Course:PATH4172019W2/Case 1

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Case 1 : School Sores

6-year-old Miriam has developed red sores around her mouth and nose. At the start of class her teacher notices the rash and calls her parents to take her home. Her parents take her to the family doctor to be examined.

She is afebrile and does not have any swollen lymph nodes. There is no rash on her hands or feet or inside her mouth. The doctor swabs the rash and sends the swab to the local Microbiology Laboratory. where the organism responsible for the infection is identified as Streptococcus pyogenes. The doctor prescribes an antibiotic and tells her parents that she needs to stay at home for a couple of days.


Q1. The Body System Questions

(i) What are the signs (objective characteristics usually detected by a healthcare professional) and symptoms (characteristics experienced by the patient, which may be subjective). Why did the doctor examine for swollen lymph nodes?

(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 is/are the antibiotic(s) of choice to treat this infection and how do they work to rid the body of the organism?

(iv) Why did the doctor suggest that Miriam stay at home for a few days? Are Miriam’s parents at risk of acquiring this infection?

Q2. The Microbiology Laboratory Questions

(i) Including the stated bacteria, 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 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 that might allow the identification of the bacteria named in this case.

Q3. Bacterial Pathogenesis Questions

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

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

(ii)  Entry: how does the bacteria enter into the human host and take up residence. What are the molecular, cellular and/or physiological factors at play in this site specificity, and in the initial adherence step, (referencing both the bacteria 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?

Q4. The Immune Response Questions

(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 with the candidate infectious agent?

Reports: School Sores

6-year-old Miriam has developed red sores around her mouth and nose. At the start of class her teacher notices the rash and calls her parents to take her home. Her parents take her to the family doctor to be examined.

She is afebrile and does not have any swollen lymph nodes. There is no rash on her hands or feet or inside her mouth. The doctor swabs the rash and sends the swab to the local Microbiology Laboratory. where the organism responsible for the infection is identified as Streptococcus pyogenes. The doctor prescribes an antibiotic and tells her parents that she needs to stay at home for a couple of days.


Q1. The Body Systems Questions

Question (i)

(i) What are the signs (objective characteristics usually detected by a healthcare professional) and symptoms (characteristics experienced by the patient, which may be subjective). Why did the doctor examine for swollen lymph nodes?

Infection with Streptococcus pyogenes can take on many forms and is often a respiratory infection or a skin infection like impetigo or cellulitis (Todar, 2012). Pharyngitis and tonsillitis are the two main respiratory infections associated with streptococcal disease, and are classified as inflammation of the pharynx or tonsils, respectively. In regards to acute skin rashes associated with streptococcal disease, there are impetigo and cellulitis. Impetigo is superficial and only involves infection of the epidermal layer of skin, whereas cellulitis is deep and spreads to the subcutaneous tissues (Todar, 2012). Additionally, S. pyogenes can present as Scarlet Fever which is a rash characterized by a red appearance of the skin accompanied with pharyngitis (Todar, 2012). The symptoms of an S. pyogenes infection can include a sore throat, a red rash on the face, crusty red lesions on the face, or an infection of the skin (Stevens and Bryant, 2016).

In Miriam’s case, she is presenting with impetigo. The signs for this include the red sores around her mouth, lack of swollen lymph nodes, lack of a systemic rash to her hands/feet and inside her mouth, and lack of fever (afebrile). However, since the rash is experienced by the patient, it is also a symptom. Therefore, the red sores double as a sign and a symptom, depending on the observer.

These observations suggest that the bacterial infection is localized to her mouth and nose and has not spread systemically, indicating that it was unlikely to be a severe skin infection such as cellulitis. Furthermore, the absence of swollen lymph nodes showed that the bacteria was likely not present in her mouth and throat, meaning that respiratory infections such as pharyngitis or tonsillitis were unlikely. By checking for this, the doctor was able to rule out some forms of infection caused by S. pyogenes. Similarly, the lack of fever, systemic rash, and pharyngitis were clear indications that the patient did not have Scarlet Fever (Todar, 2012).

References:

Stevens, D. L., & Bryant, A. E. (2016). Streptococcus pyogenes: Basic Biology to Clinical Manifestations. Oklahoma City, OK: University of Oklahoma Health Sciences Center.

Todar, K. (2012). Textbook of Bacteriology. Madison. WI: University of Wisconsin.

Question (ii)

(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.

The body system affected is the integumentary system, because Miriam has a skin infection (impetigo) (1). The skin functions as a barrier, protecting the body from the external environment (2). The two main layers of the skin are the epidermis (outer layer) and dermis (inner layer) (2). Normally, the skin acts as a permeability barrier, immune response barrier, antimicrobial barrier, antioxidant barrier, and photoprotection barrier (3). When this skin barrier is breached by bacteria either through direct invasion or by infecting already compromised skin, an infection occurs (4).

The integumentary system plays an important role in immune defence. The skin is constantly exposed to hazards and has epithelial and immune cells that exploits immune surveillance. The epidermis is host to melanocytes and immune cells such as Langerhans cells (LGs) and T lymphocytes. The dermis of the skin has a structural collagen framework hosting immune cells such as dendritic cells, T cells, NK cells, B cells, mast cells and macrophages (5). There are many pathways in skin immune response and they ensure that active defence and regulatory mechanisms are in place to maintain hemostasis.

The skin is not a sterile place and is often colonized with different types of bacteria. In normal conditions, they feed on corneocyte debris and sebum and prevent other undesirable bacteria from developing (5). However, if the number of bacteria gets too large or the surrounding environment becomes susceptible to bacterial growth, the balance of the skin microbiome is disrupted and skin infection can occur.

Impetigo is usually found to occur due to skin abrasions, insect bites, eczema or trauma (4). The preliminary step of S. pyogenes infections is adhesion to the extracellular matrix (6). Streptococcal adhesins bind to host components of ECM such as fibronectin, collagen, and laminin (6). The most prevalent adhesins are those that are covalently linked to the peptidoglycan via transpeptidases called sortase A (6). Lipoteichoic acid (LTA), which is a component of its cell wall, facilitates the first step of adhesion, and pili may also be interacting with host epithelial cells (6). S. pyogenes also have anchorless adhesins, and one such protein is alpha-enolase, which functions as a plasminogen-binding protein (6). Then, the bacterial streptokinases convert plasminogen to plasmin, which in turn degrades fibrin clots and ECM matrix to allow the bacteria to come in closer proximity to the host cell surface (6). M proteins are another type of adhesin which are virulence factors of S. pyogenes that bind to various ECM components resulting in aggregation of the bacteria, and are important for colonization, invasion of cells and evasion of phagocytosis (6). S. pyogenes have fibronectin-binding proteins that bind with high affinity to fibronectin on epithelial cells, further mediating adhesion (6). S. pyogenes bacteria use many mechanisms to gain entry into host cells. They can enter through invagination of host cell membrane or engulfment of the bacteria through growth of host cell microvilli over the bacteria (6).

The resulting lesion created by the bacteria lead to formation of papules that become vesicles surrounded by erythema, finally enlarging to form pustules surrounded by a red rash (6). Since Streptococcus pyogenes causes inflammatory lesions in the skin (1), the skin barrier’s functioning has been disturbed by the infection, meaning foreign substances including other pathogens can more easily enter the body.

References

1. Baron S. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 13, Streptococcus. [cited 2020 Jan 15]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7627/

2. Torrison J, Cameron R. Diseases of Swine [Internet]. 11th ed. [place unknown]: John Wiley & Sons, Inc; 2019. Chapter 17, Integumentary System. [cited 2020 Jan 24]. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/9781119350927.ch17#

3. Del Rosso J, Zeichner J, Alexis A, et al. Understanding the Epidermal Barrier in Healthy and Compromised Skin: Clinically Relevant Information for the Dermatology Practitioner. J Clin Aesthet Dermatol. 2016 Apr 1;9(4 Suppl 1):S2-S8.

4. Ibrahim F, Khan T, Pujalte G. Bacterial Skin Infections. Prim Care. 2015 Dec;42(4):485-499.

5.Di Meglio, P., Perera, G. K., & Nestle, F. O. (2011). The multitasking organ: recent insights into skin immune function. Immunity, 35(6), 857-869.

6. Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes: Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016. Epidemiology of Streptococcus pyogenes. [cited 2020 Jan 15]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK343616/

Question (iii)

(iii) What is/are the antibiotic(s) of choice to treat this infection and how do they work to rid the body of the organism?

Several topical antibiotics have been recommended for skin infections brought on by S. pyogenes. These antibiotics are in a cream or ointment form that can be directly applied to the local area of infection as opposed to consumed orally and transported through the bloodstream (Stevens and Bryant, 2016). A commonly prescribed topical antibiotic for S. pyogenes is mupirocin. This antibiotic can inhibit bacterial protein synthesis by reversibly binding to an aminoacyl t-RNA synthetase, thus preventing the individual amino acids from being bonded to tRNA (Parenti et al., 1987). Alternatively, retapamulin is also a topical antibiotic that has been shown to be effective against S. pyogenes infection. This antibiotic inhibits bacterial protein synthesis by binding to the 50S subunit of bacterial ribosomes, thus preventing peptide bond formation (U.S. National Library of Medicine, 2020).

Otherwise, oral antibiotics may also be prescribed, especially if topical antibiotics have not been successful in eliminating the infection. Traditionally, penicillin and erythromycin were commonly prescribed oral antibiotics for S. pyogenes. Usually for group A strep pharyngitis infections, penicillin or amoxicillin are the main antibiotics considered. (CDC, 2018). This is because treatment with penicillin limits the transmission of the bacteria. Penicillin is a bactericidal beta-lactam antibiotic that inhibits the synthesis of the bacterial cell walls (Castle, 2007). Penicillin's ability to act on peptidoglycan (PG), a component of the bacterial cell wall, makes it a bacteriostatic antibiotic. PGs are made up of linear glycan strands cross-linked by short peptides (Vollmer et al, 2008). The antibiotic binds to one or more penicillin-binding proteins, such as carboxypeptidases and transpeptidases, which are located in the bacterial cell membrane (Castle, 2007). Then, they inhibit the final step in PG synthesis in the cell wall of the bacteria, thus preventing cell wall biosynthesis and function (Castle, 2007). Penicillin block the protein parts that link PG together through penicillin-binding proteins (PBP), hence preventing bacteria from closing the small holes that occur as cells divide (Sauvage et al, 2008). PG enable bacteria to resist intracellular pressure and maintain cell shape (Sauvage et al, 2008). Due to the presence of these holes, water would rush into the bacteria, leading to lysis (ie. cell death). The cells undergo death due to the action of autolytic enzymes, such as autolysins and murein hydrolases. Alternatively, erythromycin is a bacteriostatic macrolide antibiotic that inhibits bacterial protein synthesis by binding to the 50S subunit of bacterial ribosomes, thus preventing peptide bond formation. Unfortunately, due to the emergence of drug-resistant strains of S. pyogenes, they are less frequently prescribed nowadays (Hartman-Adams et al., 2014). Other possible oral antibiotics include amoxicillin/clavulanate, a combination of a beta-lactam antibiotic and a beta-lactamase inhibitor, as well as cephalosporin, a bactericidal beta-lactam antibiotic. If the patient is allergic to penicillin, erythromycin or cephalosporin can be prescribed. Bactericidal antibiotics would kill the bacteria (such as penicillin) (Nemeth et al., 2015). Bacteriostatic antibiotics would only inhibit bacterial growth (Nemeth et al., 2015). Bactericidal antibiotics would be much more powerful as bacteriostatic antibiotics would require phagocytic cells to clear out the bacteria. Hence, for those that are ill or immunosuppressed, bactericidal antibiotics would be recommended. (Nemeth et al., 2015).

