Enteric and Anaerobe Lab Flashcards
Describe Enterobacteriaceae
These are facultative anaerobes that:
- ferment glucose
- have peritrichous or no flagella
- oxidase negative
- reduce nitrate to nitrite
Where are Enterobacteriaeceae found?
most are found as normal flora or pathogens in the vertebrate gut. A few can survive in the environment
What are the lactose fermenting enterobacteriaceae?
- E. Coli
- Klebisella pneumoniae
- Enterobacter
- Serratia
- Citrobacter
Describe Klebsiella pneumoniae
Mainly an opportunist causing:
community and nosocmial pneumonoa
- UTI
- wound infections
- meningitis
What are the nonlactose fermenters of enterbactericeae?
-Salmonella Typhi and Enteritidis
Shigella
- Proteus and Morganella
- Yersinia Pestis and enterocolitica
- Edardsiellla and Hafnia
The presence of E. coli in surface or well water is an indication of what?
animal or human fecal contamination
How can E. Coli be tested for in water?
by performing a coliform count by plating samply on media that are selective for Enterobacteriaceae and differential for lactose fermenters (note that other bugs such as Klebsiella and Enterobacter may add colonies to this test since they can also ferment lactose)
How can you distinguish between E. coli colonies and other lactose-fermenting colonies?
IMViC test (Indole production from tryptophan, Methyl red (large amount of acid from glucose), Voges-Proskauer (test that detects the production of acetylmethyl carbinol from glucose) and Citrate (utilization)
T or F. The IMViC rxns are used to distinguish between the fecal coliform E. Coli and Klebisella or Enterobacter (not neccessary fecal coliforms)
T.
How would E. Coli react to a IMViC?
Indole and Methyl Red +
VP and Citrate -
How would klebisella and Enterobacter react to a IMViC?
Indole and Methyl red -
Citrate and VP +
How can E Coli and E. Coli coliforms be differentiated?
A Colilert test, a broth-based test that distinguishes between coliforms and E. Coli based on the ability of coliform B-galactosidase to degrade ONPG subtrate and E. Coli B-glucuronidase to degrade MUG substrates
Where do anaerobes generally cause infection?
in sites continguous to a surface harboring indigious anaerobic flora when the surface has been breached and conditions favor growth
Specimens MUST be obtained from the acutal site of infection (e.g. abscess, internal body space) anf free from contaminating normal anaerobic flora. In the lab, special anaerobic collection dervies are required for collection ant special techniques are needed for ID
Strict anaerobes account for __% of bacteria in a fecal specimen
95-99%
What ANAEROBES do cause GI disease?
- Clostridium Perfringens (foodbourne diarrhea)
- C. diff
- C. botulinum (not normal flora; infent and wound botulism, an infection; foodbourne botulism in adult, an intoxication)
How should C. perfringens be ID’d?
Stool culture and suspect foods to detect high conc of C. perfringens
How should C. diff be ID’d?
Culture: Selective culture and cytotoxin production by isolates
Culture Independent: Toxin detectio, C. diff antigen detection, and/or NAAT of C. diff markers
How should C. botulinum be ID’d?
Culture: Selective culture and toxin testing by isolates from suspect food or stool (or wounds)
Culture independent: Toxin detection (mouse bioassay or toxin antigen test) in stool, serum, or suspect foods
Info on suspected diagnoses and tx are important to include with requests on the specimens being sent to labs. For ex., labs will not attempt to ID organisms that compete poorly with normal gut flora on typical enteric media (Vibrio and Yersinia)
As a general rule, stool specimens should be:
- selected from the actual site of infection
- collected appropriately and in a timely manner
Stool sampling
Stool specimens to detect enteric pathogens should be collected from from extaneous material (urine, barium preps, laxatives) and submitted to the lab ASAP
T or F. Gram +, - and anaerobes can cause bacteremia
T.
Describe a proper blood draw for bacteremia
- properly decontaminate puncture site
- collect 8-10 mL/set
- collect into appropriate media types (aerobic or anaerobic)
- collect 2-3 sets from different puncture sites
How are blood draws from suspected bacteria handled?
Almost always cultures from primary isolation and purification of potential pathogens from other flora. This generally takes 18-24 hrs (longer for slowing growing organisms). After isolation of potential pathogens, additional ID procedures and sometimes secondary isolations are needed. These may add an additional 18-24 hrs
T or F. Most enteric-related bacteria can be distinguished from each other just using a BAP
F.
Describe MacConkey Agar
It contains bile salts and crystal violet which inhibit the growth of most gram + bacteria (selects for gram neg enterics) It also has glucose and lactose and a pH indicator which permit differentiation of lactose utilizing and lactose non-utilizing bacteria (however both Lac+ and Lac- enterobacteriaceae will grow due to the presence of other nutrients)
What is one very important use of MacConkey agar/
Distinguishing between E. Coli (lactose +, red/pink) and Shigella/Salmonella (both lactose negative, white/colorless)
How is E. Coli O157 ID’d?
