Pathogenic gram-negative cocci. Neisseria. Morphology and biological properties. Laboratory diagnostics of gonococcal and meningococcal infections.
Taxonomy of medical important Gram-negative cocci:
Genus Neisseria includes about 14 species as pathogenic and non-pathogenic ones.
Pathogenic species: N. gonorrhoeae and N. meningitides
Neisseria gonorrhoeae (Gonococcus)
N. gonorrhoeae causes the venereal disease gonor¬rhea. The gonococcus was first described in gonor¬rheal pus by Neisser in 1879.
Morphology: They are Gram negative oval or spherical diplococci 0.6-0.8 μm in size, typically arranged in pairs, with the adjacent sides flattened. They are resemble to coffee corns or kidneys. Gonococci are non-motile, non-sporeforming, but they form microcapsules. In smears from the urethral discharge in acute gonorrhea, the organism appears as a diplococcus with the adjacent sides concave, being typically kidney shaped.
Gonococci such as meningococci are auxotrophs. They require special media for cultivation and do not grow on ordinary media. Growth occurs on media enriched with blood, serum or ascitic fluid.
Gonococci are more diffi¬cult to grow than meningococci. They are aerobic but may grow anaerobically also. Growth occurs best at pH 7.2-7.6 and at a temperature of 35-36 °C. It is es-sential to provide 5-10 per cent CO2.
Colonies are small, round, translucent, convex or slightly umbonate, with finely granular surface and lobate margins. They are soft and easily emulsifiable.
Biochemical reactions: They possess weak enzymatic activity. Unlike to meningococci, gono-cocci ferment only glucose and not maltose. They can liquefy gelatin.
Antigenic properties: Gonococci are antigenically heterogeneous.
1. Pili (adhesins) which are hair-like structures several mi¬crometres long, act as virulence factors by promoting attachment to host cells and inhibiting phagocytosis.
2. The outer membrane also contains lipopolysaccharide (endotoxin) which may be responsible for the toxicity in gonococcal infections
3. IgA – protease inhibits protective secretory immunoglobulins and weakens local immunity
Resistance: The gonococcus is a very delicate organ¬ism, readily killed by heat, drying and antiseptics. It is a strict parasite and dies in 1-2 hours in exudates out¬side the body. Formerly, it was highly susceptible to sulphonamides, penicillin and many other antibiotics. However, gonococci have developed resistance to one antibiotic after another.
Pathogenicity: Gonorrhea is a venereal disease which are transmitted by sexual intercourse. The only source of infection is a human - carrier or less often a patient. The first step in infection is adhesion of gonococci to the urethra or other mucosal surfaces. Pili are involved in this adhesion.
The cocci penetrate through the intercellular spaces and reach the subepithelial connective tissue by the third day after infection. The incubation period is 2-8 days. In men, the disease starts as an acute ure¬thritis with a mucopurulent discharge containing gonococci in large numbers. The infection extends along the urethra to the prostate, seminal vesicles and epididymis. Chronic urethritis may lead to stricture formation. In women, the initial infection involves the ure¬thra and cervix uteri. The vaginal mucosa is not usu¬ally affected in adults because the stratified squamous epithelium is resistant to infection by the cocci and also because of the acid pH of vaginal secretions. The in¬fection may extend to Bartholin's glands, endometri-um and fallopian tubes. Pelvic inflammatory disease and salpingitis may lead to sterility. Rarely, peritonitis may develop with perihepatic inflammation.Clinical disease is as a rule less severe in women, many of whom may carry gono¬cocci in the cervix without developing any clinical symptoms. Asymptomatic carriage of gonococci is rare in men.
Conjunctivitis may occur, usually due to autoinoculation by the patient's fingers. Blood invasion may occur from the primary site of infection and may lead to metastatic lesions such as arthritis, ulcerative endocarditis and very rarely meningitis. A nonvenereal infection is gonococcal ophthal¬mia in the newborn, which results from direct infec¬tion during passage through the birth canal. It has been controlled by the practice of instilling 1% silver nitrate solution into the eyes of all newborn babies
In the acute stage, diagnosis can be established readily but chronic cases some¬times present great difficulties. In acute gonorrhea the urethral discharge contains gonococci in large num¬bers. That is why microscopy of stained smears is reliable to diagnose acute cases.
The use of fluorescent antibody techniques for the identifica¬tion of gonococci in smears has increased the sensitiv¬ity and specificity of diagnosis by microscopy.
Culture: specimens should be inoculated on prewarmed plates, immediately on collection. In acute gonorrhea, cul¬tures can be obtained readily on chocolate agar incubated at 35-36 °C under 5-10 per cent CO2.
The complement fixation test becomes positive only some weeks after the infection is established and may remain positive for months or years after the disease has been cured. So, serological method is more often used for diagnostics of chronic infections.
Therapy and prophylaxis:
The Centers for Disease Control and Prevention, USA in 1993 recommended the following schedule for uncomplicated gonorrhea: Ceftriaxone 125 mg single IM dose or Ciprofloxacin 500 mg (or Ofloxacin 400 mg) single oral dose, plus Doxycycline 100 mg twice daily for 7 days or Erythromycin 1 g single oral dose. The regimen is The Centers for Disease Control and Prevention, USA in 1993 recommended the following schedule for uncomplicated gonorrhea: Ceftriaxone 125 mg single IM dose or Ciprofloxacin 500 mg (or Ofloxacin 400 mg) single oral dose, plus Doxycycline 100 mg twice daily for 7 days or Erythromycin 1 g single oral dose.
Control of gonorrhea consists of early detection of cases, contact tracing, health education and other general measures. As even clinical disease does not confer any immunity, vaccination has no place in prophylaxis.