Since Miriam does not exhibit signs of pharyngitis (such as swollen lynph nodes), I would recommend a topical antibiotic, either mupirocin or retapamulin, because they have been shown to be as effective as oral antibiotics while also avoiding systemic side effects such as disruptions of the gastrointestinal system (Hartman-Adams et al., 2014).

References

“Group A Strep.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 1 Nov. 2018, www.cdc.gov/groupastrep/diseases-hcp/strep-throat.html

Castle, S. (2007). xPharm: The Comprehensive Pharmacology Reference. Huntington, WV: Elsevier. Doi: https://doi.org/10.1016/B978-008055232-3.61011-6

Hartman-Adams, Holly, et al. “Impetigo: Diagnosis and Treatment.” American Family Physician, American Academy of Family Physicians, 15 Aug. 2014, www.aafp.org/afp/2014/0815/p229.html#sec-4.

Johannes Nemeth, Gabriela Oesch, Stefan P. Kuster, Bacteriostatic versus bactericidal antibiotics for patients with serious bacterial infections: systematic review and meta-analysis, Journal of Antimicrobial Chemotherapy, Volume 70, Issue 2, February 2015, Pages 382–395, https://doi-org.ezproxy.library.ubc.ca/10.1093/jac/dku379

Parenti, MA, et al. “Mupirocin: a Topical Antibiotic with a Unique Structure and Mechanism of Action.” Clinical Pharmacology, vol. 6, no. 10, 1987, pp. 761–770.

“Retapamulin.” National Center for Biotechnology Information. PubChem Compound Database, U.S. National Library of Medicine, 2020, pubchem.ncbi.nlm.nih.gov/compound/Retapamulin#section=Pharmacology-and-Biochemistry.

Sauvage E, Kerff F, Terrak M, Ayala JA & Charlier P (2008) The penicillin‐binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev 32: 234– 258.

Stevens, Dennis L., and Amy E Bryant. “Impetigo, Erysipelas and Cellulitis.” National Center for Biotechnology Information, U.S. National Library of Medicine, 10 Feb. 2016, www.ncbi.nlm.nih.gov/books/NBK333408/?report=reader.

Question (iv)

(iv) Why did the doctor suggest that Miriam stay at home for a few days? Are Miriam’s parents at risk of acquiring this infection?

The doctor likely recommended that Miriam stay at home because impetigo is highly contagious and can rapidly spread through schools and daycares, via direct contact or fomites, objects or materials likely to carry infection, such as clothes or furniture (Cole and Gazewood, 2007). It is recommended to keep children out of daycare and school for 24 hours after starting antibiotic treatment (Johns Hopkins Medicine, n.d.). This is likely because treatment with an antibiotic for 24 hours or longer eliminates contagiousness, however, antibiotics should still be taken to completion as prescribed (New York State Department of Health, 2011). Overall, bacteria can be spread via direct contact with nose and throat discharges of an infected individual, as well as contact with infected skin lesions, as this is the main reservoir for S. pyogenes (New York State Department of Health, 2011). The bacteria can also be spread through airborne droplets, such as through shared food or drink, or from coughing and sneezing (Mayo Clinic, 2018). Miriam is capable of transmitting S. pyogenes via the skin-to-skin contact when coming into close contact with others, which can cause infection if the skin sustains trauma, letting bacteria in. Skin disruption can reveal fibronectin receptors in the skin, which are required for teichoic acid adhesion of the bacteria and subsequent colonization (Lewis, 2019).

Although impetigo is more common in children, it can occur in adults, meaning that Miriam’s parents can theoretically acquire impetigo (Pereira, 2014). The following information was found for neonates; however, I believe differences in these features still exist between adults and young children. Firstly, there are microstructural differences such as a thinner stratum corneum and papillary dermis in children that can make them more susceptible to skin infections (Stamatas et al., 2010). A higher surface area-to-volume ratio and decreased subcutaneous fat stores can make neonates more susceptible to percutaneous toxins (Darmstadt and Dinulos, 2000; Afsar, 2009). Furthermore, it is encouraged to keep the child’s fingernails short to prevent scratching and spreading of the infection (Johns Hopkins Medicine, n.d.). This can be indicative that impetigo spreads more easily in children due to frequent breakages in the skin, which adults are less likely to sustain.

Overall, there are a variety of factors to consider. Host factors such as integrity of the skin barrier, production of defensins, adequate nutritional status, and more can lead to resistance of infection. Moreover, handwashing between children and caretakers greatly decreases their chance of getting impetigo (Pereira, 2014; Luby et al., 2005). Although the bacteria are capable of spreading to the skin of the parents, good hygiene and caution with insults to the skin can be greatly preventative against impetigo.

References

Afsar, F. (2009). Skin care for preterm and term neonates. Clinical and Experimental Dermatology, 34(8), pp.855-858.

Cole, C. and Gazewood, J. (2007). Diagnosis and Treatment of Impetigo. Am Fam Physician, (75), pp.859-64, 868.

Darmstadt, G. and Dinulos, J. (2000). Neonatal Skin Care. Pediatric Clinics of North America, 47(4), pp.757-782.

Johns Hopkins Medicine. (n.d.). Impetigo. [online] Available at: https://www.hopkinsmedicine.org/health/conditions-and-diseases/impetigo [Accessed 23 Jan.

2020].

Lewis, L. (2019). Impetigo. [online] Medscape. Available at: https://emedicine.medscape.com/article/965254-overview#a1 [Accessed 23 Jan. 2020].

Luby, S., Agboatwalla, M., Feikin, D., Painter, J., Billhimer, W., Altaf, A. and Hoekstra, R. (2005). Effect of handwashing on child health: a randomised

controlled trial. The Lancet, 366(9481), pp.225-233.

Mayo Clinic. (2018). Strep throat. [online] Available at: https://www.mayoclinic.org/diseases-conditions/strep-throat/symptoms-causes/syc-20350338 [Accessed 25 Jan. 2020].

New York State Department of Health. (2011). Streptococcal Infections (invasive group A strep, GAS). [online] Available at:

https://www.health.ny.gov/diseases/communicable/streptococcal/group_a/fact_sheet.htm [Accessed 24 Jan. 2020].

Pereira, L. (2014). Impetigo - review. Anais Brasileiros de Dermatologia, 89(2), pp.293-299.

Stamatas, G., Nikolovski, J., Luedtke, M., Kollias, N. and Wiegand, B. (2010). Infant Skin Microstructure Assessed In Vivo Differs from Adult Skin in

Organization and at the Cellular Level. Pediatric Dermatology, 27(2), pp.125-131.   

Q2. The Microbiology Laboratory

Question (i)

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

The key characteristics of Miriam’s infection are red sores around her mouth and nose. The fact that Miriam is afebrile, does not show swelling in her lymph nodes, and the rash hasn’t spread to other parts of her body suggests the infection is non-systemic[2.1 1]. Red sores around the mouth and nose are characteristic of Impetigo – a contagious bacterial skin infection that commonly affects infants and children[2.1 2]. Impetigo infection accounts for 10% of pediatric skin infections and is most prevalent in warm climates or summer seasons[2.1 3][2.1 4]. The bacteria causing impetigo can be easily transmitted to others by nasal discharge or skin-to-skin contact. With a lack of proper hygiene and hand-washing, children are most at risk for this disease. As a result, children diagnosed with impetigo are often withheld from school and sporting activities and are urged to avoid swimming pools, gymnasiums, and day cares[2.1 5].

Impetigo

Impetigo is an example of a primary skin infection, and is typically caused by one particular pathogen entering via a lesion in the skin[2.1 6]. These skin lesions can be caused by Staphylococcus aureus, a member of the normal skin microbiota[2.1 7]. Staphylococcus aureus contains toxins with specific proteases that can damage the outer skin layer[2.1 7]. Streptococcus pyogenes can then populate the already compromised dermal surfaces and cause skin infections such as impetigo[2.1 8]. In many cases co-infections with both Staphylococcus aureus and Streptococcus pyogenes have been observed[2.1 9]. Two forms of impetigo have been previously characterized: bullous and non-bullous impetigo.

Non-bullous impetigo:

Non-bullous impetigo comprises ~70% of impetigo cases and is most common in pre-school/school aged children[2.1 6][2.1 10]. Both Staphylococcus aureus and Streptococcus pyogenes are able to cause non-bullous impetigo[2.1 11]. It is characterized by small red blisters around the mouth, nose and occasionally the extremities. Blisters are quick to burst and emit fluid or pus, resulting in golden-coloured crusts[2.1 10]. While pain is moderate, the sores can be very itchy, and individuals are advised to avoid scratching to inhibit the infection from spreading to other people or parts of the body[2.1 10].

Bullous impetigo:

Bullous (staphylococcal) impetigo is caused by a specific strain of Staphylococcus aureus that secretes a locally-acting epidermolytic toxin, resulting in the characteristic superficial, thin-walled, bullous lesions[2.1 11]. After a lesion ruptures, a thin, transparent crust forms over the lesion, distinct from the yellowish crust in non-bullous impetigo[2.1 11]. As these lesions spread much more rapidly than non-bullous lesions, they appear on multiple parts of the body, including the trunk, legs, and arms[2.1 10].

The most common bacterial pathogens associated with impetigo, and the most likely culprits responsible for Miriam’s disease are Streptococcus pyogenes and Staphylococcus aureus.

Streptococcus pyogenes:

Streptococcal bacteria are Gram-positive cocci (round or oval shaped) and less than 2 µm in diameter[2.1 12]. They are catalase negative, hemolytic bacteria and possess Lancefield antigens[2.1 13]. Streptococci bacteria form characteristic chain colonies greater than 5mm in size and as facultative anaerobes are able to survive in both aerobic (oxygen containing) and anaerobic (oxygen lacking) conditions [2.1 12][2.1 13].

Streptococcus pyogenes are found in the normal human flora and become a threat for infection when host defense systems are compromised or when they are able to penetrate the constitutive defenses[2.1 14].6 It is a gram-positive, non-spore-forming bacterium that occurs in chains or pairs[2.1 14]. When Streptococcus pyogenes manage to infect vulnerable tissues, a variety of suppurative (pus-forming) infections can occur (such as impetigo), as well as pharyngitis, erysipelas (cellulitis) accompanied by fever) and Scarlet fever (if left untreated)[2.1 14]. S. pyogenes produces various virulence factors including adhesion proteins such as: M protein (inhibits phagocytosis), fibronectin-binding protein (Protein F) and lipoteichoic acid; hyaluronic acid as an immunological disguise and a phagocytosis inhibitor; invasins such as streptokinase, streptodornase and streptolysins; and exotoxins which cause the characteristic rash[2.1 14]. Infections caused by S. pyogenes occur mainly in the respiratory tract, bloodstream or the skin[2.1 14].

