Course:PATH4172019W2/Case 2

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Case 2: A New Partner

21-year-old Naser recently hooked up with a new sexual partner. This morning he noticed a burning pain in his penis during urination, followed by a greenish discharge. He decided to visit the student health clinic where the doctor asks Naser about his recent sexual history. He recounts that he had unprotected sexual intercourse with a new partner about one week ago.

When asked, the new partner claims no knowledge of having any sexually transmitted infections. The doctor asks Naser to provide a urine sample to send to the Microbiology Laboratory. The laboratory report come back positive for Neisseria gonorrheae. The doctor prescribes antibiotics for Naser and counsels him on safe sex practices and on the importance of encouraging his new partner to come in for testing too.


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).

Our case study patient, Naser, reports to the doctor after noticing the symptoms of burning pain in his penis during urination, followed by a greenish discharge. These presentations are often the result of urogenital Gonorrhea, a specific type of urethritis that involves the infection of urethral mucous membranes by N. gonorrhoeae. (1). This is later confirmed in the laboratory test, as it comes back positive for N. gonorrhoeae, the causative bacteria for gonorrhea.

According to the Centre for Disease Control website, N. gonorrhoeae can infect both men and women, causing infections in the genitals, rectum, pharynx and conjunctiva. However, infection is most common among young people ages 15-24 (2). Mucous infections by this bacteria are usually characterized by purulent discharge which may present as white, yellow or green (2). In males, the discharge may range from a scanty, clear or cloudy fluid to one that is copious and purulent. Discharge may be obtained for a urethral swab by milking the penis (3). Dysuria, the pain or difficulty urinating, may also be present. The intense burning and pain upon urination is caused by the inflammation of the urethral tissues, which also results in the characteristic redness, swelling and heat associated with this region. N. gonorrhoeae may also invade the prostate gland, resulting in prostatitis, or the testicles resulting in orchitis. Invasion of the testis poses the risk of sterility due to infection (1). Furthermore, men may experience painful or swollen testicles, although this is less common (2). Epididymitis, the inflammation of the epididymis, may also result. The doctor may notice the epididymis as swollen and tender to palpation (3).

The signs that a healthcare professional will notice will be through asking Naser questions - does he feel pain when he urinates? Has he had any discharge from the penis -  and if so, what color? Has he had any anal itching or discharge? A lot of what a health care professional will pay attention to in such a situation is due to the information that Naser will provide him with. Upon being provided with such information, the health care professional will make note of these symptoms experienced by the patient and make his diagnosis upon further examination of these signs.

In women, Gonorrhea is most often characterized by increased vaginal discharge and sometimes dysuria which is associated with a painful or burning sensation when urinating (1,2). Women may also experience vaginal bleeding between periods or pain after intercourse, which is termed dyspareunia, as well as severe pelvic pain (2,3). When the physician takes a swab from the cervix, he/she should recognize the sign of easily bleeding when rubbed with a cotton tip, as this is also an indication of N. gonorrhoeae infection (3).

Furthermore, it is possible for infections to ascend through the uterus to the fallopian tubes, resulting in salpingitis, or to the ovaries, resulting in ovaritis (1). This may occur in 10-20% of infected women and symptoms may range from none to severe abdominal pain and high fever (3). Infection in the ovaries may result in sterility (1). Furthermore, as many as 15% of women may develop pelvic inflammatory disease, or PID (1). PID often results from untreated gonorrhea and can result in various complications including the formation of scar tissue that blocks fallopian tubes, ectopic pregnancy (when the fertilized egg grows outside of the uterus which may result in miscarriage), infertility and chronic pelvic-abdominal pain (2). During diagnosis, physicians are recommended to maintain a low threshold for diagnosing PID because of the significant negative sequelae associated with infection (3).

Anorectal and pharyngeal infections by N. Gonorrhoea present signs and symptoms that are summarized in the table below.

Despite the various signs and symptoms that may manifest from N. gonorrhoeae infection, there are a number of cases that go asymptomatic while still maintaining infectious capability. Both men and women can be asymptomatic, however, it is most common for women to not have any symptoms (2). Even when a women does have symptoms, they are often mild and can be mistaken for a bladder or vaginal infection (2).

Individuals with inherited complement deficiencies have a markedly increased risk of acquiring systemic N. gonorrhoea infection, and are subject to recurrent episodes of infection (1).

In ocular infections by N. gonorrhoeae, corneal scarring or perforation may result. This may occur in newborns who are exposed to infected secretions in the birth canal (1). In addition, if the infection is left untreated, it can cause perforation of the globe of the eye and blindness (3). Other signs include inflammation of the conjunctiva and mucopurulent discharge from the eye (3).

Dissemination of the infection to other parts of the body can occur if the initial infection is left untreated. Some complications include dermatitis-arthritis syndrome, endocarditis and meningitis (1). In the case of dermatitis-arthritis syndrome, infection of the synovium in joints and the skin can occur. The hands and feet are the most common for infected joints, likewise for skin lesions, the result of skin infection, most often present in the extremities (3). Patients with disseminated Gonorrhea usually have no urogenital symptoms (3).

Table. 1 Summary of Signs and Symptoms of N. gonorrhoea Infection   
Signs    Symptoms   
Urogenital Infection    Males
  • Purulent discharge (1)
  • Penile redness and swelling (1)
  • Discharge expressed by milking the penis (3)
  • Swollen/tender epididymis (3)

Females

  • Salpingitis or ovaritis if infection ascends through the uterus (1)
Males
  • Discharge and dysuria (1)
  • Sensation of intense burning and pain upon urination (1)
  • Painful or swollen testicles (2)

Females

  • Discharge and dysuria (1)
  • Increased vaginal discharge (2)
  • Vaginal bleeding between periods or after intercourse (dyspareunia) (2,3)
  • Severe pelvic pain (3)
  • High fever (3)
Anorectal Infection   
  • Discharge (2)
  • Rectal bleeding (2)
  • Anal itching (2)
  • Soreness (2)
  • Painful bowel movements (2)   
Pharyngeal infection   
  • Erythematous exudate of the pharynx (4)   
  • Pain/soreness in the pharyngeal area    

References

  1. Todar K. Pathogenic Neisseriae: gonorrhea and meningitis [Internet]. Todar’s Textbook of Bacteriology. [cited 2020 Feb 7]. Available from: http://textbookofbacteriology.net/neisseria.html
  2. STD Facts - Gonorrhea [Internet]. 2019 [cited 2020 Feb 7]. Available from: https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea.htm
  3. Miller KE. Diagnosis and Treatment of Neisseria gonorrhoeae Infections. Am Fam Physician. 2006 May 15;73(10):1779–84.
  4. Prevalence and factors associated with gonorrhea infection with respect to anatomic distributions among men who have sex with men. - PubMed - NCBI [Internet]. [cited 2020 Feb 14]. Available from: https://www.ncbi.nlm.nih.gov/pubmed/30943191

(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?

According to Todar, 2012, the current recommended treatment for all gonococcal infections is with antimicrobial agents including ceftriaxone, cefixime, ciprofloxacin, or ofloxacin. As of 2010, cephalosporins have remained the foundation of gonorrhea treatment guidelines. The recommended dosage of ceftriaxone is 250 mg, and is included in recommendations for combination therapy. Combination therapy includes prescription of a cephalosporin, such as ceftriaxone, followed by administration of a second antimicrobial, such as azithromycin. The purpose of combination therapy is to treat co-occurring pathogens that frequently overlap, such as Chlamydia trachomatis. This combination therapy treatment may hinder the spread of cephalosporin antimicrobial resistance.  (5) The use of combination therapy is highly recommended by physicians as it results in sustained high blood bactericidal levels. (2) Ceftriaxone is administered through intramuscular injection, and may be injected at all anatomical sites. This allows for effective treatment of gonorrheal infections. An alternative method of treatment constitutes as a single dose of 400 mg oral Cefixime, a cephalosporin, along with a single dose of 1g oral azithromycin. (2) However, this alternative method does not result in bactericidal blood levels that are nearly as high or as sustained as intramuscular injection offers.

Ceftriaxone antibiotics are semisynthetic β-lactam antibiotics that have excellent antimicrobial activity against a significant amount of both gram-positive and gram-negative bacteria. (1) Such a broad in vitro spectrum suggests that these antibiotics may have the potential to provide adequate therapy against most bacterial infections. Furthermore, ceftriaxone antibiotics have a particularly long half-life, which allows for them to be administered less frequently than traditional antibiotics.  As a β-lactam antibiotic, ceftriaxone acts to disrupt the synthesis of the peptidoglycan layer that forms the bacterial cell wall. As the peptidoglycan is pertinent to the structural integrity of the cell wall, the antibiotic disrupts the structural integrity of the bacteria. β-lactam antibiotics work by mimicking D-Ala-D-Ala, which in turn inhibits peptidoglycan synthesis by penicillin-binding-proteins. (3) This results in disruption of N. gonorrhoeae’s cell wall, creating an osmotically unstable cell that will ultimately lead to cell lysis. Alternatively, azithromycin works by binding to the 23S rRNA of the 50s ribosomal subunit that is found in N. gonorrhoeae. By binding to the 23S rRNA, the antibiotic works to inhibit the 50S ribosomal subunit assembly. As a result, bacterial protein synthesis is inhibited, eventually killing the bacteria and ridding the body of the infection. (6)

As antibiotic resistance to penicillin has been rapidly increasing, all isolates of N. gonorrhoeae should undergo testing for beta-lactamase production and for susceptibility to determine is the strain is beta-lactamase positive. (4) According to Todar, 2012, gonorrhea cases in England have risen by 25% as drug-resistant strains are increasing as well. Therefore, effective treatment of gonorrhea with antibiotics has been compromised by the growing resistance to antibiotics. In 2008, a strain of gonorrhea was found that was resistant to all antibiotics that were recommended as treatment. This demonstrates the importance of prevention of infection with N. gonorrhoeae, as well as the importance of completing the prescribed dose of antibiotics given by a doctor. Furthermore, it brings awareness to antibiotic-resistant strains of N. gonorrhoeae as an emerging public health threat.

N. gonorrhoeae can result in ocular infections. This type of infection most commonly occurs in newborns that are exposed to gonorrheal secretions in the birth canal. (5) Consequently, silver nitrate or other antibiotics are added to the eyes of newborns in order to prevent N. gonorrhoeae from causing ocular infections.

References:

1.Epstein JS, Hasselquist SM, Simon GL. Efficacy of ceftriaxone in serious bacterial infections. Antimicrobial agents and chemotherapy. 1982 Mar 1;21(3):402-6.

2. Gonococcal Infections - 2015 STD Treatment Guidelines [Internet]. 2019 [cited 2020 Feb 7].

Available from: https://www.cdc.gov/std/tg2015/gonorrhea.htm)

3. KONG KF, Schneper L, Mathee K. Beta‐lactam antibiotics: from antibiosis to resistance and bacteriology. Apmis. 2010 Jan;118(1):1-36.

4. Ronald AR, Alfa MJ. Microbiology of the Genitourinary System. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2020 Feb 7]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK8136/

5.Todar, Kenneth. “Pathogenic Neisseriae: Gonorrhea, Neonatal Ophthalmia and Meningococcal Meningitis (page 1)  .” Todar's Online Textbook of Bacteriology , textbookofbacteriology.net/streptococcus.html.

6. ZITHROMAX (azithromycin dihydrate) Action And Clinical Pharmacology | Pfizer Medical

Information - Cana).

(iv) Why did the doctor emphasis the importance of Naser’s partner being tested (and for what?)  Would the infection present similarly, or differently, in his female partner and what samples would be taken to send to the laboratory?

As mentioned earlier, the bacteria N. gonorrhoea attaches to a host to thrive and replicate. Replication, therefore, results in the spread of this bacteria via transmission during unprotected sexual behaviours. The doctor emphasized the importance of Naser’s partner getting checked to avoid the spread of this bacteria and further infection. Although the urethra, in men, and the uterine cervix, in women, serve as the initial sites for gonococcal infection, infections of the conjunctiva, pharynx, and rectal mucosa are also quite likely. Furthermore, education to modify sexual behaviour, use of condoms and screening asymptomatic individuals in some populations are also effective measures to contain the spread of infection. If left unrecognized and therefore untreated, the disease can progress and more adverse effects can result. In addition, although there is medication to stop infection, it will not undo any permanent damage, such as PID, caused unnoticed disease progression [5].

In most women, gonococcal infection most often goes without symptoms. In select symptomatic women, Neisseria gonorrhoea causes increased vaginal discharge, painful urination, vaginal bleeding and abdominal or pelvic pain [1]. Naser’s partner would likely have an endocervical infection, the most common form of uncomplicated gonorrhea in women [2]. Naser’s partner would then present with vaginal discharge and possible dysuria.

The samples that will be taken to send to the laboratory are urine samples for both males and females. For males, a swab will be taken from the opening of the urethra, while females will get a swab sample taken of their cervix. N. gonorrhoeae can be identified by culture or nucleic acid amplification tests and, Gram stain. Nucleic acid amplification tests can be conducted on a wide range of samples, including urine, vulvovaginal, cervical and urethral swabs [3]. Squamous epithelium, which lines the adult vagina, is not susceptible to infection by the N. gonorrhoeae. However, the prepubescent vaginal epithelium which has not been keratinized by the presence of estrogen, may be infected. Hence, gonorrhea in young girls may occur as vulvovaginitis. Mucosal infections can be accompanied by a purulent discharge [4]. For females, diagnosis of N. gonorrhoeae involves examining cervical exudate. Additionally, both males and females may have swab samples taken from another suspected area of infection, such as the mouth or the rectum.

References:

[1] Ng, Lai-King, and Irene E Martin. “The Laboratory Diagnosis of Neisseria Gonorrhoeae.” The Canadian Journal of Infectious Diseases & Medical Microbiology = Journal Canadien Des Maladies Infectieuses Et De La Microbiologie Medicale, Pulsus Group Inc, Jan. 2005, www.ncbi.nlm.nih.gov/pmc/articles/PMC2095009/.

[2] Todar, Kenneth. “Pathogenic Neisseriae: Gonorrhea, Neonatal Ophthalmia and Meningococcal Meningitis (page 1). ”Todar's Online Textbook of Bacteriology, textbookofbacteriology.net/streptococcus.html.

[3] Information NC for B, Pike USNL of M 8600 R, MD B, Usa 20894. RECOMMENDATIONS FOR TREATMENT OF GONOCOCCAL INFECTIONS [Internet]. World Health Organization; 2016. https://www.ncbi.nlm.nih.gov/books/NBK379222/

[4] Pathogenic Neisseriae: gonorrhea and meningitis [Internet]. http://textbookofbacteriology.net/neisseria.html

[5] STD Facts - Gonorrhea [Internet]. 2019 [cited 2020 Feb 7]. https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea.htm

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 the 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 at the entry site and/or does it spread beyond the initial site. Are there, for instance, secondary sites of infection. Does the organism remain extracellular and/or does it enter into cells - and what are the molecular and cellular determinants of these events.

(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 do 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 this infectious agent?

Reports: A New Partner

21-year-old Naser recently hooked up with a new sexual partner. This morning he noticed a burning pain in his penis during urination, followed by a greenish discharge. He decided to visit the student health clinic where the doctor asks Naser about his recent sexual history. He recounts that he had unprotected sexual intercourse with a new partner about one week ago.

When asked, the new partner claims no knowledge of having any sexually transmitted infections. The doctor asks Naser to provide a urine sample to send to the Microbiology Laboratory. The laboratory report come back positive for Neisseria gonorrheae. The doctor prescribes antibiotics for Naser and counsels him on safe sex practices and on the importance of encouraging his new partner to come in for testing too.

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).

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.

Gonorrheal infection is generally limited to superficial mucosal surfaces lined with columnar epithelium. Specifically, N.gonorrhoea can only attach to microvilli of non-ciliated columnar epithelial cells (3). Due to the tissue specificity of the bacteria, the areas most frequently involved are the urethra, cervix, rectum, pharynx, and conjunctiva. Furthermore, if left untreated the infection may disseminate to a variety of tissues and also lead to skin and joint infections, endocarditis and meningitis.

Urethra

The physiology of the female and male lower urinary tract differs significantly. In females, the adult urethra is 3-4cm in length, it extends from the bladder neck to the external urethral orifice. The proximal urethra in women is lined by squamous epithelial cells that line the majority of the female urethra. In males the urethra is approximately 18-20cm long and connected to the bladder neck (4). The physiological function of the urethra is to act as a path for urine to exit the kidneys.

The N. gonorrhoeae infections acquired by sexual contact affect the mucous membranes of the anterior urethra in males and females. The pathogen attaches to non-ciliated epithelial cells via pili and produces endotoxin (3). This causes Inflammation of the urethral tissues results in the characteristic redness, swelling, heat, and pain in the region. In the male, complications can arise if the organism invades the prostate resulting in prostatitis, or invades the testicles resulting in orchitis (3). An infection of the testicles may cause sterility in males. The main disturbance to the normal functions of this system during an N. gonorrhoeae infection is discomfort.

The urinary tract and urine are normally sterile. Additionally, physical and chemical mechanisms are present to ensure that microorganisms do not enter the urinary tract. However, women are more susceptible to urinary infections because the female urethra is short (2).

Cervix

The cervix is part of the female reproductive system. The cervix is the lower part of the uterus connecting the main body of the uterus to the birth canal (vagina). The cervix is made of connected tissue and divided into the endocervix (inner part of cervix lining) and ectocervix (outer part of cervix)(5). The normal physiological function of this system is to hold a child while pregnant.

The N. gonorrhoeae infections acquired by sexual contact affect the mucous membranes of the endocervix in females. In the female, cervical infection may continue through the uterus to the fallopian tubes resulting in salpingitis, or to the ovaries resulting in ovaritis. 15% cases of uncomplicated cervical infections lead to pelvic inflammatory disease. The involvement of fallopian tubes or ovaries may result in sterility which would disrupt the normal functioning of the reproduction system and cervix.

Rectum

The rectum is a continuation of the colon which normal physiological function is to propel stool out of the body. The rectum can also act as a storage for small amounts of stool until the proper degree of distention needed to engage the defecation reflex is obtained (6).

Rectal infections (proctitis) with N. gonorrhoeae occur in about 1/3 of women with cervical infection. They most often result from autoinoculation with cervical discharge. In men,  rectal infections are more common when men engage in anal intercourse with other men (3). Symptoms of rectal infection in both men and women may include discharge, anal itching, soreness, bleeding, or painful bowel movements. Rectal infection also may be asymptomatic (7). These symptoms would disrupt the normal functioning of this structure by causing discomfort depending on the severity of the infection.

Pharynx

The normal physiological function of the pharynx is digestion and respiration. It receives food from the mouth and air from the nasal cavities. The pharynx is a short tube of skeletal muscle that descends into the esophagus and larynx(8).

Not only does N. gonorrhoeae colonize the pharynx, it also appears to be transmitted bidirectionally through oral-penile contact (9). Pharyngeal infections cause symptoms that are mild or absent.  Signs of infection include inflammation of the pharynx and presence of exudates. Anterior cervical lymphadenopathy (swollen lymph nodes) also may be present. Most cases of pharyngeal infection will spontaneously resolve and usually do not cause adverse sequelae(10). The disruption to the normal functioning of the system will be discomfort.

