). In N. meningitidis, LOS has an important role in pathogenesis as it is found on the surface of the outer membrane of the bacteria and contributes to adherence of meningococci to host cells (3). LOS binds to receptors of the innate immune  system  cells, which  can  trigger the  secretion of  cytokines  and  increase  the inflammatory response (3). LOS release into the bloodstream and cerebral spinal fluid (CSF) also promotes meningococcal sepsis and meningitis; this is seen in studies that have reported a positive correlation between LOS concentration in blood and CSF samples with severity of these symptoms (4). Similarly, LPS facilitates the dissemination of invasive N. meningitidis during meningitis and septicemia (blood poisoning by the bacteria) and is a key inducer of host immune responses. Overstimulation of the host immune response and inflammation can result in vascular leakage, causing poor oxygenation and blood circulation to major organs of the body, which can ultimately progress to organ failure and septic shock (4).

To combat the host immune response during multiplication and spread, N. meningitidis can produce IgA1 protease, as mentioned above. IgA1 protease specifically degrades the hinge region of host IgA and ultimately assists N. meningitidis in evading elimination by opsonization (5). It has also been found that IgA1 protease can alter levels of a major lysosomal protein to reduce levels of lysosomal constituents that are responsible for digesting foreign compounds, consequently promoting intracellular survival (6). Additionally, when N. meningitidis is taken up by epithelial cells, Porin B (PorB) proteins modulate electrochemical potential at the mitochondrial membrane to inhibit the release of cytochrome C and inhibit the activation of the intrinsic apoptosis pathway (7). This allows the bacteria to continue to persist and replicate in the host cell (7). Together, the multiple mechanisms of immune system evasion help to promote intracellular survival and multiplication. It is interesting to note that the invasion and intracellular survival of N. meningitidis is dependent on the complex interactions between the bacteria and host factors. The growth and proliferation of N. meningitidis during colonization and biofilm formation requires sufficient nutrients from the environment. Although research is ongoing regarding the metabolic constraints of N. meningitidis growth upon colonization, nutrients that are likely required for growth include glucose, maltose, and lactate (8). The metabolization of glucose has been observed to occur through either the Entner-Douderoff pathway or the pentose phosphate pathway (9).

N. meningitidis uses type IV pili to facilitate proliferation and aggregation during microcolony formation, as well as disaggregation and dissemination in the spread of infection. Following initial attachment, N. meningitidis will form apical cortical plaques which will anchor meningococcal to the attachment site, combating displacement by mucus and ciliary action (4). Cortical plaques are molecular complexes that are made of host proteins, such as transmembrane surface markers, like CD44, intracellular adhesion molecule (ICAM) 1, ezrin and moesin linkers and cortical acid (7). The bacteria will then proliferate and colonize, forming microcolonies and eventually a biofilm over the host epithelial cell surface. This process involves meningococcal interactions with the mucosa of the host upper respiratory tract (4). Once meningococci adhere to the host surface, a portion of the epithelial surface will be effaced, allowing for the formation of microcolonies (10).

Figure: B) After attachment of N. meningitidis, the epithelial surface will be effaced, and the bacteria will colonize the mucosal surface (10)

Figure: C) Microcolonies forming during colonization of the mucosal surface (10)

In addition to type IV pili, Opc, Opa, LOS, NadA, and other bacterial proteins will assist in establishing strong interactions with host receptors, such as integrins and carcinoembryonic antigen-related cell adhesion molecules (CEACAMS) on the epithelial cell surface (4). These interactions can activate signaling pathways in the host epithelial cells, causing downstream inflammatory responses that can assist in the dissemination of N. meningitidis. N. meningitidis is primarily an extracellular bacterium, however disease-causing meningococcal will also utilize the ability to survive intracellularly, as internationalization is an important aspect of pathogenesis (11). In cases of invasive meningococcal disease, N. meningitidis will cross the mucosal epithelial layer by invading the non-ciliated host columnar epithelial cells, which is when they function as intracellular pathogens. Cortical plaques will have formed underneath  bacterial microcolonies after pilus-mediated adhesion  to  the cells (12). The invasive N. meningitidis cortical plaque will recruit factors that induce formation of epithelial cell pseudopodia that will engulf the pathogen, thereby causing internalization of N. meningitidis into host cells (11).

Once internalized, N. meningitidis can either survive in membrane-bound compartments or the cytoplasm (13). N. meningitidis can survive individually or as colonies in membrane-bound compartments. In brain epithelial cells, N. meningitidis can reside in Neisseria-containing vacuoles (NCVs), which provide a hideout for replication, away from extracellular host immune defenses. NCVs interact with the endocytic pathway to acquire early and late endosomal marker proteins and a transferring receptor (13). These components allow the NCV to avoid maturation and trafficking to the phagosome for degradation. However, N. meningitidis face harsh environments in these compartments due to acidic conditions and the presence of antibacterial peptides and lysosomal digestive enzymes. Consequently, N. meningitidis are required to either adapt or alter their environment to make it more suitable for growth and replication (13). Some N. meningitidis will escape the vacuole and survive in the nutrient-rich cytoplasm. Here, the capsule remains crucial for survival and they are able to disseminate throughout the cell using actin for motility (13). 

Figure: Adhesion, proliferation, invasion into host nasopharynx epithelial cells and dissemination into blood vessels (14)

The internalized N. meningitidis are then able to cross the mucosal layer through transcytosis, entering the bloodstream for systemic dissemination (1, 15). When N. meningitidis enters the bloodstream, the infection is called meningococcemia (16). In the bloodstream, meningococci will multiply rapidly, induce a strong inflammatory responses, and activate complement and coagulation cascades (9, 17). The LOS on the surface of the bacteria is a key inducer of cellular inflammatory responses, and secretes various cytokines including IL-6 and TNF-α (tumor necrosis factor), as well as chemokines such as ROS (reactive oxygen species) and NO (nitric oxide) (17). These cytokines and chemokines can ultimately lead to endothelial damage and capillary leakage in the host, which can cause multiple organ failure.

Additionally, endothelial cells lining the vessels provide an antithrombotic and anti-inflammatory surface to allow for optimal vascular flow (18). In meningococcemia, N. meningitidis interacts, adheres and colonizes the endothelium of blood vessels in the body, which can alter vascular properties, vessel integrity, inflammation and coagulation (18). Type IV pili are important in allowing adhesion and aggregation to peripheral vascular endothelial cells (18). Following initial adhesion, bacteria proliferate and form a microcolony along the vascular wall (4, 18). This induces local surface reorganization, which results in cellular protrusions (18). Then, bacterial attachment induces the formation of cortical plaque (18). Together, this reorganization allows the microcolonies to resist higher shear forces due to blood flow (18). High bacteremia results in massive meningococci colonization of peripheral endothelial cells, which can cause damage and inflammation, increasing vascular permeability and vascular leakage (19). This has been associated with expensive thrombosis and purpura, a blanching skin rash, at these sites due to bleeding under the skin (4, 19,8).

N. meningitidis is also able to reach skin capillaries, which causes rashes and lesions that can rapidly progress to cutaneous lesions (7). These cutaneous lesions are thought to be caused by cytokine induced response to meningococcal endotoxin, which has been termed the Sanarelli-Shwartzman reaction (7). In addition to spreading to the central nervous system and the skin, meningococcemia can result in the spread of bacteria to any vascularized tissue or body (7). N. meningitidis is known to cause primary pyogenic arthritis by spreading to the pericardium and joints (7). Additionally, N. meningitidis can cause Waterhouse-Frederischen syndrome by spreading to and infecting the adrenal glands, which can cause bilateral haemorrhage and acute adrenal insufficiency (7). Meningococcemia can also result in endophthalmitis, inflammation of the inner eye cavity, and endocarditis, inflammation of the inner heart chambers (7).

By systemic dissemination through entry into the bloodstream, N. meningitidis can establish secondary sites of infection and induce damage in several areas of the body; for example, it can induce thrombosis, involving blood clots obstructing blood vessels and causing lesions, in major organs such as the heart and lungs (4). Additionally, although relatively rare, mucosal infections occur in tissues of the upper and lower airways, urogenital tract and anus (7). Thus, it is clear that once N. meningitidis spreads from the initial site of entry and enters the bloodstream, it can access many secondary sites. The secondary site that is infected is likely dependent on the antigens and outer surface proteins expressed by the specific N. meningitidis at the time and how they interact with receptors on host cells in the area. However, this is still an area of active research. Studies suggest that peripheral blood vessels are a suitable niche for invasive N. meningitidis, in part due to hemoglobin that are sources of iron for bacterial survival and growth (20). In the bloodstream, N. meningitidis may undergo metabolic adaptation to enhance virulence and promote sustained bacteremia.

Virulence factors that allow for survival as extracellular pathogens allow N. meningitidis to evade host immune responses to replicate the bloodstream and spread to secondary sites (19). The polysaccharide capsule plays a large role in virulence in the bloodstream (7). The expression of polysaccharides on the bacterial capsule is positively correlated with resistance to host-mediated killing mechanisms (21). While N. meningitidis can be either encapsulated or not, strains that cause invasive disease and reach the blood and cerebrospinal fluid are almost always encapsulated (9). This encapsulation contributes to the resistance of N. meningitidis to many components of the innate immune system, such as circulating antimicrobial peptides, phagocytosis by immune cells, and complement-mediated killing (7). Depending on the serotype, the bacterial capsule consists of sialic acid derivatives (7). For instance, serotypes B and C express homopolymers of NANA, the most common sialic acid in humans (7). This is a form of molecular mimicry that allows the bacteria to avoid the immune system (7). It is proposed that these biochemical motifs resist deposition of C3 on the bacterial surface by the alternative complement pathways and prevent membrane attack complex (MAC) formation (7). It also has been shown to provide resistance to antibody-mediated killing and inhibit phagocytosis (9). However, the mechanisms of capsule-mediated resistance is currently an area of active research (7). Bacterial adhesins, such as vitronectin, can also inhibit the formation and insertion of MAC (membrane attack complex) into the bacterial membranes (17). Interestingly, N. meningitidis is also known to express its own complement regulating proteins (7). Meningococcal factor H-binding protein (fHBP), called GNA1870 is a surface associated lipoprotein on the bacteria that recruits Factor H, and inhibitor of the alternative complement pathway, and reduces C3 deposition by the alternative complement pathway to prevent bacterial cell lysis (22, 19). Additionally, Porin protein, PorA, on the outer surface of meningococci can influence further serum resistance by binding to C4-binding proteins, which are complement regulators (17).

