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Gram-Negative Diplococci Bacteria

Gram-negative diplococci bacteria as shown above image. Gram stain of CSF  having Gram-negative diplococci indicates Neissera menigitidis whereas Gram stain of urethral discharge having such property indicates Neisseria gonorrhoeae.

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 cerebrospinal 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 a 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 the 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 antibiotics ( vancomycin, colistin, nystatin, 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 a maltose fermenter and does not produces beta-lactamases. It has three important virulence factors: 1. Polysaccharide capsule. It is antiphagocytic in nature. 2. The endotoxin of N. meningitidis is a lipopolysaccharide (LPS). It induces septic shock by causing the release of cytokines. 3. IgA protease. It cleaves the IgA antibodies present in the 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 the 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 bloodstream).

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: an acute fever with chills, malaise, prostration,  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- Fredericksen syndrome,  Meningococcal disease is favored by deficiency of the terminal complement components (C5-C9).

Epidemiology 

The human is the only reservoir of the N. meningitidis. 5-10% of adults are asymptomatic carriers.  Modes of infection are direct contact or respiratory droplets (nasopharyngeal carriers )from the nose and throat of infected people.  The prevalence of meningitis is highest in the meningitis belt of Africa (frequent epidemics occurred there). In 1996, among 150000 cases 15000 deaths were reported. Epidemic usually occurs in overcrowded areas. Inhalation of contaminated droplets adherence of organism to nasopharyngeal mucosa. Local invasion and spread from the nasopharynx to meninges through the bloodstream (directly along the perineural sheath of the olfactory nerve, cribriform plate to subarachnoid space).  In meninges, organisms are internalized into phagocytic cells. They replicate and migrate to subepithelial spaces and the 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 outbreaks.

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 tubing 3 records 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 the blood or chocolate agar and incubated at 37ºC in 5-10% Carbon dioxide (CO₂) and high humidity. After 24 hours bacterial colonies appear and the organism is tested for biochemical and agglutination reactions. Now in details, follow as-

Useful methods for laboratory diagnosis of Meningococcus are-

• Gram staining
• Culture
• Biochemical test
• Latex agglutination test
• Serotyping
• 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 the 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 have no 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 polysaccharides in the CSF.

Serotyping: Serogroups and serotypes on the basis of the 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: localized 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 Meningococcal 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. Chloramphenicol or third-generation cephalosporins 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 the nasopharynx. Following antimicrobial agents are recommended-

• Rifampicin
• Minocycline  and
• Ciprofloxacin.

Immunoprophylaxis: A vaccine containing capsular polysaccharides 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 whereas conjugate vaccine: polysaccharide antigen is conjugated to Diptheria toxoid.

Neisseria gonorrhoeae Introduction

Neisseria gonorrhoeae also called Gonococcus which causes sexually transmitted disease(STD) gonorrhea. First described by Albert Ludwig Sigesmund Neisser  in 1879 in gonorrheal pus. It resembles meningococci very closely in many properties.

• Colony characteristics of Neisseria gonorrhoeae on 5% sheep blood agar (BAP) are as follows-
• Small
• Round
• grey
• white
• opaque as shown above picture.
• They are easily emulsifiable.
• It produces four types of colonies and they are T1, T2, T3, and T4.
•  T1 and T2: Small brown colonies and possess pili. They are autoagglutinable and virulent strains. On repeated subcultures, they change to T3 and T4 respectively. They are also known as P+ and P++respectively.
•  T3 and T4: They are larger, granular, non-pigmented colonies and non-pi
• Piliated. They form smooth suspensions and are avirulent. They are also named as P-.

Note

Few large white colonies on blood agar are because of contamination. Medically important species of this genus are  Neisseria gonorrhoeae  (gonococcus) and Neisseria meningitidis ( meningococcus).

Morphology 

Gram-negative oval cocci arranged in pairs i.e. diplococci with adjacent sides concave ( pear or bean-shaped). In smear from purulent material, they are intracellular within polymorphonuclear cells( polymorphs), some cells containing as many as a hundred cocci as shown below.

 Culture Characteristics of Neisseria

This gonococcus is fastidious and thus does not grow on ordinary culture media like nutrient agar. It is aerobic but may grow anaerobically also. The optimum temperature for growth is 35-36°C and the pH is 7.2-7.6. It is essential to provide 5-10% CO₂.  Media used: a) Non-selective media: Chocolate agar, Mueller-Hinton agar Modified New York City medium b) Selective media: Thayer Martin medium with antibiotics (Vancomycin, Colistin, and  Nystatin). Vancomycin and colistin are antibacterial agents that kill Gram-positive and Gram-negative bacteria respectively ignoring the Neisseria while nystatin is an antifungal drug that makes the medium selective. Colony morphology is shown above.

