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

Families Neisseriaceae Moraxellaceae Neisseria Kingella Eikenella Simonsiella Alysiella Moraxellaceae Moraxella Acinetobacter

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

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

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

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

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

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

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

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

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

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

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

Laboratory Diagnosis: Neisseria gonorrhoeae

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

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

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

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.

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 24-72 hours

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

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

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

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

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

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

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

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)

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

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

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

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)

Virulence factors: Moraxella catarrhalis Endotoxin Pili Beta-lactamase

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

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

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

Identification of Selected Neisseria Species & Moraxella

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_10 652_606366_29104_-1_0 https://picasaweb.google.com/pia8628/0411microlab#559610 4725803822690/ Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.