The virulence of N. meningitidis varies, not only between unencapsulated and capsulated strains, but also between different disease-causing capsulated strains. The N. meningitidis genome contains large genetic islands, such as IHT-A1, IHT-A2, and IHT-B, that encode various virulence factors and outer membrane proteins (4). The specific genes present in each genetic island will differ by strain. For example, IHT-A1 encodes genes for the biosynthesis, assembly, and transport of the capsule in highly virulent, but not commensal strains (4). The host environment also influences the spread of invasive N. meningitidis; for example, an elevated temperature associated with the host inflammatory response induces upregulation of certain bacterial virulence factor genes (21). Moreover, specific inflammatory cytokines of the host, such as IL-8, can also affect the transcription of bacterial gene. Overall, there are several factors and processes that contribute to the multiplication and dissemination of invasive meningococci (21).

Upon N. meningitidis entry into the blood vessels, the vasculature is then considered to be the primary route to the brain and central nervous system (9, 17). The blood-brain barrier (BBB) is a boundary that exists between the circulating blood and the brain, to prevent substances from leaving the vasculature and entering the CNS. The BBB contains endothelial cells that serve as a line of defense against invading pathogens because they have specialized junctional complexes and highly specialized transport systems that limit entry of most molecules (22).

When circulating N. meningitidis reach the BBB, meningococcal meningitis can occur (7). Meningococcal meningitis is one of the most frequent manifestations of meningococcal disease (7). It is preceded by meningococcemia because bacteremia is believed to favour meningeal invasion by increasing the likelihood of the bacteria interacting with cells of the blood brain barrier (19). The BBB is a non-fenestrated endothelium that is meant to protect the central nervous system from pathogens (7), However, N. meningitidis is able invade the central nervous system through a process is similar to that of its mucosal epithelial invasion. N. meningitidis uses type IV pili for pilin-mediated adhesion to endothelial cells in low blood flow areas (7). If some bacteria have lowered pilus adhesion capacity, Opa proteins can also facilitate capsulate meningococci adherence (17).

The flow rate of blood and shear stress within the vasculature greatly affects meningococci adherence. The bacteria require low shear stress for initial attachment, but once that has occurred, it is able to resist high blood flow through microvilli protrusions from microcolonies (17, 22). The type IV pili of the bacteria are important in maintaining the adherence to the endothelial cells under high flow conditions (17). Specifically, PilE and PilV proteins of the type IV pili adhere to host cells and interact with β2-adrenergic receptors, which induces β-arrestin signalling (7,6). Recently, CD147, a member of the immunoglobulin superfamily, was also found to serve as a receptor for PilE and PilV to allow for adhesion to the brain or peripheral endothelial cells and vascular colonization of N. meningitidis (23).

There are several proposed routes for bacterial translocation, as the invasion is not completely understood. One potential mechanism is the transport of the pathogen across the cell (transcellularly) by passive or adhesion-induced transcytosis, which utilizes vesicles (22). A second proposed mechanism is the passage of the pathogen between endothelial cells (paracellularly), through leaky tight junctions. In this proposed mechanism, division and growth of N. meningitidis on the endothelium promotes the formation of cortical plaques (7). This activates the β2-adrenoreceptor/β-arrestin pathway and induces the recruitment of the Par3/Par6/PKCζ polarity complex, which induces the formation of ectopic intercellular junctional domains at the sites of N. meningitidis-endothelial interaction (7). As a result, there is a depletion of junctional proteins between endothelial cells, openings of intercellular contacts and overall, disorganization at the endothelial surface (7). Consequently, N. meningitidis is able to cross the blood brain barrier and enter the subarachnoid space via a paracellular pathway (7). Other mechanisms include direct damage to the endothelial barrier via LPS mediated cytotoxic effects; however, this is unlikely because this would cause hemorrhages in the sub-arachnoid space, which is not commonly seen in meningococcal meningitis (17, 22, 24). Lastly, another potential mechanism includes leukocyte-facilitated transport by infected phagocytes (22).

After passage through the BBB, N. meningitidis can replicate and colonize endothelial cells in this area (7). Meningeal cells causes pro-inflammatory cytokine release, which provokes an excessive inflammatory reaction of the meninges, resulting in meningococcal meningitis (17).

Figure: Meningococcal penetration of the BBB and interaction with the meninges (17)

Once the pathogen enters the meninges surrounding the brain and spinal cord, they can also enter the CSF that is filling the subarachnoid space. N. meningitidis tend to hone in on this particular site because the CSF is a sterile fluid, which has an absence of host defense mechanisms (9, 25). The sterility of the CSF, coupled with its low serum protein content and high nutrient composition (including glucose, sodium chloride, and urea) greatly favors the replication and dissemination of N. meningitidis throughout the meninges (25).

References

1. Pathogenic Neisseriae: Gonorrhea, Neonatal Ophthalmia and Meningococcal Meningitis [Internet]. textbookofbacteriology. 2021 [cited 13 February 2021]. Available from: http://textbookofbacteriology.net/neisseria_5.html

2. Preston A, Mandrell RE, Gibson BW, Apicella MA. The lipooligosaccharides of pathogenic gram-negative bacteria. Critical reviews in microbiology. 1996 Jan 1;22(3):139-80.

3. Zarantonelli ML, Huerre M, Taha MK, Alonso JM. 2006. Infection and Immunity. Differential Role of Lipooligosaccharide of Neisseria meningitidis in Virulence  and Inflammatory Response during Respiratory Infection in Mice. Infect Immun  74(10): 5506-5512. https://doi.org/10.1128/IAI.00655-06

4. Stephens, DS. 2020. Neisseria meningitidis. In J.E. Bennett, R. Dolin, M,J. Blaser (eds). Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. Elsevier US, New York, New York.

5. Vidarsson G, Overbeeke N, Stemerding AM, van den Dobbelsteen G, van Ulsen P, van der Ley P, Kilian M, van de Winkel JG. Working mechanism of immunoglobulin A1 (IgA1) protease: cleavage of IgA1 antibody to Neisseria meningitidis PorA requires de novo synthesis of IgA1 Protease. Infection and immunity. 2005 Oct 1;73(10):6721-6.

6. Bourdoulous, S, Nassif, X. 2006. Mechanisms of attachment and invasion. In M. Frosch, M.C.J. Maiden (eds.), Weinheim, FRG: Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. https://doi.org/10.1002/3527608508.ch13

7. Schmitz, JE, Stratton, CW. 2015. Chapter 98 - Neisseria meningitidis. In D. Liu, I. Poxton, J. Schwartzman, M. Sussman, Y. Tang. Molecular Medical Microbiology. Elsevier Ltd, Nashville, Tennessee https://doi.org/10.1016/B978-0-12-397169-2.00098-6

8. Exley RM, Goodwin L, Mowe E, Shaw J, Smith H, Read RC, Tang CM. Neisseria meningitidis lactate permease is required for nasopharyngeal colonization. Infection and immunity. 2005 Sep 1;73(9):5762-6.

9. Rouphael NG, Stephens DS. 2012. Neisseria meningitidis: biology, microbiology,  and epidemiology. Methods in molecular biology, 799:1-20. https://doi.org/10.1007/978-1-61779-346-2_1

10. Stephens DS, Greenwood B, Brandtzaeg P. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. The Lancet. 2007 Jun 30;369(9580):2196-210.

11. Stephens DS. Biology and pathogenesis of the evolutionarily successful, obligate human bacterium Neisseria meningitidis. Vaccine. 2009 Jun 24;27:B71-7.

12. Schubert-Unkmeir. 2017. Molecular mechanisms involved in the interaction  of Neisseria meningitidis with cells of the human blood–cerebrospinal fluid  barrier. Pathogens and Disease 75(2):1-10. DOI:10.1093/femspd/ftx023

13 Nikulin J, Panzner U, Frosch M, Schubert-Unkmeir A. Intracellular survival and replication of Neisseria meningitidis in human brain microvascular endothelial cells. International journal of medical microbiology. 2006 Dec 4;296(8):553-8.

14. Figure 1 | Life cycle of N. meningitidis. The human nasopharynx is the... [Internet]. ResearchGate. 2021 [cited 13 February 2021]. Available from: https://www.researchgate.net/figure/Life-cycle-of-N-meningitidis-The-human-nasopharynx-is-the-sole-natural-reservoir-of_fig1_332961532

15. Caugant DA, Brynildsrud OB. Neisseria meningitidis: using genomics to understand diversity, evolution and pathogenesis. Nature Reviews Microbiology. 2020 Feb;18(2):84-96.

16. Morse, SA. 1996. Neisseria, Moraxella, Kingella and Eikenella. In S. Baron (ed.), Medical Microbiology. University of Texas Medical Branch at Galveston, Galveston, Texas.