Staphylococcus aureus:

Staphylococcal bacteria are Gram-positive, spherical bacteria 0.5 - 1.5 µm in diameter, occur in clusters, and are normally found in bacteria cultures of the nose and skin of healthy humans[2.1 15]. They are also facultative anaerobes and grow at temperatures ranging from 15 to 45 degrees[2.1 15]. Though frequent, human staphylococcal infections usually remain localized at port of entry thanks to host system defenses[2.1 15]. It can cause a wide range of suppurative infections, food poisoning, and toxic shock syndrome[2.1 15].

S. Aureus produces various virulence factors: surface proteins to promote attachment to host tissue; invasins to aid bacterial spread; surface factors to inhibit phagocytosis; biochemical properties such as catalase to enhance survival in phagocytes; immunological disguises such as protein A and coagulase; membrane-damaging and exo-toxins aimed at damaging cell membranes and host tissues, promoting lesion formation[2.1 15].

References:

  1. Washington, JA (1996). "10". In Baron, S (ed.). Medical Microbiology. Galveston (TX): Medical Microbiology.
  2. "Impetigo". Mayo Clinic. 2019.
  3. Nardi, NM; TJ, Schaefer (2019). Impetigo. Treasure Island (FL): StatPearls Publishing.
  4. Wessels, MR (2011). "Clinical practice. Streptococcal Pharyngitis". The New England Journal of Medicine. 364(7): 648–655.
  5. "Impetigo:Overview". InformedHealth.org. 2006.
  6. 6.0 6.1 Allmon, A; Deane, K; Martin, KL (2015). "Common Skin Rashes in Children". American Family Physician. 92(3).
  7. 7.0 7.1 Hanakawa, Y; Schechter, NM; Lin, C; Garza, L (2002). "Molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin syndrome". Journal of Clinical Investigations. 110(1). doi:10.1172/JCI15766.
  8. Cunningham, MW (2000). "Pathogenesis of Group A Streptococcal Infections". Clinical Microbiology Reviews. 13(3). doi:10.1128/CMR.13.3.470.
  9. Bowen, AC; Tong, SY; Chatfield, MD; Carapetis, JR (2014). "The microbiology of impetigo in Indigenous children: associations between Streptococcus pyogenes, Staphylococcus aureus, scabies, and nasal carriage". BMC Infectious Disease. 14(1). doi:10.1186/s12879-014-0727-5.
  10. 10.0 10.1 10.2 10.3 Brazier, Y. "Impetigo: Treatment, symptoms, and causes". Medical News Today.
  11. 11.0 11.1 11.2 Aly, R. "98". In Baron, S (ed.). Microbial Infections of Skin and Nails (4th ed.). Galveston (TX): Medica Microbiology.
  12. 12.0 12.1 Patterson, M.J. (1996). Streptococcus. In Baron, S. (ed.), Medical Microbiology (4th ed.). Galveston, TX: University of Texas Medical Branch at Galveston.
  13. 13.0 13.1 Jorgensen, J. H., Pfaller, M. A., Carroll, K. C., & Ebrary, I. (2015). Chapter 22: Streptococcus. Manual of clinical microbiology (11th ed.). Chapter 22: Streptococcus. Washington, DC: ASM Press. 383-402.
  14. 14.0 14.1 14.2 14.3 14.4 Todar, K. "Streptococcus pyogenes and streptococcal disease". Online Text of Bacteriology.
  15. 15.0 15.1 15.2 15.3 15.4 Todar, K. "Staphylococcus aureus". Online Textbook of Bacteriology.

Question (ii)

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

All impetigo cases are caused by either S. pyogenes or S. aureus or a combination of both bacteria 1. However, both S. pyogenes and S. aureus are part of the indigenous flora of human skin 2.  Oftentimes, a physical examination of the observable signs, such as sores or accumulations of pus, during a doctor’s visit, along with staining and/or culturing of pus or exudate samples is adequate for diagnosis of impetigo 3. It is important that that physicians select specimens that represent the clinical disease in suspicion and collect enough specimen to stain in order to prevent misidentification 4.  

To collect a sample for further analysis, only the centre of the infected skin is swabbed to prevent collection of normal microbiota that may be found on the skin 3. Furthermore, it is important that decontamination procedures are adhered to and the infected skin is disinfected prior to swabbing 4. If pustules or vesicles are present, then a sterilized blade is used to scrape off the crust 3. These measures are taken during sample collection in order to eliminate contamination and  increase the accuracy of identification.

The swab should be contained in an air-tight, sealed and sterile tube with medium 5. Additionally, the swab should not touch the opening of the tube and the tip should b1e in contact with the medium 6. The optimal medium is 1mL of skim milk tryptone glucose broth 6.  The sample should be kept at room temperature and ideally transported to a microbiology laboratory within 3 – 4 hours 6. After transportation, the swab can be frozen and stored at -70 ˚C within 5 days of collection 6. When these samples are frozen, they can last for a long time (years), and when ready for testing, it is defrosted and incubated for 48 hours 6. The optimal temperature for incubation is 37 ˚C 6.

Skin bacteria, such as S. pyogenes, can be cultured using artificial media. Samples are often cultured on agar media with additional blood supplies. This media will allow for optimal growth of streptococci and detection of β-hemolysis 7. Usually, a selective medium is combined with general blood agar in order to accurately identify the bacteria 3. For example, in a blood agar medium, S. aureus often overgrows S. pyogenes when both are present, however when crystal violate is introduced to the medium (at 1μg/ml), S. pyogenes is selected for 3.

Importance of the Microbiology Laboratory:

Microbiology laboratory testing is necessary in determining the causative microbe, since many pathogens induce similar symptoms 3. Additionally, one infection can also be caused by multiple pathogens and identification of the correct microbes is critical for proper treatment 3. Microbiology laboratory testing is also necessary to determine the specific serotype for accurate physician diagnosis and optimal treatment selection that can eliminate the pathogen 8. Furthermore, the microbiology laboratory can perform genome analysis of conserved regions of the bacteria, such as 16S rRNA 9. This information gives physicians more confidence about their identification and diagnosis of a patient’s bacterial infection. Through microbiology laboratory testing, they can also potentially identify other pathogens that are also infecting Miriam. This allows for Miriam to be given the best and most specific treatment plan in order to eliminate the causative agent. Early testing and diagnosis decreases the likelihood that more severe infections can progress.

Laboratory testing can also determine if the infecting bacterial strain is resistant to antibiotics, in which case, another form of treatment must be considered. Antibiotic resistant can occur due the overuse of broad-spectrum antibacterial agents or the prescription of medication without confirmation from the lab or proper diagnosis. Antibiotic resistance is acquired through mutations, such.  as those that could potentially inactivate the microbial agent, or modifications to the target site on the bacteria so that it is no longer recognized by the agent. Resistance is a worldwide problem, as it reduces the ability to treat common, non-invasive infections with antibiotics. According to the World Health Organization, patients with drug-resistant bacterial infections are at high risk of death. For example, a strain of Staphylococcus aureus known as MRSA (methicillin-resistant Staphylococcus aureus) are 64% more likely to cause death in infected individuals than with non-resistant strains 10.

Since impetigo can be caused by S. aureus or S. pyogenes, or even a combination of both, laboratory testing can be helpful in determining the most effect treatment that both microbes are susceptible to.

If impetigo is not treated effectively and in a timely manner, there are several complications than can arise, including but not limited to: Staphylococcal scaled skin syndrome and post-streptococcal glomerulonephritis.

References:

1.    Nardi NM, Schaefer TJ. Impetigo. StatPearls Publishing: Treasure Island, Florida; 2019.

2.    Patterson MJ. Streptococcus. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 13.

3.    Aly R. Microbial Infections of Skin and Nails. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 98. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8301/#

4.    Washington JA. Principles of Diagnosis. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 10. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8014/

5.     Procedure: Swab for Culture & Susceptibility in Suspected Wound Infection. Procedure: Swab for Culture & Susceptibility in Suspected Wound Infection 2015 p. 1–6.

6.    Bowen AC, Tong SY, Chatfield MD, Carapetis JR. The microbiology of impetigo in Indigenous children: associations between Streptococcus pyogenes, Staphylococcus aureus, scabies, and nasal carriage. BMC Infectious Diseases. 2014;14(1).

7.    Spellerberg, B., & Brandt, C. (2016). Laboratory Diagnosis of Streptococcus pyogenes (group A streptococci). University of Oklahoma Health Sciences Center. https://www.ncbi.nlm.nih.gov/books/NBK343617/

8.    BC Centre for Disease Control. (2009). Group A Streptococcal (GAS) Disease. Retrieved from https://www.cdc.gov/groupastrep/diseases-hcp/strep-throat.html#diagnosis

9.    Pereira LB. (2014). Impetigo - review. Anais Brasileiros de Dermatologia 89(2). doi:10.1590/abd1806-4841.20142283

10.   Antimicrobial resistance [Internet]. World Health Organization. World Health Organization; [cited 2020Jan17]. Available from: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance

Question (iii)

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

Microscopic Examination:

Direct examination of the specimen is an easy and direct way to identify that the infection is in fact caused by bacteria, as opposed to a fungus or virus.[1] With more specific direct examination techniques such as microscopy, immunofluorescence, gram-staining, or other immunoassays, it may be possible to detect, and narrow-down specific microbial pathogens involved.[1] When performing a microscopic assay, a compound binocular microscope is sufficient.[1]

In Miriam’s case, the gram-staining technique allows the laboratory to confirm that the infection is in fact bacterial and can possibly identify present microorganisms. The technique involves staining bacterial cells with crystal violet (a water-soluble die), decolorization, and counterstaining (with safranin).[2] During the decolorization process (ex. with an ethanol compound), gram-positive bacteria retain the die in their cell membrane due to its increased thickness compared to gram-negative bacteria.[2] Gram-negative bacteria are instead stained by safranin during the counterstaining process.[2] Under microscopic examination, S. pyogenes would appear Gram positive, and in a chain-like formation of cocci.[3] S. aureus, however, would appear Gram positive in clustered formations of cocci.[4]

Culture:

In most cases, the causative bacteria of an infection is confirmed by isolating the organism in artificial media (either liquid broth or solid agar) and culturing it.[1] Solid media, though less sensitive to smaller amounts of microorganisms than liquid media, have the ability to provide isolated, identifiable, and quantifiable colonies.[1]

S. aureus and S. pyogenes can be cultured on sheep-blood agar at 37˚C and incubated for 48 hours.[5] Selective media for gram-positive bacteria, such as adding phenylethyl alcohol to the agar.[6] When examining the cultures, blood agar plates can be screened to detect the presence of beta-hemolytic colonies.[6] Typically, S. pyogenes colonies appear dome shaped, with a smooth, moist surface, displaying a white/grey colour, have a diameter greater than or equal to 0.5mm, and are surrounded by a zone of B-hemolysis (roughly 2-4 times the colony diameter).[6] As with direct microscopic examination, S. pyogenes will be arranged in chains of cocci. Conversely, S. aureus strains of bacteria form round, convex colonies (1-4mm diameter) with sharp borders, appearing golden in colour.

If S. aureus is identified in primary microscopic and culturing tests, Mannitol Salt Agar can be used to selectively grow S. aureus. The solid medium contains a variety of specific components: 7.5% sodium chloride, which inhibits most growth of other bacterial organisms; peptones and beef extract which supply vitamins, minerals, and amino acids for S. aureus growth; Mannitol (a fermentable carbohydrate) that when fermented, produces an acid detected by a phenol red indicator.[7] Once cultured, S. aureus produces yellow colonies surrounding yellow medium, while other staphylococci produce red colonies, and do not change the colour of the phenol red indicator.[7] Streptococci do not grow in mannitol salt agar.