Conjunctiva

The normal physiological function of the conjunctiva of the eye is to provides protection and lubrication by producing mucus and tears. It prevents microbial entrance into the eye and contributes to immune surveillance. It is highly vascularized with extensive lymphatic vessels (11).

Ocular infections by N. gonorrhoeae can cause scarring or perforation of the cornea. Ocular infections (ophthalmia neonatorum) occur most commonly in newborns who are exposed to infected secretions in the birth canal (3). The bacteria will disrupt the normal functioning of this system by reducing vision abilities.

Vagina

The vagina is part of the reproduction system in females. It is a muscular canal which acts as a conduit for menstrual flow and babies during childbirth. Squamous epithelium, which lines the adult vagina, is not susceptible to infection by the N. gonorrhoeae. However, the prepubescent vaginal epithelium which has not been affected by the presence of estrogen, may be infected. Hence, gonorrhea in young girls may occur as vulvovaginitis. Mucosal infections can be accompanied by a purulent discharge which is the main disruption to this physiological system (3).

Disseminated Infection

           When the bacteria infection is left untreated, a risk of spread to other organs is possible. If treated promptly with antibiotics these further infections can be prevented. When N. gonorrhoeae disseminates to other parts of the body in a cluster via the blood it is termed Septic emboli which can cause dermatitis-arthritis syndrome (which affects the extremities), endocarditis (affecting the heart), and meningitis (affecting the meninges) can occur (10). In dermatitis arthritis syndrome, infection occurs in the synovium (tissue that lines the joints) and the skin which presents in extremities as skin lesions (10).

References

  1. Information NC for B, Pike USNL of M 8600 R, MD B, Usa 20894. RECOMMENDATIONS FOR TREATMENT OF GONOCOCCAL INFECTIONS [Internet]. World Health Organization; 2016 [cited 2020 Feb 7]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK379222/
  2. Ronald AR, Alfa MJ. Microbiology of the Genitourinary System. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2020 Feb 7]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK8136/
  3. Pathogenic Neisseriae: gonorrhea and meningitis [Internet]. [cited 2020 Feb 7]. Available from: http://textbookofbacteriology.net/neisseria.html
  4. Abelson B, Sun D, Que L, Nebel RA, Baker D, Popiel P, et al. Sex differences in lower urinary tract biology and physiology. Biol Sex Differ. 2018 Oct 22;9(1):45.
  5. The cervix - Canadian Cancer Society [Internet]. www.cancer.ca. [cited 2020 Feb 14]. Available from: https://www.cancer.ca:443/en/cancer-information/cancer-type/cervical/cervical-cancer/the-cervix/?region=on
  6. Shafik A, Mostafa RM, Shafik I, EI-Sibai O, Shafik AA. Functional activity of the rectum: A conduit organ or a storage organ or both? World J Gastroenterol WJG. 2006 Jul 28;12(28):4549–52.
  7. Detailed STD Facts - Gonorrhea [Internet]. 2019 [cited 2020 Feb 7]. Available from: https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea-detailed.html
  8. OpenStax. 23.3 The Mouth, Pharynx, and Esophagus. In: Anatomy and Physiology [Internet]. OpenStax; 2013 [cited 2020 Feb 14]. Available from: https://opentextbc.ca/anatomyandphysiology/chapter/23-3-the-mouth-pharynx-and-esophagus/
  9. Ota KV, Fisman DN, Tamari IE, Smieja M, Ng L-K, Jones KE, et al. Incidence and Treatment Outcomes of Pharyngeal Neisseria gonorrhoeae and Chlamydia trachomatis Infections in Men Who Have Sex with Men: A 13-Year Retrospective Cohort Study. Clin Infect Dis. 2009 May 1;48(9):1237–43.
  10. Miller KE. Diagnosis and Treatment of Neisseria gonorrhoeae Infections. Am Fam Physician. 2006 May 15;73(10):1779–84.
  11. Shumway CL, Motlagh M, Wade M. Anatomy, Head and Neck, Eye Conjunctiva. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 [cited 2020 Feb 14]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK519502/
  12. Richards DM, Heel RC, Brogden RN, Speight TM, Avery GS. Ceftriaxone. A review of its antibacterial activity, pharmacological properties and therapeutic use. Drugs. 1984 Jun;27(6):469–527
  13. Spectinomycin - Infectious Diseases [Internet]. Merck Manuals Professional Edition. [cited 2020 Feb 7]. Available from: https://www.merckmanuals.com/professional/infectious-diseases/bacteria-and-antibacterial-drugs/spectinomyci
  14. Macrolides - Infectious Diseases [Internet]. Merck Manuals Professional Edition. [cited 2020 Feb 7]. Available from: https://www.merckmanuals.com/professional/infectious-diseases/bacteria-and-antibacterial-drugs/macrolides?query=azithromycin
  15. Ng L-K, Martin IE. The laboratory diagnosis of Neisseria gonorrhoeae. Can J Infect Dis Med Microbiol. 2005;16(1):15–25.

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?

According to Todar, 2012, the current recommended treatment for all gonococcal infections is with antimicrobial agents including ceftriaxone, cefixime, ciprofloxacin, or ofloxacin. As of 2010, cephalosporins have remained the foundation of gonorrhea treatment guidelines. The recommended dosage of ceftriaxone is 250 mg, and is included in recommendations for combination therapy. Combination therapy includes prescription of a cephalosporin, such as ceftriaxone, followed by administration of a second antimicrobial, such as azithromycin. The purpose of combination therapy is to treat co-occurring pathogens that frequently overlap, such as Chlamydia trachomatis. This combination therapy treatment may hinder the spread of cephalosporin antimicrobial resistance.  (5) The use of combination therapy is highly recommended by physicians as it results in sustained high blood bactericidal levels. (2) Ceftriaxone is administered through intramuscular injection, and may be injected at all anatomical sites. This allows for effective treatment of gonorrheal infections. An alternative method of treatment constitutes as a single dose of 400 mg oral Cefixime, a cephalosporin, along with a single dose of 1g oral azithromycin. (2) However, this alternative method does not result in bactericidal blood levels that are nearly as high or as sustained as intramuscular injection offers.

Ceftriaxone antibiotics are semisynthetic β-lactam antibiotics that have excellent antimicrobial activity against a significant amount of both gram-positive and gram-negative bacteria. (1) Such a broad in vitro spectrum suggests that these antibiotics may have the potential to provide adequate therapy against most bacterial infections. Furthermore, ceftriaxone antibiotics have a particularly long half-life, which allows for them to be administered less frequently than traditional antibiotics.  As a β-lactam antibiotic, ceftriaxone acts to disrupt the synthesis of the peptidoglycan layer that forms the bacterial cell wall. As the peptidoglycan is pertinent to the structural integrity of the cell wall, the antibiotic disrupts the structural integrity of the bacteria. β-lactam antibiotics work by mimicking D-Ala-D-Ala, which in turn inhibits peptidoglycan synthesis by penicillin-binding-proteins. (3) This results in disruption of N. gonorrhoeae’s cell wall, creating an osmotically unstable cell that will ultimately lead to cell lysis. Alternatively, azithromycin works by binding to the 23S rRNA of the 50s ribosomal subunit that is found in N. gonorrhoeae. By binding to the 23S rRNA, the antibiotic works to inhibit the 50S ribosomal subunit assembly. As a result, bacterial protein synthesis is inhibited, eventually killing the bacteria and ridding the body of the infection. (6)

As antibiotic resistance to penicillin has been rapidly increasing, all isolates of N. gonorrhoeae should undergo testing for beta-lactamase production and for susceptibility to determine is the strain is beta-lactamase positive. (4) According to Todar, 2012, gonorrhea cases in England have risen by 25% as drug-resistant strains are increasing as well. Therefore, effective treatment of gonorrhea with antibiotics has been compromised by the growing resistance to antibiotics. In 2008, a strain of gonorrhea was found that was resistant to all antibiotics that were recommended as treatment. This demonstrates the importance of prevention of infection with N. gonorrhoeae, as well as the importance of completing the prescribed dose of antibiotics given by a doctor. Furthermore, it brings awareness to antibiotic-resistant strains of N. gonorrhoeae as an emerging public health threat.

N. gonorrhoeae can result in ocular infections. This type of infection most commonly occurs in newborns that are exposed to gonorrheal secretions in the birth canal. (5) Consequently, silver nitrate or other antibiotics are added to the eyes of newborns in order to prevent N. gonorrhoeae from causing ocular infections.

References:

1.Epstein JS, Hasselquist SM, Simon GL. Efficacy of ceftriaxone in serious bacterial infections. Antimicrobial agents and chemotherapy. 1982 Mar 1;21(3):402-6.

2. Gonococcal Infections - 2015 STD Treatment Guidelines [Internet]. 2019 [cited 2020 Feb 7].

Available from: https://www.cdc.gov/std/tg2015/gonorrhea.htm)

3. KONG KF, Schneper L, Mathee K. Beta‐lactam antibiotics: from antibiosis to resistance and bacteriology. Apmis. 2010 Jan;118(1):1-36.

4. Ronald AR, Alfa MJ. Microbiology of the Genitourinary System. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2020 Feb 7]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK8136/

5.Todar, Kenneth. “Pathogenic Neisseriae: Gonorrhea, Neonatal Ophthalmia and Meningococcal Meningitis (page 1)  .” Todar's Online Textbook of Bacteriology , textbookofbacteriology.net/streptococcus.html.

6. ZITHROMAX (azithromycin dihydrate) Action And Clinical Pharmacology | Pfizer Medical

Information - Cana).

Question (iv)

(iv) Why did the doctor emphasis the importance of Naser’s partner being tested (and for what?)  Would the infection present similarly, or differently, in his female partner and what samples would be taken to send to the laboratory?

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.

Naser’s infection is characterized by greenish discharge and pain in the penis during urination. Since Naser has had unprotected sex, this infection is likely a sexually transmitted infection (Baron, 1996). Genitourinary infections are of two main types: 1) infections due to microbes that are a part of the resident flora and 2) primary infections as a result of sexually transmitted pathogenic microorganisms (Baron, 1996). In Naser’s case, the infection is a primary infection due to a sexually transmitted pathogen.

In men, genital infections can result in pain during urination, urethral discharge, and painful scrotal swellings (Baron, 1996). Genital infections can also cause genital ulcers or enlarged inguinal lymph nodes (Baron, 1996). Urethritis, an inflammatory infection of the urethra, is associated with urethral discharge, and is classified as either gonococcal urethritis (caused by Neisseria gonorrhoeae (N. gonorrhoeae)) or nongonococcal urethritis (caused by Chlamydia trachomatis (C. trachomatis)) (Baron, 1996). Urinary discharge and dysuria (painful urination) are symptoms observed in the majority of gonococcal urethritis patients, while patients with nongonococcal urethritis only exhibit one of these two symptoms, not both (Baron, 1996). N. gonorrhoeae and C. trachomatis are sexually transmitted (Baron, 1996).

Gonococcal urethritis

This is caused by N. gonorrhoeae (Figure 1) and common symptoms in men are dysuria and urethral discharge with a complication being epididymitis (Baron, 1996).

Neisseria gonorrhoeae

Figure 1. Image of Neisseria gonorrhoeae (Tankeshwar, 2013).

This is a gram-negative coccus, usually a diplococcus (seen in pairs), with a diameter of 0.6 to 1.0 µm. It has fimbriae, which function in adherence, and has lipopolysaccharide in the outer membrane referred to as lipooligosaccharide (LOS) (Todar, 2012). Of the Neisseria and related species, only N. gonorrhoeae is always considered pathogenic and is never considered as a part of the normal flora (Centers for Disease Control and Prevention, 2017). N. gonorrhoeae causes gonorrhea, a sexually transmitted infection (STI) that can infect females and males at various sites, such as the genital tract, rectum, eyes, throat, and joints (Mayo Clinic, 2019). N. gonorrhoeae belongs to the family of Neisseriaceae, which comprises the genera Neisseria, Moraxella, Kingella, and Acinetobacter. Within this family of bacteria, the only significant human pathogens are “N. gonorrhoeae, the agent of gonorrhea, and N. meningitidis, an agent of acute bacterial meningitidis” (Baron, 1996). Several Neisseria related species could be misidentified as N. gonorrhoeae (Centers for Disease Control and Prevention, 2017). Therefore, it is important that the appropriate differential tests are performed, and only strains of N. gonorrhoeae is identified and reported. Gonorrhea is a reportable bacterial sexually transmitted infections, which means that health workers are obligated to report a confirmed case of gonorrhea (Government of Canada, 2020). Since there are “serious social and medicolegal consequences of misdiagnosing gonorrhea”, there are regulations and criteria for laboratory tests before a case can be diagnosed and reported (Centers for Disease Control and Prevention, 2017). The of laboratory tests and basis of diagnoses will be discussed with more details in the next few questions. N. gonorrhoeae can lead to severe consequences if left untreated. For example, women may experience severe sequelae of pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy and tubal infertility while men may develop epididymitis, prostatitis and urethral stricture (LCDC Expert Working Group on Canadian Guidelines for Sexually Transmitted Diseases. Canadian STD Guidelines, 1998).

Infection mostly occurs in the superficial mucosal epithelial surfaces which are lined with columnar epithelium, including the rectum, cervix, urethra, conjunctiva and pharynx. After adhesion via pili and opa (P.II) proteins to columnar epithelial cells, the bacteria penetrate these cells and multiply in the basement membrane of the cells (Todar, 2012). In males, the infection is of the anterior urethra mucous membranes and is pyogenic and inflammatory. The bacteria enter the cells through parasite-directed endocytosis, which involves the pinching off of a phagosome containing the bacteria, followed by migration to the base of the cell and exocytosis to release the bacteria into the subepithelial tissue (Todar, 2012). The outer membrane of N gonorrhoeae is composed of proteins, phospholipids, and lipopolysaccharide (LPS). Virulence factors of N. gonorrhoeae include PilE (fimbrial protein that mediates binding to epithelial cells), P.II (outer membrane protein functioning in invasion), P.I (outer membrane porin contributing to survival in phagocytes), LOS (lipooligosaccharide, a highly branches basal oligosaccharide structure and do not have repeating O-antigen subunits, which triggers an inflammatory response, release of TNF, activation of complement, attraction of phagocytes and their subsequent lysis, leading to discharge), P. III (outer membrane protein, which produces ineffective antibodies that block bactericidal antibodies against LOS and P.I), transferrin receptors Tbp1 and Tbp2, and a lactoferrin receptor Lbp (for iron acquisition to grow) (Todar, 2012; Baron, 1996).

Nongonococcal urethritis (NGU)

The symptoms of this infection in men include painful urination, urinary discharge, underwear stains, and irritation or itching (American Sexual Health Association, n.d.). If the infection is left untreated, complications such as skin lesions, conjunctivitis, epididymitis (inflammation of the epididymis) or Reiter’s syndrome (arthritis) can occur (American Sexual Health Association, n.d.).

Figure 2. Image of Chlamydia Trachomatis.

Chlamydia trachomatis

A substantial proportion of NGU cases are attributed to C. trachomatis. It is a gram-negative obligate intracellular pathogen that has the ability to avoid destruction by the host immune system (Witkin et al., 2017). Strains are divided into 3 biovars, which are further subtyped by 18 serovars. Trachoma biovars include serovars A to C, which causes non-congenital blindness, while genital tract biovars include serovars D to K which is usually transmitted sexually (Elwell, Mirrashidi and Engel, 2016). Women are usually asymptomatic. It is transmitted via anal, oral or vaginal sexual intercourse, and can be passed from mother to child at birth (Elwell, Mirrashidi and Engel, 2016).

Figure 3. Image of Mycoplasma genitalium

It is found in two forms: as an elementary body (EB), which is the infectious extracellular form in which the bacteria cannot divide and reticulate bodies (RB), a form in which the bacteria are able to multiply (Bébéar and de Barbeyrac, 2009). The cycle involves EBs converting to RBs, division, and conversion of RBs back to EBs. C. trachomatis releases a heat shock protein Hsp60 which interacts with the host cytokine system, resulting in inflammation. Other virulence factors include a type III secretion system and a cytotoxin (Bébéar and de Barbeyrac, 2009).

Mycoplasma genitalium

This is a common pathogen causing NGU. M. genitalium is also transmitted by sexual contact and can cause watery discharge from the penis and pain during urination in men (WebMD, n.d.). However, the distinct green discharge of the patient is an indicator that the causative agent is unlikely to be M. genitalium. The bacterium M. genitalium is of the Mollicutes class and lacks a cell wall (Manhart, Broad and Golden, 2011). It is one of the smallest bacteria known and is slow growing (Gnanadurai and Fifer, 2020). It adheres to host epithelial cells which produce acute inflammatory signals, leading to inflammation in the urogenital tract (Gnanadurai and Fifer, 2020). Lipoproteins of M. genitalium bind to these cell receptors, activating pro-inflammatory signals such as chemokines, which lead to recruitment of leucocytes (Gnanadurai and Fifer, 2020). Furthermore, there is a cytopathological effect on host epithelium due to the metabolic by-products of the host immune response and M. genitalium, which include nitric oxide and reactive oxygen species (McGowin and Totten, 2017).

Ureaplasma urealyticum

Figure 4. Image of Ureaplasma urealyticum

The causative agent of U. urealyticum infections, ureaplasma are small free-living bacteria found in the normal flora of the urogenital tract. They can overgrow and inflame healthy tissue, possibly leading to infection. Symptoms of ureaplasma include discharge, rash, pain during urination and increased urge to urinate (Berry and Murrell, 2018). They have been linked to a variety of medical problems, such as bacterial vaginosis and pregnancy complications. It can also inflame the urethra and cause urethritis (Osborn and Murrell, 2018).

This spherical bacterium is a member of the Mollicutes class, is 0.2-0.3 µm in diameter, and is a commensal organism of the human urogenital tract (Dhawan and Kokkayil, 2015). U. urealyticum have evolved from a gram-positive bacterium, losing their cell wall in the process. It is sexually transmitted and uses cytoadherence proteins to attach to mucosal epithelial cells, including urethral epithelial cells (Dhawan and Kokkayil, 2015). Virulence factors of U. urealyticum include MBA, a surface exposed lipoprotein that activates nuclear factor kappa B, IgA protease activity, which destroys mucosal IgA, urease activity, used to cleave urea to produce ammonia (which can change pH and cause toxicity to host tissues), and biofilm formation, which can lead to inflammation (Dhawan and Kokkayil, 2015).

Viruses

Adenoviruses and Herpes Simplex Virus are some viruses that may cause NGU, although they are responsible for a very small percentage of cases: around 2-4% (Bradshaw, Tabrizi and Read, 2006).

Co-morbidities

Other STIs may co-occur with gonorrhea. Therefore, patients may also be tested for chlamydia, hepatitis B, human immunodeficiency virus (HIV), syphilis, and trichomoniasis (Haldeman-Englert and Holloway, 2020). Chlamydia can have very similar symptoms to gonorrhea and was discussed above. Hepatitis B is a viral infection that attacks the liver and is caused by the hepatitis B virus. The virus is transmitted through blood, semen, or other bodily fluids, for example by sexual contact, sharing needles or from mother to child, and can be potentially life-threatening (Mayo Clinic, 2017; World Health Organization, 2019).