17. Hill DJ, Griffiths NJ, Borodina E, Virji M. 2010. Cellular and molecular biology of  Neisseria meningitidis colonization and invasive disease. Clinical science 118(9):547–564. https://doi.org/10.1042/CS20090513

18. Melican, K, Dumenil, G. 2011. Vascular colonization by Neisseria meningitidis. Current opinion in microbiology. 15:50-56. https://doi.org/10.1016/j.mib.2011.10.008

19. Coureuil, M, Join-Lambert, O, Lécuyer, H, Bourdoulous, S, Marullo, S, Nassif, X. 2013., Pathogenesis of meningococcemia. Cold Spring Harb Perspect Med. 3:a012393. doi:10.1101/cshperspect.a012393

20. Capel E, Barnier JP, Zomer AL, Bole-Feysot C, Nussbaumer T, Jamet A, Lécuyer H, Euphrasie D, Virion Z, Frapy E, Pélissier P. Peripheral blood vessels are a niche for blood-borne meningococci. Virulence. 2017 Nov 17;8(8):1808-19.

21. Liu Y, Zhang D, Engström Å, Merenyi G, Hagner M, Yang H, Kuwae A, Wan Y, Sjölinder M, Sjölinder H. Dynamic niche-specific adaptations in Neisseria meningitidis during infection. Microbes and infection. 2016 Feb 1;18(2):109-17.

22. Coureuil M, Join-Lambert O, Lécuyer H, Bourdoulous S, Marullo S and Nassif X.  2012. Mechanism of meningeal invasion by Neisseria meningitidis, Virulence  3(2):164-172. https://doi.org/10.4161/viru.18639

23. Pizza, M, Rappuoli, R. 2014. Neisseria meningitidis : pathogenesis and immunity. Current opinion in microbiology. 23:68-72.

24. Doran KS, Fulde M, Gratz N, Kim BJ, Nau R, Prasadarao N, Schubert-Unkmeir A,  Tuomanen, EI, and Valentin-Weigand, P. 2016. Host-pathogen interactions in  bacterial meningitis. Acta neuropathologica 131(2): 185–209.  https://doi.org/10.1007/s00401-015-1531-z

25. Soriani M. 2017. Unraveling Neisseria meningitidis pathogenesis: from functional  genomics to experimental models. F1000Research 6:1228  

https://doi.org/10.12688/f1000research.11279.1

iv. Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case?

When N. meningitidis initially enters the host body and colonizes the nasopharynx,  the individual affected is usually asymptomatic (1). In cases where the infection proliferates, the two main presentations are meningococcal septicemia (aka meningococcemia)  and meningococcal meningitis, with the latter representing the most severe cases (2). It is possible for an individual infected with the meningococcal bacteria to be diagnosed with both meningococcal meningitis and septicemia. These disease symptoms will typically manifest between 1 to 14 days following the entry of invasive N. meningitidis into the mucosal layer (3).

In its mildest form, meningococcal disease can be a  transient bacteraemic illness with symptoms such as fever and malaise and tends to be resolved within 1-2 days (1). If  N.  meningitidis crosses the endothelial cells and the blood-brain barrier, it will likely cause meningococcal meningitis; while if it infects the bloodstream and proliferates, it will likely cause meningococcal septicemia. The wide variety of symptoms that are associated with invasive invasion of N. meningitis include headache, fever, stiff neck, vomiting, myalgia, photophobia, irritability, decreased ability to concentrate, agitation, drowsiness, cloudy CSF, and potential rash (3). Both meningococcemia and meningococcal meningitis involve damaging effects to the host, either induced by the bacteria or host immune response. The damage induced by N. meningitidis is primarily attributed to the release of endotoxins, bacterial proliferation, and indirectly through induction of the host immune response (3). When reviewing Mary’s case we can see that she is experiencing few of the mentioned symptoms such as fever, chills, headache and a stiff neck. These  symptoms and signs best correlate to those seen in meningococcal meningitis patients which is what Mary is diagnosed with.  It is unlikely that her disease has  progressed to meningococcemia or purpura fulminans because many of their signs and symptoms  are not present in her case, such as petechial rash or soft-tissue necrosis; this is better supported below.

Meningococcal Septicemia (meningococcemia)  

Mild Purpura and Petechiae presentation on the skin

Meningococcemia, which is considered a form of blood poisoning, is witnessed in cases where N. meningitidis enters the bloodstream. It is a  systemic form of the disease (4) that is characterized by severe,  widespread vascular injury with evidence of circulatory collapse and disseminated intravascular coagulation (DIC) (5). The hypotension and circulatory collapse seen with this form of the infection can result in scarring, limb and digit loss, and other disabilities in surviving hosts (3). In the blood, the N.  meningitidis bacteria can multiply to higher loads, which can lead to septicemia septic shock because of damage to the walls of the vasculature (5). When infected with meningococcemia, 40-80% of patients may present with petechiae, which are small round red, brown, or purple coloured spots on the skin (3). The cause of meningococcemia and petechiae is the rapid proliferation of invasive meningococci in the bloodstream, the adhesion of bacteria to micro-vessel epithelial cells by type IV pili causing vascular damage, and the production of endotoxins by N. meningitidis (6). The rapid proliferation of these bacteria also plays a strong role in damaging the host indirectly through destructive inflammatory response, contributing to circulatory collapse and coagulopathy, or impaired blood clotting ability (3). It’s important to note that the concentration of endotoxins in the bloodstream is positively correlated with the severity of these symptoms.

Severe Purpura and Petechiae presentation on the skin

In extreme cases of meningococcemia, purpura fulminans can be seen. Purpura fulminans are a  rare but fatal complication of meningococcal sepsis that leads to dermal and soft-tissue necrosis in  5  to  15%  of individuals with meningococcal disease (1, 7). Symptoms of purpura fulminans include sudden high fever,  weakness,  myalgia,  nausea,  vomiting,  and headache (1). When N. meningitidis travels through the blood as an intracellular organism within white blood cells after an inflammatory response, it can release powerful endotoxins.  The endotoxins can increase vascular permeability, which facilitates edema formation via extravasation of blood into the interstitium (7).  This causes damages to small and medium blood vessels of various organs, leading to organ failure due to necrosis (7). Furthermore, the release of endotoxins and rapid proliferation of N. meningitidis during meningococcemia can also induce activation of the coagulation system and down-regulate the fibrinolytic system, which normally removes clots in the bloodstream that arise sporadically and upon blood vessel repair (11). A downstream outcome of this bacterial process is thrombosis of the host, where blood clots block vessels and major organs, as well as impaired adrenal, renal, and pulmonary functioning (3). The visual presentations of petechiae and purpura can be seen in Figures 1 and 2.

Meningococcal Meningitis

Meningococcal meningitis is the form of N. meningitidis infection that has been diagnosed in Mary. This infection generally targets young adults like her and has symptoms including sudden headaches, irritability, fever, vomiting, myalgias (muscle aches), neck stiffness, cloudy CSF, and potential rash (3). A large amount of evidence indicates that activation of host immune mechanisms in response to N. meningitidis is what mainly causes damage to host tissues, although impaired CNS function may be seen in meningococcal disease as a  result of direct invasion  (5). Both direct invasion and immune defense mechanisms by the host can cause increased intracranial pressure, which can lead to cerebral herniation or cerebral edema (5).  Meningococcal meningitis is characterized by inflammation of the membranes around the brain or spinal cord, called meninges (1).  The inflammation can be acute or sub-acute, depending on whether it occurs suddenly or gradually,  and takes place when the interaction of meningococci with meningeal cells elicits the release of pro-inflammatory cytokines (8).  When N. meningitidis enters the subarachnoid space, bacterial replication occurs, and levels of LOS rise (4). The inflammatory response in the subarachnoid space in response to bacterial infection and LOS release is responsible for damage and the symptoms associated with the disease (4). LOS interacts with TLR4 and induces the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-8, and IL-10 (3,9). It also induces the release of chemokines and other inflammatory mediators (9). This increases the permeability of the blood-brain barrier to allow the influx of neutrophils and other immune cells (9). This inflammatory response ultimately results in increased blood flow and cerebral edema, where fluid builds up and causes an increase in intracranial pressure (9). This leads to symptoms of headaches and nuchal rigidity, also known as a stiff neck (9). Papilledema, the swelling of the optic nerve, can also be a result of increased intracranial pressure and cause a sensitivity to light (10).

References

1. Todar K. 2020. Pathogenic Neisseriae: Gonorrhea, Neonatal Ophthalmia and  Meningococcal Meningitis. Online Textbook of Bacteriology, Madison, Wisconsin.  http://textbookofbacteriology.net/neisseria_7.html
2. Pathogenic Neisseriae: Gonorrhea, Neonatal Ophthalmia and Meningococcal Meningitis [Internet]. textbookofbacteriology. 2021 [cited 13 February 2021]. Available from: http://textbookofbacteriology.net/neisseria_5.html
3. Bennett JE, Dolin R, Blaser MJ. Mandell, douglas, and bennett's principles and practice of infectious diseases: 2-volume set. Elsevier Health Sciences; 2014 Aug 28.
4. Signs and Symptoms. Meningococcal Meningitis. 2017.Centre for Disease Control  and Prevention. https://www.cdc.gov/meningococcal/about/symptoms.html
5. Pathan N, Faust SN, Levin M. 2003. Pathophysiology of meningococcal meningitis  and septicaemia. Archives of Disease in Childhood 88:601-607. http://dx.doi.org/10.1136/adc.88.7.601
6. Liu Y, Zhang D, Engström Å, Merenyi G, Hagner M, Yang H, Kuwae A, Wan Y, Sjölinder M, Sjölinder H. Dynamic niche-specific adaptations in Neisseria meningitidis during infection. Microbes and infection. 2016 Feb 1;18(2):109-17.
7. Bollero D, Stella M, Gangemi, EN, Spaziante L, Nuzzo J, Sigaudo G, and Enrichens  F. 2010. Purpura fulminans in meningococcal septicemia in an adult: a case  report. Annals of burns and fire disasters 23(1): 43–47. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3188232/
8. Hill DJ, Griffiths NJ, Borodina E, Virji M. 2010. Cellular and molecular biology of  Neisseria meningitidis colonization and invasive disease. Clinical science 118(9):547–564. https://doi.org/10.1042/CS20090513
9. Schmitz, JE, Stratton, CW. 2015. Chapter 98 - Neisseria meningitidis. In D. Liu, I. Poxton, J. Schwartzman, M. Sussman, Y. Tang. Molecular Medical Microbiology. Elsevier Ltd, Nashville, Tennessee https://doi.org/10.1016/B978-0-12-397169-2.00098-6
10. Morse, SA. 1996. Neisseria, Moraxella, Kingella and Eikenella. In S. Baron (ed.), Medical Microbiology. University of Texas Medical Branch at Galveston, Galveston, Texas.
11. 21. Capel E, Barnier JP, Zomer AL, Bole-Feysot C, Nussbaumer T, Jamet A, Lécuyer H, Euphrasie D, Virion Z, Frapy E, Pélissier P. Peripheral blood vessels are a niche for blood-borne meningococci. Virulence. 2017 Nov 17;8(8):1808-19.

