Enterics and NF Flashcards
Non-fermenters reservoirs
soil, water
human mucous membranes
Non-fermenters cause disease by
gaining access to sterile body sites of immunocompromised or debilitated
Suspect a Non-fermenting GNR/GNCB if
distinctive colony morphology (not always)
grows NLF on MAC or doesn’t grow at all
oxidase +
AK/NC on KIA or TSI
oxidase test
determines if isolate prod. enzyme indophenol oxidase
oxidized enzyme prod. color change from yellow===purple
indophenol oxidase oxidizes phenylenediamine oxidase to form indophenol (purple cmpd)
1. add few drops of reagent to strip of filter paper placed on slide
2. streak loopful of organism of reagent saturated paper
3. positive turns purple w/in 30 sec
BBL DrySlide oxidase test
nnnn-tetramethyl-p- phenylenediamine dihydrochloride (aromatic amine)
if organism prod. cytochrome oxidase it will oxidize cytochrome c which oxidizes reagent to form purple cmpd
1. smear organism onto rxn area of BBL DrySlide Oxidase in area of 3-4 mm
2. examine for drk purple color w/in 20 sec
Embden-Meyerhof pathway
classical metabolic pthwy used by enteric GNR
generates lactic acid and mixed acids
end prod. (strong acids) drops pH and prod. gas
pyruvic acid==other acids (lactic, propionic, formic, acetic, butyric, etc)
Entner-Douderoff pathway
most non-fermenters use
end prod. is weak acids
glucose==pyruvic acid==Krebs cycle
Hexose Monophosphate pathway
Warburg-Dickins hexose monophosphate pthwy
glucose==pyruvic acid==either goes to krebs cycle or is end prod. of mixed acids
Hugh Leifson OF media
detection of weak acid end products (used instead of KIA for NF)
low peptones, high carbohydrates
tube 1 overlay w/ oil, tube 2 no oil
tube 1 green no glucose fermented, tube 2 yellow glucose oxidized=== non fermenter and oxidizer or glucose
tube 1 green, tube 2 green non oxidizer of glucose===non fermenter and non oxidizer
Pseudomonas aeruginosa morph
most common non fermenter prod. pyocyanin (greenish blue sheen) grape juice/tortilla chip odor beta hemolytic feathered, frosted glass apperance GNR fluorescent
Pseudomonas aeruginosa key tests
oxidase + catalase + motile grows @ 42*C grows on BA and MAC as NLF LAO =/+/= OF/O + OF/F = some gelatinase + nitrate + citrate +
Pseudomonas aeruginosa pathology
exopolysaccharide capsule
noscomial infect (catheter, IV, pneumoniae)
opportunistic (Cystic Fibrosis, burns, cancer)
very resistant- always perform AST (antimicrobial susceptibility testing)
swimmers ear
Fluorescent pseudomonads
oxidase + prod. pyoverdine but not pyocyanin not grow @ 42*C gelantinase + (fluorescens)/ = (putida) septicemia in blood bank
Pseudomonas stutzeri tests
yellow wrinkled colonies on BA NLF on MAC GNR oxidase + motile grows @ 42*C LAO =/=/= oxidizes glucose, lactose (+/=), maltose (+/=), fructose reduces nitrate to nitrogen gas
Pseudomonas stutzeri
disease in immuno.
otitis media, conjunctivitis, endocarditis, meningitis, wounds
Acintobacter baumannii/calcoaceticus tests
2nd most common NF grows MAC faint pink colony lactose + oxidase = GNCB catalast + oxidizes glucose, xylose, lactose LAO =/=/= nitrate = non-motile
Acinetobacter lwoffi
nonsacchrolytic (not react)
oxidase =
LAO =/=/=
non motile
Acintobacter baumannii/calcoaceticus reservoir
environment
Acinetobacter baumannii/calcoaceticus pathology
pneumonia, bronchitis, septicemia, endocarditis, wounds, UTI
A. lwoffi pathology
septecemia, gastritis
Strenotrophomonas maltophilia morph.
3rd most common NF BAP- lavendar-green colonies smells like ammonia MAC pinpoint colonies GNR
Stenotrophomonas maltophilia tests
oxidase = ONPG + oxidizes glucose and maltose catalase + DNase + Geletinase + hydrolyzes esculin motile LAO +/=/=
Stenotrophomonas maltophilia pathology
nosocomial infec. wound endocarditis bacteremia rare UTI
Alcaligenes faecalis morph.
normal BA (wt @ 48 hrs, green discoloration)/ MAC green apple odor GNR tiny
Alcaligenes faecalis tests
nonsaccharolytic oxidase + catalase+ motile reduces nitrite but not nitrate to nitrogen gas LAO =/=/=
Alcaligenes faecalis pathology
noscomial from water source
Achromobacter xylosoxidans morph.