HIV infection is caused by the human immunodeficiency virus, which attacks the host’s immune system. The last stage of HIV infection is acquired immune deficiency syndrome (AIDS). HIV can be transmitted through infected blood, semen, or vaginal fluids (HealthLink BC, 2019). Syphilis is an infection that starts as a painless sore. Syphilis is caused by the bacteria Treponema pallidum, which is usually spread via sexual contact (Mayo Clinic, 2019). Trichomoniasis is an STI caused by infection with a protozoan parasite called Trichomonas vaginalis (Centers for Disease Control and Prevention, 2017).

References:

American Sexual Health Association. (n.d.). NGU. [online] Available at: http://www.ashasexualhealth.org/stdsstis/ngu/ [Accessed 13 Feb. 2020].

Baron, S. (1996). Medical Microbiology. 4th ed. Galveston: University of Texas Medical Branch at Galveston.

Bébéar, C. and de Barbeyrac, B. (2009). Genital Chlamydia trachomatis infections. Clinical Microbiology and Infection, 15(1), pp.4-10.

Berry, J. and Murrell, D. (2018). What is Ureaplasma?. [online] Medical News Today. Available at: https://www.medicalnewstoday.com/articles/321636 [Accessed 13 Feb. 2020].

Bradshaw, C., Tabrizi, S. and Read, T. (2006). Etiologies of Nongonococcal Urethritis: Bacteria, Viruses, and the Association with Orogenital Exposure. J Infect Dis, 193(3), pp.336-345.

Centers for Disease Control and Prevention. (2017). Characteristics of N. gonorrhoeae and Related Species of Human Origin. [online] Available at: https://www.cdc.gov/std/gonorrhea/lab/ngon.htm [Accessed 8 Feb. 2020].

Centers for Disease Control and Prevention. (2017). Gonorrhea: Identification of N. gonorrhoeae and Related Species. [online] Available at: https://www.cdc.gov/std/gonorrhea/lab/pathogenicity.htm [Accessed 7 Feb. 2020].

Centers for Disease Control and Prevention. (2017). Trichomoniasis - CDC Fact Sheet. [online] Available at: https://www.cdc.gov/std/trichomonas/stdfact-trichomoniasis.htm [Accessed 7 Feb. 2020].

Cook, S., & Soni, S. (2019). Antibiotic treatment of Mycoplasma genitalium infection. Acute pain, 10, 00.

Chlamydia Trachomatis. (n.d.). Retrieved from https://www.sciencedirect.com/topics/medicine-and-dentistry/chlamydia-trachomatis

Dhawan, B. and Kokkayil, P. (2015). Ureaplasma: Current perspectives. Indian Journal of Medical Microbiology, 33(2), p.205.

Elwell, C., Mirrashidi, K. and Engel, J. (2016). Chlamydia cell biology and pathogenesis. Nature Reviews Microbiology, 14(6), pp.385-400.

Gnanadurai, R. and Fifer, H. (2020). Mycoplasma genitalium: A Review. Microbiology, 166(1), pp.21-29.

Government of Canada. (2020). Gonorrhea. [online] Available at: https://www.canada.ca/en/public-health/services/diseases/gonorrhea.html [Accessed 13 Feb. 2020].

Haldeman-Englert, C. and Holloway, B. (2020). Gonorrhea Test (Urine). [online] University of  Rochester Medical Center. Available at: https://www.urmc.rochester.edu/encyclopedia/content.aspx?contenttypeid=167&contenti (Links to an external site.)d=gonorrhea_urine [Accessed 7 Feb. 2020].

HealthLink BC. (2019). HIV (Human Immunodeficiency Virus) Infection. [online] Available at: https://www.healthlinkbc.ca/health-topics/hw151408 [Accessed 7 Feb. 2020].

LCDC Expert Working Group on Canadian Guidelines for Sexually Transmitted Diseases. Canadian STD Guidelines. (1998). Ottawa: Health Canada, pp.57-58,140-9.

Manhart, L., Broad, J. and Golden, M. (2011). Mycoplasma genitalium: Should We Treat and How?. Clinical Infectious Diseases, 53(suppl_3), pp.S129-S142.

Mayo Clinic. (2019). Gonorrhea: Symptoms & causes. [online] Available at: https://www.mayoclinic.org/diseases-conditions/gonorrhea/symptoms-causes/syc- (Links to an external site.)20351774 [Accessed 7 Feb. 2020].

Mayo Clinic. (2017). Hepatitis B: Symptoms & causes. [online] Available at: https://www.mayoclinic.org/diseases-conditions/hepatitis-b/symptoms-causes/syc- (Links to an external site.)20366802 [Accessed 7 Feb. 2020].

Mayo Clinic. (2019). Syphilis: Symptoms & causes. [online] Available at: https://www.mayoclinic.org/diseases-conditions/syphilis/symptoms-causes/syc-20351756 (Links to an external site.) [Accessed 7 Feb. 2020].

McGowin, C. and Totten, P. (2017). The Unique Microbiology and Molecular Pathogenesis of Mycoplasma genitalium. The Journal of Infectious Diseases, 216(suppl_2), pp.S382-S388.

Osborn, C. and Murrell, D. (2018). Everything You Should Know About Ureaplasma. [online] Healthline. Available at: https://www.healthline.com/health/ureaplasma [Accessed 13 Feb. 2020].

Tankeshwar, A. (2013). Neisseria gonorrhoeae: Disease, Pathogenesis and Laboratory Diagnosis. [online] Microbeonline. Available at: https://microbeonline.com/neisseria-gonorrhoeae- (Links to an external site.)properties-disease-pathogenesis-and-laboratory-diagnosis/ [Accessed 7 Feb. 2020].

Todar, K. (2012). Todar's Online Textbook of Bacteriology. Madison: University of Wisconsin.

Ureaplasma urealyticum. (n.d.). Retrieved from https://www.sciencedirect.com/topics/medicine-and-dentistry/ureaplasma-urealyticum

WebMD. (n.d.). What Is Mycoplasma Genitalium?. [online] Available at: https://www.webmd.com/sexual-conditions/mycoplasma-genitalium#1 [Accessed 13 Feb. 2020].

Witkin, S., Minis, E., Athanasiou, A., Leizer, J. and Linhares, I. (2017). Chlamydia trachomatis: the Persistent Pathogen. Clinical and Vaccine Immunology, 24(10).

World Health Organization. (2019). Hepatitis B. [online] Available at: https://www.who.int/news-room/fact-sheets/detail/hepatitis-b [Accessed 7 Feb. 2020].

Question (ii)

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

Sample collection:

In order to make a diagnosis of urethritis, it first involves a physical examination. This includes inspection of the underwear for secretions, examination of the distal urethral meatus for urethral discharge or lesions, examination of the penis for skin lesions that may suggest other STDs, checking the inguinal lymph nodes for lymphadenopathy (swelling), examining the testes for inflammation, palpating the prostate for indication of prostatitis, and looking for perianal lesions during a digital rectal examination (11).

The necessary samples to collect for laboratory testing from Naser, a male patient, are a first-catch urine specimen, a midstream urine specimen, a blood sample, and possibly a urethral swab.

Urethral swab:

In the past, urethral swabs were used to collect samples from inside the urethra. In this process, a swab with a dacron, cytobrush or rayon tip and a wire or plastic shaft is inserted 2-3 cm into the urethra and rotated 2-3 times in order to collect enough cuboidal or columnar epithelial cell.

After collection of suspected C. trachomatis samples, storage of the samples is done in transport media such as M4 media (Lenexa, Kansas, Thermal Scientific) or sucrose phosphate glutamate buffer. To ensure viability of the organisms, culture samples are transported within 24 hours of collection to the laboratory at 4°C. In cases of transport being delayed by more than 24 hours, the storage temperature of the specimens is -70°C. (Papp et al., 2014).

After collection of suspected N. gonorrhoeae samples, a culture medium inoculated with the swab sample is placed into a CO2 enriched atmosphere for transportation. The culture media includes a base medium with bovine blood or heated (chocolatized) equine. The inoculated media are then incubated in a 5% CO2 environment at 35°C – 36.5°C and examined at time periods of 24 and 48 hours after collection. (Papp et al. 2014).

Nonetheless, since this procedure was invasive, it has mostly been replaced by urine specimen collection since both sample collection methods yield accurate results after being tested. According to a 2014 report by the CDC, the testing of first-catch urine samples for detection of C. trachomatis and N. gonorrheae is equally as effective as, and sometimes even better than, urethral swabs (Papp et al., 2014). In Naser’s case, a swab of the urethra can be collected for samples of the greenish discharge, however this is likely unnecessary if a urine specimen is collected, especially considering how uncomfortable the swab collection process is for patients. Traditional screenings of both chlamydia and gonorrhea consist of insertion of 1 or more swabs into the urethra in men (Cook, 2005). These methods of collecting specimen can be uncomfortable for patients and therefore, decrease their likelihood of regular STI screening. Therefore, promoting less invasive screening procedures, like taking urinary sample or self-collected vaginal swabs, could help remove barriers for people to screen for chlamydial or gonococcal infections (Cook, 2005).

Table 1. Various clinical specimens for the laboratory diagnosis of N. gonorrhoeae; information from (Ng and Martin, 2005).

Urine samples:

Specifically, to test for C. trachomatis and N. gonorrheae, it is traditionally recommended that a first-catch urine specimen be collected instead of a mid-stream urine specimen because it increases the probability of laboratory detection to successfully diagnose chlamydia and/or gonorrhea (Wiedbrauk, 2015). This is because a midstream sample can lead to a false negative if the bacteria have been flushed out of the urethra prior to the specimen collection. As a result, a first-catch specimen is suggested in order to accurately test for chlamydia and gonorrhea. Nonetheless, in order to test the urine for the presence of an E. coli-causing UTI, it is recommended that midstream urine specimens be collected instead of first-catch urine specimens. As a result, both first-catch and midstream urine specimens should be collected for laboratory testing.

In general, only 10-30mL is required from the patient and should be collected in a sterile specimen container. It should be transferred to the Microbiology Laboratory immediately and kept somewhere between 2-8 degrees Celsius (Samuel, 1996). If immediate transportation is unavailable, then the sample should be refrigerated within 30 minutes, as urine is unstable, and samples that are not received within 24 hours urination may be rejected. However, if samples are to be delayed, they must be stored at -70ºC.

Blood samples:

Finally, if it is suspected that Naser has gonorrhea or chlamydia, a blood sample should also be collected to test for other common sexually transmitted infections, including syphilis, and HIV (Brill, 2010). This is recommended because coinfection with the previously mentioned infections can commonly occur, thus the blood sample can be used to test for the presence of any of those infections in the blood.

This information is summarized in Table 1 above!

Importance of the Microbiology Laboratory

Since the symptoms of infection of the three mentioned possible bacterial pathogens are quite similar, the Microbiology Laboratory is a critical step in the diagnosis because the laboratory confirmation will allow the doctors to accurately treat the patient. This is because the recommended course of antibiotics is different depending on the bacterial infection present since specific bacterial strains will be more susceptible to specific antibiotics and some will even be resistant to specific antibiotics. If the infection is found to be chlamydia, the doctor would prescribe a single dose of azithromycin or doxycycline for seven days (CDC, 2015). Contrarily, if the infection is found to be gonorrhea, the doctor would prescribe a combination of ceftriaxone andazithromycin (CDC, 2019). This dual antibiotic regiment is recommended for gonorrhea because of N. gonorrheae strains that have been found to be resistant to cephalosporin antibiotics, such as ceftriaxone. Additionally, the Microbiology Laboratory will allow the patient to be quickly diagnosed so that the doctor can prescribe the necessary treatment before the patient’s infection becomes more severe. In Naser’s case, urethritis can develop into disseminated gonococcal infection, which is a dangerous progression of the pathogen. Thus, it is evident that laboratory confirmation is critical for patient care because it will guide the treatment that the doctors will prescribe to their patients and will allow them to administer the treatment more rapidly before the infection develops any further into a more dangerous form.  

References:

Brill, John R. “Diagnosis and Treatment of Urethritis in Men.” American Family Physician, American Academy of Family Physicians, 1 Apr. 2010, www.aafp.org/afp/2010/0401/p873.html.

“Chlamydial Infections - 2015 STD Treatment Guidelines.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 4 June 2015, www.cdc.gov/std/tg2015/chlamydia.htm.

Cook, R. L., Hutchison, S. L., Østergaard, L., Braithwaite, R. S., & Ness, R. B. (2005). Systematic review: noninvasive testing for Chlamydia trachomatis and Neisseria gonorrhoeae. Annals of internal medicine, 142(11), 914-925.

“Gonorrhea Treatment and Care.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 5 Nov. 2019, www.cdc.gov/std/gonorrhea/treatment.htm.

“HIV Antibody and HIV Antigen (p24).” Patient Education on Blood, Urine, and Other Lab Tests, AACC, Sept. 2019, labtestsonline.org/tests/hiv-antibody-and-hiv-antigen-p24.

Imam, Talha H. “Urethritis - Kidney and Urinary Tract Disorders.” Merck Manuals Consumer Version, Merck, 2018, www.merckmanuals.com/home/kidney-and-urinary-tract-disorders/urinary-tract-infections-utis/urethritis.

Leon-Sicairos, Nidia, et al. “Strategies of Intracellular Pathogens for Obtaining Iron from the Environment.” BioMed Research International, vol. 2015, May 2015, pp. 1–17., doi:10.1155/2015/476534.

Malhotra, Meenakshi, et al. “Genital Chlamydia Trachomatis: An Update.” Indian Journal of Medical Research, vol. 138, no. 3, Sept. 2013, pp. 303–316.

Papp, John R, et al. “Recommendations for the Laboratory-Based Detection of Chlamydia Trachomatis and Neisseria Gonorrhoeae - 2014.” Morbidity and Mortality Weekly Report (MMWR), Centers for Disease Control and Prevention, Mar. 2014, www.cdc.gov/mmwr/preview/mmwrhtml/rr6302a1.htm.

Rosen, John M, and David J Klumpp. “Mechanisms of Pain from Urinary Tract Infection.” International Journal of Urology, vol. 21, no. 1, 2014, pp. 26–32., doi:10.1111/iju.12309.

Rowley, Jane, et al. “Chlamydia, Gonorrhoea, Trichomoniasis and Syphilis: Global Prevalence and Incidence Estimates, 2016.” Bulletin of the World Health Organization, vol. 97, no. 8, 6 June 2019, pp. 548–562., doi:10.2471/blt.18.228486.

Samuel, B. (1996). Medical Microbiology (4th ed.). Galveston, TX: University of Texas Medical Branch at Galveston

“Syphilis Tests.” HealthLink BC, HealthLink BC, 2019, www.healthlinkbc.ca/medical-tests/hw5839.

Wei Tan, Chee, and Maciej Piotr Chlebicki. “Urinary Tract Infections in Adults.” Singapore Medical Journal, vol. 57, no. 9, Sept. 2016, pp. 485–490.

Wiedbrauk, Danny. “The Importance of Obtaining First Catch Urine Samples for Chlamydia Trachomatis and Neisseria Gonorrhoeae Testing.” Warde Medical Laboratory, Warde Medical Laboratory, 2015, www.wardelab.com/25-1-2.html.

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.

When getting samples to test for STDs, many different types of tests can be done to identify the pathogen involved in the infection, including laboratory and point-of care tests.

Laboratory tests can be split into culture and non-culture tests. Culture tests include Gram stain, biochemical tests and antibiotic susceptibility tests. Non-culture tests include Enzyme Immunoassays (EIA), Direct Fluorescent Antibody (DFA) and staining, molecular tests (including Nucleic Acid Hybridization (NAH), Nucleic Acid Genetic Transformation, Nucleic Acid Amplification (NAAT)) and Serology Tests. Point-of-care tests include the Leukocyte Esterase Test (LET)

CULTURE TESTS

Culturing:

N. gonorrhoeae

Specimens are streaked out onto a medium that supports bacterial growth, such as chocolatized equine or bovine blood [1]. For culturing, the bacteria need to stay alive. To promote the growth of this species, warm conditions (~36ºC) are needed with about 3-10% of added CO2 [2].

C. trachomatis

Bacteria of this kind are obligate intracellular pathogens, so their cell culture reflects this; culturing requires inoculation of a monolayer of susceptible cells [3]. Infected cells are stained with iodine and centrifugation combines this with the monolayer of cells. As mentioned earlier, if the sample can’t be tested within a day, it must be refrigerated or frozen at -70ºC [4].

U. urealyticum

A7 agar plates are used for this strain of bacteria as they help culture and identify the pathogen [5].

Gram Stain [6]:

This method builds off of cell cultures that are made using the procedures outlined from that section.

The gram stain aims to identify bacteria as part of one of two groups: gram-negative or gram-positive. This is achieved based off of the cell wall properties of the bacterium. Gram-negative bacteria possess a thin peptidoglycan layer in their cell wall, which is further protected by an outer membrane, whereas gram-positive bacteria have a thick peptidoglycan layer as they do not have this extra membrane. The first step of the procedure involves the cells being stained with a crystal violet dye, followed with the addition of Gram’s iodine solution binds to the crystal violet. A decolorizer (ex. Ethyl alcohol) is added to shrink the peptidoglycan (PG) layer, and the crystal violet-iodine complex is unable to penetrate the PG layer. As a result this complex becomes embedded in the PG layer. Gram-positive bacteria obtain the distinct violet colour as they are able to retain these complexes right away into their thick PG layer.  Gram (+) bacteria have cross-linked teichoic acids on their cell membranes, allowing them to retain the primary dye crystal violet [3]. Gram-negative bacteria do not stain violet because this procedure usually results in the destruction of the outer membrane which also prevents the violet-iodine complexes from reaching the PG layer. Furthermore, Gram (-) have a higher lipid content in cell wall due to having an extra layer, making them able to take up the counterstain and appear pink as they don’t react to the dyes [3]. They stain red/pink due to the addition of the counterstain (safranin), which does not impact the violet stain of gram-positive bacteria due to it being significantly lighter.

This is only used for N. gonorrhoeae in this scenario. U. urealyticum lacks a cell wall and therefore cannot be used in this testing method as they do not contain peptidoglycan [7]. While C. trachomatis is a gram-negative bacterium, it is an obligate intracellular pathogen, making it hard to perform a stain [8].

Biochemical Tests [9]:

There are a number of tests that can be used under this category, and they check for the presence of enzymes. One is the oxidase test, which is used to identify bacteria that possess cytochrome oxidase, an enzyme involved in the electron transport chain. The electron donor N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) is provided to undergo a redox reaction [10]. The test will become dark blue/purple (due to the presence of indophenol blue) to indicate the presence of this enzyme, as oxidation occurs. Another option is the catalase test that allows for identification of bacteria that possess catalase; an enzyme that breaks down hydrogen peroxide. In this test, a small amount of hydrogen peroxide is added, and presence of bacteria is indicated by bubbles. Another option is testing with glucose, fructose, sucrose, and maltose. A specific type of this test is the QuadFERM+ test, which detects acid production from these sugars. A medium will contain one of the listed sugars and phenol red, which is a pH indicator. The pH decreases (and the indicator will turn yellow) if the bacteria from the sample are capable of metabolizing the sugar in the medium.