Q.4 The Immune Response

i. Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

Innate immunity

Neisseria meningitidis is a gram negative, diplococcus bacteria that can infect humans. N. meningitidis typically enters the body through the nasal passage and colonizes the nasopharyngeal cavity. Here, the nasopharyngeal mucosa, which contains a complex and integrated network comprised of tissues, cells, and effector molecules, represents the first line of defense that prevents the bacteria from infecting epithelial cells (Kvalsvig & Unsworth, 2003). Colonization by N. meningitidis does not always result in an inflammatory response that causes symptoms for the host, as 10% of healthy individuals have N. meningitidis in their upper airway at any given time (Pizza & Rappuoli, 2015).  Studies have indicated that within the airway mucus, N. meningitidis can act as a commensal bacteria and it does not actively infect the underlying cells, so a strong immune reaction is not triggered (Audry et al., 2019). Mucosal immunity is a critical host mechanism as N. meningitidis infection occurs by aspiration of bacteria that attach to respiratory and squamous epithelial cells of the oropharyngeal and nasopharyngeal mucosa, and thus cross into the bloodstream through the mucosal barrier (Gasparini et al., 2015). This may occur if there is mucosal damage due to viral infection or chronic irritation of the mucosa from environmental factors like dust (Morse, 1996).

The continuous washing of saliva and mucosal secretions over the nasopharyngeal mucosal surface reduces Neisseria meningitidis bacterial adherence. Saliva contains lysozyme, which can destroy peptidoglycan and destabilise the bacterial cell, causing bacterial lysis (Fábián et al., 2012). Saliva also contains IgA which is a neutralizing antibody capable of triggering phagocytosis of bacterial cells through downstream signalling (Fábián et al., 2012). The nasopharynx is lined with two types of epithelium: a squamous epithelium and columnar respiratory epithelium (Audry et al., 2019). The nasopharynx epithelium is protected by a two-layer liquid surface of low-viscosity periciliary liquid and high-viscosity mucus which trap foreign particles and debris (Audry et al., 2019). Cilia on the surface of respiratory epithelial cells are able to bend through an ATP-consuming mechanism. This bending movement allows them to rid pathogens by working in conjunction with the low-viscosity periciliary liquid and high-viscosity mucus to expel pathogens from the body (Beule, 2011). This is known as the mucociliary clearance mechanism. Pathogens such as N. meningitidis which are bound to this mucous layer are propelled towards the pharynx, and either expelled through the mouth via coughing or shunted through the esophagus to be destroyed by the highly acidic stomach environment (Beule, 2011).

The mucous produced is largely made up of water and glycoproteins (Lane, 2009). For instance, mucin-secreting goblet cells upregulate mucin genes which are able to produce heavily glycosylated macromolecules (Beule, 2011). The viscoelasticity, as well as adhesive and cohesive properties of the mucus are mainly determined by these glycoprotein compound as they are able to obtain a highly negative charge, which draw water to create a sticky environment (Beule, 2011). In order to prevent infection, the mucous is slightly acidic, having a pH of around 5.5-6.5 (Beule, 2011). Mucus also contains immunoglobulins, enzymes, opsonins, and antimicrobial peptides (AMPs) that can prevent the growth of N. meningitidis (Lane, 2009).

Within the nasopharynx area, the epithelial cell barrier, which is directly below the mucosal layer, also contains many antimicrobial compounds such as AMPs, reactive oxygen and nitrogen species (ROS, RNS); and complement factors (Gill et al., 2010). As the epithelial cell barrier is the final barrier before the blood stream, it maintains high expression of antibacterial properties to deter pathogenic colonization (Gill et al., 2010). Two families of AMPs are evident in the epithelial cell barrier of the nasopharynx: cathelicidins and defensins (Lo et al., 2009). Within the cathelicidin family, LL-37 is the sole human cathelicidin antimicrobial peptide, whereas there are multiple human defensins such as Human B defensins 1 and 2, and Human neutrophil peptides 1 and 2 (Lo et al., 2009). These antimicrobial peptides are positively charged molecules that alter membrane stability and permeability through charge interactions and can induce cell lysis and thus death (Lo et al., 2009). They can additionally act as chemokines and opsonins, thereby enhancing phagocytosis of bacterial cells (Lo et al., 2009). Reactive Oxygen species (ROS) can damage microbial DNA and RNA by oxidation, in addition to eliciting pro-inflammatory responses (Fang, 2004). ROS can signal this inflammatory response by granuloma formation, which produces cytokines and subsequently enhances antibody mediated phagocytosis by macrophages. Reactive Nitrogen species (RNS) work alongside ROS to inhibit DNA replication by inducing DNA base damage and interrupt bacterial respiration through the inactivation of zinc metalloproteins (Fang & Vázquez-Torres, 2019).

NF-κB signalling cascade resulting in a pro-inflammatory response (Lawrence, 2009)

The innate immune response is initiated through the binding of pathogen-associated molecular patterns (PAMPS) on N. meningitidis bacterium to pathogen-recognition receptors (PRRs) on resident macrophages, neutrophils, and dendritic cells (Johswich, 2017). The innate immune response plays an important role in cytokine and chemokine release to modulate inflammation and the complement cascade, identifying and removing pathogens via phagocytosis, and activating the adaptive immune response (Johswich, 2017). A prominent PRR present on macrophages is Toll Like Receptors (TLRs) which are membrane-bound signalling receptors responsible for activation of down-stream signalling cascades, and subsequent cytokine release (Johswich, 2017). Of the 10 human TLRs, TLR 1, TLR2, TLR4 and TLR9 are capable of recognizing N. meningitidis PAMPs (Johswich, 2017). TLR1/TLR2 on human airway epithelial cells recognize PorB, which is a porin protein of N. meningitidis. Likewise, TLR1/TLR2 on monocytes can recognize outer membrane protein NhhA, which triggers the monocytes differentiation into macrophages, upregulating the presence of phagocytic cells in this area for effective bacterial clearance (Johswich, 2017).. TLR4 on macrophages and peripheral blood mononuclear cells interacts with the lipooligosaccharide (LOS) on the surface of N. meningitidis, which triggers the immune cells to release inflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 6 (IL-6) (Johswich, 2017). TLR9 is found on the endosomal membrane of immune cells and is activated by bacterial DNA, which is distinguished from host DNA by the lack of methylation on CpG sequences, stretches of DNA that contain a cysteine followed by a guanine nucleotide (Johswich, 2017). These cells take up N. meningitidis and the bacteria is translocated to the cellular endosome where the bacterial DNA interacts with TLR9, resulting in the production of inflammatory cytokines and chemokines, including IL-6, IL-8, and TNF (Johswich, 2017). In addition, intracellular, cytoplasmic PRRs such as the nucleotide oligomerization domain – like receptor (NLR), can respond to peptidoglycan present in N. meningitidis bacterial cell walls that may be released during meningococcal meningitis infection, to activate nuclear factor kappa b (NF-κB) and release cytokines (Johswich, 2017). The activation of PRRs such as TLRs or NLRs triggers signalling cascades, which results in the release of NF-κB from IκB (Liu et al., 2017). NF-κB is a transcription factor encoding several genes involved in inflammatory response and is able to relocate to the nucleus to induce the expression of pro-inflammatory genes (Liu et al., 2017). Downstream pathways of NF-κB can induce a local immune response, a systemic immune response, and can increase recruitment and production of immune cells (Liu et al., 2017). When activated, the NF-κB pathway can induce the transcription of pro-inflammatory cytokines (ex. IL-8), chemokines (ex. MCP-1) and mediators in various innate immune cells (Liu et al., 2017). These inflammatory mediators can act indirectly by promoting the differentiation of inflammatory T-cells or can directly engage in the induction of inflammation (Liu et al., 2017).

The three methods of activating complement, all of which result in bacterial cell lysis (Schneider, 2007).

