Neisseria gonorrhoeae: morphology, characteristics, pathogenesis, diagnosis and treatment

November 20, 2020 Gaurab Karki Bacteriology 0




Neisseria gonorrhoeae: morphology, characteristics, pathogenesis, diagnosis and treatment
Neisseria gonorrhoeae: morphology, characteristics, pathogenesis, diagnosis and treatment

Neisseria gonorrhoeae:

  • Neisseria gonorrhoeae; commonly termed as gonococcus (gonococci in plural).
  • In 1879, the gonococcus was first described by Neisser in gonorrheal pus.
  • Members of the genus Neisseria dominate the mucous membranes of human and other animals.
  • Neisseria gonorrhoeae is responsible to cause the sexually transmitted disease gonorrhoeae.
  • Gonococci are suited to grow on mucous membranes and hence are not able to tolerate drying.
  • Their fragility restricts the transmission to direct contact between mucous membranes or the exchange of contaminated secretions.

Morphology of Neisseria gonorrhoeae:

  • Under the microscope, it appears as a gram-negative coccus which is present in pairs (diplococci) with the flattening of the adjacent sides.
  • The diameter ranges from 0.6-1 μm.
  • The diplococci have kidney/coffee bean shape.
  • It is a non-spore forming bacteria and is able to move using twitching motility.
  • Gonococci exhibit pili on their surface.
  • Pili enhances the binding of the cocci to the mucosal surfaces and promotes virulence by restricting phagocytosis.
  • Human red blood cells are agglutinated by piliated gonococci, but not red cells from other mammals.

Cultural and biochemical characteristics for identification of Neisseria gonorrhoeae:

  • Gonorrhoeae is a fragile organism with strict environmental and nutritional requirements.
  • At pH 7.0-7.4 and at a temperature of 35-36 °C, growth occurs best.
  • It is an aerobe meaning it needs oxygen to grow, however it can grow in anaerobic conditions as well.
  • The supply of 5-10 percent CO2 is needed.
  • They grow rapidly on chocolate agar and Mueller-Hinton agar.
  • The Thayer-Martin medium (chocolate agar containing antimicrobials such as vancomycin, colistin and nystatin) is a common selective medium that inhibits most contamination, including nonpathogenic Neisseria.
  • Trimethoprim lactate may be introduced to the Thayer-Martin medium to suppress swarming Proteus species that are sometimes present in cervicovaginal and rectal specimens.
  • Neisseria gonorrhoeae shows positive oxidase test (having cytochrome c oxidase) and is catalase positive as well, i.e. it can convert hydrogen peroxide to oxygen.
  • The acid production takes place only from glucose and not from maltose, and also it does not ferment lactose or sucrose.
  • It tests negative for nitrate reduction test.
  • In enzyme substrate test, it tests positive for hydroxyprolylaminopeptidase.

Colonial morphology of Neisseria gonorrhoeae:

  • In a humid aerobic environment enriched with 5-10% CO2, colonies are thin, round, translucent, convex or slightly umbonate, with finely granular surface and lobate margins after incubation for 24 hours.
  • The colonies of Neisseria gonorrhoeae are pinkish brown.
  • They are easily emulsifiable and soft.
  • The colonies are larger (1.5-2.5 mm) after 48 hours, often with a crenated margin and an opaque elevated center.
  • The transparent, golden-brown pigmentation noticeable in 48 hr cultures after incubation results from cell autolysis.
  • With gonococcal colonies, substantial variance in size exists and the colony out-line is irregular in most culture media, unlike the circular colonies of N. meningitidis.
  • Growth is slower on the Thayer-Martin medium.
  • Even if colonies are similar to those on the MNYC medium, they are typically smaller.
  • Types of gonococci:
  • On the basis of colonial appearance, auto-agglutinability and virulence, Kellogg classified gonococci into four types (T1-T4).
  • T1 and T2 form small brown colonies and possess several numbers of fimbriae (pilated types P1 and P2). They are virulent and auto-agglutinable.
  • Types T3 and T4 are non-piliated (P-), are avirulent type and form smooth suspensions.
  • The fresh isolates obtained from acute cases of gonorrhea normally form T1 and T2 colonies.
  • They transition to T3 or T4 colonial morphology in serial subculture.
  • The forms T1 and T2 are often referred to as P+ and P++, while T3 and T4 are known as P-.

