Neisseria

                The genus Neisseria consists of Gram negative, aerobic, non-sporulating, nonmotile, oxidase positive cocci typically arranged in pairs(Diplococci).They occur in CSF, urinogenital tract, commensals of mouth or the upper respiratory tract. They are saccharolytic, catalase positive, G+C content 47-52mol %. Pathogenic Neisseria species are fastidious organisms, requiring enriched media for optimal recovery.

N. meningitidis (meningococcus) – occur in cerebrospinal fluid (CSF). It causes meningococcal meningitis (cerebrospinal fever), occur as localised outbreaks or epidemics.

Morphology & Cultural characteristics -  They are Gram negative oval or spherical cocci, 0.6-0.8µm in size, arranged in pairs with adjacent sides flattened. They are nonmotile, some fresh isolates are capsulated. They are typically seen in large numbers inside polymorphonuclear leucocytes.

Growth occurs on media enriched with blood or serum. They are strict aerobes, optimum temperature for growth is 35-36⁰C, optimum pH is 7.4-7.6, requires high humidity and 10% CO2. On solid media after incubation of 24 hours colonies are small, translucent, round, convex, bluish grey, smooth glistening surface and entire edges.Weak hemolysis on blood agar, smooth and rough type of colonies are found. Commonly used media are Blood agar, Chocolate agar, Mueller-Hinton Starch Casein hydrolysate agar, selective medium is Thayer-Martin medium. They are catalase, oxidase positive. Glucose and maltose are fermented, but not sucrose and lactose, producing acid but not gas.

They are killed by heating at 55⁰C in 5 minutes, highly susceptible to dessication and oxidation.  Weak disinfectants such as 1% phenol or 1% mercuric chloride kill them in 1-2 minutes.

Meningococci possesses a polysaccharide capsule and on the basis of immunologic specificity of capsular polysaccharide it has been subdivided into 13 serogroups. Most meningococcal infections are caused by strains of groups A, B, C. It has outer membrane proteins and polysaccharides which are antigenic.They are susceptible to heat,dessication,alterations in pH and to disinfectants.They are uniformly sensitive to Penicillin and resistant strains have emerged.

Pathogenic Determinants

1. Capsular polysaccharide- contributes to the invasive properties of the pathogen by inhibiting phagocytosis. But in the presence of specific antibody and complement,  the organisms are readily destroyed by phagocytic leukocytes.
2. Endotoxins- These organisms produce large amounts of LPS-containing OM and during division, vesicles of this material are released extracellularly, which induces an inflammatory response an cause vascular necrosis.
3. IgA1 protease- All strains of meningococci produce an  IgA1 protease, excreted into extracellular environment. These enzymes are neutral endopeptidases with a substrate specifity for human IgA1 immunoglobulin. It cleaves the heavy chain in the hinge region and produce intact Fc and Fab fragments. IgA is the predominant immunoglobulin involved in mucosal defence. IgA1 protese cleaves the immunoglobulin molecule and inactivates it.
4. Transferrin-bound iron as a sole source of iron- Meningococci can use transferrin-bound iron as a sole source of iron while nonpathogenic species lack the ability to use iron from transferrin.
5. Pili- they possess pili on their surface which allow intimate contact with the host cell and organisms release endotoxin. Pili may also have antiphagocytic effect.
6. OM proteins- divided into 5 classes,class 5 associated with attachment to the host cell.

Pathogenicity- Cerebrospinal meningitis and meningococcal septicemia are the two main types of meningococcal disease. Meningococci are strict human parasites inhabiting the nasopharynx.

Bacterial meningitis is an inflammation of meninges,the membrane that cover the brain and spinal cord. Meningitis causes necrosis (death of tissues in the infected area), clogging of  blood vessels, increased pressure with in the skull, decreased cerebrospinal fluid flow and impaired central nervous system function. The early symptoms are headache, fever, chills. Death occurs from shock and other serious complications within hours of the appearance of symptoms. In meningococcal meningitis the organisms colonize the nasopharynx, spread to the blood and make their way to the meninges, where they grow rapidly. Sometimes it invades all parts of the body and death occurs within hours from endotoxin shock. The immediate death is usually due to blood clotting followed by massive hemorrhage in the adrenal glands leading to fatal deficiency of essenrial adrenal hormones. A lesser degree of hemorrhaging is sometimes seen in meningitis patients who develops a skin rash.

The meningococcal disease can be divided into 3 stages:

              1^st stage- The organism appear in nasopharynx leading to nasopharyngeal infections,which is usually asymptomatic with minor inflammation. This will induce the formation of protective antibodies with in a week. The infection in the nasopharynx may extend into adjacent areas giving rise to conjunctivitis, pneumonia etc.

