MLAB 2434: MLAB 2434: MicrobiologyMicrobiologyKeri Brophy-MartinezKeri Brophy-Martinez

The Gram-Negative Cocci

FamiliesFamiliesNeisseriaceae

◦ Neisseria◦ Kingella◦ Eikenella◦ Simonsiella◦ Alysiella

Moraxellaceae◦ Moraxella◦ Acinetobacter

General Characteristics:General Characteristics:NeisseriaNeisseria Species Species

Aerobic, gram-negative diplococci

NonmotileOxidase positiveCatalase positiveFastidious, capnophilic

NeisseriaNeisseria Species and Species and Moraxella Moraxella catarrhaliscatarrhalis

Habitat◦Upper respiratory tract◦Genitourinary tract◦Alimentary(Digestive) tract

Primary pathogens:◦N. gonorrhoeae◦N. meningitidis

Virulence FactorsVirulence 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

Clinical Conditions:Clinical Conditions: Neisseria gonorrhoeae 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:Clinical Infections: Neisseria gonorrhoeae 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 ConditionsClinical Conditions:: Neisseria gonorrhoeae 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 ConditionsClinical Conditions:: Neisseria gonorrhoeae 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:Laboratory Diagnosis:Neisseria gonorrhoeaeNeisseria gonorrhoeae

Clinical specimens◦Genital sites Female: endocervix Male: urethra

◦Anal◦Oral/pharyngeal◦Eye◦Blood/joint fluids

Specimen CollectionSpecimen Collection

◦Dacron/ Rayon swabs preferred◦Swabs transported in Amies medium with

charcoal◦Inoculate media within 6 hours of collection,

avoid drying

Transport MediaTransport Media

Transgrow or JEMBECJEMBEC= James E Martin Biological Environmental Chamber

JEMBEC

Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria gonorrhoeae

Morphology

◦Gram-negative, kidney-bean–shaped diplococci

Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria gonorrhoeae

Media SelectionMedia 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

IncubationIncubation

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:Laboratory Diagnosis:Neisseria gonorrhoeaeNeisseria 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: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria 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

Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria 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: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria 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: Antimicrobial Resistance: Neisseria gonorrhoeaeNeisseria 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 meningitidisCommensal of carriers in the nasopharynxCross 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

MeningitisHighly fatal (25% even if treated)

◦ Encapsulated strains A, B, C, Y, W-135

Virulence Factors:Virulence Factors:Neisseria meningitidisNeisseria meningitidis

PiliPolysaccharide capsuleCellular membrane proteinsLipooligosaccharide/endotoxin

Clinical InfectionsClinical Infections:: Neisseria meningitidis 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:Clinical Infections: Neisseria meningitidis Neisseria meningitidis

Hemorrhage in the adrenal glands in Waterhouse-Fridericksen syndrome

Laboratory Diagnosis:Laboratory Diagnosis:Neisseria meningitidisNeisseria 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

Neisseria meningitidis growing on sheep blood agar (right) and chocolate agar (left)

Laboratory Diagnosis: Laboratory Diagnosis: Neisseria meningitidisNeisseria meningitidis

Examine cultures on blood agar & chocolate agar after incubation in increased CO2

Colony Morphology

◦Small

◦Tan-grey color

◦Smooth

Laboratory Diagnosis: Laboratory Diagnosis: Neisseria meningitidisNeisseria meningitidis

◦ Oxidase-test positive

◦ Conventional CTA carbohydrates for biochemical identification (glucose+ and maltose+)

◦ Immunologic methods

Antibiotic Therapy:Antibiotic Therapy: Neisseria meningitidisNeisseria meningitidis

PenicillinOther options: rifampin or

sulfonamide

Vaccine◦For use with people aged 11-55◦Does not protect against all serotypes

Nonpathogenic Nonpathogenic Neisseria speciesNeisseria species

Normal flora of upper respiratory tract

Some members◦Neisseria cinera◦Neisseria lactamica◦Neisseria mucosa◦Neisseria sicca◦Neisseria subflava

Moraxella catarrhalisMoraxella catarrhalis

Previously known as Branhamella catarrhalis

Normal commensal of the respiratory tractHas 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:Virulence factors:Moraxella catarrhalisMoraxella catarrhalis

EndotoxinPiliBeta-lactamase

Laboratory Diagnosis:Laboratory Diagnosis:Moraxella catarrhalisMoraxella catarrhalis

Direct smear from an otitis media sample showing intracellular gram-negative diplococci

Laboratory Diagnosis:Laboratory Diagnosis:Moraxella catarrhalisMoraxella 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 :Laboratory Diagnosis :Moraxella catarrhalisMoraxella catarrhalis

Oxidase positiveCatarrhalis Disc

◦ Positive= blue-grenAll CTA sugars negative

Produce beta- lactamase

Identification of Identification of Selected Selected NeisseriaNeisseria Species & Species & MoraxellaMoraxella

Species GrowthBAP R.T T/M

Acid productionGluc Mal Lac Suc

N. gonorrhoeae

N. meningitidis

N. lactamica

N. sicca

M. catarrhalis

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ReferencesReferences

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.