Virulence factors of Neisseria gonorrhoae:

  • Being typical gram-negative bacteria, Neisseria gonorrhoeae has thin peptidoglycan layer that is sandwiched between the inner cytoplasmic and outer membrane.
  • A true carbohydrate capsule does not enclose the outer surface.
  • Gonococci are antigenically diverse and are able to modify their surface structures invitro.
  • They likely do so in vivo as well in order to prevent host defense.
  • The following are included in the surface structures:
  • Pili:
    • Pili are hair-like appendages that reach out of the gonococcal surface up to several micrometers.
    • By facilitating attachment to host cells and inhibiting phagocytosis, they serve as virulence factors.
    • Pili is composed of repeating subunits of protein (pilins), whose expression is regulated by the pil gene complex.
    • At the amino terminal end, pilin proteins have a conserved region and at the exposed carboxyl terminus, a highly variable region is present.
    • Pili goes through antigenic and phase variations.
  • Por protein (Protein I):
    • The Por protein is an essential outer membrane protein present on all gonococcal strains.
    • It forms pores or channels in the outer membrane.
    • Even if the protein I shows considerable heterogeneity among different strains, protein I of single strain is antigenically constant.
    • Two types of Por proteins have been described (PorA and PorB), each with a range of antigenic variations.
    • Only one type of Por is expressed by each strain of gonococcus, but the Por of various strains is antigenically distinct.
    • Any single strain carries either IA or IB only, but not both.
    • Utilizing monoclonal antibodies to protein I epitopes, gonococci can be categorized into multiple serovars, AI to 24 and BI to32.
  • Opa proteins (Protein II):
    • Opa are the opacity proteins that are variably expressed on gonnacoccal strains that is responsible for phenotypes of various colony.
    • These proteins promote bacterial attachment to each other along with eukaryotic cells and also for the clumping of cocci seen in urethral exudate smears.
  • Rmp (Protein III):
    • Highly conserved Rmp proteins (reduction-modifiable proteins, formerly protein III) are the third group of proteins in the outer membrane.
    • These proteins induce antibodies which inhibit the serum bactericidal activity against N. gonorrhoeae.
  • Lipo-oligosaccharide (LOS):
    • This antigen has endotoxic activity and is composed of lipid A and a main oligo-saccharide identical to gram-negative lipopolysaccharide (LPS).
  • Other proteins:
    • The other important gonococcal proteins are IgA protease which degrades secretory IgA and beta-lactamase which degrades penicillin.
    • Fbp (iron-binding protein), similar to Por in molecular weight, is expressed when the supply of iron available is restricted, for example, in human infection.

Resistance showed by gonococcus:

  • Gonococcus is a very sensitive organism, readily destroyed by drying, soap and water, and many other washing agents or antiseptic agents when used and diluted correctly.
  • Organisms can remain viable in pus-contaminating linen or other fabrics for a day or so.
  • In cultures, the coccus dies in 3-4 days at room temperature.
  • The most effective method for long-term gonococcus storage is freeze-drying, but storage at-70 ° C or in liquid nitrogen might be more convenient for intermediate storage.

Pathogenesis of Neisseria gonorrhoeae:

  • Gonorrhea is a sexually transmitted disease.
  • Gonorrhea is a condition that is essentially limited to humans’ mucus-secreting epithelial cells.
  • The first stage in the infection is the adhesion of gonococci to the urethra or other mucosal surfaces.
  • For the first attachment, the existence of pili is important.
  • As the adhesion is quick and firm, the micturition after exposure offers no protection against infection.
  • By the third day following infection, the cocci penetrate into the intercellular spaces and enter the subepithelial connective tissue.
  • Gonococci presumably penetrate between columnar epithelial cells.
  • Stratified squamous epithelium is comparatively resistant to infection.
  • The period of incubation is 2-8 days.
  • Gonococci invade the genitourinary tract, eye, rectum, and throat mucous membranes, causing acute suppuration that can lead to tissue invasion; which are followed chronic inflammation and fibrosis.

