MLAB 2434: Microbiology Keri Brophy-Martinez

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

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

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

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

5. Virulence Factors • Fimbrae (common pili)- • 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:Neisseriagonorrhoeae • 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:Neisseriagonorrhoeae • 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:Neisseriagonorrhoeae: • 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:Neisseriagonorrhoeae: • 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:Neisseriagonorrhoeae • 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. Transport Media Transgrow or JEMBEC JEMBEC= James E Martin Biological Environmental Chamber JEMBEC

13. Laboratory Diagnosis: Neisseriagonorrhoeae • Morphology • Gram-negative, kidney-bean–shapeddiplococci

14. Laboratory Diagnosis: Neisseriagonorrhoeae

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:Neisseriagonorrhoeae • 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: Neisseriagonorrhoeae • Oxidase Test • Test on filter paper or directly on plate • Oxidase reagent =Dimethyl or tetramethyl oxidase reagent • Violet-purple color indicates a positive result

19. Laboratory Diagnosis: Neisseriagonorrhoeae • Carbohydrate utilization • Cystine trypticase agar (CTA) • Contain 1% of a single carbohydrate • Glucose, maltose, lactose, sucrose • Phenol red is pH indicator • Read in 24-72 hours

20. Laboratory Diagnosis: Neisseriagonorrhoeae • 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: Neisseriagonorrhoeae • 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:Neisseriameningitidis • Pili • Polysaccharide capsule • Cellular membrane proteins • Lipooligosaccharide/endotoxin

24. Clinical Infections:Neisseriameningitidis: • 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:Neisseriameningitidis Hemorrhage in the adrenal glands in Waterhouse-Fridericksen syndrome

26. Laboratory Diagnosis:Neisseriameningitidis • 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: Neisseriameningitidis • 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: Neisseriameningitidis • Oxidase-test positive • Conventional CTA carbohydrates for biochemical identification (glucose+ and maltose+) • Immunologic methods

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

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

31. Moraxellacatarrhalis • 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:Moraxellacatarrhalis • Endotoxin • Pili • Beta-lactamase

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

34. Laboratory Diagnosis:Moraxellacatarrhalis • 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 :Moraxellacatarrhalis • 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. • http://www.awinhospitalproducts.com/product/35-collection-swab-amies-medium-w-charcoal-wwcsam-3450 • https://new.fishersci.com/ecomm/servlet/fsproductdetail_10652_606366_29104_-1_0 • https://picasaweb.google.com/pia8628/0411microlab#5596104725803822690/ • Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.