MOD1 Neisseria + Moraxella

Neisseria gonorrhoeae pathogenicity

STI

spread by sexual intercourse

never non-pathogenic

80% of infc men → symptomatic 1-14 days post contact, 20% asymptomatic

symptoms of Neisseria gonorrhoeae

men: infl of urethra + dysuria + purulent discharge

untreated → prostatitis + epididymitis

women: vaginal discharge + burning + urinary frequency

untreated → salpingitis, endometritis, peritonitis

pharyngeal + rectal infcs can occur with no apparent signs of infc

gonococcal conjunctivitis

eye infc caused by N.gonorrhoeae

bloodshot eyes, purulent discharge, periorbital edema

untreated → corneal ulceration, perforation, blindness

gonococcal infc in neonates

due to exposure to infc cervical secretions from delivery

neonatal conjunctivitis, scalp abscesses, meningitis, arthritis, bacteremia, pharyngitis, rhinitis, vaginitis, urethritis

disseminated gonococcal infection DGI

bacteria spread from localized site to bloodstream

rash, arthritis, tenosynovitis (infl of tendon sheath), endocarditis, meningitis

non-selective isolation media for N.gonorrhoeae

doesnt grow well on BAP → nutrient arent available from intact RBC

use CHOC

selective/inhibitory gonococcal media examples

specimens often mucous membrane sites → abundance of normal flora

• modified thayer martin MTM

• new york city NYC

• martin lewis ML

modified thayer martin MTM

selective plate

CHOC base w enrichment supplement

1. IsoVitaleX → enhance growth

2. colistin/polymyxin E → inhibition of GNOs + nonpath Neisseria spp

3. vancomycin → inhibition of GPOs

4. nystatin → inhibition of yeast

5. trimethoprim lactate → inhibit swarming Proteus spp

new york city agar NYC

not CHOC, translucent plates

contains lysed horse RBCs, plasma, yeast, glucose

allows for growth of other urogenital pathogens → Mycoplasma spp + Urealplasma spp

1. vancomycin → GPO inhibition (lower conc than MTM)

2. colistin → inhibition of GNO + nonpath Neisseria spp

3. amphotericin B → antifungal

4. trimethoprim lactate → inhibition of Proteus spp

martin lewis ML

similar to MTM

1. anisomysin → antifungal

2. vancomycin → inhibition of GPO (most concentrated)

expiration of selective media

more selective agar = shorter the shelf-life

antimicrobial action decreases over time

collection requirements for Neisseria spp

sensitive to drying + extreme temp changes

for culture → planted immediately

screening for Neisseria spp

use nucleic acid amplification test NAAT

detects presence of N.gonorrhoeae

highly sensitive + specific (>90%)

cultures for Neisseria spp

not routine → only performed if has hx

tx failure, sexual exposure/assault, eye swabs of neonates

non-nutrient holding media for culture

prevents dehydration of specimen + maintains viability of N.gonorrhoeae in semi-solid media w/o growth

buffer inhibitory substances (ex. fatty acids) from cotton/wooden swabs → inhibits growth of organism

ex. elute-swab (Eswab)

elute-swab Eswab + advantages

flocked swab → most of the specimen gets released into the liquid media surrounding the swab

better organism recovery

better analytical sensitivity + reproducibility

swab can be used with automated planting instruments

one swab can be used for multiple tests

oxidase test reagent

detects cytochrome oxidase enzyme

reagent reduced → colourless // oxidized → purple

routinely use dimethyl reagent → tetramethyl reserved for orgs that may test neg w dimethyl

unstable + readily oxidized by air

oxidase + ascorbic acid

stabilizes oxidase reagent

decreases sensitivity

0.2% → stable for 1 week, less sensitive

0.02% → stable for 1 day, more sensitive » make fresh reagent daily

oxidase test QC

PC: Pseudomonas aeruginosa = purple/red

NC: Escherichia coli = colourless/beige

oxidase filter paper method

reagent added to filter paper

colour change to purple/blue black = positive

oxidase swab method

dip into reagent

touch suspect colony

observe for colour change

oxidase flood — direct plate method

drop of reagent on colony

observe for purple colour in 15s

disadvantage: colonies rapidly killed after exposure → difficult to subculture

oxidase false positive

using nichrome loops to pick colonies

contain iron → oxidizes reagent

oxidase false negative

old cultures → reduced enzyme activity

oxidase erroneous results

inhibitory/selective media + media high in carbs

test is v sensitive to pH changes

N.gonorrhoeae cellular morphology

Gram negative diplococci GNDC

0.8-1.0um

no spores, flagella, capsules

may be seen intracellularly → phagocytosed by PMN

growth requirements for N.gonorrhoeae

obligate aerobes → requires O2 for growth, but grows better in inc CO2 (3-7%) — little to no growth in anaerobic condition

