Module 1 Flashcards
What types of nonselective media are there?
Chocolate agar- RBCs are lysed and release nutrients, supplements are added (supplement B, Iso-Vitatex)
Selective Neisseria media but with out antimicrobials (specific for Neisseria)
What is the purpose of selective media?
Prevents the overgrowth of contaminating normal flora
What types of selective media are there?
Modified Thayer Martin (MTM)
New York City (NYC)
What does MTM agar contain? What does each do?
Vancomycin- inhibits G+
Colistin- inhibits G- and no pathogenic Neisseria
Nystatin- inhibits yeast
Trimethoprim lactate- inhibits Proteus spread
What does NYC agar contain?
Vancomycin
Colistin
Amphotericin B- inhibits yeast
Trimethoprim lactate
What type of media is ideal for isolation of vancomycin resistant strains of Neisseria?
Combo of selective and nonselective media
What types of transport media are there?
Non nutrient holding media- without growth, Amie’s transport medium, up to 6hrs
Growing transport media- used when there’s a delay, container with CO2 generating system, more recovery of N. gonorrhoea, vancomycin resistant strains may not be isolated
What is the genus test for Neisseria and Moraxella? How does the test work?
Oxidase positive
Amine turns positive when oxidized by enzyme
Reagent- dimethyl or tetramethyl-para-phenylenedianire (Kovac’s oxidase reagent ~1%)
Why can oxidase reagent only stand for a few hours after prep? How can it be stabilized?
Readily oxidized by oxygen
Ascorbic acid- reduces sensitivity
What are the methods for performing the oxidase test?
Filter paper method- reagent on paper, rub colonies on
Alternate procedure- drop of reagent on colonies
Positive- purple
What can cause false positives or negatives in oxidase testing?
False neg- old colonies, inhibitory/selective media, carb utilization media
False pos- nichrome wire, MAC
What is the QC for the oxidase test?
PC- Neisseria or Moraxella catarrhalis
NC- Staph, Strep, Enterobacteriaceae
What are the cellular characteristics of N. gonorrhoea?
G- cocci, kidney beans in pairs, 0.8-1.0um
Intracellular G- cocci
What are the growth requirements for N. gonorrhoea?
Obligate aerobes
Capnophiles (3-7%)
35°C
Moist environment required
Enriched media required (chocolate agar)
72hrs of incubation
Colonies- 0.5-3mm, grey, translucent, entire, gummy, older colonies tend to stick
What tests are used for Neisseria species ID?
Carb utilization- determines what carbs bacteria use, phenol red detects acid produced by oxidative mech
Chromogenic substrate test- detects specific enzymes, Gonochek II
Ag-Ab tests- confirmatory, coagglutination, direct fluorescent Ab, test for Protein I
Gene probes- detects unique NA sequences, ssDNA probe, positive = high RLU
What are the ID results for N. gonorrhoea?
Carb utilization- glucose +
Chromogenic substrate test- no colour change, reagent added, red-pink colour (proline aminopeptidase enzyme)
Coagglutination with Protein I antisera (N. gonorrhoea)
What causes false negatives in carb utilization tests?
Extended incubation can cause reversion and loss of acid pH
Utilization of peptone in the presence of O2
What sugars are used for reference in carb utilization testing?
CTA sugars
What rapid carb utilization test kits are there?
QuadFerm+kit
API NH
What colours indicate what antibiotics in chromogenic substrate tests?
Blue- N. lactamica (beta galactosidase)
Yellow- N. meningitidis (glutamylaminopeptidase)
No colour, reagent, red-pink- N. gonorrhoea (proline aminopeptidase enzyme)
What can cause false negatives in Ag-Ab testing?
Some strains lack Protein I
What is the antimicrobial susceptibility of N. gonorrhoea?
Originally all isolates were sensitive to penicillin
B lactamase resistant cephalosporins are effective (ceftizoxime and ciprofloxacin)
How is penicillin resistance caused in N. gonorrhoea?
Plasmid- codes for B lactamase, PPNG
Genetic info on chromosome- changes the structure of PBPs and outer membrane permeability (resistance without B lactamase)
What is the pathogenicity of N. gonorrhoea?
STI- urethral inflammation, dysuria, purulent discharge, prostatitis and epididymitis, salpingitis, endometriosis, peritonitis, infertility
Rectal infection
Purulent conjunctivitis
Occasionally spreads to bloodstream- DGI
What are the growth requirements of N. meningitidis?
Aerobic
Capnophilic (3-7%)
50% humidity
35°C
Nutrient media not recommended, good growth on MTM and NYC
SPS anticoagulant is inhibitory to some strains
What are the colonial characteristics of N. meningitidis?
Moist
Smooth
May be mucoid
No hemolysis
Translucent
Older colonies tend to autolyse
What are the species ID results for N. meningitidis?
Oxidase +
Carb- glucose + and maltose +
Coagglutination and latex reagents
Polyvalent meningococcal antisera
What is the antimicrobial susceptibility of N. meningitidis?
Most strains are susceptible to penicillin
Resistance due to plasmid induced B lactamase
What is the pathogenicity of N. meningitidis?
Nasopharynx of ~5% of healthy individuals
Children and young adults are most susceptible
Petechial rash
Elevated WBC in spinal fluid
Fulminate form AKA Waterhouse Friderichsen syndrome
Endocarditis, pneumonia, arthritis, conjunctivitis
What are the characteristics of other Neisseria species?
G- cocci in pairs, not usually intracellular
O2 required, but not increased CO2
Grows well on nutrient agar without enrichment, don’t grow on selective media
Dry, hard, wrinkled colonies
What is the species ID for other Neisseria species?
Oxidase +
N. lactamica- glucose + maltose + lactose +
N. species- may be positive for sucrose, negative for all others
What are the cellular characteristics of Moraxella catarrhalis (M. cat)?
G- cocci in pairs, resist decolourizing
Found intra and extracellularly
What are the growth conditions of M. cat?
Aerobe, doesn’t require increased CO2
35°C
Grows on nutrient agar without enrichment (optimum growth on blood and chocolate agar)
What are the species ID results of M. cat?
Strongly oxidase and catalase positive
Carb utilization- doesn’t produce any acid
Lipase pos
DNase pos (Neisseria are neg)
Butyrate esterase test- pos (most Neisseria neg)
What is the antimicrobial susceptibility of M. cat?
Most strains are resistant to vancomycin, colistin, trimethoprim and clindamycin
Suitable antimicrobials- amoxicillin with clavlanate, cotrimoxazole, erythromycin, tetracycline, second gen cephalosporins
~80% produce B lactamase
What is the pathogenicity of M. cat?
Normal respiratory flora
Otitis media and maxillary sinus infection
Conjunctivitis, pneumonia, bronchitis, tonsillitis, endocarditis, meningitis, septicaemia
Why generas belong to the family Neisseriaceae?
Acinetobacter- G- rods
Kingella- G- coccobacilli
Neisseria- G- cocci
Moraxella- G- cocci and coccobacilli