Another PRR that’s important for the immune response to N. meningitidis is mannose-binding lectin (MBL). MBL is a protein found in serum that binds to mannose and N-acetylglucosamine which are found on microbial cell surfaces (Jack et al., 2001). MBL activates the complement system after it interacts with its ligand, via MBL-associated serine protease (MASP)-1 and MASP-2 (Jack et al., 2001). This is known as the lectin binding pathway of complement. The complement system involves many proteins and molecules that act as a cascade resulting in bacterial killing. MASP-2 cleaves C4 and C2, creating C4b2a, which acts as a C3 convertase (Schneider et al., 2007). The C3 convertase cleaves C3 and creates C3a and C3b (Schneider et al., 2007). C3b can bind to the surface of N. meningitidis and act as an opsonin, increasing the ability of phagocytic cells to uptake the bacteria (Schneider et al., 2007). C3b also acts as part of the C5 convertase, which cleaves C5 into C5a and C5b and results in the formation of the membrane attack complex (MAC) (Schneider et al., 2007). MAC can insert into bacterial cell membranes, disrupting the lipid bilayer and causing cell lysis and death. MAC is very important in the immune response to N. meningitidis, as individuals with mutations in the genes for the proteins that make up this complex have a 50-fold increase in their risk of developing a systemic infection (Jack et al., 2001).

Complement can be activated via two other pathways, the classical pathway and the alternative pathway. The N. meningitidis shares its specific capsular structure and polysaccharide component with other commensal bacteria like N. lactamica and other non-pathogenic Neisseria species (Kvalsvig & Unsworth, 2003). Therefore, antibodies may be present in individuals that can target N. meningitidis as well (Kvalsvig & Unsworth, 2003), (Gasparini et al., 2015). The concentration of these antibodies, typically IgG and IgM, can vary from person to person, however, if there is a sufficient amount of these antibodies at the time of N. meningitidis infection, they can bind to the antigenic portion of the bacteria and trigger the classical complement pathway (Rouphael & Stephens, 2012). Lastly, the alternative pathway can be activated through the interaction of a spontaneously hydrolyzed complement component C3, with microbial surface structures (Charles A Janeway et al., 2001). The hydrolyzed product reacts with Factor B and Factor D, other components of the complement pathway, to generate C3 convertase (Charles A Janeway et al., 2001). The alternative pathway is thus able to auto-activate via C3 tickover (Charles A Janeway et al., 2001).

Adaptive Immunity

Neisseria meningitidis typically colonizes within the nasopharyngeal epithelium, thus inflammatory molecules such as macrophages, dendritic cells, neutrophils, and the complement system activation are concentrated to this region in early infection. Generally, it is within the nasopharyngeal epithelium that dendritic cells phagocytose Neisseria meningitidis bacterium. DCs are a crucial connection between the innate and adaptive immune response. Once DCs engulf N. meningitidis, they are able to migrate to the lymph nodes, the spleen and other lymphoid organs in order to present the pathogen to T cells and B cells (Alberts et al., 2002). T cells are responsible for mounting the cellular adaptive response, whereas B cells are responsible for the humoral adaptive response (Alberts et al., 2002).

The process of dendritic cell antigen presentation is as follows:

The process of dendritic cells taking up and presenting antigen on MHCII (18.2 Major Histocompatibility Complexes and Antigen-Presenting Cells, 2021)

1. Dendritic cells uptake Neisseria meningitidis bacterial proteins into vesicle compartments
2. The vesicle merges with endosome + lysosome, thus causing the breakdown of Neisseria meningitidis proteins into peptides.
3. MHC II molecules are constitutively synthesized in the Golgi Body, and are then transported to vesicles in the cytosol.
4. MHC II vesicles can recognize and dock onto peptide vesicles.
5. Peptides capable of associating with MHC II load onto the molecule, however MHC II will only load the specific peptide with high affinity.
6. MHC II + specific peptide moves to the cell surface.Specific T Cell receptor (TCR) recognizes the MHCII peptide complex, causing subsequent activation of CD4+ T cells and B cells specific to Neisseria meningitidis (Gasparini et al., 2015), (Raziuddin et al., 1991),(Rouphael & Stephens, 2012).

Activation of CD4 T cells produces T helper cells and launches a Th1 response, as N. meningitidis is an extracellular bacteria (Alberts et al., 2002). CD4 T cells migrate to the infection site via chemokine and cytokine gradients (“The Immune System by Peter Parham,” 2015).. The Th1 response is characterized by the secretion of pro-inflammatory molecules like IFN-y, IL-6, IL-12, and TNF-alpha which culminate into an increase in phagocytosis via macrophages (“The Immune System by Peter Parham,” 2015).

T helper cells also help with the activation of B cells in the lymphoid organs (Alberts et al., 2002). B cells can directly take up pathogens which enter the lymph node or other lymphoid organs and present pathogen particles on their surface. This allows T helper cells to interact with B cells, causing them to proliferate, differentiate and secrete antibodies against N. meningitidis (Gasparini et al., 2015). CD4 T cells primarily help activate B cells and aid in Ig class switching so B cells can begin the production of IgM, IgG and IgA antibodies against N. meningitidis (Rouphael & Stephens, 2012).  IgA is secreted by B cells into the blood and binds to surface components of the bacteria to reduce further invasion (“The Immune System by Peter Parham,” 2015). IgG is also secreted into the blood to opsonize and neutralize bacteria and can activate complement (“The Immune System by Peter Parham,” 2015). IgM is the first to be secreted in the blood, yet, cannot enter affected tissues well due to its size ((“The Immune System by Peter Parham,” 2015). IgM can bind to microorganisms and activate complement (“The Immune System by Peter Parham,” 2015). B cells can also be activated directly by recognizing the polysaccharides in the N. meningitidis capsule  – however response to these antigens by B cells only results in the production of short lived IgM antibodies (Gasparini et al., 2015).

If the bacteria passes through the blood-cerebral spinal fluid (CSF) barrier and translocates into the central nervous system, like in the case of Mary, an immune response is triggered in the subarachnoid space (Doran et al., 2016). Like in the blood stream, high levels of inflammatory cytokines are produced, changing the vasculature of the meninges to upregulate adhesion molecules and allowing immune cells, especially neutrophils, to enter the subarachnoid space (Doran et al., 2016). Antibodies and complement also enter the CSF, resulting in a significant immune response in the central nervous system (Doran et al., 2016).

References:

18.2 Major Histocompatibility Complexes and Antigen-Presenting Cells—Microbiology | OpenStax. (Jan 05, 2021). Retrieved February 24, 2021, from https://openstax.org/books/microbiology/pages/18-2-major-histocompatibility-complexes-and-antigen-presenting-cells?query=dendritic&target=%7B%22index%22%3A0%2C%22type%22%3A%22search%22%7D#fs-id1167660275280

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). T Cells and MHC Proteins. Molecular Biology of the Cell. 4th Edition. http://www.ncbi.nlm.nih.gov/books/NBK26926/

Audry, M., Robbe-Masselot, C., Barnier, J.-P., Gachet, B., Saubaméa, B., Schmitt, A., Schönherr-Hellec, S., Léonard, R., Nassif, X., & Coureuil, M. (2019). Airway Mucus Restricts Neisseria meningitidis Away from Nasopharyngeal Epithelial Cells and Protects the Mucosa from Inflammation. MSphere, 4(6). https://doi.org/10.1128/mSphere.00494-19

Beule, A. G. (2011). Physiology and pathophysiology of respiratory mucosa of the nose and the paranasal sinuses. GMS Current Topics in Otorhinolaryngology - Head and Neck Surgery, 9, Doc07. https://doi.org/10.3205/cto000071

Charles A Janeway, J., Travers, P., Walport, M., & Shlomchik, M. J. (2001). The complement system and innate immunity. Immunobiology: The Immune System in Health and Disease. 5th Edition. http://www.ncbi.nlm.nih.gov/books/NBK27100/

Doran, K. S., Fulde, M., Gratz, N., Kim, B. J., Nau, R., Prasadarao, N., Schubert-Unkmeir, A., Tuomanen, E. I., & Valentin-Weigand, P. (2016). Host–pathogen interactions in bacterial meningitis. Acta Neuropathologica, 131, 185–209. https://doi.org/10.1007/s00401-015-1531-z

Fábián, T. K., Hermann, P., Beck, A., Fejérdy, P., & Fábián, G. (2012). Salivary Defense Proteins: Their Network and Role in Innate and Acquired Oral Immunity. International Journal of Molecular Sciences, 13(4), 4295–4320. https://doi.org/10.3390/ijms13044295

Fang, F. C. (2004). Antimicrobial reactive oxygen and nitrogen species: Concepts and controversies. Nature Reviews Microbiology, 2(10), 820–832. https://doi.org/10.1038/nrmicro1004

Fang, F. C., & Vázquez-Torres, A. (2019). Reactive nitrogen species in host–bacterial interactions. Current Opinion in Immunology, 60, 96–102. https://doi.org/10.1016/j.coi.2019.05.008

Gasparini, R., Panatto, D., Bragazzi, N. L., Lai, P. L., Bechini, A., Levi, M., Durando, P., & Amicizia, D. (2015). How the Knowledge of Interactions between Meningococcus and the Human Immune System Has Been Used to Prepare Effective Neisseria meningitidis Vaccines. Journal of Immunology Research, 2015. https://doi.org/10.1155/2015/189153

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ii. Host damage: what damage ensues to the host from the immune response?

The immune response must be a highly regulated process, as under-activation can result in ineffective killing of the pathogen, however, over-activation can result in damage to the host. N. meningitidis has the ability to cause septicemia, which is a life-threatening condition where the body responds to infection in the blood. Once the bacterium gets into systemic circulation, the inflammatory response continues to be active, which can cause damage to the host in a variety of body sites. From the bloodstream, N. meningitidis is able to gain access to the central nervous system (CNS), which can lead to severe neurological consequences, and even death.

a)      Systemic Circulation

While the complement system is a critical host immune response, it has been linked to both an inappropriate inflammatory response and over-activation of the host clotting cascade. Firstly, complement is a key trigger of the inflammatory response in systemic circulation. A downstream product of the complement system is C5, which is cleaved into C5a and C5b by host convertases (Schneider et al., 2007). C5a contributes to the high levels of pro-inflammatory cytokines circulating in the blood stream. For instance, C5a can act on polymorphonuclear leukocytes (PMNs) and monocytes in circulation to secrete cytokines (i.e. IL-1, IL-6, IL-8, TNF-α) and oxidative species (Schneider et al., 2007). Neutrophils can undergo degranulation and release inflammatory molecules and enzymes, such as proteases, which can degrade host tissue (Pathan, Faust & Levin, 2003).  The activated inflammatory components in the bloodstream, including cytokines, oxidative species and histamine, cause endothelial damage and capillary leakage which can result in not only a skin rash, but also shock in more severe cases (Kvalsvig & Unsworth, 2003). This is because as vascular permeability increases and damage ensues, capillaries can lose albumin and electrolytes, causing fluid to be lost from the vasculature. The venous return to the heart is thus slowed and impaired, thereby causing decreased cardiac output, leading to shock.