Neisseria meningitidis (Meningococcus):
Meningococcus was first described and isolat¬ed in 1887 by Weichselbaum from the spinal fluid of a patient.
N. meningitidis causes meningococcal meningitis (formerly also known as cerebrospinal fever) which may occur sporadically, as localised outbreaks or as epidemics, and also septicemia.
Meningococci are Gram negative oval or spherical cocci 0.6-0.8 μm in size, typically arranged in pairs, with the adjacent sides flattened. In smears from lesions, the cocci are more regular and generally intracellular. They are nonmotile. Most fresh isolates are capsulated.
Such as gonococci meningococci have exact¬ing growth requirements and do not grow on ordinary media. They are strict aerobes, no growth occurring anaerobically. The optimum temperature for growth is 35-36 °C. No growth takes place below 30 °C. Opti¬mum pH is 7.4-7.6. Growth is facilitated by 5-10 per cent CO2 and high humidity. On solid media, after incubation for 24 hours, the colonies are small (about 1 mm in diameter) translu¬cent, round, convex, bluish grey, with a smooth glis¬tening surface and with entire edges. Weak hemolysis occurs on blood agar. Growth is poor in liquid media, producing a granular turbidity with little or no surface growth.
Blood agar, chocolate agar and Mueller-Hinton starch casein hydrolvsate agar are the media common¬ly used for culturing meningococci. Modified Thayer -Martin (with vancomycin, colistin and nystatin) is a useful selective medium.
They are catalase and oxidase positive. Glucose and maltose are fermented, but not sucrose or lactose, producing acid but no gas (gonococci ferment glucose but not mal¬tose).
Antigenic properties and classification:
Meningo¬cocci are capsulated, unlike other neisseriae. Based on their capsular polysaccaride antigens, meningococci are classified into at least 13 serogroups, of which Groups A, B and C are the most important. Group A is usually associated with epidemics and Group C mostly with localised outbreaks, while Group B causes both epidemics and outbreaks. Groups 29-E, W-135 and Y also frequently cause meningitis.
Meningococci are very delicate organ¬isms, being highly susceptible to heat, dessication, alterations in pH and to disinfectants. They were uniformly sensitive to penicillin and other antibiotics, but resistant strains have emerged and become com¬mon in many areas.
1. Endotoxin (LPS)is released by auto¬lysis. The vascular endothelium is particularly sensi-tive to the endotoxin.
2. Pili (adhesins) are responsible to attachment to nasophayngeal epithelium.
3. Capsule protects from phagocytosis.
Cerebrospinal meningitis and menin¬gococcal septicemia are the two main types of menin-gococcal disease. Meningococci are strict human parasites inhabiting the nasopharynx. Infection is usu¬ally asymptomatic. In some, local inflammation en¬sues, with rhinitis and pharyngitis. The manner in which the cocci spread from the nasopharynx to the meninges is controversial. On reaching the central nervous system, a suppurative le¬sion of the meninges is set up, involving the surface of the spinal cord as well as the base and cortex of the brain. The cocci are invariably found in the spinal flu¬id, both free and within the leucocytes. Case fatality is variable but in untreated cases may be as high as 80 per cent.
Meningococcemia presents as acute fever with chills, malaise and prostration. Typically a petechial rash occurs early in the disease. A few develop fulminant meningococcemia (for¬merly called Waterhouse-Friderichsen syndrome) which is an overwhelming and usually fatal condition, characterised by shock, disseminated intravascular coagulation and multisystem failure.
The human nasopharynx is the only reservoir of the meningococcus. Asymptomatic na-sopharyngeal carriers rarely contract the illness but serve to infect their contacts. Transmission is essen¬tially by airborne droplets or less often by fomites. Meningitis is common in children between 3 months and 5 years of old. Epidemics usually occur in semi-closed communities living in crowded conditions, as in jails and ships formerly, and in army camps in re¬cent times.
In meningo¬coccal meningitis, the cocci are present in large num¬bers in the spinal fluid and, in the early stage, in the blood as well. Demonstration of meningococci in the nasopharynx helps in the detection of carriers.
Examination of CSF:
1. Microscopy. The fluid will be under pres¬sure and turbid, with a large number of pus cells. Under microscopy CSF contains a large number of meningococci inside polymorphs but often extracellularly al¬so.
2. Detection of capsular antigen with serological tests. They may be demonstrated by precipitation using meningococcal antisera.
3. Culture method: CSF is inoculated on blood agar or chocolate agar plates and incubated at 35-36 °C under 5-10 per cent. CO2. Colonies appear after 18-24 hours and may be identified by morphol¬ogy and biochemical reactions.
Blood culture: In meningococcemia and in early cases of meningitis, blood culture is often positive.Cultures should be incubated for 4-7 days.
Nasopharyngeal swab: This is useful for the detection of carriers. Collected specimens are cultivated and identified as it is described for CSF culture.
Retrospective diagnose of meningococcal infection may be obtained by detection of antibodies with complement fixation test.
Treatment and prophylaxis:
Intravenous penicillin G is the treatment of choice. Chloramphenicol is equally effective. One of the later cephalosporins (Ceftriaxone, Ceftazidime) may be used for the initiation of treatment before the etiology of meningitis is known.
Monovalent and polyvalent vaccines are availa¬ble containing the capsular polysaccharides of groups A, C, W-135 and Y. The vaccines induce good immu¬nity after a single dose in older children and adults but are of little value in infants. The immunity is group specific. There is no Group B vaccine available at present. As attack rates are very high in the households or close contacts of meningococcal patients, they should be provided with chemoprophylaxis.