35degs — sensitive to cold shock

humidity required

use enriched media → CHOC, MTM, NYC, ML

incubated for 72hr before determining if culture is neg for GC

N.gonorrhoeae colonial morphology

gray, translucent, entire

0.5-3mm (depends on incubation time)

gummy/sticky, matte on agar

colonies can break apart or pushed along agar

carbohydrate utilization

cystine trypticase agar CTA sugars → semi-solid media supplemented w carb

acid production via oxidation → semi solid keeps small amount of acid produced near top of tube

colour change from red (alkaline pH 7.4) to yellow (acid pH 6.8)

carbohydrate utilization results for N.gonorrhoeae

glucose positive

maltose negative

lactose negative

sucrose negative

carbohydrate utilization test limitations + false negatives

limitation: need heavy inoculum for test

false negative: production of alkaline end products

• over incubation = reversion of acid endpoint

• utilization of peptones in media

analytical profile index API

mini ID kits w substrate in cupules → inoculated w bact suspension

incubation in O2

acid production (pos rxn) due to carb utilization → indicator turns yellow

protein 1: co-agglutination test

monoclonal ab attached to killed staph cells that are directed to specific protein 1 on outer membrane of N.gonorrhoeae

protein 1: co-agglutination test false negative

rare strains of N.gonorrhoeae that do not have protein 1 → false neg

protein 1: direct fluorescence antigen DFA

fluorescent monoclonal antibodies to protein 1

slide + bacteria + antisera + 15 min

observe using fluorescent microscope

N.gonorrhoeae → apple-green diplococci

mechanism of resistance of N.gonorrhoeae

plasmid-mediated resistance → penicillinase-producing N.gon (PPNG)

penicillin binding proteins PBP altered → genetic resistance on chromosome

Neisseria meningitidis cellular morphology

capnophilic GNDC

may be found intracellularly in neutrophils

some strains can be capsulated + mucoid

N.meningitidis pathogenicity

colonizes in nasopharynx → non pathogenic

causative organism of meningococcal meningitis

growth requirements for N.meningitidis

incubate 35deg

humidity 50%

susceptible to cold shock

grows well on BAP/CHOC/MTM/NYC

blood culture bottles + SPS

sodium polyanethole sulfonate → anticoag

inhibits antimicrobial systems of blood

used in culture bottles → ensure org isnt killed by specimen itself

N.meningitidis on BAP vs CHOC

BAP: grey/white, non-hemolytic, wet/mucoid, opaque

CHOC: grey, may be wet/mucoid/capsulated, 2-3mm

carbohydrate utilization results for N.meningitidis

glucose positive

maltose positive

lactose negative

sucrose negative

N.meningitidis pathogenicity

healthy carrier rate → 10% in nasopharynx

bloodstream → blood-brain barrier BBB → CNS = serious disease

virulence factors → polysaccharide capsule, pili, adhesins → helps invasion + adhesion to CN tissue

how can N.meningitidis be contracted

accidental parenteral inoculation

exposure to mucous membranes to infectious droplet/nuclei/aerosols

ingestion

N.meningitidis symptoms

purpura → hemorrhaging of blood = bruising

petechial skin rash → pin point red spots from hemorrhaging + disseminated intravascular coagulation DIC

waterhouse-friderichsen syndrome WFS

fulminate (rapid + severe onset) form of meningococcal sepsis

characterized by bilateral adrenal hemorrhage, septicemia, disseminated intravascular coagulation DIC

can be fatal if untreated

non-pathogenic Neisseria spp

normal flora

usually dry, hard, opaque, wrinkled, light yellow

may also be grey, moist → resemble other Neisseria spp

do not grow on selective media

lumped in w pharyngeal flora → dont need to report

if isolated from sterile site → Neisseria spp non-gonorrhoeae/non-meningitidis isolated

Moraxella catarrhalis growth requirements

aerobe → grows better in CO2

mesophilic temp → 35deg

most grows on nutrient agar w/o enrichment

many grows on selective media

Moraxella catarrhalis colonial morphology

1-3mm, grey-white, non-hemolytic, opaque, entire, convex, dry/matte

“friable”

carbohydrate utilization results for Moraxella catarrhalis

asaccharolytic → doesnt oxidize any sugars

glucose negative

maltose negative

lactose negative

sucrose negative

fructose negative

other tests for ID of M.catarrhalis

lipase — 100% positive

DNase — positive

butyrate esterase — positive

differentiate M.catarrhalis vs asaccharolytic Neisseria spp

Moraxella spp pathogenicity

75% of children + 1-3% of healthy adults → carriers

children: causes otitis media (middle ear infc), sinus infc, respiratory infc, pneumonia

adults+children: conjunctivitis, pneumonia, bronchitis, tonsilitis, endocarditis, meningitis, septicemia