Secondly, complement also plays a role in over-stimulating the clotting cascade causing systemic intravascular coagulation, which can be damaging to the host. Specifically, the complement cascade is also linked to Disseminated Intravascular Coagulation (DIC), which is when blood clots form in the vasculature thus blocking small blood vessels. While C5a can upregulate pro-inflammatory cytokines leading damage to host tissue, C5a can also directly upregulate thrombin, which is an enzyme integral to clot formation. The pathways of inflammation and coagulation are tightly interrelated, and uncontrolled inflammation evident in the host immune response to N. meningitidis bacterium induces the blood clotting cascade as a host-defense mechanism to contain pathogens from spreading within the body. The process of DIC occurs when multiple blood clots form in small blood vessels, thus compromising blood flow. In addition, the process of DIC consumes platelet and clotting factors over and above normal levels within the small vessels, which can compromise regular blood clotting, thus leading to uncontrollable and severe internal bleeding (Lupu et al., 2014).

The combination of increased vascular permeability and increased coagulation can lead to inadequate tissue perfusion and can result in organ failure and necrosis of extremities in very serious infections (Kvalsvig & Unsworth, 2003). The changes in vasculature, including decreased vascular tone and increased vessel leakage can result in hypotension and hypovolaemia, leading to septic shock (Pathan, Faust & Levin, 2003). There are a variety of proinflammatory molecules released during septic shock which have been found to impair cardiac function, including nitric oxide, IL-1β, and TNF-α. In addition, due to the multiple dysregulated processes within the microvasculature, blood flow back to the heart can be slowed, which results in a decrease in cardiac output. This decrease in blood delivery to organs and tissues can result in organ damage and failure (Coureuil et al., 2013). As a result of decreased cardiac output and reduced tissue perfusion, renal, pulmonary and gastrointestinal consequences can occur and, in some cases, severe damage can occur. For instance, reduced blood flow to the gastrointestinal tract occurs early in septic shock, however in prolonged cases, decreased blood supply can result in ischemic ulceration and perforation later on in the infection (Pathan, Faust & Levin, 2003).

b)      Central Nervous System

In the CNS, the inflammatory response also plays a role in host cell damage, which is very serious as vital structures such as the brain are housed in the CNS. Cerebral blood flow is impaired due to inflammation of the vessels (vasculitis) from cytokine production that supply blood to the brain (van der Flier et al., 2003). The infiltration of neutrophils and the release of vasoconstrictive agents can cause narrowing of the lumen of the vasculature, which not only reduces blood supply, but also causes vasospasms. This inadequate blood supply can lead to necrosis in various regions of the brain (van der Flier et al., 2003).

Figure1: Bacteria in the blood can reach CSF and the brain parenchyma by first breaching the BBB (van de Beek, 2016).

Once in the blood vessels, N. meningitidis’ primary entry site into the CNS is breaching the blood brain barrier (BBB), as seen in Figure 1 (Borkowski, Schroten & Schwerk, 2020). The production of reactive oxygen species (ROS), proteolytic enzymes, and toxic cytokines from the innate response itself damages the barrier function of the BBB (Borkowski et al., 2014). Not only does the BBB damage grant access for N. meningitidis into the CNS, disturbances to the endothelial cells of the BBB results in cerebral edema in 30% of cases in cerebral herniation in approximately 7% of cases (van der Flier et al., 2003). Once N. meningitidis has breached the BBB, it now has access to the cerebral-spinal fluid (CSF), thereby causing inflammation of the meninges, which are the membranous coverings of the brain and spinal cord, also known as meningitis. When N. meningitidis comes in contact with meningioma cells, this leads to the release of pro-inflammatory and cytokines and chemokines (i.e. TNFα, IL-6, IL-8, MIP-2α), as well as the upregulation of genes for apoptosis (Borkowski, Schroten & Schwerk, 2020).

This inflammatory response is not isolated to the meninges however, as it can affect the spinal cord, ventricles, and brain parenchyma. In addition to damage caused by neutrophils undergoing apoptosis, neutrophils, macrophages including microglia, and dendritic cells all produce nitric oxide (NO), which is a cell death signal that alongside ROS forms tissue damaging intermediates. This formation of ROS/NO intermediates ultimately causes cell degradation and death to healthy neighbouring cells, thereby increasing permeability to enhance white blood cell migration, and ultimately resulting in enhanced bacterial invasion and damage (Zwijnenburg et al., 2006).  Dying parenchymal cells also lose membrane integrity and release TLR ligands, which induce “danger signals”. These danger signals cause further inflammatory damage through activating dendritic cells and promoting the generation of immune responses to antigens at the site of dying cells (Rock & Kono, 2008).

Many of Mary’s signs and symptoms can be attributed to the infection and resulting inflammatory response in the blood and CNS. Firstly, it was noted by the doctor that Mary had low blood pressure. This can be due to the changes in vasculature caused by the host immune response, such that loss in vascular tone and decreased fluids can lead to low blood pressure. In addition, decreases in cardiac output can also lead to decreased blood pressure.  This could indicate that Mary is going into septic shock. In addition, Mary experienced sensitivity to light. As meningitis causes inflammation of areas surrounding the brain, this can interfere with neurological processes leading to photophobia. The headache caused by meningitis infection can also amplify the sensitivity to light. Lastly, the meninges not only cover the brain, but also the spinal cord which runs through the neck (Sinicropi, 2017). Thus, as the meninges become inflamed due to infection in the CNS, then this can result in the stiff neck we see in Mary’s case.

References

Borkowski, J., Schroten, H., Schwerk, C. (2020). Interactions and Signal Transduction Pathways Involved during Central Nervous System Entry by Neisseria meningitidis across the Blood–Brain Barriers, International Journal of Molecular Sciences, 21(22) 8788. https://doi.org/10.3390/ijms21228788

Borkowski, J., Li, L., Steinmann, U., Quednau, N., Stump-Guthier, C., Weiss, C., Findeisen, P., Gretz, N., Ishikawa, H., Tenenbaum, T., Schroten, H., & Schwerk, C. (2014). Neisseria meningitidis elicits a pro-inflammatory response involving IκBζ in a human blood-cerebrospinal

Coureuil, M., Join-Lambert, O., Lécuyer, H., Bourdoulous, S., Marullo, S., & Nassif, X. (2013). Pathogenesis of Meningococcemia. Cold Spring Harbor Perspectives in Medicine, 3(6). https://doi.org/10.1101/cshperspect.a012393

Kvalsvig, A. J., & Unsworth, D. J. (2003). The immunopathogenesis of meningococcal disease. Journal of clinical pathology, 56(6), 417–422. https://doi.org/10.1136/jcp.56.6.417

Lupu, F., Keshari, R. S., Lambris, J. D., & Coggeshall, K. M. (2014). Crosstalk between the coagulation and complement systems in sepsis. Thrombosis Research, 133(0 1), S28–S31. https://doi.org/10.1016/j.thromres.2014.03.014

Pathan, N., Faust, S. N., & Levin, M. (2003). Pathophysiology of meningococcal meningitis and septicaemia. Archives of disease in childhood, 88(7), 601–607. https://doi.org/10.1136/adc.88.7.601

Rock, K. L., & Kono, H. (2008). The inflammatory response to cell death. Annual Review of Pathology, 3, 99–126. https://doi.org/10.1146/annurev.pathmechdis.3.121806.151456

Schneider, M. C., Exley, R. M., Ram, S., Sim, R. B., Tang, C. M. (2007). Interactions between Neisseria meningitidis and the complement system, Trends in Microbiology, 15(5), 233-240.

Sinicropi, S. (2017). How Meningitis Causes Neck Pain and Stiffness. Accessed from https://www.spine-health.com/conditions/neck-pain/how-meningitis-causes-neck-pain-and-stiffness#:~:text=Most%20commonly%20in%20meningitis%2C%20the,neck%20pain%20and%20nuchal%20rigidity.

van de Beek, D., Brouwer, M., Hasbun, R., Koedel, U., Whitney, C, G., Wijdicks, E. (2016). Community-acquired bacterial meningitis, Nature Reviews Disease Primers. https://doi.org/10.1038/nrdp.2016.74

van der Flier, M., Geelen, S. P., Kimpen, J. L., Hoepelman, I. M., & Tuomanen, E. I. (2003). Reprogramming the host response in bacterial meningitis: how best to improve outcome?. Clinical microbiology reviews, 16(3), 415–429. https://doi.org/10.1128/cmr.16.3.415-429.2003

Zwijnenburg, P. J. G., van der Poll, T., Roord, J. J., & van Furth, A. M. (2006). Chemotactic Factors in Cerebrospinal Fluid during Bacterial Meningitis. Infection and Immunity, 74(3), 1445–1451. https://doi.org/10.1128/IAI.74.3.1445-1451.2006

iii. Bacterial evasion: how does the bacteria attempt to evade these host response elements.