            2^nd stage- In a small percentage of cases the meningococci may enter the bloodstream from posterior nasopharynx. This stage is known as meningococcaemia. The patient develops fever, malaise and petechial skin lesions due to the infection in capillaries. The organisms may also cause lesions in the joints and lungs,and may cause bilateral haemorrhages in the adrenals. This is known as Waterhouse-Friderichsen syndrome. Meningococci may also result in a condition known as disseminated intravascular coagulation,in which blood clots may block circulation to the extremities, necessitating amputation.

            3^rd stage- meningococci infect the meninges cause severe headache, stiff neck,vomiting, delirium and confusion.

Laboratory Diagnosis - CSF- It is diagnosed by culturing CSF. The fluid is turbid, so thick with pus and will be difficult to draw with a syringe, becuse of a large number of polymorphonuclear leukocytes. In centrifuged Gram stained smears, the meningococci seen mainly inside leukocytes. Strains causing meningitis are usually encapsulated and may show a distinct pink halo surrounding the cells. The CSF is inoculated on blood agar or chocolate agar for the biochemical reactions.

            Blood culture- Specimen of blood is inoculated into blood culture bottle of  Trypticase-Soy broth, incubated at 35-36⁰C in 5-10% CO₂ for 4-7 days, with daily subcultures on blood agar.

            Pus aspiration & swabs- Thayer-Martin selective medium is used for the culture of materials expected to yield a mixture of organisms(pus, aspirates,throat, nasopharyngeal and genital swabs). Vancomycin, nystatin,colistin are present in the medium to inhibit the Gram positives,yeast,Gram negatives respectively.

            Serological diagnosis- Specific antibodies to capsular polysaccharide may be demonstrated by hemagglutination test.

Penicillin is the drug of choice, also third generation cephalosporins and ampicillin used. Vaccines available to type A and C, not to B. The risk can be decreased by prevention of overcrowding.The human nasopharynx is the only reservoir of the organism. Transmission by airborne droplets or by fomites.

N. gonorrhoeae- causes the veneral disease gonorrhea,isolated from urethral discharge of patients.

Morphology & Cultural characteristics - The organism appears as diplococcus with adjacent sides concave or kidney-shaped. It is found predominantly in polymorphic cells(neutrophils). They possess pili on their surface, which facilitates adhesion of cocci to mucosal surface and promote virulence by inhibiting phagocytosis.

They are aerobic or facultatively anaerobic. Best growth at pH 7.2-7.6, temperature 35-36⁰C. It is essential to provide 5-10% CO2. They grow well on Chocolate agar, Mueller-Hinton agar, Theyer-Martin medium. Colonies are small, round translucent convex or slightly umbonate, with finely granular surface and lobate margins. They are soft and easily emulsifiable. It ferments only glucose.

Pili and hair-like structures act as virulence factors by promoting attachment to host cells and inhibiting phagocytosis. It will undergo antigenic variation. The outer membrane of gonococci contain many different proteins. The OM also contains LPS(endotoxin) which may be responsible for the toxicity in gonococcal infections. It is a very delicate organism,readily killed by heat, drying and antiseptics. It is a strict parasite and dies in 1-2 hours outside the body. Now it has developed resistance to the antibiotics such as Penicillin.

Pathogenicity -  Gonococci produce an endotoxin that damages the mucosa in Fallopian tubes and releases enzymes such as proteases and phospholipases that are important in pathogenesis. They also produce an extracellular protease that cleaves IgA, the immunoglobulin present in secretions.They adhere to neutrophils and are phagocytized by them. Phagocytosis kills some of the bacteria, but survivors multiply inside PMNLs.

Gonorrhea is veneral disease and acquired by sexual contact. The infection starts by the adhesion of gonococci to the urethra or other mucosal surfaces by means of pili. Then it penetrates through the intercellular spaces and reach the subepithelial connective tissue. The incubation period is 2-8 days.Transfer of organisms by contaminated hands or fomites from genitals,results in eye infections. Also organisms present in birth canal enter the eyes as a baby is born. The resulting infection causes keratitis, an inflammation of cornea,which progress the perforation and destruction of cornea and blindness. Tetracyclines are the effective treatment. A few drops of 1% AgNO3 solution in the eyes immediately after birth kills gonococci but irritate eyes. So Ophthalmia neonatorum is a nonveneral disease.

Laboratory Diagnosis - The  specimen  swabs  collected  should  be  either  inoculated  directly  on  to   growth   medium  or transported to the laboratory in a  transport  medium  such   as   Amies  medium  with  charcoal  and  plated with in 6 hours. The specimens include synovial fluid, blood, conjunctival swab, oropharynx, anal canal, urethra, vagina, Bartholin’s glands. The best way to prevent gonorrhea is to avoid sexual contact with infected individuals, detection of cases, health education etc..

            Control of gonoorrhoea consists of early detection and treated.