Antibiotic Susceptibility Tests [3]:

This works for N. gonorrhoeae, C. trachomatis, and U. urealyticum. The bacteria are inoculated in media broth and incubated overnight. They are then added to solutions that have various antibiotics and concentrations and growth is measured. The minimum inhibitory concentration (MIC) is the concentration at which noticeable bacterial growth is inhibited, and this is noted for each of the solutions. C. trachomatis utilizes increasing concentrations of antibiotics.  Fluorescent labeled anti-chlamydial antibodies can be found [11].

NON-CULTURE TESTS

Enzyme ImmunoAssay (EIA) Tests [1]:

The bacteria are detected using monoclonal or polyclonal antibodies that are labeled with a particular enzyme. Once the antibodies bind their target, the enzyme helps transform the substrate into a coloured product that can be detected. First, the sample with the target antigen will be added into a tube/well that has the corresponding antibody. Then, enzyme is added to attach to the antibody and the substrate to illicit colour is added as well (this is the substrate for the enzyme). Finally, a coloured product is created once the antibody and antigen have successfully binded each other. For chlamydia, antigens such as LPS are used, but for gonorrhea, the antigen gonozyme is often used.

Direct Fluorescent Antibody (DFA) Tests [1]:

This can be used for both C. trachomatis and N. gonorrhoea. It involves the use of fluorescent labeled monoclonal antibodies that bind to certain bacterial antigens in the specimen. Again, LPS may be used as an antigen for C. trachomatis, whereas N. gonorrhoeae will have its own particular antigens. The specimen sample will be put on a microscope slide and the fluorescently-labeled antibody will be added. Fluorescence can then be observed under the microscope.

Molecular Tests (3 Kinds Listed Below)

Extraction of genetic material through cell lysis or cell disruption is done to remove membrane lipids by detergents or surfactants, RNA with RNAses and proteins by proteases [12]. Purification is then needed for the removal of reagents used during cell lysis.

1, Nucleic Acid Hybridization Tests:

This test involves the extraction and purification of genetic materials; this is done the same way as described for NAAT. It can detect N. gonorrhoeae or C. trachomatis from one specimen, but it doesn’t distinguish between these two so further testing should be employed for specific results [1]. A DNA or RNA probe, which is complementary to a specific sequence of the genetic material for one of these pathogens, will hybridize with any complementary sequences in the sample. This causes fluorescence. The Gen-Probe hybridization assay is commonly used to detect gonorrhea/chlamydia [12]. It works by first lysing the bacterial cell in order to access the rRNA. The target sequences will be pulled out in beads, and a lysate will be poured over them to allow DNA to hybridize with the rRNA. This DNA will then become double-stranded, and the RNA will bind to the fluorescent probe. RNA probes are also available for hybridization to targeted DNA sequences [1].

2. Nucleic Acid Genetic Transformation Tests:

This works only for N. gonorrhoeae in particular [1]. A gonococcal mutant will exhibit growth when it is transformed by the DNA from the sample specimen if the sample contains this bacteria.

3. Nucleic Acid Amplification Tests (NAAT):

This test can identify both N. gonorrhoeae and C. trachomatis from the same sample [1]. It operates by detecting and amplifying certain nucleic acid sequences, DNA or RNA, within the pathogen. This enables them to pick up on the bacteria, and amplification can occur through a number of ways, depending on the target sequences. For example, PCR may be used to amplify the genetic sequences if the target within N. gonorrhoeae is a 201 base-pair sequence located in the gene for cytosine methyltransferase [12]. For C. trachomatis, the target sequence used most of the time is found within the cryptic plasmid of this bacterium [12]. In order to perform this test, the genetic materials of the pathogen must be isolated, and this can occur via methods such as cell lysis or destruction of membrane lipids. Furthermore, the genetic material must be purified with ethanol.

Serology Tests [3]:

These tests are not recommended for C. trachomatis identification. This is because the antibodies produced during a previous infection with this species cannot be separated from the antibodies made during the current one. It can be used to identify chlamydia in neonates however.For ureaplasma, agglutination tests on blood samples may be done [11].

Leukocyte Esterase Test

These are quick tests that patients may do within 30 mins. For this specific test, a dipstick is used to test urine samples for urinary tract inflammation and presence of white blood cells in urine. The presence of esterase would show a change in color on the dipstick [11].

References:

  1. Johnson, R. E. (2002). Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections -- 2002. Atlanta, GA: Centers for Disease Control and Prevention.
  2. Todar, K. (2012). Textbook of Bacteriology. Retrieved February 4, 2020.
  3. Chernesky, M. A. (2005). The laboratory diagnosis of chlamydia trachomatis infections. The Canadian Journal of Infectious Diseases & Medical Microbiology, 16(1), 39-44 DOI: 10.1155/2005/359046
  4. Samuel, B. (1996). Medical Microbiology (4th ed.). Galveston, TX: University of Texas Medical Branch at Galveston
  5. Shepard, M. C. and Lunceford, C. D. (1987). Differential agar medium (A7) for identification of Ureaplasma urealyticum (human T mycoplasmas) in primary cultures of clinical material, 3(6), 613-625.
  6. Bruckner, M. Z. (2016). Gram Staining. Retrieved February 7, 2020, from http://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html
  7. Cunningham, S. A., Mandrekar, J. N., Rosenblatt, J. E., & Patel, R. (2013). Rapid PCR Detection of Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum. International Journal of Bacteriology. DOI: https://doi.org/10.1155/2013/168742
  8. Chlamydia infections (2019). Retrieved February 6, 2020 from https://www.amboss.com/us/knowledge/Chlamydia_infections
  9. Saginur, R., Clecner, B., Portnoy, J., and Mendelson, J. (1982). Superoxol(catalase)test for identification of Neisseria gonorrhoeae. Journal of Clinical Microbiology, 15(3), 475-477.
  10. Acid Detection Test. (2008). Retrieved February 06, 2016, from http://www.cdc.gov/std/gonorrhea/lab/tests/acid.htm
  11. Laboratory Tests for Venereal Diseases. (n.d.). Retrieved February 06, 2016, from http://www.labome.com/method/Laboratory-Tests-for-Venereal-Diseases.html
  12. Gross, G., & Tyring, S. K. (2011). Sexually transmitted infections and sexually transmitted diseases. Retrieved February 5, 2020.

Question (iv)

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

Gram Stain Results:

Neisseria gonorrhoeae:

A gram-stain can identify N. gonorrhoeae if it is red-stained, which shows gram-negative diplococci bacteria.

Figure 1.

Figure 1.


https://phil.cdc.gov/details_linked.aspx?pid=1948


Chlamydia Trachomatis:

Chlamydia Trachomatis is not seen in gram stains although it has a gram-negative cell wall. Stain shows absence of organisms and many WBCs.

Figure 2.

Figure 2.
  https://www.labce.com/spg513605_chlamydia_trachomatis.aspx

Culture Test Results:

Neisseria gonorrhoeae:

Culturing test on chocolate agar: Shows translucent colonies.

Figure 3.

Figure 3.

https://laboratoryinfo.com/chocolate-agar/


Chlamydia Trachomatis:

Light microscopy image of HeLa cells infected with C. trachomatis showing visible inclusion bodies.

Figure 4.

Figure 4.


http://ispub.com/IJMB/2/2/11219

NAAT Results:

Neisseria gonorrhoeae:

NAAT result (PCR result) of a positive gonococcal culture shows a 390 bp product of amplification of N. gonorrhoeae DNA.

Figure 5.

Figure 5.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC495310/pdf/jclinpath00419-0075.pdf


Chlamydia Trachomatis:

NAAT result (PCR result) of amplification of C. trachomatis serovars shows a 241 bp product.

Figure 6.

Figure 6.
 https://www.sciencedirect.com/science/article/pii/089085089290031R?via%3Dihub

Direct Fluorescent Antibody (DFA) Test Results:

Neisseria gonorrhoeae:

Antibodies against N. gonorrhoeae have an attached fluorescent dye. These antibodies bind to the bacterium and fluoresce.

Figure 7.


dfa_gonococcus_02.jpg

[2]


Chlamydia Trachomatis:

DFA test of C. trachomatis showing elementary bodies bound by a monoclonal antibody, viewed under a fluorescence microscope.

Figure 8.

Figure 8.
 https://www.researchgate.net/figure/Direct-fluorescence-assay-DFA-staining-for-the-detection-of-C-trachomatis-M-hominis_fig1_263430394

Enzyme Immunoassay (EIA) Results:

Neisseria gonorrhoeae:

Positive N. gonorrhoeae result shows a colored product detected by a spectrophotometer, or fluorescence in the case of a fluorescence microscope.

Figure 9.

Figure 9.

[3]


Chlamydia Trachomatis:

Enzyme immunoassay result showing monoclonal antibodies bound to LPS of C. trachomatis, visualized with a fluorescence microscope.

Figure 10.

Figure 10.
  https://www.genetex.com/Product/Detail/Chlamydia-trachomatis-LPS-antibody-1685/GTX36870#datasheet

Biochemical Test Results:

Neisseria gonorrhoeae:

1) Oxidase test: A positive result shows a bacterial colony stained purple.

Figure 11.


GCOxidc.jpg

[4]

2) Catalase test: A positive result shows bubbling or gas release due to bacterial catalase reacting with 3% hydrogen peroxide.

Figure 12.


GCcatc.jpg

[5]

3) Glucose, Sucrose, Maltose and Fructose test:

Acid production results from fermentation of glucose only. This causes a decrease in pH, causing the culture to change into a yellowish color.

Figure 13.1.


GCacid.jpg

[6]

A polysaccharide negative result, shown by the absence of purple to black staining, because N. gonorrhoeae does not produce polysaccharide from sucrose.

Figure 13.2.

PolGC.jpg

[7]

Antibiotic Susceptibility Results:

In the case of resistance to an antibiotic, there is bacterial growth on the culture plate. In the case of antibiotic susceptibility, there are zones of inhibition, as seen below.

Figure 14.


KB_test.jpg

[8]


Nucleic Acid Genetic Transformation Results:

Neisseria gonorrhoeae:

This test only applies to N. gonorrhoeae. A gonococcal mutant shows growth when it is transformed by the DNA from the sample specimen if the sample contains these bacteria (1).


Nucleic Acid Hybridization Results:

Neisseria gonorrhoeae:

For a positive test result, hybridization of the DNA probe with a complementary rRNA sequence in the sample occurs. The labeled probes then emit a quantifiable signal (1).

Leukocyte Esterase Test:

This test is positive if it detects the esterase enzyme produced by PMNL’s (polymorphonuclear neutrophils) in urine (2). Detection of PMNL’s indicates an inflammatory response that may be attributed to an infection (2). The results are determined after placing a dipstick in the urine and then observing the color of the dipstick (2).

Figure 15.

Figure 15.


https://encrypted-tbn0.gstatic.com/images?q=tbn%3AANd9GcQsqlH1dto7pMdV1c5lJWVgcpnBlmIheX5g5MpgVHmErbaLGSSo


References:

  1. Johnson, R. E. (2002). Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections -- 2002. Atlanta, GA: Centers for Disease Control and Prevention
  2. Bartelsman M, de Vries HJC, van der Loeff MFS et al. Leucocyte esterase dip-stick test as a point-of-care diagnostic for urogenital chlamydia in male patients: A multi-center evaluation in two STI outpatient clinics in Paramaribo and Amsterdam. BMC Infect Dis. 2016 Nov 3;16(625):1-8.

Q3. Bacterial Pathogenesis Questions

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

Question (i)

(i)  Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How might our patient have come in contact with this bacteria.
Figure 1: N. gonorrheae gram stain (pink because the staining indicates gram negative).

Our patient Naser had unprotected sex with a partner and this was most likely how the bacterial infection was transmitted to Naser. His sexual partner denies any symptoms, however, asymptomatic infection with N. gonorrhoea is common. Naser’s symptoms of purulent discharge from the urethra and pain when urinating is common for male symptoms of this infection. Although Naser’s infection was positive for N. gonorrhoea, 50-70% of individuals with gonorrhea infection are co-infected with Chlamydia trachomatis [Q3-1 1]. N. gonorrhoea is a gram-negative diplococcus that infects mucous secreting epithelial cells that utilizes glucose [Q3-1 2].

Urinary tract normal flora:

The urinary tract is home to its own microbiota which aid with pathogen exclusion and metabolic processes. Normal flora genera include as Lactobacillus, Sneathia, Veillonella, Corynebacterium Prevotella, Streptococcus and Ureaplasma which can be found through urethral swabbing[Q3-1 3]. This N. gonorrhoea infection is a result of the N. gonorrhoea pathogen outcompeting the normal flora for space, allowing invasion and multiplication within the host urinary tract.

Global Neisseria gonorrhoea:

Neisseria bacteria has pathogenic strains and commensal. The two pathogenic strains are N. gonorrheae and N. meningitidis (causing the sexually transmitted disease and meningitis respectively). The commensal Neisseria strains normally make up a large portion of human nasal and oropharyngeal commensals. Neisseria is not limited to residing on and in humans and can also colonize various other mammals and non-mammalian hosts (such as primates, cats, dolphins, dogs, birds and insects). N. gonorrhoea causes 78 million infections each year, and with growing antibiotic resistant the bacterial infections are a global health concern[Q3-1 3]. N. gonorrhoea has an increased incidence in poor communities and developing countries resulting from diagnostic tests being largely unavailable (due to cost and accessibility of initial or follow-up visits)[Q3-1 4]. As a result, care and treatment can be incomplete. There is an estimated 33 to 106 million new cases that occur each year, out of the 498 million new cases of curable STI – which also includes syphilis, chlamydia, and trichomoniasis[Q3-1 4]. Unfortunately, due to growing antimicrobial resistance, in some countries, the only remaining monotherapy option for N. gonorrhoea treatment is an injectable extended-spectrum cephalosporin (ESC) ceftriaxone[Q3-1 5]. This bacterium will normally colonize urogenital mucosa but can less frequently infect the conjunctiva, anorectal area, nasopharyngeal area or ovarian/uterine regions[Q3-1 6].

N. gonorrheae Characteristics:

N. gonorrheae is fastidious and grows under specific conditions. The bacteria will die if exposed to dehydration, or a temperature that is not within physiological range, meaning the bacteria cannot live successfully outside the host[Q3-1 6].

There are a variety of molecular characteristics that N. gonorrhoea use as virulence factors to thrive in the host. Some of these virulence factors include:

  • Surface proteins with various functions and sugars called lipoogliosaccharides (LOS).
  • Pili which mediate adherence, movement and DNA exchange
  • Opa proteins which interact with the host immune system
  • Porins which are outer membrane proteins that facilitate entry of nutrients

All of these virulence factors exhibit antigenic variation, meaning they change their protein sequence constantly in order to evade the host immune recognition responses [Q3-1 5].

N. gonorrheae Transmission:

Transmission occurs in multiple ways involving the genitalia. Transmission can occur from mother to neonate during delivery through the vaginal canal, or sexual contact through vaginal, anal or oral sex. Transmission via sexual contact occurs via sexual networks, occurring due to participation in unprotected sex with multiple partners. The most well understood transmission is from males (with bacteria adhering to sperm during ejaculation) to their sexual partner. Female transmission to their sexual partners is less well understood. The adult female vagina is more resistant to N. gonorrhoea infection compared to the male genitalia (with up to 35% of women participating in vaginal intercourse with an infected male being resistant to acquiring the infection)[Q3-1 7]. This resistance is due to the vaginal pH of 4.5 or lower being protective against successful survival of N. gonorrhoea in the vagina[Q3-1 7]. Unfortunately, for the females that do acquire gonorrhoea infection, the cervicovaginal microbiome of females secrete bacterial enzymes sialidases which desialylate N. gonorrheae’s surface, allowing it to successfully enter urethral epithelial cells, then allowing transmission from females to other females or males [Q3-1 5].

Due to sex differences in cell structure of male and female urogenital tract cells, different microenvironments and surface receptors exist, leading to different survival mechanisms for N. gonorrhoea [Q3-1 6].

N. gonorrheae Disease Presentation:

Once successful transmission of N. gonorrheae occurs, disease presentation can look very different based on the type of transmission. The disease presentation of Naser in the case was one of many routes of transmission.

Maternal to neonate transmission can result in unique disease presentation in the infant being conjunctivitis leading to ophthalmia neonatorum as well as joint or bloodstream infections leading to a variety of other complications if untreated. Thankfully, this transmission can be prevented if maternal infection is identified and the mother is provided prophylactic antibiotic treatment pre-birth and the infant is treated with antibiotics once born [Q3-1 8].

Anal transmission can yield proctitis, which is infection in the rectum. For many, proctitis can be asymptomatic, however if left untreated can lead to abscess formation. Unfortunately, proctitis disproportionately affects men who have sex with men (MSM)[Q3-1 9].

References:
  1. Aminomethyl Spectinomycins as Therapeutics for Drug-Resistant Gonorrhea and Chlamydia Coinfections Michelle M. Butler, Samanthi L. Waidyarachchi, Kristie L. Connolly, Ann E. Jerse, Weirui Chai, Richard E. Lee, Stephan A. Kohlhoff, Dean L. Shinabarger, Terry L. Bowlin Antimicrobial Agents and Chemotherapy Apr 2018, 62 (5) e00325-18
  2. Evangelista AT, Beilstein HR. 1993. Cumitech 4A: Laboratory Diagnosis of Gonorrhea. Coordinating ed. C. Abramson. American Society for Microbiology, Washington D.C.
  3. 3.0 3.1 Gottschick, C., Deng, Z., Vital, M. et al. The urinary microbiota of men and women and its changes in women during bacterial vaginosis and antibiotic treatment. Microbiome 5, 99 (2017).
  4. 4.0 4.1 Newman, Lori; Rowley, Jane; Vander Hoorn, Stephen; Wijesooriya, Nalinka Saman; Unemo, Magnus; Low, Nicola; Stevens, Gretchen; Gottlieb, Sami; Kiarie, James; Temmerman, Marleen; Meng, Zhefeng (2015). Global Estimates of the Prevalence and Incidence of Four Curable Sexually Transmitted Infections in 2012 Based on Systematic Review and Global Reporting. PLOS ONE. 10 (12): e0143304.
  5. 5.0 5.1 5.2 Hill, S. A., Masters, T. L., & Wachter, J. (2016). Gonorrhea - an evolving disease of the new millennium. Microbial cell (Graz, Austria), 3(9), 371–389. doi:10.15698/mic2016.09.524
  6. 6.0 6.1 6.2 Quillin, S., Seifert, H. Neisseria gonorrhoeae host adaptation and pathogenesis. Nat Rev Microbiol 16, 226–240 (2018).
  7. 7.0 7.1 McLaughlin SE, Ghanem KG, Zenilman JM, Griffiss JM. Risk of Gonococcal Infection During Vaginal Exposure is Associated With High Vaginal pH and Active Menstruation. Sex Transm Dis. 2019;46(2):86–90.
  8. Heumann CL, Quilter LA, Eastment MC, Heffron R, Hawes SE. Adverse Birth Outcomes and Maternal Neisseria gonorrhoeae Infection: A Population-Based Cohort Study in Washington State. Sex Transm Dis. 2017;44(5):266–271.
  9. Hamlyn E, Taylor C. Sexually transmitted proctitis. Postgrad Med J. 2006;82(973):733–736.