N. meningitidis entry into CNS through transcellular and paracellular mechanism across the endothelial layer (Herold et al., 2019).

The host immune response is quite effective in limiting infection caused by Neisseria meningitidis bacterium, as the rate of carrier development of clinical disease is low. It is evident however that Neisseria meningitidis bacterium have evolved several evasion strategies towards innate and adaptive host immune responses (Lo et al., 2009). Key evasion strategies include capsule switching, molecular mimicry, genome plasticity temperature regulated responses, and pili mediated adhesion.

Bacterial Capsule

All pathogenic strains of Neisseria meningitidis are encapsulated, which suggest that the bacterial polysaccharide capsule is an important virulence factor. The bacterial capsule is encoded by the capsule biosynthesis operon (css). The css operon can influence capsular biosynthesis and the LOS sialyation pattern by altering gene transcription and inducing phase and antigenic variation of surface antigens (Gasparini et al., 2015). The variation in surface antigens is effective in avoiding of host recognition of bacteria, and thus evasion of complement mediated immunity. Neisseria meningitidis bacterial capsules (particularly serotype B) are considered intrinsically antiphagocytic as the capsule contains sialic acid, a component of host polysaccharides. The inclusion of sialic acid therefore mimics the host polysaccharide, and thus the host immune system recognizes Neisseria meningitidis recognizes as “self”, allowing the bacterium to evade phagocytosis (Talà et al., 2014).  The polysaccharide capsule is a homopolymer of α2-8-linked sialic acid, which mimics host cell adhesion molecule, NCAM-1 (Gasparini et al., 2015). Neisseria meningitidis is also poorly immunogenic, as the polysaccharide layer hides antigen epitopes from antibodies, thus eliciting low antibody titres and allowing the bacterium to avoid both innate and adaptive immune recognition (Gasparini et al., 2015). N. meningitidis are also able to cluster together to produce outer membrane vesicles (OMVs) which allow them to hide their surface antigens which might provoke an immune response (Gasparini et al., 2015).

Neisseria meningitidis bacteria have a polysaccharide capsule that aids with evasion from host responses (Stephens et al., 2007).

The capsule of Neisseria meningitidis also resists complement deposition, in two ways. The first is the incorporation of sialic acid into the Neisseria meningitidis bacterial capsule which increases the affinity of C3b from the complement cascade with factor H, which is a host negative regulator of complement (Gasparini et al., 2015). As C3b is required for C3 convertase in the alternative complement pathway, factor H can effectively dislodge C3b from C3 convertase, actively downregulating complement activation. The second way that Neisseria meningitidis can resist complement deposition involves capsular LOS. Capsular LOS contains special complement deposition sites that bind C3b and C4b of the complement cascade with high affinity (Lewis & Ram, 2014). The binding of these complement proteins precents formation of the MAC complex required for lysis of bacterial cells (Lewis & Ram, 2014). Furthermore, class 1 outer membrane proteins have the ability to resist engulfment by neutrophils through Fcy, C1 and C3 receptor downregulation in these neutrophils (van Deuren et al., 2000) . In addition, IgA1 proteases create a break in the hinge of IgA1 allowing for the release of Fab-alpha fragments that can inhibit IgG and IgM contact, evading adaptive immune mechanisms (van Deuren et al., 2000). Stains that do not express a bacterial capsule have been found to be highly sensitive to complement and membrane disruption, emphasizing its role in bacterial longevity and invasion (Schneider et al., 2007).

Molecular Mimicry

Molecular mimicry and molecular decoys are also utilized by Neisseria meningitidis to evade the host immune response. The basis of molecular mimicry outlines that potential antigens on bacterial cells have similar structures to markers on human cells, and thus immune cells interpret the antigens as “self” and no immune response is elicited (Lewis & Ram, 2014). As mentioned above, Neisseria meningitidis contains sialic acid within its polysaccharide capsule, which allows the bacteria to avoid host immune system recognition (Gasparini et al., 2015). Another example of molecular mimicry involves the LOS of Neisseria meningitidis, which contains an alpha carbohydrate chain with a lacto-N-neotetrase (LNT) like moiety (Lewis & Ram, 2014).. The LNT forms the backbone of the host blood group antigens A, B and H, and thus immune cells are unable to distinguish between self and pathogen (Lewis & Ram, 2014). This prevents detection and phagocytosis of bacteria cells by the immune response.

Neisseria meningitidis also use molecular decoys to prevent cell death. FprB is an outer membrane iron transporter in Neisseria meningitidis bacterium critical for iron uptake into the periplasm (Gasparini et al., 2015). FprB contains an antigenic subdomain that host antibodies such as IgG and IgM can bind to, which is separate from the transport domain (Gasparini et al., 2015). IgG and IgM antibodies bind to the antigenic subdomain instead of the bacterium antigens that would elicit an immune response, however as the transport domain is separate from this region, Neisseria meningitidis bacterium can still transport iron into the cell (Saleem et al., 2013). The binding of IgG and IgM bind to the FprB antigenic subdomain instead of the bacterium antigens allows the bacteria to avoid activation of the adaptive immune response while maintaining cellular iron transport (Saleem et al., 2013).

Genome Plasticity

Genome plasticity is evident in Neisseria meningitidis bacteria and is a contributor to evasion of the host response. The Neisseria meningitidis bacterial genome has regions of high variability which are thus prone to mutation (Gasparini et al., 2015). The DNA within these regions encode genes for adherence, entry, and colonization, thus Neisseria meningitidis has highly variable surface proteins, and massive antigenic variation. By constantly changing antigenic targets, Neisseria meningitidis bacteria can avoid antibody binding and activation of the immune response (Pollard & Goldblatt, 2001). An example of a highly variable surface protein is PorA, a cation selective outer membrane transporter found amongst all serogroups of Neisseria meningitidis (Gasparini et al., 2015). Due to its ubiquity, one may assume that PorA would be a candidate target for antibody and vaccine selection, however Neisseria meningitidis has evolved mechanisms to deter immune response against PorA (Pollard & Goldblatt, 2001). Neisseria meningitidis bacteria can generate multiple variants of PorA by 1) altering nucleotide lengths 2) incising or excising mobile DNA elements of the gene and 3) introducing point mutations (Lo et al., 2009). These multiple variants limit antibody recognition by changing previously recognized epitopes to new, unknown structures, and allow evasion of both the innate and adaptive immune response (Lo et al., 2009).

N. meningitidis also encodes a number of genes within its genome that aid in host evasion. For example, N. meningitidis encodes an IgA protease which prevents IgA in the mucous from binding to the bacterium (Finlay & McFadden, 2006). This protease uses an autotransporter process which enables the enzyme to be secreted from the bacterium (Finlay & McFadden, 2006). In addition to overcoming immunoglobulins, N. meningitidis must also overcome other host cellular secretions. As discussed earlier, immune cells, such as macrophages and neutrophils, can produce ROS and AMPs as part of the innate response (Gasparini et al., 2015). N. meningitidis encodes catalase in their katA gene, which is regulated by OxyR. In addition, superoxide dismutase is coded by SodB and SodC. Catalase and superoxide dismutase play a very important role in neutralizing the toxic response of ROS, thus protecting the bacterium from destruction (Gasparini et al., 2015). Other genes such as aniA, CycP, nirK, nsrR, and norB, encode for denitrifying enzymes which further promote the survival of N. meningitidis by downregulating the production of NO-dependent cytokines when needed, such as TNF-α, IL-8 and IL-10 (Gasparini et al., 2015). N. meningitidis are also able to upregulate receptors which allow them to sequester nutrients from the host, thus promoting their survival and overcoming host responses. For instance, outer membrane receptors allow the bacterium to acquire nutritional metal, including zinc and iron. The ZnuD receptor of N. meningitidis enables it to take up zinc, allowing it to specifically avoid killing by neutrophils (Gasparini et al., 2015).

Additionally, Neisseria meningitidis has multiple mechanisms for DNA repair, as the oxidative burst response from neutrophils and monocytes can result in bacterial damage (Silhan et al., 2012). The bacteria therefore encodes specialized proteins to fix DNA damage such as Neisserial exonuclease; which recognizes and removes DNA bases and DNA lesions caused by oxidative damage, and Neisserial apurinic/apyrimidinic endonuclease; which excises abasic and damaged DNA residues for the DNA backbone (Silhan et al., 2012). The bacteria also utilizes nitric oxide from the host environment during respiration, denitrifying the environment and impacting the host ability to produce cytokines that are dependent on nitric oxide, including IL-8 and TNF, which are important for the innate immune response (Gasparini et al., 2015).These repair mechanisms optimize Neisseria meningitidis bacteria survival.

Temperature Response

Neisseria meningitidis bacteria can sense temperature changes within host cells and utilize this signal as a warning for an upcoming immune response. The increase in host temperature as a result of fever can cause additional stress on bacteria, potentially staving off an infection. However, a change in host temperature as a result of fever may signal for Neisseria meningitidis bacteria to increase the expression of proteins utilized to evade a host immune response, evident in the historical observation that meningococcal infections are often preceded by an infection that causes fever (Gasparini et al., 2015). Specifically, bacterial RNA thermometer genes, located close to the 5’ untranslated region of the css operon, are able to sense an increase in temperature. At low temperatures, the RNA thermometer gene expresses mRNA transcripts that encode a protein complex (Gasparini et al., 2015). This protein complex binds to cellular ribosomes, and prevents translation of bacterial proteins used for host evasion. With increasing temperatures, as induced by the inflammatory response, the protein complex undergoes temperature-mediated conformational changes, disassociating from the ribosome and allowing for translation of the capsule biosynthesis operon, subsequently upregulating the production of immune evasion proteins (Gasparini et al., 2015). An example of an evasion protein is cssA, which is a gene required for sialic acid biosynthesis, a critical compound involved in molecular mimicry and thus host immune evasion (Lappann et al., 2016).

