1. MLAB 2434: Microbiology Keri Brophy-Martinez
The Gram-Negative Cocci

2. Families Neisseriaceae Moraxellaceae Neisseria Kingella Eikenella
Simonsiella
Alysiella
Moraxellaceae
Moraxella
Acinetobacter

3. General Characteristics: Neisseria Species
Aerobic, gram-negative diplococci
Nonmotile
Oxidase positive
Catalase positive
Fastidious, capnophilic

4. Neisseria Species and Moraxella catarrhalis
Habitat
Upper respiratory tract
Genitourinary tract
Alimentary(Digestive) tract
Primary pathogens:
N. gonorrhoeae
N. meningitidis

5. Virulence Factors Fimbrae (common pili)- Lipooligosaccharide: Capsule
enhance the ability of bacterial cells to adhere to host cells and to each other
Lipooligosaccharide:
endotoxin involved in damage to host tissue
Capsule
Cell membrane proteins
IgA protease-
cleaves IgA on mucosal surfaces

6. Clinical Conditions: Neisseria gonorrhoeae
Pyogenic (pus-producing) infection of columnar and transitional epithelial cells
urethral, endocervix, anal canal, pharynx, and conjunctiva
Incubation period: 2 to 7 days
Transmitted by sexual contact

7. Clinical Infections: Neisseria gonorrhoeae
Disease in the male
95% show symptoms of acute infection
Symptoms include dysuria, urethral discharge
Complications include epididymitis and urethral stricture, and prostatitis
Disease in the female
20% to 80% are asymptomatic
Symptoms include:
Burning or frequency of urination, vaginal discharge, fever and abdominal pain
Complications include pelvic inflammatory disease (PID), sterility and ectopic pregnancy

8. Clinical Conditions: Neisseria gonorrhoeae:
Disseminated gonococcal disease
Acute form has the following symptoms: fever, chills, malaise, intermittent bacteremia, and skin lesions
If untreated will progress to septic joint form of the disease (inflamed joints, swollen, hot, full of pus and fluid)
Gonococcal arthritis occurs as a result of disseminated gonococcal bacteremia

9. Clinical Conditions: Neisseria gonorrhoeae:
Disease in children
In infancy, an eye infection (ophthalmia neonatorum) may occur during vaginal delivery
May cause blindness if not treated
Infection is preventable with the application of antibiotic eye drops at birth
Extragenital infections
Pharyngitis
Anorectal infections

10. Laboratory Diagnosis: Neisseria gonorrhoeae
Clinical specimens
Genital sites
Female: endocervix
Male: urethra
Anal
Oral/pharyngeal
Eye
Blood/joint fluids

11. Specimen Collection Dacron/ Rayon swabs preferred
Swabs transported in Amies medium with charcoal
Inoculate media within 6 hours of collection, avoid drying

12. JEMBEC= James E Martin Biological Environmental Chamber
Transport Media
Transgrow or JEMBEC
JEMBEC= James E Martin Biological Environmental Chamber
JEMBEC

13. Laboratory Diagnosis: Neisseria gonorrhoeae
Morphology
Gram-negative, kidney- bean–shaped diplococci

14. Laboratory Diagnosis: Neisseria gonorrhoeae

15. Media Selection Chocolate agar
Subject to overgrowth of normal flora
Thayer-Martin agar is chocolate agar with vancomycin, colistin, and nystatin
MTM contains the above plus trimethoprin
Specimen MUST be plated on warmed media ASAP

16. Incubation
Inoculated culture media must be incubated at 350 C in 3% to 5% CO2 or candle jar
Candle jar must use white wax candles

17. Laboratory Diagnosis: Neisseria gonorrhoeae
Colony morphology on modified Thayer-Martin (MTM) agar
Small, beige- gray
Translucent, smooth
Fresh growth must be used for testing, because N. gonorrhoeae produces autolytic enzymes

18. Laboratory Diagnosis: Neisseria gonorrhoeae
Oxidase Test
Test on filter paper or directly on plate
Oxidase reagent =Dimethyl or tetramethyl oxidase reagent
Violet-purple color indicates a positive result
The next step in the identification process is to perform an oxidase test. The oxidase test is useful to differentiate various GN bacteria. The test is performed on filter paper or directly on the plate. In the filter paper method, the oxidase reagent is placed on the filter paper and a colony is rubbed onto the reagent with an applicator stick. A positive reaction, which is a violet-purple color should develop within 10 seconds. This method is seen on the picture on the left. If the oxidase reagent is dropped directly on the plate, a positive reaction will result in the colony turning violet-purple, then changing to black.