Gonococcal Infection in men:

  • A few days after unprotected vaginal or anal sexual intercourse, acute urethritis in males is the most common clinical presentation.
  • Discharge or dysuria normally occurs within 1 week of exposure, but there are never any signs or symptoms for as many as 5-10 percent of patients.
  • The discharge is characteristically purulent, and in the gram stain of the exudate, gram-negative intracellular diplo-cocci can be easily seen.
  • Inguinal lymphadenopathy is often present, and on the penile shaft and corona, occasionally frank lymphangitis may develop.
  • There may be an asymptomatic condition in men up to several weeks after infection.
  • Chlamydial urethritis or non-gonococcal urethritis (NGU) is the differential diagnosis of gonococcal urethritis due to non-chlamydial etiology such as Mycoplasma genitalium.
  • With a history of receptive rectal intercourse, symptomatic anorectal gonococcus disease exists in men.
  • Around 50 percent have symptoms, including pain in the rectum, discharge, constipation, and tenesmus.
  • Some acute sexually transmitted rectal infections (STIs), such as herpes simplex, chlamydia proctitis and syphilitic proctitis, are included in the differential diagnosis.
  • Women who have endocervical gonorrhea and who have not usually had receptive rectal intercourse might also have anorectal diseases.
  • In these cases, contamination is believed to have occurred by the tracking of secretions throughout the perineum.
  • Indeed, up to 30% of such women also have rectal infections that coexist, but they are usually asymptomatic.
  • In either sex, Gonococcal pharyngitis can follow orogenital contact.
  • Conjunctivitis will typically occur by finger auto-inoculation.

Gonococcal infection in women:

  • The endocervix is the main site of infection in women and spreads to the urethra and vagina, causing mucopurulent discharge to occur.
  • In adults, the vaginal mucosa is typically not affected because the stratified squamous epithelium is immune to cocci infection and also due to the acid pH of vaginal secretions, but in prepubertal girls, serious vulvovaginitis can occur.
  • Asymptomatic carriage is prevalent in females, especially in the endocervical canal.
  • Vaginal discharge, dysuria, and abdominal pain are widely experienced by symptomatic patients.
  • Bartholin’s glands, endometrium, and fallopian tubes can be affected by the infection.
  • Gonococci ascend into the fallopian tubes during menstruation or after instrumentation, particularly at the end of pregnancy, to give rise to acute salpingitis, which may be accompanied by pelvic inflammatory disorders and a high risk of sterility if handled inadequately.
  • Occasionally, peritoneal spread occurs and can result in perihepatic inflammation (Fitz-Hugh-Curtis syndrome).
  • Clinical disease is less severe in women, many of whom may bear cervical gonococcus without any clinical symptoms.
  • Asymptomatic carriage of gonococci is rare in men.

Disseminated Gonococcal Infection:

  • An unusual complication is disseminated gonococcal infection (DGI, gonococcal septicemia).
  • Constitutional signs, fevers, chills, and a distinct set of syndromes consisting of oligoarticular septic arthritis, tenosynovitis, and rash characterize the condition.
  • The rash consists of pustular lesions on the extensor surfaces of the upper and lower extremities that are normally sparse (<20).
  • There are two typical clinical developments.
  • Tenosynovitis and rash patients are more likely to have positive blood cultures.
  • In patients with septic arthritis, in just 50 percent of cases, healthy blood and/or synovial fluid cultures are present.
  • In concert with the outcomes of genital and mucosal site cultures, the diagnosis is made clinically.
  • Any young sexually active patient presenting with fever, dermatitis and rheumatological symptoms should be suspected of having a DGI diagnosis.