Pili

OMPs facilitate tight adherence to the epithelial surface, and also suppression of the immune response and passage into systemic infection (Gray-Owen & Blumberg, 2006).

The type IV pili of N. meningitidis play an important role in the initial adhesion process, specifically PilE, PilC and PilQ (Gasparini et al., 2015). To assist in adhesion, N. meningitidis utilizes the interaction of pili, and class 5 outer-membrane proteins (OMPs) such as Opc or Opa; furthermore, antigenic and phase variation in pilin subunits affects the adhesiveness of the bacteria in an effort to increase immune evasion. It is thought that CD46, present on epithelial cells, is a possible receptor for host-pathogen pilin interactions (Pollard & Goldblatt, 2001). OMPs serve to mediate the binding to epithelial-cell membrane surface receptors; particularly, Opc has been found to be critical in the successful invasion of non-capsulated organisms through the interaction with heparan sulphate proteoglycans or integrins present on the epithelial cell surface (Pollard & Goldblatt, 2001)

Pili also mediate a number of processes while N. meningitidis is in systemic circulation. Once N. meningitidis reaches the BBB, the type IV pili interact with endothelial receptors – this interaction with receptors such as β2-adrenergic receptor causes the tight junctions holding epithelial cells together to be downregulated, thus allowing N. meningitidis to enter the CNS (Gasparini et al., 2015). While N. meningitidis can access the CNS through this paracellular pathway, N. meningitidis can also pass through endothelial cells of the BBB transcellularly. The bacteria can replicate in vacuoles inside the cells and they are protected from intracellular death by their capsule (Borkowski et al., 2020). The Opc and Opa proteins of N. meningitidis allow the bacterium to hijack cell cycle checkpoints in order to replicate and enter the CNS (Borkowski et al., 2020).

References

Borkowski, J., Schroten, H., & Schwerk, C. (2020). Interactions and Signal Transduction Pathways Involved during Central Nervous System Entry by Neisseria meningitidis across the Blood–Brain Barriers. International Journal of Molecular Sciences, 21(22), 8788. https://doi.org/10.3390/ijms21228788

Finlay, B. B., & McFadden, G. (2006). Anti-immunology: Evasion of the host immune system by bacterial and viral pathogens. Cell, 124(4), 767–782. https://doi.org/10.1016/j.cell.2006.01.034

Gasparini, R., Panatto, D., Bragazzi, N. L., Lai, P. L., Bechini, A., Levi, M., Durando, P., & Amicizia, D. (2015). How the Knowledge of Interactions between Meningococcus and the Human Immune System Has Been Used to Prepare Effective Neisseria meningitidis Vaccines. Journal of Immunology Research, 2015. https://doi.org/10.1155/2015/189153

Gray-Owen, S. D. & Blumberg, R. S. (2006). CEACAM1: contact-dependent control of immunity. Nature Reviews Immunology, 6, 433-466. https://doi.org/10.1038/nri1864

Herold, R., Schroten, H., & Schwerk, C. (2019). Virulence Factors of Meningitis-Causing Bacteria: Enabling Brain Entry across the Blood-Brain Barrier. International Journal of Molecular Sciences, 20(21). https://doi.org/10.3390/ijms20215393

Lappann, M., Otto, A., Brauer, M., Becher, D., Vogel, U., & Johswich, K. (2016). Impact of Moderate Temperature Changes on Neisseria meningitidis Adhesion Phenotypes and Proteome. Infection and Immunity, 84(12), 3484–3495. https://doi.org/10.1128/IAI.00584-16

Lewis, L. A., & Ram, S. (2014). Meningococcal disease and the complement system. Virulence, 5(1), 98–126. https://doi.org/10.4161/viru.26515

Lo, H., Tang, C. M., & Exley, R. M. (2009). Mechanisms of avoidance of host immunity by Neisseria meningitidis and its effect on vaccine development. The Lancet Infectious Diseases, 9(7), 418–427. https://doi.org/10.1016/S1473-3099(09)70132-X

Pollard, A. J., & Goldblatt, D. (2001). Immune response and host-pathogen interactions. Methods in Molecular Medicine, 66, 23–39. https://doi.org/10.1385/1-59259-148-5:23

Saleem, M., Prince, S. M., Rigby, S. E. J., Imran, M., Patel, H., Chan, H., Sanders, H., Maiden, M. C. J., Feavers, I. M., & Derrick, J. P. (2013). Use of a molecular decoy to segregate transport from antigenicity in the FrpB iron transporter from Neisseria meningitidis. PloS One, 8(2), e56746. https://doi.org/10.1371/journal.pone.0056746

Silhan, J., Nagorska, K., Zhao, Q., Jensen, K., Freemont, P. S., Tang, C. M., & Baldwin, G. S. (2012). Specialization of an Exonuclease III family enzyme in the repair of 3′ DNA lesions during base excision repair in the human pathogen Neisseria meningitidis. Nucleic Acids Research, 40(5), 2065–2075. https://doi.org/10.1093/nar/gkr905

Stephens, D. S., Greenwood, B., & Brandtzaeg, P. (2007). Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. The Lancet, 369(9580), 2196–2210. https://doi.org/10.1016/S0140-6736(07)61016-2

Talà, A., Cogli, L., De Stefano, M., Cammarota, M., Spinosa, M. R., Bucci, C., & Alifano, P. (2014). Serogroup-Specific Interaction of Neisseria meningitidis Capsular Polysaccharide with Host Cell Microtubules and Effects on Tubulin Polymerization. Infection and Immunity, 82(1), 265–274. https://doi.org/10.1128/IAI.00501-13

van Deuren, M., Brandtzaeg, P., & van der Meer, J. W. M. (2000). Update on Meningococcal Disease with Emphasis on Pathogenesis and Clinical Management. Clinical Microbiology Reviews, 13(1), 144–166.

iv. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria ?

Quick administration of treatment, such as broad-spectrum antibiotics, is crucial to the recovery of Neisseria meningitidis. Penicillin is typically used to treat meningococcal meningitides, however, ampicillin and ceftriaxone are also helpful (1). These antibiotics, administered intravenously, can cross the blood brain barrier when the meninges are inflamed and can kill the bacteria present in the brain, the blood stream and the rest of the body (2). Treatment stops the proliferation of N. meningitidis and lowers the endotoxin concentration within hours and, after a few days, the patient will start to feel better and symptoms will subside (1). Even with rapid treatment, about 8-15% of patients die because of the damage inflicted upon the host during the first 24-48 hours of infection (3). The bacteria is completely removed from the host via antibiotics and the host immune response, however, it is possible that N. meningitidis can remain in the host and colonize in the nasopharynx without causing symptoms or infection (4). This happens due to the commensal nature of N. meningitidis strains also known as ‘carriage’ which occurs in about 10-35% in young adults in the US and Europe (5).

The damage and complications which the immune response and the bacteria itself have caused will factor into the level of recovery an individual can achieve post infection. Continuous inflammation of the brain can result in permanent nerve damage and produce long term issues concerning muscle weakness, balance, seizures, vision, headaches and, most commonly,  hearing loss (4). Endothelial damage adds to the inflammatory response and can lead to edema and brain herniation which can cause neurological deficits and/or death because of the increased, prolonged cranial pressure (6). Other long-term problems can arise from complications during infection. If a patient experiences septic shock, this can result in acute myocardial failure and leave survivors with long term myocardial dysfunction relating to contractility (7). Impaired organ diffusion due to increased blood volume in vessels can lead to infarction of organs and limbs which can result in long term dysfunction of the affected organs and even limb loss (7). Ultimately, the level of recovery is dependent on the time it takes to receive treatment and the amount of damage inflicted upon the patient before treatment.

Reinfection of the same N. meningitidis serotype is unlikely in healthy adults given that the adaptive immune response is responsible for producing memory B and T cells, as well as antibodies that can combat reinfection before invasion and the manifestation of symptoms (8). Unfortunately, even individuals with normal antibody levels against N. meningitidis may still become infected. Memory cells secrete IgA antibodies which do not bind complement and are manipulated by N. meningitidis to prevent IgM and IgG antibodies from binding to the bacteria and activating complement (9). Individuals who do not produce serum bactericidal antibodies as a result of infection were shown to contract the disease, therefore, confirming the importance of these antibodies in the protection from subsequent infections (8). Moreover, those who have immunodeficiencies can have difficulty acquiring long term protection from reinfection. For instance, individuals with complement deficiencies specifically concerning fragments C2 and C5-C9 can only produce a reduced complement cascade and overall have less ability to kill bacteria (10). The uncertain long-term immunity and the severity of the disease are both factors as to why available vaccinations against certain serotypes of N. meningitidis are important to take (1).

References

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2. La M, C P, Ae B, Ac R, N R, S S, et al. Detection of Ciprofloxacin-Resistant, β-Lactamase-Producing Neisseria meningitidis Serogroup Y Isolates - United States, 2019-2020. MMWR Morb Mortal Wkly Rep. 2020 Jun 19;69(24):735–9.
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7. Pathan N. Pathophysiology of meningococcal meningitis and septicaemia. Archives of Disease in Childhood. 2003 Jul 1;88(7):601–7.
8. Gasparini R, Panatto D, Bragazzi NL, Lai PL, Bechini A, Levi M, et al. How the Knowledge of Interactions between Meningococcus and the Human Immune System Has Been Used to Prepare Effective Neisseria meningitidis Vaccines. J Immunol Res [Internet]. 2015 [cited 2021 Feb 21];2015. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553322/
9. Morse SA. Neisseria, Moraxella, Kingella and Eikenella. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2021 Feb 10]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7650/
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