Meningococcus: Introduction, Culture characteristics, Pathogenesis, Laboratory Diagnosis and Treatment

Meningococcus is the common name of Neisseria meningitidis and causes meningitis (infections of the lining of the brain and spinal cord) and bloodstream infections (bacteremia or septicemia). The genus Neisseria is named after the German bacteriologist Albert Neisser, who discovered Neisseria gonorrhoeae, the pathogen that causes the human disease gonorrhea in 1879. Later, in 1887 Weichselbaun isolated meningococcus from the cerebro-spinal fluid (CSF) of a patient. Meningococcus spreads through the exchange of respiratory and throat secretions like spit (e.g. by living in close quarters, kissing). Antimicrobial agents as well as vaccine both are available to save life from this bacterial infections.

Introduction of Meningococcus

Meningococcus is the common name of Neisseria meningitidis and causes meningitis (infections of the lining of the brain and spinal cord) and bloodstream infections (bacteremia or septicemia). The genus Neisseria is named after the German bacteriologist Albert Neisser, who discovered Neisseria gonorrhoeae, the pathogen that causes the human disease gonorrhea in 1879. Later, in 1887 Weichselbaun isolated meningococcus from the cerebro-spinal fluid (CSF) of a patient. Meningococcus spreads through the exchange of respiratory and throat secretions like spit (e.g. by living in close quarters, kissing). Antimicrobial agents as well as vaccine both are available to save life from this bacterial infections.

Scientific classification of Meningococcus

Domain: Bacteria

Phylum: Proteobacteria

Class: Betaproteobacteria

Order: Neisseriales

Family: Neisseriaceae

Genus: Neisseria

Species: Neisseria meningitidis

The Neisseria are Gram negative diplococci.

N. meningitidis and N. gonorrhoeae are the pathogenic species of this genus.

General characteristics of  Neisseria meningitidis

Following are the general features of N. meningitidis-

  1. They are Gram-negative spherical , oval or cocci , bean-shaped (adjacent sides flattened), in pairs  or diplococci.
  2. Do not possess flagella or spores
  3. They are capsulated and possess pili.
  4. Strict parasites, do not survive long outside of the host
  5. They are strict aerobes and thus can not grow anaerobically.
  6. Oxidative metabolism
  7.  Produce enzymes catalase and oxidase
  8. Pathogenic species require enriched complex media and CO2.

Morphology of Neisseria meningitidis

  1. They are Gram-negative spherical , oval or cocci ,  0.8 x 0.6 µm  in size arranged in pairs with the adjacent sides flattened. sides flattened).
  2. They do not possess flagella or spores.
  3. They are capsulated and possess pili.

Cultural characteristics of Neisseria meningitidis

They do not grow on ordinary media like nutrient agar but have exacting growth requirements but can grow in non selective media like blood agar, Chocolate agar and Mueller-Hinton starch casein hydrolysate agar. Growth is improved by addition of blood or serum. Growth is also improved by incubation in the presence of 5- 10 % CO2. Growth temperature is 35-36⁰C and pH ranges of 7.4-7.6. Colonies are 1-2 mm in diameter, convex, grey and transparent. No hemolysis in blood agar. Selective media Modified Thayer-Martin medium with antbiotics ( vancomycin, colistin, nustatin and trimethoprim) and New-York City medium.  Colony characteristics on Modified Thayer-Martin medium are as follows-

  • Color: Bluish grey
  • Shape: Round
  • Size: About 1 mm
  • Surface: Smooth
  • Elevation: Convex
  • Opacity: Translucent
  •  Consistency: Butyrous

Biochemical properties of N. meningitidis 

They are oxidase-positive; i.e., they possess the enzyme cytochrome and produce oxidase. Meningococcus is maltose fermenter and does not produces beta lactamases. It has three important virulence factors: 1. Polysaccharride capsule. It is antiphagocytic in nature. 2. The endotoxin of N. meningitidis is a lipopolysaccharide (LPS). It induces septic shock by causing release of cytokines. 3. IgA protease. It cleaves the IgA antibodies present in respiratory mucosa.

Pathogenesis of Meningococcus 

• Humans are the only natural hosts and they are transmitted by airborne droplets, They colonize the nasopharynx and become transient flora of the upper respiratory tract. From the nasopharynx, the organism can enter the bloodstream and spread to meninges and grow in the cerebrospinal fluid (CSF). N. Meningitidis is the most common cause of meningitis in persons between the ages of 2 and 18 years. Outbreaks of meningitis are most common in winter and early spring, and favored by close contact between individuals. They cause meningitis and meningococcemia (multiplication of bacteria in the blood stream).

Clinical Features 

Febrile illness : Mild and self limiting

Pyogenic meningitis : High fever, stiff neck, Kernig’s sign ( an indicator of subarachnoid hemorrhage or meningitis), severe headache, vomiting, photophobia, chills

Meningococcemia : acute fever with chills, malaise, prostation,  Waterhouse-Friderichsen syndrome (WFS)- is a group of symptoms resulting from the failure of the adrenal glands to function normally as a result of bleeding into the gland, Disseminated intravascular coagulation (DIC)

Other Syndromes : Pneumonia, arthritis, urethritis, respiratory tract infection,  Waterhouse- Fridericksen syndrome,  Meningococcal disease is favoured by defieciency of the terminal complement components (C5-C9).