Question (ii)

(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).
Figure 2: Cartoon of Neisserial Cell Wall
Initial Adhesion Step

Neisseria gonorrheae enters the human body by sexual transmission, eye contamination during vaginal delivery, or transmission through fomites [Q3-2 1]. The gonococci also has a unique mechanism by which they enter urogenital cells and cervical epithelial cells. For urogenital cells such as in the urethra, N. gonorrheae is internalized due to the interaction of LOS and the asialogylcoprotein receptors[Q3-2 2]. For cervical epithelial cells in the cervical genital tract, endometrium, and fallopian environments, the interaction between complement receptor 3 (CR3) and the lutropinchoriogonadotropic hormone receptor internalizes the bacteria[Q3-2 2].

Pili

Prior to endocytosis, initial host cell adhesion is dependent on the type IV pili[Q3-2 2]. These pili (hair-like appendages) extend beyond the cell surface and help the bacteria adhere to these columnar cells[Q3-2 3] These pili are 50-80 angstroms in width, several microns in length, and are composed of repeating “pilin” protein subunits.2 Pili traverse the cell membrane via the outer-membrane protein “PilQ”[Q3-2 4]. PilC, located at the tip and in the inner membrane of the bacteria, is significant to the actual adhesion to host cells[Q3-2 5] [Q3-2 6]. Binding of the pili is thought to involve CD46, complement component C4 binding protein (C4BP), and complement receptor 3 (CR3).[Q3-2 7]

To avoid being targeted by antibodies made by the host, the pili also undergo phase and antigenic variation[Q3-2 3]. This variation is achieved through areas of extreme antigenic variability (from P+ (pilus positive) to P- (pilus negative) [Q3-2 8]) in the pilin subunit, even within a single strain of N. gonorrheae, despite co-occurring areas of intrastrain antigenic similarity near the amino terminus[Q3-2 4]. When N. gonorrheae without pili are grown in culture, they are much less able to attach to human mucosal surface and substantially less virulent,[Q3-2 4] lending evidence to the importance of pili to N. gonorrheae’s pathogenesis.

Pili play an integral role in cellular invasion, as the attachment of pili to non-ciliated epithelial cells initiates entry and transport through the cell into subepithelial space via interaction with the CD46 receptor on the epithelial cell membrane surface, complement component C4 binding protein (C4BP). and complement receptor 3 (CR3).[Q3-2 4][Q3-2 7].

Cellular Invasion Step
Figure 3: Pathogenesis of Neisseria gonnorheae via the columnar epithelial cells. Taken from Neisseria gonorrhoeae host adaptation and pathogenesis, p. 228

All gonococci attach only to the microvilli of nonciliated columnar epithelial cells, regardless of what area of the body the bacteria infects in a process called parasite-directed endocytosis[Q3-2 3]. This endocytosis is “parasite-directed” as it involves host cells that aren’t normally phagocytic, is initiated by microbial factors, and does not occur unless the gonococci are viable [Q3-2 3]. The endocytosis is mediated by host cell actin, microtubule, and clathrin-dependent processes, as drugs that antagonize these processes inhibit the engulfment of N. gonorrheae [Q3-2 3]. Endocytosis mediated by ASGP-R (a host epithelial receptor) results in endosomal fusion, acidification, and subsequent clathrin-coat disassembly and uncoupling of the AGP-R-ligand complex, thereby internalizing the gonococcus[Q3-2 4]. During endocytosis, the mucosal cell membrane retracts, and gonococci are pinched off in a membrane-bound vacuole, which is then rapidly transported to the base of the cell, and gonococci are released via exocytosis into subepithelial tissue[Q3-2 3]. Throughout the entire endo- and exocytosis process, gonococcal cells are not destroyed within the vacuoles[Q3-2 3]. Though the mechanism is not known, the gonococcus while within the epithelial cell (i.e. before it is deposited in the subepithelial space) induces antiapoptotic events, prolonging the life of the epithelial cell and therefore conserving its own survival for a prolonged period of time.[Q3-2 4]

Opa

Opa proteins are integral outer membrane proteins. They are also the reason why colonies appear opaque due to intergonoccal aggregations when viewed via phase contrast microscopy[Q3-2 5]. Opa proteins belong to a multigene family, where a single gonococcal cell can have up to 12 genes constitutively transcribed [Q3-2 9]. Opa protein expression can undergo phase variation; this is due to the changing numbers of pentameric repeat units (-CTTCTT-) which are located in the leader peptide encoding region, which results in on/off switching of expression. A single cell is capable of expressing either several Opa proteins or none[Q3-2 5]. Opa expression, unlike pili, is not required for the initial attachment of bacteria to host. However, Opa proteins are very involved in cell invasion, and certain variants of Opa greatly promote such invasion, for example heparin-related compounds, CD66 and carcinoembryonic antigen-related cell adhesion molecules (CAECAMs).[Q3-2 4] As there are CAECAM receptors on B and T cells of the immune system, Opa proteins can bind these receptors resulting in a downregulation of immune responses.[Q3-2 4] Binding of the CAECAM receptor sends a priming signal within the PMNs that activates adhesion receptors without release of inflammatory mediators, thereby enhancing the survival of N. gonorrheae.[Q3-2 4]

Membrane Proteins that Help N. gonorrheae inhibit host immune response:

Part of successful residency in the host cell is the ability for the pathogen to remain undetected by immune cells, and/or to block or delay the host’s immune response to allow time to multiply and spread. This requires “teamwork” between the gonococcus and the host cell. To accomplish this, N. gonorrheae has multiple surface proteins with various anti-immune functions, as well as a wide range of virulence determinants.

Porins

Alongside these host entry mechanisms, Neisseria gonorrhoea possess a porin protein, Por, on the outer membrane[Q3-2 10]. It is thought that this invasion mediates penetration of the host cell membrane[Q3-2 10]. Por is the most abundant protein in gonococcus, it takes up approximately 60% of total protein content[Q3-2 5]. Por is a channel that allows acqueous solutes to pass through the hydrophobic membrane[Q3-2 4] and simultaneously a key invasin for N. gonorrheae.[Q3-2 3] It does this by causing a change in membrane potential in neutrophils, without causing their respiratory burst of inflammatory mediators.[Q3-2 4] Porin also inhibits phagosome maturation and downregulates immune cell’s cell surface receptors that are important to immune function, for example complement receptors.[Q3-2 4]

There are two major antigenic classes of Por proteins, PorB1A and PorB1B, where many genetic variants of each class exist.[Q3-2 4] Primarily PorB1A (and sometimes PorB1B) are associated with normal human serum’s bactericidal (specifically non-immune) effects, giving Por1BA-expressing gonococci an advantageous outlet for bacteremia.[Q3-2 4] It is also Por1BA that promotes invasion into the epithelial cell.[Q3-2 4]

Another unique feature of Por proteins is that they are able to transfer into an infected cell’s mitochondria, leading to the formation of porin channels into the mitochondrial inner membrane, causing increased permeability[Q3-2 11]. This action can lead to the release of cytochrome c and other proteins, leading to eventual apoptosis of the infected cell[Q3-2 10][Q3-2 11].

RMP

Another important membrane protein found on all N. gonorrheae is RMP (Protein III) – a protein that does not undergo phase variation, and is found in complex with Por and LOS who work in conjunction to block bactericidal antibodies against Por and LOS [Q3-2 3]. As its homology is similar to that of an Omp protein in Escherichia coli, antibodies against Rmp can arise either from previous E. coli infection or via direct N. gonorrheae infection[Q3-2 3]. These antibodies have the ability to block bactericidal antibodies directed against Por and LOS, and thus facilitate infection with N. gonorrheae[Q3-2 3].

LOS

Gonococcal LPS is composed of Lipid A and core polysaccharides yet lacks the repeating O-antigens – therefore gonococcal LPS has been designated as lipopolysaccharide (LOS). LOS oligosaccharide composition is more variable in both length and carbohydrate content [Q3-2 5]. The outer membrane of a single gonococcal cell contains a heterogenous variety of LOS molecules which has a drastic effect on the bacterium’s virulence and pathogenesis.[Q3-2 3];Gonococcal LOS damages the mucosal endothelium in the fallopian tube organ cultures by stimulating the release of proteases and phospholipases.[Q3-2 3] Additionally, LOS stimulates the production of tumor necrosis factor (TNF) in the fallopian tube area, further damaging host tissue.[Q3-2 3] Damaging host tissue impedes the host’s ability to combat infection by N. gonorrheae, and allows the bacterium to maintain and progress host tissue colonization. Furthermore, gonococci can use CMP-NANA (cytidine monophospho-N-acetylneuraminic acid) from the host in vivo to sialylate the oligosaccharide component of its own LOS, which increases antigenic similarity between neissaral LOS and antigens on human erythrocytes, effectively increasing its resistance to host serum.[Q3-2 3] However, sialylated LOS are less invasive than non-sialylated LOS antigens.[Q3-2 3] This increased resistance results in neutrophil recruitment to the site of infection which engulfs the bacteria and aids in the transport of the pathogen.[Q3-2 2][Q3-2 10]

Extracellular proteases

Finally, strains of N. gonorrheae can produce two types of extracellular proteases that cleave the heavy chain of immunoglobulin A1 (IgA1), specifically the prolyl-seryl peptide bond, which impedes the host immune response from eliminating the pathogen.[Q3-2 3] Furthermore, fragments cleaved from IgA1 can bind to the gonococcal cell surface, effectively blocking the Fc-mediated functions of intact immunoglobulins. Furthermore, IgA proteases also cleave LAMP1 – a major lysosome associated with membrane protein. This LAMP1 cleavage leads to lysosome modification, which helps the bacteria’s survival by avoiding lysosomal break down/killing.

References
  1. Government of Canada. Pathogen safety data sheet: infectious substances – Chlamydia trachomatis. Retrieved from: [1]
  2. 2.0 2.1 2.2 2.3 Quillin SJ, Seifert HS. Neisseria gonorrhoeae host adaptation and pathogenesis. Nat Rev Microbiol. 2018 Apr;16(4):226–40.
  3. 3.00 3.01 3.02 3.03 3.04 3.05 3.06 3.07 3.08 3.09 3.10 3.11 3.12 3.13 3.14 3.15 3.16 Morse SA. Neisseria, Moraxella, Kingella and Eikenella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 14. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7650/.
  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 4.13 4.14 Bennett, J. E., Dolin, R., Blaser, M. J., Mandell, G. L., Douglas, R. G., & undefined, undefined undefined. (2015). Neisseria gonorrhoeae (Gonorrhea) . In Mandell, Douglas, and Bennetts Principles and practice of infectious diseases. Philadelphia: Elsevier / Saunders.
  5. 5.0 5.1 5.2 5.3 5.4 Hill, S. A., Masters, T. L., & Wachter, J. (2016). Gonorrhea - an evolving disease of the new millennium. Microbial cell (Graz, Austria), 3(9), 371–389. doi:10.15698/mic2016.09.524
  6. Jonsson AB, Nyberg G, Normark S. Phase variation of gonococcal pili by frameshift mutation in pilC, a novel gene for pilus assembly. EMBO J. 1991;10(2):477–488.
  7. 7.0 7.1 Virji M. Pathogenic neisseriae: surface modulation, pathogenesis and infection control. Nat Rev Microbiol. 2009 Apr;7(4):274–86.
  8. Griffiss JM, Lammel CJ, Wang J, Dekker NP, Brooks GF. Neisseria gonorrhoeae coordinately uses Pili and Opa to activate HEC-1-B cell microvilli, which causes engulfment of the gonococci. Infect Immun. 1999;67(7):3469–3480.
  9. Gray-Owen SD, Dehio C, Haude A, Grunert F &Meyer TF(1997). CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes. EMBO J. 16(12):3435-45.
  10. 10.0 10.1 10.2 10.3 Todar, K. Online Textbook of Bacteriology. Madison, WI: Kenneth Todar, University of Wisconsin-Madison Dept. of Bacteriology. Web. 5 Feb. 2017.
  11. 11.0 11.1 Judd RC (1989) Protein I: structure, function, and genetics. Clin Microbiol Rev.; 2 Suppl():S41-8.

Question (iii)

(iii)  Multiplication and Spread: does the organism remain at the entry site and/or does it spread beyond the initial site. Are there, for instance, secondary sites of infection. Does the organism remain extracellular and/or does it enter into cells - and what are the molecular and cellular determinants of these events.

Initial Infection

Neisseria gonorrhoeae infections are generally limited to  superficial mucosal surfaces found in the cervix, urethra, rectum, pharynx, and conjunctiva (1).  Squamous epithelium, which is a single layer of epithelial cells, is generally not susceptible to infection due to the lack of proper receptors for the pathogen (1). However, there is a risk that the prepubertal vaginal epithelium is infected as the body has not yet started to produce estrogen, which will keratinize the epithelium and prevent the bacteria from adhering (1).

N. gonorrhoeae is both an extracellular and intracellular organism. However, it is an obligate human pathogen and does not have reservoirs outside the human host (2). A micro-colony forms upon the host’s non-ciliated columnar epithelial cells and this occurs approximately one to two hours after the infection has been established (3). This results in cytoskeletal rearrangement in the host and protein aggregation allows for colonization of N. gonorrhoeae (3). This colonization results in over 100 diplococci, which will mediate pilus attachment to host receptor CD46 for tight binding (3).

Dissemination

In most cases of infection, N. gonorrhoeae causes non-complicated mucosal infections. However, more serious sequelae can develop such as salpingitis (acute inflammation of the fallopian tubes), pelvic inflammatory disease (infection of the upper part of the female reproductive system) or occasionally, bacteremic infections (4,5). Deeper infections can also occur in the endocervix of females when the pathogen travels across thin layers of epithelial cells and could possibly secondarily infect the lamina propria (6). The lamina propria is a thin layer of connective tissue that is beneath the epithelium. It is a component of the mucosa along with the epithelium and basement membranes. Once the pathogen penetrates the lamina propria, then it can target immune cells such as macrophages and dendritic cells (7). In macrophages, the pathogen is able to survive inside the phagosome and regulate apoptosis and the production of inflammatory cytokines (7). The bacteria also modifies macrophages so that they are less capable of T cell activation (7). When it infects dendritic cells, they are less capable of signalling for T cell proliferation (7). N. gonorrhoeae also changes the adaptive immune response by suppressing T helper cell proliferation, cytokine production and programmed cell death by neutrophil NETs (7). Furthermore, N. Gonorrhoeae can possibly spread from the mucous present in the endocervical canal through the endometrium to the fallopian tubes and travel to the pelvic peritoneum (1). This spread of infection can result in further complicated infections such as peritonitis and salpingitis (1). Signs and symptoms that can arise from these conditions include: pelvic and abdominal pain, fevers, and cervical motion tenderness (1).

Its ability to infect migrating immune cells allows for systemic dissemination into the periphery systems through the lymphatic system or the circulatory system (1). Dissemination can arise from gonococcal bacteremia, which is the presence of the pathogen in the blood (1). The ability to spread beyond the initial infection site through the circulatory system is due to the presence of hemoglobin receptors on all N. gonorrhoeae (8). This can most commonly lead to dermatitis-arthritis syndrome (1). This is the swelling of tendon sheaths and symptoms include: fever, chills, skin lesions, and arthralgias (1). Complications could lead to the patient developing a septic joint (1). Furthermore, there is a higher rate of expression of the hemoglobin receptor on N. gonorrhoeae during infection of women in the early phases of their menstrual cycle (9). This increases the likelihood of pelvic inflammatory disease development. In approximately 15% of women with uncomplicated infections, it may develop into pelvic inflammatory disease (PID) (1). PID has significantly damaging consequences to the host such as decreased fertility and increased probability of ectopic pregnancies, meaning the egg is fertilized in an abnormal place (1). In addition to the increased expression of hemoglobin receptors, it was observed that in women, Opa protein expression also varies with the menstrual cycle and if the patient is taking oral contraceptives (10). At mid-cycle, the bacteria isolated from the cervix expressed Opa proteins whereas there was no expression during menstruation (10). Interestingly enough, bacteria isolated from infected fallopian tubes are also Opa negative, even though an Opa expressing organism can be isolated from the cervix of the same patient (10). This observation could be explained by the possibility that there is a higher presence of proteolytic enzymes during menstruation due to the increased cervical secretions compared to the follicular phase (10). As a result, non-Opa expressing cells may be selected because of the extreme sensitivity Opa proteins have to trypsin-like enzymes (10).

In males, N. gonorrhoeae isolated from the urethra generally all co-express pili and several other Opa proteins (4). Gonorrhea can lead to major complications in men such as infertility, prostatitis, and orchitis (11). N. gonorrhoeae can lead to sterility as it causes a small, coiled tube in the rear portion of the testicles where the epididymis is located to become inflamed (11). In addition, there is an increased risk of developing HIV/AIDS (11). Prostatitis in men is also known to cause infertility and chronic pelvic pain(12). Symptoms include painful urination and pain in the groin (12). It is likely caused by the dissemination of N. gonorrhoeae into the prostate gland from the urinary tract or directly from the rectum (12).

How Infection and Spread Occurs

1.  Neisseria gonorrhoea expressing their Opa proteins and pili on their outer membrane, interacts with mucosal. The pili’s hair-like appendages provide the initial contact with the host’s receptors on the surface of the mucosal cells.

2.     The pili are then retracted to allow for more intimate attachment, which is then enhance further by Opa-mediated attachment of the bacteria with the CD66 antigens located on the mucosal cells

3.     Following  Opa-mediated attachment, the bacteria is engulfed and internalized into the mucosal cells

4.     Following its internalization, some bacteria are transcytosed (relocated) to the basolateral side of the mucosal epithelium.

5.     Depending on which Opa protein is being expressed, gonococci can also reside and survive inside of neutrophils.

6.     Following transcytosis, gonococci can enter into the bloodstream, where heavy sialyation of lipoooligosaccharide renders the bacteria serum resistant.

Survival Tactics and Nutrient Acquisition

N. gonorrhoeae is well adapted to survive and multiply in the mucosal surfaces of the human host cells. They exploit human iron stores for growth using TonB-dependent transporters (TdTs) (1). These receptors serve to transport iron and other nutrients into the cell from the host environment to ensure survival and multiplication. N. gonorrhoeae can also be found in the bloodstream in order to extract the iron bound to blood proteins transferrin, lactoferrin, and haemoglobin through interacting with their membrane receptors (13). During times of low iron, the gonococci express additional transferrin receptors, Tbp1 and Tbp2, and a lactoferrin receptor, Lpb, on their cell membrane to increase iron acquisition (14). Although these receptors are made by the host cell, they are manipulated by the pathogen for its own benefit (15).