A special MacConkey agar in which sorbitol is substituted for lactose because more E. Coli CAN ferment sorbitol (pink/red) while O157 cannot (white)
What is EMB agar used for?
semi-selective and differential plating medium for preliminary ID of enteric-related GRAM NEGATIVE rods
Similar in utility to MacConkey
Why cant Gram + grow on EMB agar?
they are inhibited by dyes (eosin, methylene blue)
Describe SS (Salmonella-Shigella) agar
Highly selective for these two due to high conce of bile salts that inhibit growth of coliforms from E. Coli and gram + bacteria
How does Salmonella appear on SS agar? Shigella?
Salmonella will not ferment lactose, but produce hydrogen sulfide (H2S) gas. The resulting bacterial colonies will appear colorless with black centers.
Shigella do not ferment lactose or produce hydrogen sulfide gas, so the resulting colonies will be colorless.
How does E. Coli appear on SS agar if it survives?
they will be pink due to lactose presence
The use of gram stains in enteric infections
Generally not used.. cultures preferred
What is the composition of KIA SLants (Kligler’s Iron Agar)?
Peptone, beef extract, two sugars (low glucose, high lactose), sodium thiosulfate, phenol red (yellow if pH less than 6.8), ferrous sulfate
Rxna observed at 18-24 hr of growth
What is detected on a KIA Slants agar?
- Fermentation of glucose and lactose
- H2S
- Gas (H2)
Describe the results of fermentation of glucose and lactose in a KIA
The glucose conc is 1/10 that of lactose. Both slant and deep into agar are inoculated. If only glucose is fermented, the small amount of acid produced is oxidated and also neutralized by the oxidative breakdown of peptides to amines causing the slant to turn back to the original red (alkaline) color. If lactose is fermented, the slant will turn yellow.
In the butt of the tube, where O2 tension is low, the acid rxn in maintained (yellow) if the organism can ferment glucose or glucose and lactose
Describe the results of H2S presence in a KIA
Ferrous sulfate (FeSO4) is present which reacts with hydrogen sulfide produced by the bacteria to form ferrous sulfide (FeS) which forms a black precipitate at the bottom of the tube
Describe the results of H2 presence in a KIA
Presence demonstrated by cracks and bubbles in the deep portion of the tube
What does an alkaline (red) slant and an alkaline (red) butt on a KIA mean?
No carb fermentation, characteristic of nonfermentative bacteria like Pseudomonas aeruginosa
What does an alkaline (red) slant and an acid (yellow) with no Gas butt on a KIA mean?
Glucose is fermented but lactose is not. Shigella
What does an alkaline (red) slant and an acid + H2S (black) butt and Gas on a KIA mean?
glucose fermented but lactose is not fermented. H2S formed. Either Salmonella, Citrobacter, or Proteus
What does an acid (yellow) slant and an acid (yellow) butt with gas on a KIA mean?
Lactose and glucose are fermented . Either E. Coli, Klebsiella, or Enterobacter
What is an oxidase test used for?
to ID bacteria that can produce cytochrome C Oxidase to differeniate between the families of Pseudomonadaceae (ox +) and Enterobacteriaeceae (ox -)
What happens on an oxidase test in the presence of Ox +?
purple color appears on a reagent
What does an indole test detect?
conversion of tryptophan to indole
When does a positive indole test occur in time?
at 24 hr of growth (Kovacs reagant reacts with newly formed indole to produce a red product)
An Indole test is positive (red) for ______ and negative for ________
Positive for Shigella (some strains)
Negative for Salmonella
Flat, dry colonies of oxidase negative, lactose fermenting, gram negative rods that are indole positive are called ______
E. Coli
What is a ‘positive’ urease test?
conversion of urea to ammonia increases pH to produce red color on the slant portion of the agar
What is a ‘positive’ motility test?
Growing bacteria spreading out from an inoculation line in the middle of a semi-solid agar
_____ and _______ are nonmotile and do not spread from the site of inoculation
Klebsiella. (Some yersinia are only motile at room temp)
What aerobe will only show spreading on a motility test near the top of the agar surface when oxygen is plentiful?
Pseudomonas
What is serotyping useful for in the GI?
Subgrouping of Salmonella and Shigella are ID
How does serotyping work?
It detects the presence of specific antigens on the surface of the bacteria by adding a specific antiserum to a suspension of the organisms. If the antigens are present, the Abs bind the bacteria causing agglutination, or clumping, of the bacterial cells.
Expensive and not routinely done
How is Salmonella subtyped?
H and O antigens.. O antigens are LPS antigens and H antigens are flagellar antigens
Serotyping is useful in ID of Salmonella serovar Typhi using _____
capsular Vi antigen
ABX susceptibility is determined using the ______ method
Kirby Bauer
Kirby Bauer Test
medium: Mueller Hinton agar, pH 7.2-7.4, 4 mm thick
Incubation: 16-18 hr at 35-37C)
What antigen can be used to test for C. diff?
GDH
What does a negative GDH mean?
negative for toxigenic C. diff
What does a positive GDH mean?
Need to do a Toxin A (enterotoxin)/b (cytotoxin) assay or cytotoxin neutralization test
What does a positive Toxin A (enterotoxin)/b (cytotoxin) assay or cytotoxin neutralization test mean?
Positive for toxigenic C. diff
What does a negative Toxin A (enterotoxin)/b (cytotoxin) assay or cytotoxin neutralization test mean?
need to do NAAT assay or toxigenic culture.
This is the last step. Positive= positive for C. diff and negatve is a definite negative for C. diff