Immunologic Assays:

Lancefield Antigen Test:

Once streptococci are identified in microscopic and culturing techniques, further laboratory techniques can be applied to definitively identify the streptococcal species. A common technique to identify S. pyogenes is the Lancefield antigen test, where Lancefield antigens on the bacterium’s surface are identified through antibody introduction.[6] This technique is an example of an immunologic agglutination technique, where the specific antibody for a given bacteria’s antigens is bound to a latex particle, or to a heat-killed, treated protein A-rich strain of S. aureus.[1] These coated antibodies are then used to detect specific antigen presence in a given antigen specimen. Agglutination is a great way to confirm suspected bacteria post-culture.[5] However, it is important to note that the presence of Lancefield Antigens are not limited to S. pyogenes, appearing also on Streptococcus anginosus and Streptococcus dysgalactiae, therefore further testing is needed for a reliable species identification.[6]

PYR test:

Another method to identify specific bacterial species are using ELISA (Enzyme-linked immunosorbent assay) tests. The most frequently used ELISA method is to use an antigen-specific antibody fixed to a solid phase (a latex or metal bead, or the inside surface of a well in a plastic tray). If that microbe’s antigen is present in the specimen, it binds to the antibody.[1] Then, a second antigen-specific antibody is added, that is bound to an enzyme that changes colour when bound to a specific substrate.[1] In effect, the addition of the second antibody sandwiches the microbe’s antigen between the solid phase, and the enzyme-conjugated antibody.[1] When the enzyme’s colour-changing substrate is added, the reaction is completed, and if the microbe in question is present in the specimen, there will be an observed colour change.[1]

In the case of identifying S. pyogenes, a version of ELISA called “PYR testing” is employed.[6] L-pyrrolidonyl-B-naphthylamide (PYR) is hydrolyzed to B-naphthylamide by an enzyme found on S. pyogenes surface called pyrrolidonyl aminopeptidase, which produces a red colour when cinnamaldehyde is added.[6]

Susceptibility test:

Bacitracin susceptibility is a widely used screening method for identification of S. pyogenes as S. pyogenes are highly sensitive to Bacitracin. When sub-cultured on a sheep blood agar plate with a disk containing 0.04U of bacitracin is added and left to incubate overnight, a zone of inhibition surrounding the bacitracin disk indicates the susceptibility of the strain and confirms S. pyogenes as the causative bacterium.[6]

Catalase test:

The catalase test is an important method for differentiating catalase-positive staphylococci from catalase-negative streptococci.[3][4] Catalase is produced by bacteria that respire using oxygen, protecting them from toxic by-products produced from oxygen metabolism such as hydrogen peroxide.[8] The catalase enzyme neutralizes the harmful effects of H2O2 to the bacterium, such that the enzyme expedites the breakdown of hydrogen peroxide into water and oxygen, experimentally showcased by rapid formation of bubbles.[9] In the catalase test, the slide/drop method is popular as it requires a small specimen sample.[9] A microscope slide is placed in a petri dish, followed by a small amount of specimen (already well-isolated) placed onto the slide.[9] A dropper is then used to administer a drop of 3% H2O2 solution onto the slide.[9] If bubbles immediately start to form, the organism in question is S. aureus.

Coagulase test:

If the suspected causative bacterium is S. aureus, further testing is required to confirm the specific species of staphylococcus present. A common test to determine this designation is the coagulase test, as S. aureus are coagulase-positive, while other species of staphylococcal bacteria are coagulase-negative.[10] Coagulase negative staphylococcal bacteria are most common on healthy skin flora, therefore the presence of coagulase positive S. aureus possibly indicates sign of infection.[11] Coagulase is an enzyme that converts fibrinogen to fibrin in blood plasma. S. aureus produces both bound coagulase, which is identified via slide test, and free coagulase, identified via tube test.[10]

In the slide test, bound coagulase (“clumping factor”) causes agglutination via converting fibrinogen to fibrin directly when the specimen is suspended on a glass slide and mixed with plasma.[10] Positive results are showcased via agglutination in less than 10 seconds.[10] Confirmation of negative slide test results can be done via tube coagulase test, which detects staphylocoagulase reacting with coagulase-reacting factor (CRF) which indirectly converts fibrinogen to fibrin.[10]A suspension of the bacterium is incubated with plasma at body temperature, and within 4 hours, the formation of a clot indicates a positive test, confirming the presence of S. aureus.

16sRNA Sequencing:

While the aforementioned techniques are useful in determining whether the impetigo infection was caused by S. pyogenes or S. aureus, they do not give information on the specific strain of the causative species. Strains within a given species may exhibit biochemical variability that may affect the virulence, and treatment of the infection at hand.[12] 16SrRNA sequencing allows us to determine the specific strains and specific mutations within the causative bacteria, helping physicians to prescribe more specific, narrow-spectrum antibiotics that ultimately make treatment more effective.[13]

In all bacteria, rRNA is an extremely conserved sequence, acting as a scaffold to hold together large and small subunits of the bacteria’s ribosome.[13] Prokaryotic ribosomes have 3 types of rRNA: 23S, 16S and 5S.[12][13] In terms of bacterial taxonomic classification, the 16S rRNA is the easiest and most affordable to sequence, the most universally distributed among species, and contains sufficient phylogenetic information for strain identification.[12]

To perform 16SrRNA sequencing, the first step is to isolate the DNA, and use PCR amplification using primers (commercially available) targeted to the 16S rRNA genes.[12][13] The isolated sequence is then compared to the library of isolated 16S gene sequences to identify the specific strain using bioinformatic technology, and a variation analysis.[13]

References:

  1. 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Washington JA. Principles of Diagnosis. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 10. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8014/
  2. 2.0 2.1 2.2 Bruckner MZ. Gram Staining [Internet]. Microscopy. 2016 [cited 2020Jan17]. Available from: https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html
  3. 3.0 3.1 Todar K, Madison. [Internet]. Streptococcus pyogenes and streptococcal disease. [cited 2020Jan17]. Available from: http://textbookofbacteriology.net/streptococcus.html
  4. 4.0 4.1 Todar K, Madison. [Internet]. Staphylococcus aureus. [cited 2020Jan17]. Available from: http://textbookofbacteriology.net/staph_6.html
  5. 5.0 5.1 Bowen AC, Tong SY, Chatfield MD, Carapetis JR. The microbiology of impetigo in Indigenous children: associations between Streptococcus pyogenes, Staphylococcus aureus, scabies, and nasal carriage. BMC Infectious Diseases. 2014;14(1).
  6. 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 Spellerberg B, Brandt C. Laboratory Diagnosis of Streptococcus pyogenes (group A streptococci) 2016 Feb 10. In: Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK343617/#
  7. 7.0 7.1 Aryal S, Panja S, Subedi N, Hatsuharu. Mannitol Salt Agar for the isolation of Staphylococcus aureus [Internet]. Microbiology Info.com. 2019 [cited 2020Jan17]. Available from: https://microbiologyinfo.com/mannitol-salt-agar-for-the-isolation-of-staphylococcus-aureus/
  8. Catalase Test - Virtual Interactive Bacteriology Laboratory. [cited 2020Jan17]. Available from: http://learn.chm.msu.edu/vibl/content/catalase.html
  9. 9.0 9.1 9.2 9.3 Reiner K. Catalase Test Protocol [Internet]. ASMscience. American Society of Microbiology; 2010 [cited 2020Jan17]. Available from: https://www.asmscience.org/content/education/protocol/protocol.3226
  10. 10.0 10.1 10.2 10.3 10.4 Khanal S, Nirmal, Kathy, Gull, Acharya T, Sukhadia H, et al. Coagulase Test: Principle, procedure and interpretation [Internet]. Learn Microbiology Online. 2019 [cited 2020Jan17]. Available from: https://microbeonline.com/diagnostic-tests-biochemical-tests-coagulase-test/
  11. Pereira LB. Impetigo - review. Anais Brasileiros de Dermatologia. 2014;89(2):293–9.
  12. 12.0 12.1 12.2 12.3 Lal D, Verma M, Lal R. Exploring internal features of 16S rRNA gene for identification of clinically relevant species of the genus Streptococcus. Annals of Clinical Microbiology and Antimicrobials. 2011;10(1):28.
  13. 13.0 13.1 13.2 13.3 13.4 16S/18S/ITS Amplicon Sequencing [Internet]. CD Genomics-the genomics service company. [cited 2020Jan23]. Available from: https://www.cd-genomics.com/16S-18S-ITS-Amplicon-Sequencing.html

Other References Used:

14. Allmon A, Deane K, Martin KL. Common Skin Rashes in Children. American Family Physician. 2015Aug1;92(3):211–6.

15. Impetigo [Internet]. Mayo Clinic. Mayo Foundation for Medical Education and Research; 2019 [cited 2020Jan17]. Available from: https://www.mayoclinic.org/diseases-conditions/impetigo/symptoms-causes/syc-20352352

16. Aly R. Microbial Infections of Skin and Nails. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 98. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8301/#

17. Brazier Y. Impetigo: Treatment, symptoms, and causes [Internet]. Medical News Today. MediLexicon International; 2017 [cited 2020Jan17]. Available from: https://www.medicalnewstoday.com/articles/162945.php

18. Procedure: Swab for Culture & Susceptibility in Suspected Wound Infection. Procedure: Swab for Culture & Susceptibility in Suspected Wound Infection 2015 p. 1–6.

19. Baron EJ, Miller JM, Weinstein MP, Richter SS, Gilligan PH, Thomson RB, et al. A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2013 Recommendations by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)a. Clinical Infectious Diseases. 2013Oct;57(4).

20. Antimicrobial resistance [Internet]. World Health Organization. World Health Organization; [cited 2020Jan17]. Available from: https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance

Question (iv)

(iv) What are the results expected from these tests that might allow the identification of the bacteria named in this case.

Results

As Streptococcus pyogenes is named in this case, Table 1 depicts the expected results from several tests that identifies this bacteria. Likewise, Table 2 depicts the expected results from tests that would identify Staphylococcus aureus.