Gonococcal Diseases in children:

  • Ophthalmic Neonatorum:
    • Ophthalmia neonatorum in newborns is a nonvenereal infection.
    • Babies born to infected women may suffer from ophthalmia neonatorum, in which gonococcus coats the eyes as the baby passes through the birth canal.
    • Within a few days of birth, a severely purulent eye discharge with periorbital edema develops.
    • When untreated, ophthalmia easily leads to blindness.
    • The practice of instilling 1 percent silver nitrate solution into the eyes of all newborn babies (Crede’s method) has controlled this.
    • Alternatively, it is possible to use topical erythromycin; this has the benefit of being active and less toxic against Chlamydia.
  • Vulvovaginitis:
    • Vulvovaginitis can be caused by gonococci in pre-pubertal children.
    • This happens either through bad hygiene conditions or by sexual abuse.
    • It should always be carefully invested and placed in communication with social workers and other practitioners capable of coping with this difficult situation.

Epidemiology of Neisseria gonorrhoeae:

  • Gonorrhea exists in humans only and no other reservoir is yet known.
  • Although a proportion of those infected, especially women, may remain asymptomatic, it is never discovered as a normal commensal.
  • Gonococcal disease has historically been a key factor in areas where there has been limited access to healthcare services.
  • This is reflected in the high rates of gonorrhea in the developing world, particularly in sub-Saharan Africa and Asia, as well as in the rates currently rising in the former Eastern Bloc/Soviet Union.
  • Typically, acute gonorrhea is quickly detected and treated, and was well managed until the 1960s in most of the world.
  • The occurrence of new cases worldwide was estimated at 16 million in1970, making it one of the most common infectious diseases.
  • In the 1980s, with the AIDS scare, there was a significant decrease in gonorrhea incidence, although this has not been sustained.
  • A higher frequency of gonorrhea was found in patients belonging to group B of the blood, the basis for this is yet not understood.

Laboratory diagnosis of Neisseria gonorrhoeae :

  • In the acute stages, the diagnosis can be easily carried out. However, the chronic cases are difficult to be diagnosed.
  • Specimens:
  • Specimens in Men:
    • In acute gonorrhea, gonococci are present in large numbers in the urethral discharge.
    • The meatus is washed with a saline-soaked gauze and a discharge sample obtained with a platinum loop for culture or directly on a slide for smears.
    • Purulent discharge can be conveyed and collected with a swab in the anterior urethra.
    • There may be no urethral discharge in chronic infections.
    • The morning drop of secretion may be investigated or some exudate after prostatic massage may be collected.
    • In cases where no urethral discharge is available, it may also be possible to demonstrate gonococci in centrifuged urine deposits.
    • Anal canal – rectal culture in homosexual males.
  • Specimens in Women:
    • Urethral, cervical and rectal specimens should always be examined in women.
    • A single well-taken endocervical swab can detect about 90% of women’s gonococcal infections.
    • A high vaginal swab is not appropriate.
    • Infection of the throat also happens and should be checked where possible.
  • Specimens in both male and female:
    • Blood, swabs of skin lesions, or pus aspirated from a joint.
    • Conjunctival swab, particularly in the context of neonatal ophthalmia.
  • Transport of specimens:
    • For culture, specimens should be inoculated, immediately upon collection, on pre-warmed plates.
    • If this is not feasible, specimens should be obtained with swabs impregnated with charcoal and transmitted to the laboratory in the Stuart’s transport medium.
  • Direct microscopy:
    • Gram staining is performed with a few extracellular species that are typical of gonococcal infection.
    • The smear is confirmed as positive, showing characteristic kidney-shaped gram-negative diplococci lying inside polymorphonuclear leucocytes.
    • About 95 percent of males infected would develop a positive smear.
    • It must be stressed that the diagnosis of gonorrhea by smear analysis in women is inaccurate since some of the typical genital flora has a morphology that is essentially identical.
    • The use of fluorescent antibody techniques in the smear identification of gonococci is more sensitive and specific diagnosis by microscopy.
  • Culture:
    • In acute gonorrhea, cultures can be acquired commonly on chocolate agar or Mueller-Hinton agar incubated at 35-36°C along with 5-10% CO2.
    • However, it is best to use a selective medium such as the Thayer-Martin medium in chronic cases where mixed infection is normal and when examining lesions such as proctitis.
    • After 24 hours of incubation, the plates are examined and morphology and biochemical reactions identify the growth.
    • Incubation of primary isolation plates is continued for 48 hours and the above-mentioned procedures re-examine cultures until any specimen can be negatively reported.
    • In a humid aerobic environment enriched with 5-10 percent CO2, colonies are thin, round, translucent, convex or slightly umbonate, with fine granular surface and lobate margins after incubation for 24 hours.
    • They are easily emulsifiable and soft. The colonies are larger (1.5-2.5 mm) after 48 hours, often with a crenated margin and an opaque center elevated.
    • Smear is obtained from the colony and Gram staining is done.
    • Gonococci are pair-arranged gram-negative cocci (diplococci) with concave (pear or bean shaped) adjacent sides.
  • Identification:
    • Gonorrhoeae are preliminarily defined on the basis of the isolation of oxidase-positive, gram-negative diplo-cocci that are selective for pathogenic Neisseria species and grow on chocolate blood agar.
    • N. gonorrhoeae is oxidase positive.
    • It only ferments glucose with acid.
  • Genetic probes:
    • Gonorrhoeae were created in clinical specimens to directly detect bacteria.
    • Tests are sensitive, precise, and rapid (results are available in 2 to 4 hours) using these probes.
  • Serological diagnosis:
    • It is not really possible to obtain gonococci in culture from some chronic cases as well as from patients with metastatic lesions like arthritis.
    • In such cases, serological tests are essential.
    • Complement fixation tests
    • Immunoblotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay) tests.
    • However, for routine diagnostic purposes, there was no serological test found to be useful.
    • Such tests are neither sensitive nor precise and are not recommended for use.