As the gonococci are not destroyed upon entering the vacuole within the endothelial cell, it is hypothesized that they replicate within the vacuole as intracellular parasites, although the mechanism is unknown (14).

References

  1. Morse SA. Neisseria, Moraxella, Kingella and Eikenella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 14. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7650/
  2. “Neisseria Gonorrhoeae.” Neisseria Gonorrhoeae - an Overview | ScienceDirect Topics, www.sciencedirect.com/topics/immunology-and-microbiology/neisseria-gonorrhoeae.
  3. Griffiss JM, Lammel CJ, Wang J, Dekker NP, Brooks GF (1999) Neisseria gonorrhoeae coordinately uses Pili and Opa to activate HEC-1-B cell microvilli, which causes engulfment of the gonococci. Infect Immun. 67(7):3469-80.
  4.  Hill, S. A., Masters, T. L., & Wachter, J. (2016). Gonorrhea - an evolving disease of the new millennium. Microbial cell (Graz, Austria), 3(9), 371–389. doi:10.15698/mic2016.09.524
  5. Easmon CS & Ison CA Easmon. (1987). Neisseria gonorrhoeae: a versatile pathogen. Journal of clinical pathology. 40(9), 1088–1097. doi:10.1136/jcp.40.9.1088
  6. Rarick, Matthew, et al. “Evidence for Cross-Regulated Cytokine Response in Human Peripheral Blood Mononuclear Cells Exposed to Whole Gonococcal Bacteria in Vitro.” Microbial Pathogenesis, Academic Press, 19 Apr. 2006, www.sciencedirect.com/science/article/pii/S0882401006000325.
  7. Quillin, Sarah Jane, and H Steven Seifert. “Neisseria Gonorrhoeae Host Adaptation and Pathogenesis.” Nature Reviews Microbiology, vol. 16, no. 4, 2018, pp. 226–240., doi:10.1038/nrmicro.2017.169.
  8. Edwards, J. L., and M. A. Apicella. “The Molecular Mechanisms Used by Neisseria Gonorrhoeae To Initiate Infection Differ between Men and Women.” Clinical Microbiology Reviews, vol. 17, no. 4, 2004, pp. 965–981., doi:10.1128/cmr.17.4.965-981.2004.
  9. Anderson, J. E., P. A. Leone, W. C. Miller, C. Chen, M. M. Hobbs, and P. F. Sparling. 2001. Selection for expression of the gonococcal hemoglobin receptor during menses. J. Infect. Dis. 184:1621-1623
  10. Draper DL, James JF, Brooks GF & Sweet RL (1980). Comparison of virulence markers of peritoneal and fallopian tube isolates with endocervical Neisseria gonorrhoeae isolates from women with acute salpingitis. Infect Immun. 27(3):882-8.
  11. “Gonorrhea.” Mayo Clinic, Mayo Foundation for Medical Education and Research, 6 Dec. 2019, www.mayoclinic.org/diseases-conditions/gonorrhea/symptoms-causes/syc-20351774.
  12. Brookings, Claire, et al. “Sexually Transmitted Infections and Sexual Function in Relation to Male Fertility.” Korean Journal of Urology, vol. 54, no. 3, 2013, p. 149., doi:10.4111/kju.2013.54.3.149.
  13. Cornelissen, CN, Kelley M, Hobbs MM, Anderson JE, Cannon JG, Cohen MS, Sparling PF. The transferrin receptor expressed by gonococcal strain FA1090 is required for the experimental infection of human male volunteers. Molecular Microbiology. 2018. 27: 611-616. doi:10.1046/j.1365-2958.1998.00710.x
  14. Todar, K. (n.d.). Pathogenic Neisseriae: Gonorrhea, Neonatal Ophthalmia and Meningococcal Meningitis. Retrieved from http://textbookofbacteriology.net/neisseria.html
  15. Turner PC, Thomas CE, Elkins C, Clary S, Sparling PF. Neisseria gonorrhoeae Heme Biosynthetic Mutants Utilize  Heme and Hemoglobin as a Heme Source but Fail To Grow within Epithelial Cells. Infect Immun. 1998 Nov;66(11):5215–23.

Question (iv)

(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?

During Neisseria gonorrheae infection, both direct and indirect damages are possible. Direct damages are caused by the pathogen whereas Indirect damages are caused by the host immune response. Given this, host damage caused by Neisseria gonorrheae is mainly indirect and accounts for the majority of the sings and symptoms experienced by patients with gonorrhea[1]. It has been observed that Neisseria gonorrheae can induce elevated levels of proinflammatory cytokine production in response to infection, especially in the genital tract [2]. There is also stimulation of the release of chemokines IL-6, IL-8, IL-1B, IL-17 and interferon-gamma[3]. This leads to an influx of neutrophils to the site of initial infection [3]. The recruitment of major role players in the innate immune response, alongside many pro-inflammatory factors produced in the genital tract, characterize many of the symptoms associated with the infection.

Symptoms

Symptoms usually occur within two to fourteen days after exposure. However, some people infected with gonorrhea never develop noticeable symptoms [4]. It’s important to remember that a person with gonorrhea who doesn’t have symptoms, a non-symptomatic carrier, is still contagious [5]. A person is more likely to spread the infection to other partners when they don’t have noticeable symptoms.

Men may not develop noticeable symptoms for several weeks while some men may never develop symptoms at all. Our patient, Naser, had come in based on one of the symptoms of experiencing painful urination. Painful urination is a common sign of a urinary tract infection (UTI) and STI’s. In fact, the first noticeable symptom in men is often a burning or painful sensation during urination. As it progresses, other symptoms may include:

  • greater frequency or urgency of urination
  • a pus-like discharge (or drip) from the penis (white, yellow, beige, or greenish)
  • swelling or redness at the opening of the penis
  • swelling or pain in the testicles
  • a persistent sore throat

Many women don’t develop any overt symptoms of gonorrhea. When women do develop symptoms, they tend to be mild or similar to other infections, making them more difficult to identify [6]. Gonorrhea infections can appear much like common vaginal yeast or bacterial infections. Resulting symptoms include:

  • discharge from the vagina (watery, creamy, or slightly green)
  • pain or burning sensation while urinating
  • the need to urinate more frequently
  • heavier periods or spotting
  • sore throat, fever
  • pain upon engaging in sexual intercourse
  • sharp pain in the lower abdomen
Pathogenic Components of Neisseria gonorrheae

The two components of Neisseria gonorrheae responsible for causing this damage are: peptidoglycan fragments and lipooligosaccharide (LOS). Both factors can activate the alternative complement pathway resulting in formation of the membrane attack complex. They also contain pathogen associated molecular patterns (PAMPs) recognizable by TLRs, and RLRs. Both mechanisms result in the rapid, non-specific activation of innate immunity and induce an inflammatory response.

Peptidoglycan fragments:

As gram-negative cocci Neisseria gonorrheae do not contain an outer peptidoglycan cell wall, however during infection, they release peptidoglycan fragments which can activate the innate immune response [7]. PAMP receptors, NOD1 and NOD2 are able to recognize bacterial peptidoglycan epitopes and promote CD8+ cytotoxic T cell selection in the thymus [8]. Furthermore, NOD activation results in NF-κB upregulation leading to localized inflammation [9]. Macrophages express CD14 which also recognizes peptidoglycan [10]. Alongside toll-like receptor 4 (TLR4) signalling, CD14 will cause the NF-κB transcription factor to be upregulated leading to the production of inflammatory cytokines and interleukins [10]. The Peptidoglycan fragments further damage the host (indirectly) by inducing an increased expression of inducible nitric oxide synthase (iNOS) as a result of IL-1 expression [11].

Lipooligosaccharides (LOSs):

Without even having to enter host cells, Neisseria gonorrheae are able to initiate an inflammatory response through cell contact with host immune cells. LOS expressed on the surface of Neisseria gonorrheae is an endotoxin which evokes a potentially harmful host inflammatory response [12]. LOS binds to Lipopolysaccharide binding protein (LBP) in the serum [13]. The LOS-LBP complex then has high affinity for CD14 on macrophages, which as stated above, induces a local inflammatory response through upregulation of NF-κB (with the help of TLR4) [13]. Although epithelial cells around the primary site of infection do not express CD14, they are able to conjugate with soluble CD14 (sCD14) and induce a similar response [14]. Epithelial cells can then release a multitude of cytokines, chemoattractants, interleukins, growth hormones, and tumor necrosis factor-alpha (TNF-α) to further potentiate the innate immune response and increase inflammation.

LOSs can also conjugate with sialic acid creating a sialylated LOS capsule which is resistant to host defense mechanisms [15].

Overall activation of innate immunity through PAMPS, pattern recognition receptors (TLRs, RLRs, NODs), and CD molecules results in the activation of many effector cells such as neutrophils and macrophages. When activated these cells release many proinflammatory cytokines, degranulate releasing toxic granules (neutrophils), and can perform respiratory bursts (macrophages) releasing reactive oxygen species (ROSs) [16]. In the case of Naser, the dysuria he experiences is most likely a result of inflammation in his urethra (urethritis). The green discharge he observes is likely dead bacteria, tissue, and immune cells. Both of these symptoms are a result of host innate immunity in response to infection with Neisseria gonorrhea.

References
  1. Morse SA. Neisseria, Moraxella, Kingella and Eikenella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 14. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7650/
  2. Chen, Adrienne, and H. Steven Seifert. 2011. “Neisseria Gonorrhoeae-Mediated Inhibition of Apoptotic Signalling in Polymorphonuclear Leukocytes.” Infection and Immunity, vol. 79, no. 11, pp. 4447–4458., doi:10.1128/iai.01267-10.
  3. 3.0 3.1 Quillin, Sarah Jane, and H Steven Seifert. 2018. “Neisseria Gonorrhoeae Host Adaptation and Pathogenesis.” Nature Reviews Microbiology, vol. 16, no. 4, pp. 226–240., doi:10.1038/nrmicro.2017.169.
  4. "Gonorrhea (the clap) Symptoms". www.std-gov.org. 2 April 2015.
  5. Hill SA, Masters TL, & Wachter, J. (2016). Gonorrhea - an evolving disease of the new millennium. Microbial cell (Graz, Austria), 3(9), 371–389. doi:10.15698/mic2016.09.524
  6. Centers for Disease Control and Prevention. (2018).  SRD. Retrieved from https://www.cdc.gov/std/stats18/default.htm
  7. Todar, K. Online Textbook of Bacteriology. Madison, WI: Kenneth Todar, University of Wisconsin-Madison Dept. of Bacteriology. Web. 5 Feb. 2017.
  8. Martinic MM, Caminschi I, O'Keeffe M, et al. The Bacterial Peptidoglycan-Sensing Molecules NOD1 and NOD2 Promote CD8+ Thymocyte Selection. J Immunol. 2017;198(7):2649–2660. doi:10.4049/jimmunol.1601462
  9. Caruso R, Warner N, Inohara N, Núñez G. NOD1 and NOD2: signaling, host defense, and inflammatory disease. Immunity. 2014;41(6):898–908. doi:10.1016/j.immuni.2014.12.010
  10. 10.0 10.1 Da Silveira Cruz‐Machado S, Carvalho‐Sousa CE, Tamura EK, Pinato L, Cecon E, Fernandes PACM, De Avellar MCW, Ferreira ZS, Markus RP. TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway. Journal of Pineal Research. 2010. 49: 183-192. doi:10.1111/j.1600-079X.2010.00785.x
  11. Woodhams KL, Chan JM, Lenz JD, Hackett KT, Dillard JP. 2013. Peptidoglycan Fragment Release from Neisseria meningitidis. Infect Immun. 81(9):3490–8.
  12. Becker Y. Chlamydia. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 39. Retrieved from: https://www.ncbi.nlm.nih.gov/books/NBK8091/
  13. 13.0 13.1 Hailman E, Lichenstein HS, Wurfel MM, et al. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med. 1994;179(1):269–277. doi:10.1084/jem.179.1.269
  14. Faure E. Bacterial Lipopolysaccharide Activates NF-kappa B through Toll-like Receptor 4 (TLR-4) in Cultured Human Dermal Endothelial Cells. Differential expression of TLR-4 and TLR-2 in endothelial cells. Journal of Biological Chemistry 275.15 (2000): 11058-1063. doi: 10.1074/jbc.275.15.11058
  15. Wetzler LM, Barry K, Blake MS, Gotschlich EC. Gonococcal lipooligosaccharide sialylation prevents complement-dependent killing by immune sera. Infect Immun. 1992;60(1):39–43.
  16. Rock KL, Kono H. The inflammatory response to cell death. Annu Rev Pathol. 2008;3:99–126. doi:10.1146/annurev.pathmechdis.3.121806.151456

Q4. The Immune Response Questions

Question (i)

(i)  Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.
Figure 1. Schematic representation of the surface structure of Neisseria gonorrhoeae (22)

Clinical Manifestation of N. gonorrheae

        Gonorrhoea is any manifestation of infection by Neisseria gonorrheae, a gram-negative diplococcus bacterium that is closely related to other human Neisseria species (21). There are symptomatic or asymptomatic localized infections caused by N. gonorrheae, such as urethritis, cervicitis, proctitis, pharyngitis and conjunctivitis (17). Disseminated infections can be caused via extension to adjacent organs (i.e. pelvic inflammatory disease and epididymitis) or via bacteremic spread (i.e. skin lesions, tenosynovitis, septic arthritis, endocarditis and meningitis) (17). Diseases in humans caused by N. gonorrheae are generally connected to opportunistic infections in compromised individuals (17). N. gonorrheae infection causes inflammation and local immunity, however, it is unknown whether secretory immune response is protective (1). Host immune responses also vary across strains of N. gonorrhea since not everyone who is exposed will acquire the disease (1). This is largely due to the variations in size or virulence as well as the nonspecific resistance or specific immunity of the host (17).

INNATE – Anatomical Barrier + First Line of Defense

Figure 2. Human anatomical barrier and first line of immune defenses.

        After sexual contact, the Neisseria gonorrheae breaches the host’s anatomical barrier by crossing the mucosal epithelia and affecting the mucous membranes of the urethra in males and the endocervix and urethra in females (1). However, in addition to these locations, the infection is capable of disseminating to a variety of tissues, including oral and anal tissues (1). N. gonorrheae is limited to these locations since it only attaches to microvilli of nonciliated columnar epithelium at these superficial mucosal surfaces and multiply on the basement membrane (1, 17). Vertical transmission is also possible from a genital infection to the conjunctiva of neonates through direct contact (17).

        As part of our anatomical barrier, mucous serves as one of the first line of immune defenses against pathogens by trapping and eliminating them before they can reach the epithelial surface (2). Through surface charge and associated antibodies (i.e. IgM and IgA), mucous impedes pathogen penetration (2). Examples of mucous relevant to infections caused by N. gonorreheae are normal vaginal discharge and penile discharge. Another aspect of our first line of immune defense is the normal bacteria flora that resides within and/or on us. When an exogenous bacterial species is introduced, disease does not always occur (3). Since N. gonorrheae does not exist as normal flora, its ability to cause disease is governed by inhibitory and/or synergistic interrelationships with other commensal bacteria in the genitourinary tract, since competition may always occur (3). When exposed to N. gonorrheae, some other anatomical barriers come into play at the genitourinary tract, including genital pH and urine characteristics (i.e. pH, osmolarity and urea concentration) in both males and females (1). Urine also contains bactericidal and bacteriostatic components that can disrupt bacterial structures and induce bacterial death (1). Furthermore, hormones at certain times of the female menstrual cycle may also have an impact on bacterial colonization (1). Once our surface anatomical barriers are breached, first line of immune defenses at the mucosal surfaces come into play.

        At the mucosal surfaces of both male and female genitourinary tracts, antimicrobial peptides (AMPs) are used as a first-line defense against pathogen invasion (4). Cells in mucosal epithelia, including dendritic cells (DCs), macrophages, neutrophils and NK cells secrete AMPs such as defensins, secretory leukocyte protease inhibitor (SLPI), lysozyme, lactoferrin, elafin and cathelicidin (2). Microbial invasion, replication and colonization can be limited and disrupted within host cells by these peptides (2). Furthermore, AMPs independently modulate the immune system, dampen inflammation and help maintain tissue homeostasis (2). Once both anatomical barriers and first line defenses are breached, the innate immune system is going to come into play in trying to eliminate the pathogen.

INNATE – The Complement System

        When a pathogen breaches the host’s epithelial barriers and initial AMP defenses, it next encounters a major component of the innate immune system – the complement system.

Complement is a collection of soluble proteins and effector molecules present in blood and other bodily fluids detecting and facilitating destruction of pathogens (5).

Figure 3. A simplified view of the complement system and its three main pathways (5).

        There are three complement activation pathways: 1) lectin pathway, 2) classical pathway and 3) alternative pathway. Although they are initiated differently and have different mechanisms of action, all three pathways have similar functions and converge at the generation of enzyme C3 convertase (5). This leads to the cleavage of C3, forming C3a and C3b (5). C3 convertase can then further bind to C3b to generate C5 convertase; this cleaves C5, liberating C5a and C5b (5). In the lectin pathway, C3a and C5a recruit phagocytic cells to the site of infection and promote inflammation (5). Inflammation leads to dilation and increased permeability of blood vessels, causing increased blood blow and leakage of fluid and blood proteins into the infected tissues (5). Through this process, many inflammatory mediators activate immune cells and recruit them into an immune response (5). In the classical pathway, C3b binds to the pathogen surface and acts as an opsonin, enabling phagocytic cells to phagocytose and destroy the C3b-coated microbes (5). In the alternative pathway, a membrane-attack complex (MAC) is formed; it functions by disrupting bacterial cell membrane, causing cell lysis (5).

        The complement system is important in host defense, since individuals with inherited complement deficiencies have an increased risk of acquiring systemic neisserial infections and are subject to recurring episodes of systemic gonococcal infections (1). Neisserial lipopolysaccharide (LPS) is referred to as lipooligosaccharide (LOS) (1). During the growth of N. gonorrheae, outer membrane fragments – “blebs” – containing LOS are released (1). LOS and peptidoglycan (PG) are further released by the autolysis of cells during infection (1). Both LOS and PG activate host classical and alternative complement pathways, with the alternative pathway being predominantly activated (6). LOS also stimulates the production of tumour necrosis factors (TNFs) that can cause bacterial cell damage (1).

INNATE – Cellular

        Major functions of the innate immune system largely depend on pattern recognition receptors (PRRs) that are on many immune cells, specifically the toll-like receptors (TLRs). PRRs are able to detect and interact with evolutionarily conserved structures on bacteria known as pathogen-associated molecular patterns (PAMPs). Upon the interaction of PRRs with PAMPs, intracellular signalling cascades are triggered, causing activation of proinflammatory cytokines (i.e. IL-1, IL-6, TNFa) and chemokines (i.e. IL-8), leading to what we know as the early/innate host response (7). The effects of the signalling cascades are also crucial for the subsequent activation of adaptive immunity (7). Host cells also respond to bacterial PG fragments within outer membrane vesicles via cytoplasmic NOD-like receptors (NLRs), which also contribute to the secretion of additional pro-inflammatory cytokines (20). Cytokines are signalling molecules, whereas chemokines are chemoattractants. They attract cells that have specific chemokine receptors, such as neutrophils and monocytes, out of the bloodstream and into the infected tissue (5). Neutrophils are recruited first, followed by the migration of monocytes, which rapidly differentiate into macrophages once they are in the infected tissue (5). Together, these immune cells reinforce and sustain the innate immune response. If inflammation persists, eosinophils may also migrate into the infected tissue and contribute to the destruction of the invading bacterium, although they are mostly important for parasitic infections and allergic reactions (5).