Table 1 : Streptococcus pyogenes Expected Test Results
Test Result Description Image
Gram Stain Gram positive Purple stained, cocci-shaped bacteria in long chains or pairs GramStain Streptococcus-pyogenes.png
Blood Agar Test β-hemolysis    White-greyish colonies with distinct margins BloodAgar Streptococcus-pyogenes.jpg
Lancefield Antigen Determination    Positive Binding of Lancefield group A antigen to Anti-Strep A antibodies on strip Lancefield Streptococcus-pyogenes.png
PYR Test    Positive Bright red colour PYR Streptococcus-pyogenes.png
Bacitracin Susceptibility    Positive Zone of inhibition around bacitracin Bacitracin Streptococcus-pyogenes.png
Catalase Test Negative No bubbles Catalase Streptococcus-pyogenes.png
Table 2: Staphylococcus aureus Expected Test Results
Test Result Description Image
Gram Stain Gram positive Purple stained, cocci-shaped bacteria in clusters Staphylococcus aureus Gram.jpg
Blood Agar Test

Positive for β-hemolysis

Large, round, golden colonies Beta Hemolysis Staphlococcus Aureus.jpg
Lancefield Antigen Determination Negative Lack of binding of Lancefield Group A antigen to anti-strep antibodies on strip (as seen by right side of figure). Lancefield Streptococcus-pyogenes.png
PYR Test Negative Lack of red pigment (orange colour). PYR-negative.png
Bacitracin Susceptibility Negative No zones of inhibition (as seen by top half of agar plate) Bacitracin+susceptibility.jpg
Catalase Test Positive Presence of bubbles due to O2 gas (top slide in right figure) Catalase Streptococcus-pyogenes.png
Coagulase Positive Cloudiness indicative of generation of fibrin (+) Coagulase test.jpg

Q3. Bacterial Pathogenesis Questions

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

Question (i)

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

S. pyogenes is one of the world most common pathogens . It is estimated that 5-15% of humans harbour this bacterium [3.1 1]. Diseases caused by S. pyogenes can range from mild skin infection to life threatening systematic infection. It usually resides on the skin or in the respiratory tract without any signs of disease. It can be part of the normal flora, however when the host is immune compromised, or when the organism can breach the constitutive defense. Streptococcus pyogenes colonizes epithelial surfaces, primarily of the throat and skin, but also colonizes other surfaces such as the vagina and rectum, from where it can cause a remarkably wide array of superficial, invasive, and immune-mediated diseases [3.1 2].

Group A B-hemolytic streptococci (which S. pyogenes belongs to), are spread by respiratory secretion and fomites. Both respiratory and skin infection incidents peak at childhood, the age Mariam is at. Because of how it spreads, it can be spread very fast in settings like schools or daycares. Many kids might have it and be asymptomatic but act as carriers and transfer the organism to others.

Geographically, the regions of the world with low income and poor infrastructure continue to suffer a high burden of Streptococcus pyogenes (group A streptococci) diseases with millions of deaths yearly [3.1 3]. This is because there is 1) less surveillance for this disease by the government 2) because the symptoms that the individual faces might be mild at first, and due to the limited resources and high cost of medical professionals, they might not seek help or medication. This makes the disease persist and advance to the more severe forms such as the development of rheumatic heart disease (RHD), due to rheumatic fever, which is fatal and a consequence of untreated or under-treated streptococcal infection such as strep throat or scarlet fever [3.1 4]. Lastly, the high burden of this disease in low income countries can also possibly be described by the genetics of that population, making them more or less susceptible to the circulating clones of this organism [3.1 5].

High income countries are not free of this organism. There have been declining rates reported [3.1 3], however it still remains an important health problem. In high income countries, the prevalence of RHD is much lower – because usually the disease is detected sooner; the majority of S. pyogenes-associated deaths are attributed to the clinical manifestations associated with invasive disease [3.1 5]. S. pyogenes has different clones with different genetic variations, changing their virulence. The increase in the incidence of invasive S. pyogenes infections has frequently been associated with specific clones, which raises the possibility that the rise of particularly virulent clones was responsible for this re-emergence—in particular, the MT1 clone which is dominant among invasive S. pyogenes isolates in most developed countries [3.1 3].

It is known that Streptococcus pyogenes will rapidly die outside human host [3.1 6]. However, compared to planktonic streptococci, ones that form a biofilm are highly tolerant to desiccation and can also still colonize tissues in vivo [3.1 6], based on a study done by Marks et al. This allows the organism to remain infectious and capable of colonizing new host in any geography, wet or dry. Additionally, bacteria that make biofilms are very resistant to antimicrobial agents and can survive under harsh environmental conditions. Furthermore, studies have shown that streptococci, especially S. pyogenes, have the ability to persist for long periods on environmental surfaces, which can contribute to the rapid spread [3.1 6].

S. pyogenes has some important virulence factors that make it suitable for surviving within the host long-term without being recognized. Some factors are explained below:

For adherence/attachment:
  1. M proteins: It is supported externally to the cell wall on fimbriae. Different serotypes of M proteins interact with different host cell receptors, allowing for adhering to happen because of this interaction. The M proteins of different serotype can mediate streptococcal entry into various epithelial cells, the differential expression of individual M serotype on S. pyogenes is thought to confer cell tropism on bacteria for targeting host cells or tissues by their recognition of distinct cellular receptors [3.1 7].
  2. Lipoteichoic acid (LTA) and protein F: LTA is also supported externally to the cell wall on fimbriae. Both LTA and protein F have an important role for attachment to host epithelial cells. Both are cell wall components of GAS, and are crucial for adherence to fibronectin on the surface of human epithelial cells [3.1 8].
It can escape phagocytosis by 2 means:
  1. Hyaluronic acid capsule: Is chemically similar to that of host connective tissue and so it acts as an immunological disguise and inhibits phagocytosis – because it is non-antigenic and the host recognizes it as “self”.
  2. M-protein: S. pyogenes’s very important virulence factor because it helps evade phagocytosis. It does so by binding fibrinogen (glycoprotein complex that circulates in the blood of vertebrates) from serum and blocking the binding of complement that targets the bacterium for phagocytosis. Blocking of complement is done through binding of H factor, which inhibits the activation of complement pathway and deposition of C3b, which is an opsonization factor.
Invasins and exotoxins mediate the invasion and survival of the organism in certain host environments:

Invasins: interact with mammalian blood and tissue components in a way that kill host cells and provoke a damaging inflammatory response [3.1 1] by host immune system. Examples are:

  1. Streptokinase: Participates in fibrin lysis. Fibrin is made by fibrogens and participate in forming blood clots. It is an enzyme.
  2. Streptodornase (DNase B): posses deoxyribonuclease activity as well as ribonuclease activity – which means it can degrade both DNA and RNA. It is also an enzyme.
  3. Hyaluronidase: the “spreading factor” because it can digest host connective tissue as well as the organism’s own capsule, which allows the organism to spread through the body of host. Its an enzyme.
  4. Streptolysins: Are responsible for killing leukocytes. It is a cytotoxin and can either be oxygen-stable (S) or oxygen-labile (O).

Exotoxins, such as pyrogenic (erythrogenic) toxin cause the rash of scarlet fever and systemic toxic shock syndrome. This is because these toxins act as superantigens. They bind to MHC class II molecules directly and stimulate 20% of T-cells (normally 1/10000 would get stimulated). When T-cells get stimulated they produce cytokines, but this massive number of T-cells stimulated means that there will be a massive cytokine storm, which is the reason for rash and in sever cases systemic toxic shock syndrome [3.1 5].

References:
  1. 1.0 1.1 Todar K, Madison. Streptococcus pyogenes and streptococcal disease.
  2. Walker MJ, Barnett TC, Mcarthur JD, Cole JN, Gillen CM, Henningham A, Sriprakash KS, Sanderson-Smith ML, Nizet V. 2014. Disease Manifestations and Pathogenic Mechanisms of Group A Streptococcus. Clinical Microbiology Reviews 27:264–301.
  3. 3.0 3.1 3.2 Efstratiou A. 2017. Epidemiology of Streptococcus pyogenes. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. U.S. National Library of Medicine.
  4. Rheumatic Heart Disease. Johns Hopkins Medicine.
  5. 5.0 5.1 5.2 Baron S. Medical Microbiology. 4th edition. University of Texas Medical Branch at Galveston.S.l.
  6. 6.0 6.1 6.2 Marks LR, Reddinger RM, Hakansson AP. 2013. Biofilm Formation Enhances Fomite Survival of Streptococcus pneumoniae and Streptococcus pyogenes. Infection and Immunity 82:1141–1146.
  7. M Protein. M Protein - an overview | ScienceDirect Topics.
  8. Lipoteichoic Acid. Lipoteichoic Acid - an overview | ScienceDirect Topics.

Question (ii)

Entry: how does the bacteria enter into the human host and take up residence. What are the molecular, cellular and/or physiological factors at play in this site specificity, and in the initial adherence step, (referencing both the bacteria and the host).

Transmission of S. pyogenes occurs through the inhalation of respiratory droplets, skin contact, or contact with contaminated surfaces, while it is less commonly transmitted through food [3.2 1].

Once transmitted, S. pyogenes first adhere to the extracellular matrix (ECM) proteins on host cells using its pili. Part of the normal flora, the bacterium needs to withstand competition from other normal flora bacteria, electrostatic & mechanical forces, and physiologically responses that can displace or physically remove them from the host tissue [3.2 2]. The adhesion/ attachment of S. pyogenes to the host would be difficult due to the human body's first line of defense, namely the mucous lining, which can trap and eliminate pathogens via the mucociliary escalator [3.2 2]. Because the ECM is often exposed by trauma/injury, it is an optimal target for streptococcal adhesion [3.2 2]. The pili on S. pyogenes use a unique intramolecular covalent linkage system that can withstand high tensile forces in the first steps of adherence to the host's ECM. Within the pili, there are collagen-binding proteins that interact with the ECM [3.2 2]. First, the ancillary protein 2 (AP2) contacts the ECM from the tip, and this interaction is reversible, allowing the bacterium to find specific tissue [3.2 3]. Then ancillary protein 1 (AP1), located on intervals along the shaft of the pilus, more firmly attaches the bacterium to the host cell [3.2 3]. It is found in vivo that the hyaluronic filled capsule of S. pyogenes interacts with CD44, a host cell-surface glycoprotein, which helps to localize of the host cell during infections [3.2 2] [3.2 4].

Attachment of S. pyogenes is mediated by LTA, M proteins, and fibronectin-binding proteins. The most widely accepted model for S. pyogenes attachment is a two-step model [3.2 5]. The first step involves a weaker, low cellular specificity interaction mediated by LTA to overcome the electrostatic repulsions, and the second step includes an irreversibly binding of host receptors to S. pyogenes surface molecules [3.2 5]. Firstly, LTA uses its glycolipid ends to form ionic complexes with the bacterium's own surface protein to undergoes conformation changes to aid attachment by exposing specific lipid ends [3.2 6]. These exposed lipid ends to interact with host cell membrane receptors. After that, the M protein is involved in the second step of attachment. The M protein is a strong anti-phagocytic virulent factor of S. pyogenes by polymorphonuclear leukocytes and a group of adhesins that interact with a diverse set of target cells [3.2 7]. The M protein ranges in size (from 41kDa to 80kDa in molecular weight) [3.2 7]. It is a multifunctional protein, meaning multifunctional domains may be found on the cell surface of a single streptococcus, making the cell surface extremely complicated and able to bind to a variety of cells in the body [3.2 7]. A variety of binding abilities among different strains of M protein such as M6 (keratinocytes), M3, M18 (collagen-binding proteins), and collagen binding protein, cpa suggest that the M proteins can help attachment through interactions with ECM proteins [3.2 7]. Thirdly, fibronectin-binding proteins, which also help create strong adhesion by binding integrin receptors on the target cell, are affected by environmental factors. The primary adherence occurs in regions with a higher partial pressure of O2, such as the respiratory tract and skin [3.2 8].

After successful colonization, the bacteria multiply extracellularly to form small colonies and to develop biofilm-like structures that prevent them from being detected by host defenses [3.2 9]. Biofilms are found to have an increased ability to withstand antibiotics and evade phagocytosis [3.2 9].