Treatment of Gonorrhea:

  • Penicillin is no longer the antibiotic of preference for treatment of gonorrhea.
  •  Since the development and wide-spread use of penicillin, gonococcal resistance to penicillin has gradually risen, owing to the selection of chromosomal mutants, so that many strains now need high concentrations of penicillin G for inhibition (MIC 2 μg/ml).
  • Empirical therapy:
    • It is troublesome to select successful empirical therapy since the incidence of antibiotic resistance in gonococci is increasing.
    • The Centers for Disease Control and Prevention (CDC) is currently promoting the use of ceftriaxone, cefixime, ciprofloxacin, or ofloxacin as an initial treatment for uncomplicated cases of gonorrhoea.
    • For infections complicated by dual Chlamydia infections, doxycycline or azithromycin should be added.

Penicillinase Producing Gonococci (PPNG):

  • Gonococci generating β-lactamase (penicillinase) emerged in1976, making treatment with penicillin ineffective.
  • These penicillinase-producing gonococci (PPNG) were isolated in the United States and England from widely separated areas and have spread widely.
  • Penicillinase development is plasmid-mediated in gonococci.

Chromosomally Mediated Resistance gonococci (CMRNG):

  • Isolation of penicillin resistant N. gonorrhoeae that do not produce β-lactamase have been carried out.
  • This chromosomal mediated resistance (CMRNG) is not limited to penicillin alone, but also applies to tetracyclines, erythromycin, and aminoglycosides, resulting from cell surface changes that prevent the antibiotic from entering the gonococcal cell.
  • In Africa, Southeast Asia, Australia, and some US cities, resistance to fluroquinolones like ciprofloxacin has also become prevalent.

Control of gonorrhea:

  • Gonorrhoea prevention consists of early cases identification, contact tracing, health education, and other general steps.
  • The rate of transmission is significantly decreased by barrier methods of contraception, condoms in particular.
  • Owing to the increase in gonococcus antibiotic resistance, chemoprophylaxis is of minimal benefit.
  • Since even clinical disease does not grant any immunity, vaccination has no place in prophylaxis.