Biochemical Reactions

• Oxidase test: Positive

• Gonococci ferment only glucose but not maltose.

Antigenic Structure  of Neisseria

1. Pili: These are hair-like structures extending from the surface. They enhance the attachment of the organism to host cells and resist phagocytosis.
2. Lipopolysaccharide: Endotoxic.
3. Outer membrane proteins: 3 types a) Protein I (por)- it is a porin & helps in adherence. b) Protein II (opa)- helps in adherence. c) Protein III (rmp)- it is associated with protein I.
4. Other Protein
   IgA1 protease: Splits and inactivates IgA.

Pathogenesis

Gonococci adhere to epithelial cells of the urethra or another mucosal surface through pili penetrate through the intercellular space reach the subepithelial connective tissue and causes inflammation which leads to clinical manifestations. The incubation period is 2-8 days. Source of infection: 1. Asymptomatic carriers 2. Patients Mode of infection: 1. Venereal infection (sexual contact) 2. Non-venereal infection. In men: The disease starts as acute urethritis with a mucopurulent discharge that extends to the prostate, seminal vesicles, and epididymis. In some, it may become chronic urethritis leading to stricture formation. The infection may spread to the periurethral tissues, causing abscesses and multiple discharging sinuses while in women: The initial infection is urethritis and cervicitis but vaginitis does not occur in adult females ( vulvovaginitis can occur in prepubertal girls). The infection may extend to Bartholin’s glands, endometrium, and fallopian tubes causing Pelvic Inflammatory Disease (PID). Rarely peritonitis may develop with perihepatic inflammation (Fitz-Hugh-Curtis syndrome). Common in both the sexes: Proctitis, pharyngitis, conjunctivitis, bacteremia which may lead to metastatic infection such as arthritis, endocarditis, meningitis, pyemia, and skin rashes. In neonates: Ophthalmia neonatorum (nonvenereal gonococcal conjunctivitis in the newborn) results from direct infection during passage through the birth canal.

 Laboratory Diagnosis  of Neisseria

• Specimens: A) In men: a) Acute infection- Urethral discharge b) Chronic infection- i) Morning drop ii) Discharge collected after prostatic massage iii) Centrifuged deposit of urine

B) In women: i) Urethral discharge ii) Cervical swabs

C) In both the sexes: Blood, CSF, synovial fluid, throat swab, rectal swab, and material from skin rashes.

• Transport: If there is a delay in processing then the specimens should be sent in “ Stuart’s transport medium”.
• Direct microscopy: Gram staining: Smear provides presumptive evidence of gonorrhea in men. Gram-negative diplococci are found. But it is unreliable in women because some of the genital normal flora have similar morphology. Fluorescent antibody stain of gonococcus can also be observable under the fluorescence microscope.
• Culture: Media used: Colony morphology: Gram’s smear: Reveals Gram-negative cocci in pairs with adjacent sides concave.
• Biochemical reactions: 1)  Oxidase test: Positive2) Gonococci ferment only glucose but not maltose.
• Serology:  Serology tests can be performed using the following tools like complement fixation test, precipitation, passive agglutination, immunofluorescence, and radioimmunoassay.

 Treatment

Previously Penicillin was the drug of choice but resistance developed rapidly. Penicillin resistance is due to the production of the penicillinase enzyme and the strains are called penicillinase-producing Neisseria gonorrhoeae (PPNG). Now ceftriaxone or ciprofloxacin plus doxycycline or erythromycin are useful drugs.

Epidemiology

Gonorrhea is an exclusively human disease. The only source of infection is a human carrier or less often a patient.  Asymptomatic carriage in women makes them a reservoir to spread the infection among their male contact. Gonorrhea is a venereal disease (STD).

Prophylaxis of Neisseria

Early detection of cases, tracing of contacts, health education, General measures, and vaccination has no role in prophylaxis.

 Non-Gonococcal  (Non-specific)) Urethritis

Urethritis is due to causative agents other than gonococcus.

Causative agents

 Bacterial

Chlamydia trachomatis ( most common)

Mycoplasma hominis

Ureaplasma urealyticum

Gardnerella vaginalis

Acinetobacter lwoffi

Viral

Herpes simplex

Cytomegalovirus

Fungal

Candida albicans

Parasitic ( protozoal)

• Trichomonas vaginalis NGU can be a part of Reiter’s syndrome- a clinical condition characterized by urethritis, arthritis & conjunctivitis.
• Tetracycline is the effective drug for both Chlamydia trachomatis and Ureaplasma urealyticum infections.

Commensal Neisseria

Commensal of Genus Neisaseria few species are  N. lactamica N. cinerea N. flavescens N. sicca and N. subflava.

Bibliography

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