References:
  1. Efstratiou A, Lamagni T. Epidemiology of Streptococcus pyogenes. In: Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016 [cited 2020 Jan 24]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK343616/
  2. 2.0 2.1 2.2 2.3 2.4 Rohde M, Cleary PP. Adhesion and invasion of Streptococcus pyogenes into host cells and clinical relevance of intracellular streptococci. In: Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016 [cited 2020 Jan 17]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK333420/
  3. 3.0 3.1 Walden M, Crow A, Nelson MD, Banfield MJ. Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125. Proteins. 2014 Mar;82(3):517–27.
  4. Cywes C, Stamenkovic I, Wessels MR. CD44 as a receptor for colonization of the pharynx by group A Streptococcus. J Clin Invest. 2000 Oct 15;106(8):995–1002.
  5. 5.0 5.1 Ryan PA, Juncosa B. Group A Streptococcal Adherence. In: Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016 [cited 2020 Jan 17]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK333427/
  6. Beachey EH, Simpson WA, Ofek I, Hasty DL, Dale JB, Whitnack E. Attachment of Streptococcus pyogenes to mammalian cells. Rev Infect Dis. 1983 Oct;5 Suppl 4:S670-677.
  7. 7.0 7.1 7.2 7.3 Fischetti VA. M Protein and Other Surface Proteins on Streptococci. In: Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016 [cited 2020 Jan 24]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK333431/
  8. Kreikemeyer B, Klenk M, Podbielski A. The intracellular status of Streptococcus pyogenes: role of extracellular matrix-binding proteins and their regulation. Int J Med Microbiol. 2004 Sep 24;294(2):177–88.
  9. 9.0 9.1 Marks LR, Reddinger RM, Hakansson AP. Biofilm Formation Enhances Fomite Survival of Streptococcus pneumoniae and Streptococcus pyogenes. Infect Immun. 2014 Mar 1;82(3):1141–6.

Question (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.

S. pyogenes is gram-positive bacterial pathogen that typically colonize the throat or skin and are responsible for a number of suppurative infections and nonsuppurative complications [3.3 1]. This organism tends to reside extracellularly but can also be a capable intracellular bacterium [3.3 2]. LaPenta et al. demonstrated in 1994 with cell culture infections model that S. pyogenes can enter non-phagocytic human epithelial cells [3.3 3].

Figure 1. Electron micrographs showing attachment and internalization of streptococci by human pharyngeal cells [3.3 4].

Intracellular invasion can be achieved by the activation of spreading factors, such as hyaluronidase, which can digest both host connective tissue and the organism’s capsule [3.3 5]. Invasion intracellularly also requires invasins such as M protein and/or fibronectin-binding proteins; substances coated with either protein are efficiently internalized by epithelial cells; cytoskeletal rearrangement also occurs within the cell to accommodate for the process [3.3 4]. Invasins not only interact with mammalian blood and tissue elements to kill host cells, they also induce damaging inflammatory responses. Their abilities in dissolving host fibrin and intercellular ground substance allow the bacterium to spread throughout tissues [3.3 1]. In addition to that, laminin has been shown to induce ingestion of S. pyogenes, independent of serum and fibronectin [3.3 4]. Another invasion mechanism involves the formation of invagination in host cell membrane at site of invasion through integrin protein clustering [3.3 2]. Having multiple mechanisms and route to the interior of epithelial cells strongly indicates that intracellular invasion plays a key role in this bacterium’s pathogenesis [3.3 4].

S. pyogenes may find intracellular environment suitable for survival since it is a good place to avoid host defense mechanisms. Two other theories have also been proposed for the role of internalization in disease pathogenesis. The first is that internalization may lead to carriage and persistence even after antibiotic therapy [3.3 2]. The second is that internalization may lead to invasion of deeper tissues [3.3 4].

If S. pyogenes enters the host through skin, it may cause infections ranging from superficial to deep layers of the skin and/or muscle [3.3 1]. This organism can remain at the entry site or spread beyond the initial site to cause secondary sites of infection. Impetigo is infection at the superficial keratin layer, usually defined by crusty lesions [3.3 6]. Erysipelas is infection at the superficial epidermis, displaying clear borders of infection and a bright red skin colour [3.3 6]. Cellulitis is infection at the subcutaneous tissue, displaying pinkish hue with a less defined border [3.3 6]. Necrotizing fasciitis is infection of deeper subcutaneous tissue and fascia, characterized by extensive and rapid spreading necrosis of skin and tissue [3.3 7]. Myositis and myonecrosis affects the muscle and cause localized purulent infections [3.3 7]. If left untreated, the bacterium can also spread to the bone, the brain and the heart to cause joint or bone infections, meningitis and endocarditis, respectively [3.3 1]. S. pyogenes has enhanced bacterial invasion and movement through normal tissue planes because it binds plasminogen to its surface receptor proteins [3.3 4]. This is a potential common mechanism utilized by invasive bacteria to cross tissue barriers since plasminogen facilitates adherence and internalization into host cells [3.3 4]. Once plasminogen is bound to the cell surface, it can be converted to plasmin by bacterial streptokinase [3.3 2]. Plasmin is a very strong serine protease that degrades extracellular matrix (ECM) proteins [3.3 2]. This allows the bacterium to come into closer contact with host cell surface [3.3 2]. Furthermore, ECM metalloproteases or collagenases would be activated to facilitate invasion and to gain access into deeper soft tissue [3.3 4]. Studies have identified alpha-enolase and G3P-dehydrogenase to be important cell surface plasmin-binding receptors that facilitate dissemination through both epithelial and endothelial barriers [3.3 2].

If S. pyogenes enters the host through the respiratory tract, it may translocate via the bloodstream to the middle ear, sinuses and lungs to cause otitis media, sinusitis and pneumonia, respectively [3.3 1]. In addition to the above, S. pyogenes can also enter the bloodstream and cause bacteremia, which can result in life-threatening systemic responses such as scarlet fever and streptococcal toxic shock syndrome [3.3 1].

Figure 2. Areas in the body where S. pyogenes can cause secondary infections [3.3 1].

The bacteria hone in on these particular secondary sites because they are able to avoid destruction by the host’s immune system due to the anti-phagocytic properties of the surface exposed M protein and hyaluronic acid (HA) capsule [3.3 7]. There are two proposed mechanisms for their anti-phagocytic properties. The first mechanism involves the binding of H factor, which inhibits the activation of complement pathway [3.3 4]. This inhibits the deposition of C3b, an opsonization factor [3.3 4]. The second mechanism involves the binding of fibrinogen to the surface of M protein, which inhibits the activation of complement via alternate pathway [3.3 4]. This also reduces the amount of C3b bound to the bacterium, causing reduced phagocytosis [3.3 4]. There are still many other surface molecules under investigation that can contribute to resistance of phagocytosis [3.3 4].

An additional property that allows S. pyogenes to be able to migrate to a variety of secondary sites is its ability to bind to CD44. This bacterium has surface HA capsules, which is a major ligand for CD44 [3.3 8]. CD44 is expressed ubiquitously throughout the human body and is a transmembrane cell surface molecule [3.3 8]. It is found in a variety of tissues such as the lungs and epidermis, which can help explain pneumonia and skin conditions such as impetigo [3.3 8].

References:
  1. 1.0 1.1 1.2 1.3 1.4 1.5 1.6 Todar KG. 2012, posting date. Streptococcus pyogenes and Streptococcal Disease. In Todar K (ed), Todar’s Online Textbook of Bacteriology. University of Wisconsin-Madison Dept. of Bacteriology, Madison, WI.
  2. 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Rohde M, Cleary PP. 2016. Adhesion and invasion of Streptococcus pyogenes into host cells and clinical relevance of intracellular streptococci. In Ferretti JJ, Stevens DL, Fischetti VA (ed), Streptococcus pyogenes: Basic Biology to Clinical Manifestations. University of Oklahoma Health Sciences Center, Oklahoma City, OK.
  3. LaPenta D, Rubens C, Chi E, Cleary PP. 1994. Group A streptococci efficiently invade human respiratory epithelial cells. Proc Natl Acad Sci U S A. 91:12115-12119.
  4. 4.00 4.01 4.02 4.03 4.04 4.05 4.06 4.07 4.08 4.09 4.10 4.11 4.12 Cunningham MW. 2000. Pathogenesis of Group A Streptococcal Infections. Clin Microbiol Rev. 13:470-511.
  5. Patterson MJ. 1996. Streptococcus. In Baron S (ed), Medical Microbiology, 4th ed. University of Texas Medical Branch at Galveston, Galveston, TX.
  6. 6.0 6.1 6.2 Stevens DL, Bryant AE. 2016. Impetigo, Erysipelas and Cellulitis. In Ferretti JJ, Stevens DL, Fischetti VA (ed), Streptococcus pyogenes: Basic Biology to Clinical Manifestations. University of Oklahoma Health Sciences Center, Oklahoma City, OK.
  7. 7.0 7.1 7.2 Stevens DL, Bryant AE. 2016. Severe Group A Streptococcal Infections. In Ferretti JJ, Stevens DL, Fischetti VA (ed), Streptococcus pyogenes: Basic Biology to Clinical Manifestations. University of Oklahoma Health Sciences Center, Oklahoma City, OK.
  8. 8.0 8.1 8.2 Senbanjo LT, Chellaiah MA. 2017. CD44: A Multifunctional Cell Surface Adhesion Receptor is a Regulator of Progression and Metastasis of Cancer Cells. Front Cell Dev Biol. https://doi.org/10.3389/fcell.2017.00018

Question (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?

Streptococcus pyogenes is capable of producing both direct and indirect damage to the host. Direct damage is caused by toxins and enzymes produced by the bacteria while indirect damage is caused by the host immune response to the bacteria.  

Soluble extracellular growth products and toxins of Streptococcus pyogenes play a large role in direct damage to the host. Streptolysin S is an oxygen-stable leucocidin (pore-forming cytotoxin that kills leukocytes) [3.4 1]. Streptolysin O is an oxygen-labile leucocidin [3.4 1]. As cytolysin exotoxins, Streptolysin S affects neutrophils, platelets, and subcellular organelles while Streptolysin O leads to an immune response [3.4 2]. NADase is leukotoxic [3.4 1]. Hyaluronidase (original “spreading factor") can digest host connective tissue hyaluronic acid, and the organism's own capsule to aid in cellular invasion [3.4 2] [3.4 1]. Streptokinases are involved in fibrin lysis [3.4 1]. Streptodornases A-D possess deoxyribonuclease activity, protecting bacteria from neutrophil extracellular traps (NETs) by digesting the neutrophil DNA used to capture bacteria [3.4 1] [3.4 3]. Streptodornases B and D also possess ribonuclease activity [3.4 1].

Streptococcus pyogenes produces three main streptococcal pyrogenic exotoxins (SPE): types A, B, C [3.4 4]. These toxins are superantigens that work in a mechanism similar to staphylococci. As antigens, they don’t require processing by antigen presenting cells; they instead stimulate T cells by binding MHC Class II molecules directly and non-specifically [3.4 4]. Superantigens stimulate about 20% of T cells as opposed to 1/10,000 T cells by conventional antigens; this results in extensive and damaging cytokine release [3.4 4]. Overproduction of cytokines produces the signs of cytokine-release syndrome (e.g. fever, rash, nausea, chills low blood pressure) due to abnormal interaction between toxin, macrophage, and T cells [3.4 4] [3.4 5].