19. Laboratory Diagnosis: Neisseria gonorrhoeae
Carbohydrate utilization
Cystine trypticase agar (CTA)
Contain 1% of a single carbohydrate
Glucose, maltose, lactose, sucrose
Phenol red is pH indicator
Read in hours

20. Laboratory Diagnosis: Neisseria gonorrhoeae
Immunologic methods
Use colonies from primary plate
Organisms do not need to be viable
Fluorescent antibody technique
Coagglutination
Non-culture methods
Use direct patient specimen
ELISA, nucleic acid probe, and PCR testing
Expensive; usually used in high-risk populations with large volume of testing
Unable to perform on all sources

21. Antimicrobial Resistance: Neisseria gonorrhoeae
PPNG = Penicillinase Producing Neisseria gonorrhoeae
First seen in 1976
Plasmid-mediated
Beta-lactamase testing should always be done on N. gonorrheoae
Treatment = Penicillin
Tetracycline if beta-lactamase positive strain. Can also use cephalosporins and flouroquinolones

22. Neisseria meningitidis
Commensal of carriers in the nasopharynx
Cross the epithelium and enter the circulatory system
Primarily affects the immunocompromised, young children, trauma victims
Leads to septicemia and localization to the meninges causing inflammation of the brain
Meningitis
Highly fatal (25% even if treated)
Encapsulated strains A, B, C, Y, W-135

23. Virulence Factors: Neisseria meningitidis
Pili
Polysaccharide capsule
Cellular membrane proteins
Lipooligosaccharide/endotoxin

24. Clinical Infections: Neisseria meningitidis:
Bacterial meningitis
Transmission is by respiratory droplets and requires both close contact (ex: dormitories, military barracks, in institutions) and lack of specific antibody (susceptibility)
Symptoms include fever, headache, stiff neck, nausea, vomiting, and purulent meningitis with increased WBCs
Serotypes B and C most common in US
Other infections include meningococcemia, pneumonia, purulent arthritis, & endophthalmitis
May be seen in genital tract with oral-genital contact

25. Clinical Infections: Neisseria meningitidis
Hemorrhage in the adrenal glands in Waterhouse-Fridericksen syndrome

26. Laboratory Diagnosis: Neisseria meningitidis
Identification
Examine direct smear from CSF for intra & extra cellular g- dc
Examine smear for halo
Other body sites include nasopharyngeal swabs, sputum, and urogenital specimens
Gram-stained smear of CSF showing the extra cellular and intracellular gram-negative diplococci

27. Laboratory Diagnosis: Neisseria meningitidis
Examine cultures on blood agar & chocolate agar after incubation in increased CO2
Colony Morphology
Small
Tan-grey color
Smooth
Neisseria meningitidis growing on sheep blood agar (right) and chocolate agar (left)

28. Laboratory Diagnosis: Neisseria meningitidis
Oxidase-test positive
Conventional CTA carbohydrates for biochemical identification (glucose+ and maltose+)
Immunologic methods

29. Antibiotic Therapy: Neisseria meningitidis
Penicillin
Other options: rifampin or sulfonamide
Vaccine
For use with people aged 11-55
Does not protect against all serotypes

30. Nonpathogenic Neisseria species
Normal flora of upper respiratory tract
Some members
Neisseria cinera
Neisseria lactamica
Neisseria mucosa
Neisseria sicca
Neisseria subflava

31. Moraxella catarrhalis
Previously known as Branhamella catarrhalis
Normal commensal of the respiratory tract
Has become an important opportunistic pathogen
Predisposing factors
Advanced age, Immunodeficiency, Neutropenia, Other debilitating diseases
Clinical infections
Pneumonia
Sinusitis
Otitis media (3rd most common cause)

32. Virulence factors: Moraxella catarrhalis
Endotoxin
Pili
Beta-lactamase

33. Laboratory Diagnosis: Moraxella catarrhalis
Direct smear from an otitis media sample showing intracellular gram-negative diplococci

34. Laboratory Diagnosis: Moraxella catarrhalis
Colonies appear smooth with a grayish- white color
When colonies pushed with loop, they “scoot” across media
Moraxella catarrhalis growing on chocolate agar after 24 hours of incubation

35. Laboratory Diagnosis : Moraxella catarrhalis
Oxidase positive
Catarrhalis Disc
Positive= blue-gren
All CTA sugars negative
Produce beta- lactamase

36. Identification of Selected Neisseria Species & Moraxella

37. References
Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams and Wilkins.
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Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.