Figure 4. Mechanisms of action of neutrophils.

        The LPS of N. gonorrheae, LOS, is a known ligand and PAMP of TLR-4. The recognition and binding of LOS to TLR-4 is responsible for eliciting Th17 responses (7). These responses recruit a huge influx of neutrophils (7). In response to N. gonorrheae and all opacity protein (Opa)-expressing bacteria, neutrophils undergo an aggressive opsonin-independent response driven by the innate decoy receptor CEACAM3; CEACAM3 is exclusively expressed by human neutrophils (8). This process drives a potent binding, phagocytic and oxidative killing of the bacterium (8). Gonococci that express certain Opas interact with neutrophils in the absence of antibodies (17). Once the bacteria are internalized by neutrophils into intracellular vesicles, they are destroyed by degradative enzymes and other antimicrobial substances stored in the cytoplasmic granules of neutrophils (6). NADPH oxidase pumps in neutrophils pump electrons into their phagocytic vacuoles, inducing a

Figure 5. Mechanism of action of macrophages (5).

charge across the membrane that must be compensated (9). Compensation is done through the movement of ions, which produces conditions conducive to microbial killing and digestion (9). For intracellular bacteria, phagocytosis is most effective; for extracellular pathogens, however, neutrophils would utilize neutrophil extracellular traps (NETs) (5). This occurs when certain activated neutrophils undergo a unique form of cell death in which the nuclear chromatin, rather than being degraded as occurs during apoptosis, is released into the extracellular space and forms a fibril matrix (5). Reactive oxygen intermediates (ROIs) are required for the formation of NETs (5).

        After monocytes differentiate into macrophages at the site of infected tissue, they elicit a variety of defense mechanisms to combat infections caused by intracellular bacteria, such as N. gonorrheae. Some of these include induction of toxic anti-microbial effectors such as nitric oxide and ROIs, and the production of AMPs and cytokines (10). Internally, the formation of phagolysosome from the fusion of phagosome and lysosome expose bacteria to lysozyme, which can disrupt and damage bacterial structures, leading to bacterial cell death (10). Macrophages typically survive longer than neutrophils do and can also phagocytose pathogens opsonized by complement proteins (6). The activation of complement system, phagocytic functions of various immune cells, and cellular and bacterial death can possibly explain the purulent discharge experienced by Naser (1).

INNATE – Bridge to Adaptive Immunity

Figure 6. Presentation of antigen peptides onto both MHC I and MHC II by APCs.

        Innate immunity is the first line of defense against non-self-pathogens (19). It has non-specific defense mechanisms that activate immediately or within hours of an antigen’s appearance in the body (19). The adaptive immune response is more complex than the innate immune response, therefore it takes longer for it to mount. However, it is antigen specific and retains memory of pathogens that makes future responses more efficient. Antigen-presenting cells (APCs) in secondary lymphoid tissues are responsible for initiating adaptive immune responses. Initiation occurs when B or T lymphocytes encounter an antigen that they have a receptor for (5). Peptides of the antigen must be presented on APCs for it to interact with B or T lymphocytes (5). DCs, macrophages and B cells are all APCs, with DCs being professional APCs (5). As professional APCs, DCs have cell-surface proteins called co-stimulatory molecules that can be triggered upon activation of PRRs (5). This supports the ability of naïve lymphocyte proliferation and differentiation into final and fully functional forms (5). Regular APCs such as macrophages and B cells are less able to do so (5).

        At the site of infection, DCs’ PRRs are first stimulated by bacteria’s PAMPs. This signals DCs to engulf the pathogen and to degrade it intracellularly (5). Different PAMP-PRR interactions lead to different cytokines being produced, which affects the final differentiation of naïve T cells (5). After engulfment of bacteria, DCs undergo editing and maturation in which NK cells mediate (11). Only DCs that have undergone NK-mediated “quality control” can become fully mature and capable of inducing effector T cell responses (11). During DC maturation, antigen processing occurs when the engulfed pathogen gets broken down into peptide fragments (5). These peptide fragments are then presented on Major Histocompatibility Complexes (MHC), activating antigen receptors on lymphocytes (5). There are two types of MHCs, MHC I and MHC II. MHC I presents antigen peptides of the endogenous pathway while MHC II presents antigen peptides of the exogenous pathway (5).

         Collectively, the APCs and their antigen presenting mechanisms can be considered a transition from the innate to adaptive immune responses. Stimulation of the innate immune system bridge the adaptive immune system, resulting in the generation of pathogen-specific adaptive cellular and humoral immunity.

ADAPTIVE – Cellular

        After APCs present antigen peptides on their MHC complexes to naïve T cells, the T cells will be induced to proliferate and differentiate into specific effector T cells (5). Effector response is elicited upon encounter of their specific antigen (5). There are two main classes of effector T cells – CD8 and CD4 T cells. The former primarily recognize antigen peptides presented on MHC I and differentiate into cytotoxic effector T cells that recognize and kill infected cells (5). The latter recognize antigen peptides presented on MHC II and differentiate into different effector subsets with different immunological functions based on cytokine environments (5). The main effector subsets for CD4 T cells are Th1, Th2, Th17, Tfh and Treg (inhibit extensive immune activation) (5). Once the T cells have been activated and have differentiated into their respective effector subsets, they will exit the lymph node and home to the site of infection to elicit their respective responses (5).

Figure 7. CD4 effector T cell subsets, their specialization of different target cells and eradication of different classes of pathogens (5).

        Cytotoxic T cells release perforin, granzyme and IFN-g (5). Perforin helps deliver granzyme into the target bacterial cell while granzymes are proteases that trigger apoptosis in the target bacterial cell (5). IFN-g is a cytokine that functions primarily as an activator of macrophages, in addition to activating NK cells and neutrophils (5). Fas ligand is a membrane-bound effector molecule that is found on cytotoxic T cells (5). When Fas ligand binds to Fas, the receptor for Fas ligand, it activates apoptosis in the target bacterial cell (5).

        Th1 cells are specialized to activate macrophages that are infected by or have ingested pathogens (5). This response is the most suitable CD4 T cell effector response for intracellular bacteria N. gonorreheae. The activated macrophages will respond with phagocytosis, eliminating the bacteria (5). These Th1 cells also express Fas ligand, meaning that they are capable of inducing apoptosis in target bacterial cells (5). Th2 cells promote immune responses to parasitic infections and/or allergic responses (5). Th17 cells promote acute inflammation by recruiting neutrophils to sites of infection (5). Neither Th2 nor Th17 are very effective or suitable against infections caused by N. gonorrheae, since it is an intracellular bacterium. However, as stated above, Th17 responses are elicited upon LOS on N. gonorrheae binding to host TLR-4.

        The key factors in Th17 differentiation are cytokines, especially IL-17, IL-22, IL-6 and TGF-b; these cytokines activate cells with their receptors (12). These cells (typically endothelial and stromal cells) secrete other inflammatory cytokines, including TNF-a, granulocyte colony stimulating factors (GCSF), granulocyte-monocyte colony-stimulating factor (GMCSF) and IL-8, recruiting defense proteins, phagocytic cells, including neutrophils, from the bone marrow to the inflammatory sites (12). In response to simulation with IL-17 and IL-22, epithelial cells at mucosal surfaces upregulate secretion of AMPs, such as defensins, S100 proteins and lipcalin-2 (12). In addition to the inappropriate Th17 response, N. gonorrheae suppresses Th1 and Th2 responses (12). This is achieved through a mechanism involving TGF-b and Tregs (12). Lack of or the inhibition of TGF-b alleviates the suppression of specific anti-gonococcal responses, allowing Th1 and Th2 responses to emerge, together with immune memory and protective immunity (12). However, genital tract tissues are naturally abundant in TGF-b, therefore fostering an immunosuppressive environment, causing the host to become susceptible to infection caused by N. gonorreheae (12). In addition to that, the presence of N. gonorrheae upregulates TGF-b, causing Th1 and Th2 responses to be further suppressed (12). The suppression of favoured CD4 T cell responses and the elicitation of a non-effective response contributes to N. gonorrheae’s pathogenesis and its evasion of host immune responses.

ADAPTIVE - Humoral

        Humoral immune responses protect our body’s extracellular spaces. Antibodies produced by B cells facilitate the destruction of extracellular pathogens and their products to prevent the spread of intracellular infections (5). Antibodies are generally Y shaped and have a variation region (fab) and a constant region. There are three major ways in which antibodies contribute to immunity: neutralization, opsonization and complement activation (5). They help neutralize pathogens by binding to bacterial cells and their toxins, preventing their ability to enter and infect cells (5). Antibodies facilitate opsonization by binding to Fc receptors on immune cells via their constant regions (5). They can also activate proteins of the classical pathway of the complement system by binding to pathogens (5).

        With the help of T helper cells, naïve B cells become activated through interactions with antigens that they are specific for (5). Activation begins when B cell receptor (BCR) binds to and processes antigen for presentation on the B cell’s MHC II (5). A T helper cell that has previously differentiated in response to the same pathogen recognizes the presented antigen peptide and activates the B cell through co-stimulatory molecules (5). Activated B cells are then capable of differentiating into memory B cells and antibody-secreting plasma cells due to different interleukins produced by T helper cells (5). Although some microbial antigens can activate B cells directly without the help of T helper cells, the antibodies produced by these B cells tend to have lower affinity and are less functionally versatile (5). This is due to the fact that immunoglobulin class switching to any isotype other than IgM depends on the interaction of antigen-stimulated B cells with helper T cells and other cells in the peripheral lymphoid organs (5). Class switching of antibodies is very important, since different isotypes have different effector functions. In addition to that, affinity

Figure 8. Adaptive immunity humoral VS cell-mediated response (14).

maturation is the process that most antibody responses undergo; this process produces antibodies of greater affinity for their target antigen via somatic hypermutation of variable regions genes (5).

        Both mucosal and systemic antibodies are produced with the infection caused by N. gonorrheae (1). IgA and IgG are the primary mucosal antibodies; they react with certain structural aspects of the bacterium, such as Por, Opa, Rmp, LOS and some iron-regulated proteins (1). Anti-pilus antibodies inhibit the pilus-mediated attachment of homologous gonococcal strain (17). In general, IgA responses are brief and decline rapidly after treatment; IgG levels decline slower (1). Gonococcal virulence factors like pili, Por, Opa, Rmp and LOS activate serum antibodies during infection (17). The main IgG subclass that reacts with various gonococcal antigens is IgG3, followed by IgG1 and IgG4; IgG2 responses are minimal (17). Anti-Por antibodies are bactericidal to gonococcus and the natural human serum includes opsonic anti-Por IgG (17). Uncomplicated genital infections are usually caused by serum sensitive strains of N. gonorrheae (1). They are killed by bactericidal activity mediated by systemic IgM and IgG antibodies that recognize bacterium LOS (1). Disseminated infections are usually caused by serum resistant strains of N. gonorrheae (1). Their resistance is partially mediated by IgA antibodies that block IgG-mediated bactericidal activity of the serum (1). Antibodies to different surface-exposed antigens are present in cervical and urethral secretions of patients infected with N. gonorrheae and therefore they likely play a role in the opsonophagocytosis of the bacteria (17).

        Numerous studies indicate that saccharide epitopes found in the LOS of N. gonorrheae can cause antibody responses in the majority, but not all humans, in response to mucosal infection with the bacteria (13). Studies have also shown that N. gonorrheae is capable of suppressing specific antibody responses, which contributes to its pathogenesis and evasion of host immune responses (12). With that said, uninfected individuals often have serum antibodies that interact with gonococcal antigens (1). These antibodies likely come from colonization/infection with other gram-negative bacteria that possess cross-reactive antigens (1). These are the only know “natural antibodies” and are very rare. The presence and absence of these antibodies may be important in individual natural resistance and susceptibility to infection, respectively (1).

References

1.    Todar KG. 2012, posting date. Pathogenic Neisseriae: Gonorrhea, Neonatal Ophthalmia and Meningococcal Meningitis. In Todar K (ed), Todar’s Online Textbook of Bacteriology. University of Wisconsin-Madison Dept. of Bacteriology, Madison, WI.

2.    Nguyen PV, Kafka JK, Ferreira VH, Roth K, Kaushic C. 2014. Innate and adaptive immune responses in male and female reproductive tracts in homeostasis and following HIV infection. Cell Mol Immunol. 11:410-427.

3.    Bryan L, Gilles RGM. 2001. Understanding the Bacterial Flora of the Female Genital Tract. Clin Infect Dis. https://doi.org/10.1086/318710.

4.    Yarbrough VL, Winkle S, Herbst-Kralovetz MM. 2014. Antimicrobial peptides in the female reproductive tract: a critical component of the mucosal immune barrier with physiological and clinical implications. Hum Reprod Update. 21:353-377.

5.    Murphy K, Weaver C. 2017. Janeway’s Immunobiology. In Zayetz E (ed), Janeway’s Immunobiology, 9th ed. Garland Science, Taylor & Francis Group, New York, NY.

6.    Densen P. 1989. Interaction of Complement with Neisseria meningitidis and Neisseria gonorrhoeae. Clin Microbiol Rev. 2:S11-S17.

7.    Mogensen TH. 2009. Pathogen Recognition and Inflammatory Signaling in Innate Immune Defenses. Clin Microbial Rev. 22:240-273.

8.    Sintsova A, Sarantis H, Islam EA, Sun CX, Amin M, Chan CHF, Stanners CP, Glogauer M, Gray-Owen SD. 2014. Global Analysis of Neutrophil Responses to Neisseria gonorrhoeae Reveals a Self-Propagating Inflammatory Program. PLoS Pathog. https://doi.org/10.1371/journal.ppat.1004341

9.    Segal AW. 2005. How Neutrophils Kill Microbes. Annu Rev Immunol. 23:197-223.

10.  Weiss G, Schaible UE. 2015. Macrophage defense mechanisms against intracellular bacteria. Immunol Rev. 264:182-203.

11.  Moretta A, Marcenaro E, Parolini S, Ferlazzo G, Moretta L. 2008. NK cells at the interface between innate and adaptive immunity. Cell Death Differ. 15:226-233.

12.  Liu Y, Feinen B, Russell MW. 2011. New Concepts in Immunity to Neisseria Gonorrhoeae: Innate Responses and Suppression of Adaptive Immunity Favor the Pathogen, Not the Host. Front Microbiol. https://doi.org/10.3389/fmicb.2011.00052

13.  Lovett A, Duncan JA. 2019. Human immune Responses and the Natural History of Neisseria gonorrhoeae Infection. Front. Immunol. https://doi.org/10.3389/fimmu.2018.03187

14.  Aryal S. 2018. Differences between Humoral Immunity and Cell mediated Immunity. Online Microbiology Notes. https://microbenotes.com/differences-between-humoral-immunity-and-cell-mediated-immunity/

15.  CDC. 2019. Gonorrhea Detailed Fact Sheet. U.S. Department of Health and Human Services, Atlanta, GA.

16.  Dinarello CA. 2000. Proinflammatory cytokines. Chest. 2:503-508.

17.  Morse SA. 1996. Neisseria, Moraxella, Kingella and Eikenella. In Baron S (ed), Medical Microbiology, 4th ed. University of Texas Medical Branch at Galveston, Galveston, TX.

18.  InformedHealth.org. The innate and adaptive immune systems [Internet]. Institute for

19.  Quality and Efficiency in Health Care (IQWiG). U.S. National Library of Medicine; 2010 [cited 2020Feb8]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK315831/

20. Hill SA, Masters TL, Wachter J. Gonorrhea - an evolving disease of the new millennium. Microbial cell (Graz, Austria). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354566/. Published September 5, 2016. Accessed February 10, 2020.

21. Smith NH, Holmes EC, Donovan GM, Carpenter GA, Spratt BG. 1999. Networks and groups within the genus Neisseria: analysis of argF, recA, rho, and 16S rRNA sequences from human Neisseria species. Mol Biol Evol 16:773–783.

22.  Shaughnessy J, Ram S, Rice PA. Biology of the Gonococcus: Disease and Pathogenesis. In: Christodoulides M, editor. Neisseria gonorrhoeae: Methods and Protocols. New York, NY: Humana Press; 2019.

Question (ii)

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

For Neisseria gonorrhoeae, asymptomatic infection occurs in <15% of male urethral infections and closer to 60% of female cervical infections [Q4-2 1]. With urethral infections, damage occurs through inflammation, and the sloughing of epithelial cells, both of which cause the common complication of urethral stricture, which involves scarring and narrowing of the urethra, restricting flow of urine from the bladder. Local genital structures such as the Mullerian glands and Cowper glands can sometimes be infected. More commonly, ascending infection along anatomical routes can lead to male complications of prostatitis, epididymitis, or orchitis , and female complications of pelvic inflammatory disease, including endometritis, salpingitis, and tubo-ovarian abscesses. Unilateral or bilateral conjunctivitis is possible in those exposed to infected secretions, with cases of neonatal infection due to contact with infected mucous tissue during birth. In approximately 0.1% to 0.3% of cases more virulent strains of N gonorrhoeae may be invasive and hematogenously spread to cause septic arthritis, meningitis, endocarditis, and osteomyelitis [Q4-2 2].

In order to continue infection, the gonococci needs to infect epithelial cells and mucosal surfaces. In doing so, tumor necrosis factor from phagocytes and gonococcal products, such as peptidoglycan and lipo-polysaccharide, damage ciliated epithelial cells of mucosal surfaces, which may lead to complications such as pelvic inflammatory disease or epididymitis [Q4-2 3].  If the infection is left untreated, complete loss of ciliary action can occur [Q4-2 4]. Cytotoxicity of ciliated cells is attributed to gonococcal peptidoglycan, LOS, and the induction of increased production of inflammatory cytokine TNF. The gonococcal LOS (endotoxin) causes direct cytotoxicity by causing ciliostasis and sloughing of ciliated epithelial cells. TNF-α and other cytokines mediate the cytotoxicity of gonococcal infections, where LOS induces production of tumor necrosis factor alpha (TNF-α) [Q4-2 5], a endogenous pyrogen. TNF-α, a member of the TNFSF family, is made soluble by action of proteases, is important in the acute phase of inflammation and infection, with signaling through NF-κB and serves as a pro-apoptotic signal through the TNF receptor death domain [Q4-2 6]. Both of these end results cause damage to cells and harm tissue integrity. LOS can activate complement, which also contributes to the acute inflammatory response, while coincidently suppressing the antibody response and harming CD4+ T cells. This damage may also lead to a coinfection with chlamydia, and other bacterial species, where the incidence of Chlamydia coinfection is 15–25% among males and 35–50% among females [Q4-2 7]. These coinfections can heighten inflammatory symptoms at infection sites, leading to greater overall host damage from the diseases. The PMN influx, in addition to cytokine release from the urethral epithelium, subsequently potentiates the clinical symptoms associated with disease.