Some of the antibodies produced during infection to certain streptococci strains also cross-react with host tissues[3.4 4]. These antibodies can indirectly damage host tissues even after the organism has been cleared, causing autoimmune complications [3.4 4].

Figure 1: Illustration of the anatomical relationship between skin structures and different S. pyogenes infections. (4)
Figure 1: Illustration of the anatomical relationship between skin structures and different S. pyogenes infections. [3.4 1]

Streptococcus pyogenes is one of the most diverse pathogens that presents itself in a variety of clinical forms. Most commonly, it takes the form of pharyngitis (strep throat), scarlet fever (rash), impetigo (superficial skin layer infection), or cellulitis (deeper layer skin infection) [3.4 4]. Invasive, toxigenic infections can produce necrotizing fasciitis, myositis, and streptococcal toxic shock syndrome [3.4 4]. Streptococcus pyogenes infection can also lead to severe non-suppurative sequelae (condition that is a consequence of previous disease/injury) such as acute rheumatic fever and acute glomerulonephritis that begin 1-3 weeks after an acute streptococcal illness [3.4 4].

Streptococcus pyogenes is one of the most significant bacterial causes of skin and soft tissue infections [3.4 6]. Streptococcal impetigo manifests on exposed areas of the body from infection of epidermal skin layers, most often on the lower facial regions [3.4 4] [3.4 6]. This can result from toxins and enzymes produced by Streptococcus pyogenes and their associated host cell damage. Another potential cause could be from the host’s own immune defense mechanisms. The host innate immune response often involves the production of antimicrobial peptides (AMPs) as a first line of defense to kill pathogens via induction of cell membrane damage; in the process, AMPs may also damage the host’s cell membranes [3.4 7]. Streptococcal impetigo lesions remain localized, but often appear in multiples [3.4 6]. This can be linked to the red sores around Miriam’s mouth and nose that are not present on her hands, feet, or in her mouth.

References:
  1. 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 Deng JC. The Nasal Microbiota: Diversity, Dynamicity, and Vaccine-Mediated Effects. Los Angeles: David Geffen School of Medicine at University of California, Los Angeles; 2015.
  2. 2.0 2.1 Patterson MJ. Streptococcus. In: Baron S, editor. Medical Microbiology. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.
  3. Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, Kotb M, et al. DNase Expression Allows the Pathogen Group A Streptococcus to Escape Killing in Neutrophil Extracellular Traps. Current Biology. 2006 Feb 21; 16 (4): 396–400.
  4. 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Todar K. Streptococcus pyogenes and Streptococcal Disease. Wisconsin: University of Wisconsin Department of Bacteriology; 2012.
  5. Breslin S. Cytokine-Release Syndrome: Overview and Nursing Implications. Clinical Journal of Oncology Nursing. 2007 Jan; 11(0): 37–41.
  6. 6.0 6.1 6.2 Stevens DL, Bryant AE. Impetigo, Erysipelas and Cellulitis. In: Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes: Basic Biology to Clinical Manifestations. Oklahoma City: University of Oklahoma Health Sciences Center; 2016.
  7. Cole JN, Nizet V. Bacterial Evasion of Host Antimicrobial Peptide Defenses. Microbiology Spectrum. 2016 Feb; 4(1).

Q4. The Immune Response Questions

Question (i)

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

S. pyogenes (otherwise known as group A Streptococcus) is a nonmotile, gram-positive bacterium, it is an exogenous secondary invader (1). S. pyogenes infects humans following viral disease, when the immune system is compromised or when constitutive defenses (normal flora and nonspecific defense mechanisms) are penetrated – often at the upper respiratory tract (2). The main route of transmission in humans is through direct physical contact and respiratory droplets. The upper respiratory tract and skin are major reservoirs for S. pyogenes infections (3). Around 5-15% of individuals harbor the bacterium in the respiratory track without signs of disease, however this pathogen can cause a variety of infections including scarlet fever, pharyngitis, impetigo, cellulitis, necrotizing fasciitis and toxic shock syndrome, as well as the sequelae of rheumatic fever and acute poststreptococcal glomerulonephritis (1) . S. pyogenes is most commonly a respiratory infection known as pharyngitis, or a skin infection termed ‘pyoderma.’

The first line of defense in a host is the anatomical barriers. An example is human skin, which is an effective barrier against streptococci. Non-specific defense mechanisms also prevent the bacteria from penetrating beyond the superficial epithelium of the upper respiratory tract eg. mucocillary movement, coughing, sneezing and epiglottal reflexes (2).

The second line of defense is the innate responses. Despite the importance of the innate immune system for host defense, the TLRs and PAMPs involved in functional recognition of S. pyogenes are not defined (4). However, epithelial cells secrete bactericidal AMPs and neutrophil chemotactic factors, such as IL‐8, whereas tissue resident macrophages phagocytose colonizing bacteria and produce TNF‐α, which in turn, activates neutrophils and monocytes (6).

Neutrophil activation and degranulation at the site of infection have recently been emphasized to be an important contributor to the disease pathology of S pyogenes (5). In Herwald et al.’s study, they found that the major trigger of this response is the M protein that is able to form a complex with fibrinogen and bind to neutrophil surfaces (6). This interaction results in the activation of neutrophils and the induction of vascular leakage following a massive release of granule proteins by the neutrophils (6). Neutrophils macrophages and dendritic cells secrete a numerous soluble inflammatory mediators, such as antimicrobial peptides; eicosanoids (including PGE2 and leukotriene B4); chemokines; and proinflammatory cytokines (7). S. pyogenes also induces an extensive formation of NETs from neutrophils, however, S. pyogenes produces DNase B which is a deoxyribonuclease which acts as a way to escape the NETs (8).

There is also the host phagocytic system where organisms are opsonized by activation of the classical or alternate complement pathway then killed via phagocytosis (2). Macrophages produce reactive oxygen species which kill and digest pathogens, however, S. pyogenes does not produce superoxide dismutase to inactivate the oxygen metabolites dependent mechanisms of the phagocyte (hydrogen peroxide and superoxide), so phagocytosis is fatal for it, however, the bacteria has developed a mechanism to evade opsonization and phagocytic system and survive.

T helper type 1 (Th1) and T helper type 17 (Th17) responses also contribute to the innate immunity against S. pyogenes (7). Upon GAS recognition, DCs promote Th1 maturation by the secretion of Th1‐polarizing cytokines, such as IL‐12, whereas epithelial cells and macrophages contribute to Th17 maturation by the secretion of the Th17‐polarizing cytokines IL‐6, TGF‐β‐1, and IL‐1β. Th17 cells secrete IL‐17A, which promotes epithelial cell‐mediated recruitment and activation of neutrophils and macrophages. Th1 cells produce IFN‐γ, which activates neutrophils and regulates neutrophil recruitment to the infection site (6).

The adaptive responses involved in this infection include the development of type-specific antibody to a plethora of antibodies against the many streptococcal cellular and extracellular components. For example, antibodies to the M protein of the fimbrae are produced following respiratory and skin infections. The anti-streptococcal antibodies can enhance the phagocytosis of the pathogen greatly and allow the pathogen to be killed. On a cellular level, epithelial cells have α5β1 integrin receptors that are triggered upon invasion by engagement of fibronectin and high-affinity fibronectin-binding proteins. Fc receptors on macrophages bind to the antibody Fc region, inducing phagocytosis and killing streptococci bacterium. This is how the immune system recognizes group A streptococci and uses opsonization by complement and type-specific antibody against S. pyogenes molecules capable of generating opsonic antibodies.

Humoral responses contribute to the development of antibodies to induce immunity. B cells produce IgA in respiratory secretions and IgG in the serum. These two antibody classes are important for preventing recurring infections (1).  IgG antibodies that bind to the M protein of S pyogenes and promote phagocytosis. These M protein antibodies are type specific, long lasting and protective. Furthermore, Antibodies against the erythrogenic toxin involved in scarlet fever are also long lasting (1). Additionally, there is development of antibodies to a variety of streptococcal components including streptokinase, streptolysin O (SLO), DNAase, hyaluronidase and the major surface protein, M protein (9).

1.    Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016 [cited 2020 Jan 24]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK333424/

2.    Streptococcus pyogenes and streptococcal disease [Internet]. [cited 2020 Jan 17]. Available from: http://textbookofbacteriology.net/streptococcus.html

3.    Brouwer S, Barnett TC, Rivera-Hernandez T, Rohde M, Walker MJ. Streptococcus pyogenes adhesion and colonization. FEBS Lett. 2016 Nov;590(21):3739–57.

4.     Fieber C, Janos M, Koestler T, Gratz N, Li X-D, Castiglia V, et al. Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2. PLoS ONE [Internet]. 2015 Mar 10 [cited 2020 Jan 24];10(3). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355416/

5.    Johansson L, Thulin P, Low DE, Norrby-Teglund A. Getting under the skin: the immunopathogenesis of Streptococcus pyogenes deep tissue infections. Clin Infect Dis Off Publ Infect Dis Soc Am. 2010 Jul 1;51(1):58–65.

6.    Herwald H, Cramer H, Mörgelin M, Russell W, Sollenberg U, Norrby-Teglund A, et al. M protein, a classical bacterial virulence determinant, forms complexes with fibrinogen that induce vascular leakage. Cell. 2004 Feb 6;116(3):367–79.

7.     Soderholm AT, Barnett TC, Sweet MJ, Walker MJ. Group A streptococcal pharyngitis: Immune responses involved in bacterial clearance and GAS-associated immunopathologies. J Leukoc Biol. 2018;103(2):193–213.

8.    Fieber C, Kovarik P. Responses of innate immune cells to group A Streptococcus. Front Cell Infect Microbiol. 2014;4:140.

9.     Ashbaugh CD, Wessels MR. Absence of a cysteine protease effect on bacterial virulence in two murine models of human invasive group A streptococcal infection. Infect Immun. 2001 Nov;69(11):6683–8.

10.   Patterson MJ. Streptococcus. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2020 Jan 17]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7611/

Question (ii)

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

The host immune response contributes to a number of inflammatory complications which can lead to both localized and invasive S pyogenes infections. Localized skin infections may clinically manifest as impetigo, the infection of the epidermal layers of skin; cellulitis, subcutaneous tissue infection; and erysipelas, infection of the dermis (1). Necrotizing fasciitis, infection of the fascia, is more invasive and can rapidly disseminate to underlying muscles causing myositis. This may occur in 5% of patients with S pyogenes infection (1). In a 2006 study, Bryant et al. found that patients with previous skeletal muscle injuries are 6 times more likely to get necrotizing fasciitis. The researchers explain that this is potentially due to the increased production of cellular vimentin following injury which enhances the binding of S pyogenes to skeletal muscles (2).

In a study conducted by Norrby-Teglund A et al., a correlation was found between host inflammation and the severity of streptococcal tissue infection (3). This may be explained by the damaging inflammatory response triggered by host cell damage following bacterial invasion. During phagocytosis, neutrophils and macrophages generate Reactive Oxygen Species (ROS), which are highly reactive oxygen-containing molecules used to facilitate clearance of bacteria at the site of infection. In this process, host cells are often destroyed, through the damage of its proteins and DNA. As a result, host tissue epithelial integrity is disrupted and there is an increase in inflammation (4). Furthermore, neutrophil activation and degranulation at the site of infection have recently been emphasized to be an important contributor to the disease pathology of S pyogenes (5). In Herwald et al.’s study, they found that a major trigger of this response is the M protein, a cell surface virulence factor involved in colonization and resistance to phagocytosis (5). As the M protein is able to form a complex with fibrinogen and bind to neutrophil surfaces, this interaction results in the massive release of granule proteins by neutrophils. The released products act on vascular permeability, leading to vascular leakage and further inflammation (5).