Epidemiology 

The human are only reservoir of the N. meningitidis. 5-10% adults are  asymptomatic carriers.  Modes of infection is direct contact or respiratory droplets (nasopharyngeal carriers )from the nose and throat of infected people.  Prevalence of meningitis is highest in meningitis belt of Africa (frequent epidemics occurred there). In 1996, among 150000 cases 15000 deaths reported. Epidemic usually occurs in overcrouded araea. Inhalation of contaminated droplets adherence of organism to nasopharyngeal mucosa. Local invasion and spread from nasopharynx to meninges through blood stream (directly along perineural sheath of olfactory nerve, cribriform plate to subarachnoid space).  In meninges, organisms are internalized into phagocytic cells. They replicate and migrate to subepithelial spaces and incubation period is 3-4 days. Meningitis is more common in children below the age of 5 years and in males. Serogroup A, B and C are responsible for outbreak.

Laboratory diagnosis of Meningococcus

They are frequently isolated from samples such as blood and CSF. Other specimens petechial lesions, nasopharyngeal swab -especially to detect carrier may also  be used. Examination of CSF:  CSF should be turbid in this infection.The collected CSF is divided into 3 tubes: -Tube no 1 for chemical analysis( biochemistry) for glucose and protein estimation, tube 2 for microbiological tests ( microbiology) for Gram stain, latex agglutination test and culture- sensitivity while tube 3 record overall appearance -cell count ( WBCs in hematology). Microscopy: Gram stained smear of CSF deposit commonly shows Gram negative intracellular diplococci. White cell count increases to several thousand per cubic mm or µl with 90-99%  polymorphonuclear cells (PMNs) . Biochemical tests: Glucose is markedly diminished  while CSF protein is markedly raised.  CSF Culture: Inoculated into blood or chocolate agar and incubated at 37ºC in 5-10% Carbon dioxide (CO2) and high humidity. After 24 hours bacterial colonies appear and organism is tested for biochemical and agglutination reaction. Now in details, follow as-

Useful methods for laboratory diagnosis of Meningococcus are-

  • Gram staining
  • Culture
  • Biochemical test
  • Latex agglutination test
  • Serotypng
  • Molecular test
  • Electrophoresis

Gram staining:  The diagnosis is suggested by the finding of gram negative bacteria bean shaped capsular ( mark of evidence in Gram stain) diplococci as shown above image but capsular mark is not recovered gram stain from culture.

Culture: The organism is cultured on blood agar or chocolate agar incubated at 37°C in a 5% CO2 atmosphere. Colonies are 1-2 mm in diameter, convex, grey and transparent and n o hemolysis as shown above picture.

Biochemical test: Following biochemical tests are important for Meningococcus  identification-

Catalase test: Positive

Oxidase test :Determines the presence of cytochrome oxidase. It is Positive in N. meningitidis. Grow the isolate(s) to be tested for 18-24 hours on a blood agar plate at 35-37°C with 5% CO2. Dispense a few drops of Kovac’s oxidase reagent. Tilt the plate and observe colonies for a color change to purple. Positive reactions will develop within 10 seconds in the form of a purple color. Another method-Use a platinum wire or wooden stick to remove a small portion of a bacterial colony (preferably not more than 24 hours old) from the agar surface and rub the sample on the filter paper or commercial disk. Observe inoculated area of paper or disk for a color change to deep blue or purple within 10 seconds as shown above image.

Fermentation test: Ferments glucose and maltose with acid production

Nitrate  test : negative

Colistin resistant

Do not  ferment lactose, sucrose and fructose

Gamma-glutamyl aminopeptidase test:  positive

DNAase  test: Positive

Latex agglutination test: It detects capsular polysaccharide in the CSF.

Serotyping: Serogroups and serotpes-on the basis of specificity of capsular polysaccharide antigens divided into 13 serogroups . These are A,B,C,D,X,Y,Z,W -135,29-E,H,I,K and L. Serogroups A,B,C,X,Y,W 135 : most commonly associated with meningococcal disease Group A: epidemics Group C: localised outbreaks Group B: both epidemics and outbreaks.

Based on the outer membrane protein serogroups further divided into serotypes- About 20 serotypes have been identified.

Molecular test: For Detection of Meningiococcal DNA. Polymerase Chain Reaction (PCR) is very useful.

Electrophoresis:  For detection of soluble polysaccharide antigen , counter current immunoelectrophoresis (CIEP) is used.

 Treatment and Prevention

Penicillin G or sulphonamides are the drugs of choice. Cholramphenicol or third generation cephalosporin such as cefotaxime or ceftriaxone are recommended for patients who are allergic to penicillin. Meningococcal vaccine, is available which contains the capsular polysaccharide.

Prophylaxis

Chemoprophylaxis :It is indicated for the close contacts of patients for eliminating the bacteria from nasopharynx. Following antmicrobal agents are recommened-

  • Rifampicin
  • Minocycline  and
  • Ciprofloxacin.

Immunoprophylaxis: A vaccine containing capsular polysaccharide of serotypes A and C : for infants below 2 years and a quadrivalent vaccine constituted by polysaccharides of serotypes A,C,Y and W-135 : for children and adults where as  conjugate vaccine: polysaccharide antigen is conjugated to diptheria toxoid.

References

  1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  2. Clinical Microbiology Procedure Hand book, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  3. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
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  7.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  8. Topley & Wilsons Principle of Bacteriology, Virology and immunology. Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
  9. Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshank
  10. https://www.slideshare.net/binuenchappanal/neisseria-66046847
  11. https://en.wikipedia.org/wiki/Neisseria
  12. District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Update
  13. https://www.cdc.gov/meningococcal/about/index.html
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