References Q4-2:

  1. Pariser, H. (1976-04-01). "Asymptomatic gonorrhea". Cutis. 17 (4): 723–726. ISSN 0011-4162. PMID 828089.
  2. Workowski, Kimberly A.; Bolan, Gail A. (2015-06-05). "Sexually Transmitted Diseases Treatment Guidelines, 2015". MMWR. Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports. 64 (RR-03): 1–137. ISSN 1057-5987. PMC 5885289. PMID 26042815. Retrieved 2020-02-07.CS1 maint: PMC format (link)
  3. "Pathogenic neisseriae: surface modulation, pathogenesis and infection control | Nature Reviews Microbiology". Retrieved 2020-02-07.
  4. Marrazzo, Jeanne M.; Apicella, Michael A. (2020). "Neisseria gonorrhoeae (Gonorrhea)". Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases (9 ed.). Elsevier, Inc. p. 212. Retrieved 2020-02-07.
  5. Patrone, Julia B; Stein, Daniel C (2007-01-26). "Effect of gonococcal lipooligosaccharide variation on human monocytic cytokine profile". BMC Microbiology. 7: 7. doi:10.1186/1471-2180-7-7. ISSN 1471-2180. PMC 1797046. PMID 17257430. Retrieved 2020-02-15.CS1 maint: PMC format (link)
  6. Shaughnessy, Jutamas; Ram, Sanjay; Rice, Peter A. (2019). "Biology of the Gonococcus: Disease and Pathogenesis". In Myron Christodoulides (ed.) (eds.). Neisseria gonorrhoeae: Methods and Protocols. Methods in Molecular Biology. New York, NY: Springer. pp. 1–27. ISBN 978-1-4939-9496-0. Retrieved 2020-02-15.CS1 maint: uses editors parameter (link)
  7. Hsu, Katherine; Ram, Sanjay; Darville, Toni (2020). "Neisseria gonorrhoeae (Gonococcus)". Nelson Textbook of Pediatrics (21 ed.). Elsevier Inc. p. 219. Retrieved 2020-02-07.

Question (iii)

(iii)  Bacterial evasion: how do the bacteria attempt to evade these host response elements.

While the immune response to Neisseria gonorrhoeae is not fully understood, the bacteria’s extensive antigenic variability and resistance to complement likely allows it to evade destruction by the host’s immune defenses. It’s hypothesized that Neisseria gonorrhoeae may be able to avoid inducing any protective immune responses at all (25). N. gonorrhoeae possesses many surface structures with the ability to undergo phase and antigenic variation, molecular mimicry by surface glycolipids of human antigens, and horizontal gene exchange; these may help directly inhibit the adaptive and innate immune responses (26).

There are two ways main strategies that Neisseria gonorrhoeae utilizes to defend itself: 1) expression of virulence factors that defend against oxidative and non-oxidative antimicrobial components and 2) modulation of phagocytic ability of phagocytes and their ability to release antimicrobial components to kill ingested bacteria (23). N. gonorrhea’s outer membrane protein porin (Por) is a virulence factor that allows the bacterium to survive inside vacuoles of phagocytes (e.g. neutrophils) by avoiding degradation after ingestion (24).

Gonococci can also hijack the host response by mechanisms that involve the control of macrophage activity (14). During innate immunity, phagocytic cells such as macrophages and PMNs represent the earliest line of defense when an invader enters the body, however there has been debate over the role of PMNs in clearing N. gonorrhoeae infection. In the paper by Escobar et.al, it is mentioned that PMN cannot clear infections by N. gonorrhoeae, since gonococcus can survive in the PMN and suppress oxidative bursts that are meant to kill it (14). By entering a PMN without killing it, gonococci can spread to other tissues within the host and even spread from one host to another (14). The exact mechanism of how this occurs is still under review. The bacteria can also avoid phagocytosis mediated by opsonic antibodies. The membrane molecules such as lipooligosaccharide (LOS), Pili, Opa, and Porin (Por) are the main targets to generate antibodies against gonococci (14) but the gonococcus varies these antigens so that they are not recognized by the opsonic antibodies to therefore evade successive IgG Fc-receptor-mediated phagocytosis.

Antigenic variation occurs when the molecular constitution of an expressed structure changes at high frequency, thus allowing a single bacterium to express more than one form of the structure, each form being recognized as antigenically distinct by the host immune system (22). For example, at any one time, a gonococcus may express zero, one, or several different Opa proteins, though each strain has 10 or more genes for different Opas (1). This genetic variation is helpful in bacterial evasion because since Opa is an outer membrane protein, if the immune system mounts an antigen-specific response (via T or B cells) to a certain Opa protein, it may not recognize the other versions of Opa and thus won’t be effective against those versions. When T and B cells get activated by a certain antigen, they develop specificity for that exact antigen and will bind to it. This variation allows the bacteria to put on “different masks” that confuse the immune response because it won’t recognize the bacteria as one that it has already been exposed to before; as a result, the immune system must mount a new response to that different antigen during exposure.

The same can be said about LOS since gonococci can express several antigenic types of LOS whose expression can be altered (via an unknown mechanism) to evade the immune response mounted against specific antigens; N. gonorrhoeae LOS makes up about 50% of the outer membrane mass (15). The resulting heterogeneity in antigenic epitopes that can be recognized by antibodies makes it more challenging for antibodies and memory cells to recognize the bacterium after each infection (15). Por protein also exhibits phase variation. Phase variation can be defined as the phenomenon observed when the expression of a surface component reversibly switches on and off (22). Since gonococci can either express or not express (phase variation) the Por gene, it is even harder for the immune system to recognize it. Even if it was possible to have antibodies against all known variants of the Por proteins, an infecting gonococcus might still be able to escape the immune system, because these antibodies are rendered useless if the Por gene is not even being expressed by the bacterium.

Figure 1: N. gonorrheae host adaptation, virulence factors and pathogenesis (28)

Additionally, gonococcal LOS is also involved in the resistance of N. gonorrhoeae to the bactericidal activity of normal human serum. Oligosaccharides containing epitopes defined by specific monoclonal antibodies are associated with a serum-resistant phenotype. Soluble LOS that are liberated during bacteremia from bacterial cell lysis can enhance the effects of bacterial endotoxins; this accounts for their ability to persist during bacteremia (3). Gonococci can also utilize host-derived cytidine monophospho-N-acetylneuraminic acid (CMP- NANA), to sialylate the oligosaccharide component of its LOS (post-translational modification that changes LOS), converting a serum-sensitive organism into a serum-resistant one (1). This will make the bacteria resistant to complement-mediated killing by serum, which may also result in impaired opsonization and phagocytosis (since complement helps with the phagocytosis) (10). Complement activation on pathogen surfaces occurs via three pathways: classical, lectin, and alternative complement pathways. LOS sialic acid inhibits the classical and alternative pathways, allowing the infection to go on without being noticed by the host immune response (10). This leads to prolonged inflammation and advancement of the infection. IgM is responsible for the “killing” of unsialylated “serum-sensitive” gonococci in non-immune normal human sera (NHS). But when LOS sialylation occurs, it decreases IgM and significantly reduces IgG binding to bacterial surfaces by compromising the binding site of the antibody through this sialylation modification. Sialylation also decreases the access of antibodies to gonococci surface protein PorB by blocking antibody engagement with C1q, which is needed as the first step to initiation of the classical pathway.

As for the alternative pathway, sialylation of the lacto-N-neotetraose (LNnT) structure of gonococcal LOS enhances human factor H (FH) binding to N. gonorrhoeae (10). FH, which is a transcription regulator, downregulates the alternative pathway of complement by providing cofactor activity for factor I-mediated C3b cleavage and accelerating decay against the alternative pathway C3-convertase, C3bBb (10). C3 serves as the central component in the complement pathway. As a result, the non-immune normal human serum will not be able to clear the infection, and as mentioned above, the serum-resistant phenotype is associated with N. gonorrhea’s ability to disseminate in the body. On the other hand, disseminating gonococcal strains resist complement killing by normal human serum (NHS) even in the absence of sialylation of gonococcal lipooligosaccharide (LOS) (10). Although organisms with sialylated LOS are less invasive, they are more resistant to host immune responses and bactericidal effects of serum (3). Furthermore, there is antigenic similarity between N. gonorrhea’s LOS and antigens present on human erythrocytes (1). This similarity to self results in an ineffective immune response to these LOS antigens because the body will not receive a signal that it is being exposed to a foreign peptide, thus maintaining immuno-tolerance in the host (1).

Figure 2: Mechanisms used by Neisseria gonorrhoeae to avoid the effects of antibody (29)

Additionally, PorB itself can resist proteolytic digestion by trypsin and α-chymotrypsin because it lacks a portion of loop 5 that contains the cleavage sites for these enzymes, making the cleavage site non-recognizable for these enzymes (10). Since PorB proteasomes activate host Toll-Like Receptor 2 (TLR2) signaling and act as immune system stimulators, N. gonorrhea’s ability to resist proteolytic digestion will result in a lack of signaling to TLRs and the bacteria will thus go unnoticed (10). An additional role for PorB in aiding the evasion of the immune system comes from the recommendation events of Por genes. The folding pattern of recombinant PorB may cause a decrease in dendritic-cell induced T-cell proliferation/activation because they cannot recognize the antigenic site (T-cell receptor) for PorB, therefore offsetting the stimulating effect of TLR2 (10).

As a result of long association exclusively with humans, Neisseria gonorrhoeae has become extremely well adapted to the human immune system and has learned to suppress the generation of potentially protective specific antibody responses. N. gonorrhoeae exploits an environment that is already rich in TGF-β, a pleiotropic cytokine with many functions (immune regulation and important for control of immune responses to developing fetus in female reproductive tract) (25). It’s hypothesized that N. gonorrhoeae enhances TGF-β production and therefore promotes Th17-dependent (T-helper 17 cells) responses and innate defense mechanisms that are favourable to the gonococci’s survival (i.e. inappropriate CD4 T cell response that facilitates the bacterium’s’ escape of host immune defenses), all while simultaneously suppressing the development of Th1- and Th2-driven adaptive immune responses (e.g. specific antibodies that could eliminate it) (25).

Strains of Neisseria gonorrhoeae generate two distinct extracellular IgA1 proteases that cleave the heavy chain of human immunoglobulin A1 (IgA1) at different sections in the hinge region (3). Type 1 protease cleaves a prolyl-seryl peptide bond and type 2 protease cleaves a prolyl-threonyl bond in the hinge region of the heavy chain (3). This region is missing in human IgA2, so this isotype is not susceptible to cleavage. Each gonococcal isolate develops only one of these two enzymes (3). Split products of IgA1 have been seen in genital secretions of women infected with Neisseria gonorrhoeae, suggesting that gonococcal IgA1 protease is present and active during genital infection (3). Fab (antigen-binding fragments) fragments of IgA1 may bind to the gonococcal cell surface and block the Fc-mediated functions of intact immunoglobulins, therefore, further reducing the possibility that IgA can have an effect on this infection (3).

Another mechanism of invasion is by antibodies to Rmp (outer membrane protein III) that are found in all strains of N. gonorrhoeae (1). Rmp does not undergo antigenic variation and is found in a complex with Por and LOS (1). It shares partial homology with the OmpA protein of Escherichia coli and antibodies to Rmp (induced either by a neisserial infection or by colonization with E. coli) tend to block bactericidal antibodies directed against Por and LOS (3). Antibodies tend to target non-sialylated LOS and Por on bacterial surfaces, however, induced IgG antibodies that react with Rmp often block bactericidal antibodies targeted against Por and LOS as mentioned (3). In fact, anti-Rmp antibodies may increase susceptibility to infection by N. gonorrhoeae (3).

Studies have also shown that the human adaptive immune response is minimal during N. gonorrhoeae infections (27). While there is a moderate increase in antibodies, responses are usually short and show no relation to previous documented infections (3). Pathogen-host interactions during infectious disease are traditionally thought of as two-way reactions (i.e. host against the pathogen and vice versa) with the outcome dependent on which one ultimately wins (25). Evidently, the Neisseria gonorrhoeae pathogen has become remarkably well-adapted to its exclusive human host since it has been able to proactively direct the host to respond in ways that are beneficial to its own survival, but detrimental to the host.

References:

1.   Medical Microbiology. 4th Edition. S.l.: University of Texas Medical Branch at Galveston.

2.     Mubaiwa TD, Semchenko EA, Hartley-Tassell LE, Day CJ, Jennings MP, Seib KL. The sweet side of the pathogenic Neisseria: the role of glycan interactions in colonisation and disease. Pathogens and Disease. 2017;75(5). doi:10.1093/femspd/ftx063.

3.     Todar K, Madison. Pathogenic Neisseriae: gonorrhea and meningitis. http://textbookofbacteriology.net/neisseria.html. Accessed February 7, 2020.

4.     Hill SA, Masters TL, Wachter J. Gonorrhea - an evolving disease of the new millennium. Microbial cell (Graz, Austria). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354566/. Published September 5, 2016. Accessed February 10, 2020.

5.     Reinholdt J. IgA and Mucosal Homeostasis. Madame Curie Bioscience Database [Internet]. https://www.ncbi.nlm.nih.gov/books/NBK6628/. Published January 1, 1970. Accessed February 10, 2020.

6.     Complement System Made Easy- Immunology- Classical Alternate & Lectin pathway. YouTube. https://www.youtube.com/watch?v=d6qFPegEYV0. Accessed February 7, 2020.

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8.     Diagnostica E. Complement System - overview. Complement system Overview. http://www.complementsystem.se/Complement System. Published February 10, 2020. Accessed February 10, 2020.

9.     Lamm ME. Interaction of antigens and antibodies at mucosal surfaces. Annual review of microbiology. https://www.ncbi.nlm.nih.gov/pubmed/9343353. Published 1997. Accessed February 10, 2020.

10.  Shaughnessy J, Ram S, Rice PA. Biology of the Gonococcus: Disease and Pathogenesis. Methods in molecular biology (Clifton, N.J.). https://www.ncbi.nlm.nih.gov/pubmed/31119614. Published 2019. Accessed February 10, 2020.

11.  Tumor Necrosis Factor. Tumor Necrosis Factor - an overview | ScienceDirect Topics. https://www.sciencedirect.com/topics/medicine-and-dentistry/tumor-necrosis-factor. Accessed February 10, 2020.

12.  Research on gonorrhea uncovers new immune system trigger. ScienceDaily. https://www.sciencedaily.com/releases/2015/06/150611144444.htm. Published June 11, 2015. Accessed February 10, 2020.

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14.  Escobar A, Rodas PI, Acuña-Castillo C. Macrophage-Neisseria gonorrhoeae Interactions: A Better Understanding of Pathogen Mechanisms of Immunomodulation. Frontiers in immunology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309159/. Published December 21, 2018. Accessed February 10, 2020.

15.  Lovett A, Duncan JA. Human Immune Responses and the Natural History of Neisseria gonorrhoeae Infection. Frontiers in immunology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389650/. Published February 19, 2019. Accessed February 10, 2020.

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17.  Hill SA, Masters TL, Wachter J. Gonorrhea - an evolving disease of the new millennium. Microbial cell (Graz, Austria). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354566/. Published September 5, 2016. Accessed February 14, 2020.

18.  Nguyen PV, Kafka JK, Ferreira VH, Roth K, Kaushic C. Innate and adaptive immune responses in male and female reproductive tracts in homeostasis and following HIV infection. Cellular & molecular immunology. https://www.ncbi.nlm.nih.gov/pubmed/24976268. Published September 2014. Accessed February 14, 2020.

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Question (iv)

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

Due to its high antigenic variability and adaptive nature, it is challenging to remove N. gonorrhoeae entirely from the body depending solemnly on the host’s immune system(1). Antibiotics are often needed for the complete removal of bacterium from the body(1). The CDC recommends dual therapy with a combination of antibiotics for N. gonorrhoeae infection, including ceftriaxone, cefixime, ciprofloxacin, or ofloxacin(1,2). Not receiving treatment can lead to severe complications such as pelvic inflammatory disease and infertility(1). Although the bacteria can be removed entirely with the right treatment, the permanent damage done by the disease, such as damage to the reproductive tract, cannot be repaired(2). Growing antimicrobial resistance of N. gonorrhoeae had made gonorrhea treatment option become limited and is a growing concern(2). As a “superbug”, N. gonorrhoeae is resistant to all classes of antibiotics, and there is only one remaining recommended treatment option(1).

Immunity to N. gonorrhea is expected to be strain-specific, therefore reinfection can easily occur(1). Being infected before does not lower an individual’s susceptibility, severity, or duration of the diseases(3). Consequently, the sexual partner of the patient needs to be checked to prevent future infections(2). This lack of immunity is mainly due to the antigenic variation of the bacterium via mechanisms such as allelic polymorphism, genetic recombination, phase-variable expression, and horizontal gene transfer (HGT)(3). Variation in antigenic factors causes the major surface molecules on the bacterium to be different, which causes memory B cells, T cells, and antibodies produced in previous infections no longer effective or applicable(3). Moreover, anti-gonococcal antibodies can be detected regardless of infection history, as immunoglobulin levels are similar between previously infected and non-infected individuals(1). Hedges et al. found there was only a slight IgA1 increase for females and IgG for males between previously infected individuals and non-infected individuals(4). Usually, serum antibodies are derived from infection with other Gram-negative bacteria with cross-reactive antigens(1). These natural antibodies differ per person, and therefore gonorrhea susceptibility also varies per person. However, natural resistance to this organism is very rare(5). Currently, stable antigens have been identified, and protective vaccines may be feasible to generate long immunity in the future(6).

File:Figure 1 Antigenic variation of N.gonorrhoeae.png

References

1. Pathogenic Neisseriae: gonorrhea and meningitis [Internet]. [cited 2020 Feb 7]. Available from: http://textbookofbacteriology.net/neisseria_4.html

2. Detailed STD Facts - Gonorrhea [Internet]. 2019 [cited 2020 Feb 7]. Available from: https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea-detailed.htm

3. Liu Y, Feinen B, Russell MW. New Concepts in Immunity to Neisseria Gonorrhoeae: Innate Responses and Suppression of Adaptive Immunity Favor the Pathogen, Not the Host. Front Microbiol [Internet]. 2011 [cited 2020 Feb 14];2. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2011.00052/full

4. Hedges SR, Mayo MS, Mestecky J, Hook EW, Russell MW. Limited Local and Systemic Antibody Responses to Neisseria gonorrhoeae during Uncomplicated Genital Infections. Infect Immun. 1999 Aug;67(8):3937–46.

5. Lovett A, Duncan JA. Human Immune Responses and the Natural History of Neisseria gonorrhoeae Infection. Front Immunol [Internet]. 2019 [cited 2020 Feb 7];9. Available from: https://www.frontiersin.org/articles/10.3389/fimmu.2018.03187/full

6. Gottlieb SL, Jerse AE, Delany-Moretlwe S, Deal C, Giersing BK. Advancing vaccine development for gonorrhoea and the Global STI Vaccine Roadmap. Sex Health. 2019 Sep 3;