Individuals who develop IgG directed towards the M protein have enhanced release of granule protein heparin binding protein (HBP), a potent inducer of vascular permeability and pulmonary lesion formation (6). These individuals are subsequently more susceptible to suffering pathologically excessive inflammatory responses to the M protein-fibrinogen-IgG complexes (6).

Figure 1. Cell surface structure and products involved in S pyogenes virulence (1).

Invasins and toxins released by S pyogenes may also lead to significant host damage by reacting with blood and tissue components in a way that kills host cells and provokes an inflammatory response (1). These include hyaluronidase which can digest host connective tissue hyaluronic acid; streptokinases with lyse fibrin and proteases that cause soft tissue necrosis or toxic shock syndrome (1). An important exotoxin, streptolysin O (SLO), has been found to modulate responses initiated by polymorphonuclear leukocytes (PMNs), which are immune cells containing granules such as neutrophils. SLO forms large pores on host cell membranes and disrupts the membrane integrity of PMNs, macrophages and epithelial cells. While this further contribute to inflammation and tissue damage, it also mediates the progression of microvascular thrombosis and ischemic tissue necrosis (6). This allows the bacteria to spread among tissues by dissolving host fibrin and other intercellular substances (1).

This large repertoire of bacterial product mediates the pathogenesis of S pyogenes (see Figure 1). However, it has been found that even antibodies to these products are relatively insignificant in protecting the host (1).

Furthermore, streptococcal pyrogenic exotoxins (SPE) have also been recognized as an important player in invasive infections. Three types of SPE have been recognized: types A, B and C (1). Pyrogenic toxins are superantigens released by S pyogenes, meaning they do not require processing by antigen presenting cells (APCs) as conventional peptide antigens do. Instead, S pyogenes superantigens can directly and non-specifically bind to MHC II on APCs and the T cell Receptors (TCR) of T cells (7), resulting in the stimulation of about 20% of host T cells (1). This is in contrast to normal T cell activation where only 1/10,000 T cells are stimulated. For this, there is a massive cytokine release which results in systemic disease manifestations including streptococcal toxic shock syndrome, fever and rash (1). Furthermore, the highly virulent M1 strain, producing type A SPE, has been associated with invasive infections (1). Recent studies show that SPE type A is highly associated with toxic shock syndrome after subcutaneous infections but rarely associated with toxic shock syndrome after pharyngitis (7).

1-3 weeks after an acute infection, S pyogenes can cause 2 kinds of serious non suppurative sequelae: acute rheumatic fever and acute glomerulonephritis. While acute rheumatic fever is only seen following pharyngeal infections, acute glomerulonephritis can follow either pharyngeal or skin infections. Acute rheumatic fever results from an autoimmune response due to the cross-reactive, heart related antigens in S pyogenes. This can lead to permanent damage in heart valves, although less than 1% of pharyngeal infections result in rheumatic fever. Fortunately, our case-study patient only shows signs of a skin infection, and thus our main concern is acute glomerulonephritis resulting from the deposition of antigen-antibody-complement complexes on the basement membrane of the kidney glomeruli.  Acute glomerulonephritis recurrences are uncommon and subsequent prophylaxis treatments are unnecessary. This is dissimilar to acute rheumatic fever where life-long antibiotic prophylaxis is recommended following a single case (1)

References:

1.     Todar K. Streptococcus pyogenes and streptococcal disease [Internet]. Todar’s Textbook of Bacteriology. 2008 [cited 2020 Jan 15]. Available from: http://textbookofbacteriology.net/streptococcus.html

2.     Bryant AE, Bayer CR, Huntington JD, Stevens DL. Group A Streptococcal Myonecrosis: Increased Vimentin Expression after Skeletal-Muscle Injury Mediates the Binding of Streptococcus pyogenes. J Infect Dis. 2006 Jun 15;193(12):1685–92.

3.     Norrby-Teglund A, Thulin P, Gan BS, Kotb M, McGeer A, Andersson J, et al. Evidence for Superantigen Involvement in Severe Group A Streptococcal Tissue Infections. J Infect Dis. 2001 Oct 1;184(7):853–60.

4.     Henningham A, Döhrmann S, Nizet V, Cole JN. Mechanisms of group A Streptococcus resistance to reactive oxygen species. FEMS Microbiol Rev. 2015 Jul;39(4):488–508.

5.     Herwald H, Cramer H, Mörgelin M, Russell W, Sollenberg U, Norrby-Teglund A, et al. M Protein, a Classical Bacterial Virulence Determinant, Forms Complexes with Fibrinogen that Induce Vascular Leakage. Cell. 2004 Feb 6;116(3):367–79.

6.     Tsatsaronis JA, Walker MJ, Sanderson-Smith ML. Host Responses to Group A Streptococcus: Cell Death and Inflammation. PLoS Pathog [Internet]. 2014 Aug 28 [cited 2020 Jan 22];10(8). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148426/

7.     Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016 [cited 2020 Jan 24]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK333424/

Question (iii)

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

One of the most significant factors in the success of S. pyogenes is its ability to colonize and rapidly multiply, spreading itself throughout the host while simultaneously evading phagocytosis by the immune system. S. pyogenes is able to successfully colonize the epithelium of the host through the use of adhesins. This ability to adhere to the epithelium of the host is significant as it allows for the bacterial cell to come into close contact with the host cell through specific adhesin-receptor interactions. (1) The cell surface of S. pyogenes has various protein adhesins that allow for it to colonize distinct tissue sites on the host, resulting in a successful infection of the pathogen in the host. This adhesion to the host epithelium is a pertinent virulence factor that results in the pathogen not being cleared by the immune system, as S. pyogenes is able to adhere to and effectively colonize the host. One the colonization is established, the pathogen can multiply and spread at rapid rates.

The cell surface of S. pyogenes is responsible for a significant amount of the bacterium’s determinants of virulence. Its chemically-diverse and complex cell surface closely resembles those of human cardiac skeletal, smooth muscle, neuronal tissues, and heart valve fibroblasts. (3) Streptococcus pyogenes uses molecular mimicry as a  means to suppress and evade the host immune response. Thus, due to the close resemblance of the pathogen’s cell surface to that of the host, S. pyogenes is able to evade the immune responses, thereby going undetected. (3) Moreover, S. pyogenes is able to use molecular mimicry to evade the host immune response through its Hyaluronic Acid (HLA) capsule. The HLA capsule is chemically similar to the host connective tissue, which is also composed of HLA. (3) This capsule enables S. pyogenes to establish virulence in the host. Thus, the pathogen can successfully disguise its antigens, going undetected by the host.

S. pyogenes possesses M proteins, which function as significant virulence factors associated with both colonization in the host and resistance to phagocytosis. The M protein exists as an alpha helical coiled‐coil dimer, (1) and is responsible for the survival of the pathogen as it inhibits phagocytosis by binding fibrinogen from the serum. The M protein therefore blocks the binding of complement to the underlying peptidoglycan, thus inhibiting a successful complement cascade. Furthermore, there have been several studies that have demonstrated the contributions of M proteins to the adherence of S. pyogenes with the host cells. (1) This is significant in successful pathogenic colonization of the host.

Another mechanism that S. pyogenes employs to evade the innate immune responses is through the expression of SLO, a pore-forming cytolysin. (2) S. pyogenes that has already been phagocytosed can increase its survival through the expression of SLO, which functions to inhibit the transport of the pathogen to the lysosome. This allows for survival of S. pyogenes as it is unable to be effectively phagocytosed by the host. Another significant function of SLO is its ability  to induce cell death in both macrophages and neutrophils. This greatly weakens the host immune system’s abilities to evade S. pyogenes, allowing for further evasion of the immune response.

S. pyogenes further promotes evasion of the immune system through blocking the complement system. The pathogen obstructs complement deposition by cleaving C5a, an important inducer in the recruitment of neutrophils. (2) Following this, streptococcal inhibitor of complement, or SIC, works to inhibit complement-mediated formation of the MAC. SIC then decreases neutrophil production of antimicrobial peptides. Inhibition of complement causes ineffective phagocytosis and membrane attack of S. pyogenes, allowing for its survival in the host. In addition, M proteins play a further role in blocking complement. These M proteins bind to fibrinogen from the serum, consequently blocking the binding of complement to the peptidoglycan. This results in inhibited phagocytosis of S. pyogenes. (3) Moreover, the M protein can bind to host Factor H, which is an influential inhibitor of both complement deposition and phagocytosis, allowing the pathogen to evade the host immune responses. (2)

Figure 2: GAS evasion of innate immunity (2)

References:

  1. Brouwer S, Barnett TC, Rivera‐Hernandez T, Rohde M, Walker MJ. Streptococcus pyogenes adhesion and colonization. FEBS letters. 2016 Nov 1;590(21):3739-57.
  1. Fieber C, Kovarik P. Responses of innate immune cells to group A Streptococcus. Frontiers in cellular and infection microbiology. 2014 Oct 2;4:140.
  2. Todar K. Streptococcus pyogenes and streptococcal disease. Online Textbook of Bacteriology. 2008.

Question (iv)

Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections with the candidate infectious agent?

This bacterium is quite often termed as “flesh-eating” due to the way in which it invades skin and soft tissues and in some situations even causes full destruction of whole limbs and organs (Cunningham M.W., 2000). The initial diagnosis is often misunderstood or missed entirely due to delayed symptoms. In the assigned case, Miriam should be cleared upon completing the antibiotic course. The standard treatment of S. pyogenes is penicillin, which is uniformly effective in the treatment of Group A streptococcal disease. (Todar, 2012). In some cases the infection by S. pyogenes is complicated further and the patient may become febrile and the diagnosis and treatment may change. S. pyogenes is present in our normal gut flora, and therefore, will most likely not be entirely removed.

If adaptive immunity is successful in producing antibodies, future immunity to the same strain of bacteria will be provided. Due to the nature of the bacterium and its’ relationship with the host, the human immune responses against S. pyogenes consist of a vigorous Th1 cellular memory response in combination with IgG1/IgG3-dominated humoral immunity that increases with age (Mortenson R., 2015). However, since the patient in our case was administered the antibiotics course fairly early on, it is possible that her adaptive immunity may not have had the chance to develop antibodies against the infectious strain of S. pyogenes.

References

Cunningham, M. W., "Pathogenesis of Group A Streptococcal Infections." Clinical Microbiology Reviews, Volume 13, Issue 3, 470–511.

Mortensen, R., Nørrelykke Nissen, T., Blauenfeldt, T., Christensen J. P., Andersen, P. & Dietrich, J. "Adaptive Immunity against Streptococcus pyogenes in Adults Involves Increased IFN-γ and IgG3 Responses Compared with Children." The Journal of Immunology, Volume 195, Issue 4, 2015, 1657-1664.

Todar, K. (2012). Streptococcus pyogenes and Streptococcal Disease. Retrieved from http://textbookofbacteriology.net/streptococcus.html