grows on BA and MAC
green discoloration of agar
GNR
Achromobacer xylosoxidans tests
oxidase + catalase + motile oxidizes glucose/ xylose nitrate + LAO =/=/=
Achromobacter xylosoxidans pathology
rarely isolated from blood, CSF, resp., urine, wounds
Burkholderia pseudomallei morph.
wrinkled, yellow-brown colonies on BA
NLF on MAC (turns pink w/ age due to lactose oxidation)
oxidizes xylose, mannitol, maltose
GNR may have bipolar staining
Burkholderia pseudomallei tests
oxidase + motile nitrate + LAO =/+/= grow @ 42*C R to polymixin
Burkholderia pseudomallei reservoir
water/ soil southeast Asia, N. Australia, Mexico
Burkholderia pseudomallei pathology
melioidosis from inhalation/ cut- disseminated abscess formation with skin pustules, septic arthritis, osteomyelitis, spleen, liver, kidneys, and prostate
known as Vietnam time bomb
considered potential bioterrorist agent
Burkholderia cepaia morph.
BA: yellow pigment (or yellow-green)
mucoid in cystic fibrosis
sweet earthy odor
tiny colonies @ 24hr
Burkholderia cepacia tests
oxidase + (weak and slow) motile glucose and lactose oxidizer LAO +/=/= ONPG +
Burkholderia cepacia pathology
opportunistic of lungs in cystic fibrosis patients or with chronic granulomatous disease (CGD)
[phagocytes not prod. bactericidal anions so get B. cepacia]
Elizabethkingia meningosepticum morph.
yellow colony w/ green/lavendar discoloration on BA
grows on MAC
fruity odor
Elizabethkingia meningosepticum tests
oxidase + catalase + LAO =/=/= indole + DNase + esculin+ H2S + ONPG +
Elizabethkingia meningosepticum pathology
newborns: meningitis or septicemia
adults: pneumonia, endocarditis, bacteremia, meningitis
Chryseomonas and Flovimonas characteristics
rarely cause path. oxidase = motile yellow coloines dry wrinkled grow on MAC Chryseomonas- yellow, deep yellow, orange Pseudomonas (Flavimonas) oryzihabitans
Moraxella spp.
rarely cause path. GNCB pinpoint colonies on BA poor to no growth on MAC oxidase + catalase + non motile not oxidize glucose: asaccharolytic (incapable of breaking down carbs for E) Id not performed
Oligella
from urinary tract O. ureolytic: urea +, motile, nitrate + O. urethralis: urea =, non motile, nitrate = but nitrite + no oxidize glucose (asacchrolytic) oxidase + catalast = PDA +
Shewanella (Pseudomonas) putrefaciens
yellow-brown-orange color oxidase+ motile H2S+ on TSI/KIA nitrate + LAO =/=/+ GNR big rods
Chromobacterium violaceum reservoir
fresh water/ soil in tropics/subtropics
SE USA
trans. by ingest contam. water/ thru broken skin
in immuno.
Chromobacerium violaceum charact.
grow BA, CHOC, MAC violet pigment (violacein) @ room temp ammonium cyanide odor (almonds) pigment iterfere w/ testing grow anaerobically no pigment oxidase - (v) no grow in high NaCl
Nitrate test
determine if bacteria reduce nitrate
nitrate a (0.8% sulfanilic acid in 5N acetic acid) and nitrate b (0.6% nn-dimethyl-alpha-naphthylamine in 5N acetic acid) reagents
1. innoculate broth incubate 18-24 & 42-48hrs 35-37*C
2. examine for growth and gas
3.if gas nitrate was reduced to nitrogen gas. if growth take 2mL of broth into new tube. Add 5 drops nitrate a and 5 drops nitrate b and mix. If pink/red w/in 2 mins. = +
4. if negative add zinc dust. if pink/red color w/in 2 mins nitrate is present & wasn’t reduced so = negative result. If no color change w/in 2 mins. nitrate reduced beyond nitrite and test= positive
MAC
selective & differential
ID GN bacilli
bile salts & crystal violet select for gram negative inhibit gram positive
lactose sole carbo. (differential)
pH indicator neutral red
LF prod acid and turn pH red so prod red/pink colonies
peptones, beef extract energy source for NLF
Normal enteric flora Enterobacteriacea
cause infec. in other areas of body, especially sterile sites Salmonella Shigella Yersinia entercolitica E. coli O157:H7 Plesiomonas
normal enteric flora (non-Enterobacteriacea)
Campylobacter spp.
Aeromonas
Vibrios
Antigenic serotype Salmonella/Shigella
O: somatic antigen
H: flagellar antigen
K: capsular antigen
Vi: in virulent strains of Salmonella
E. coli types
O157:H7
enterotoxigenic E. coli
enteropathogenic E. coli
enteroaggregative E. coli
Escherichia coli morph.
GNR
med. grey colony on BA can be beta hemolytic
LF on MAC
Escherichia coli tests
motile indole + ornithine decarboxylase+ MIO +/+/+ O157:H7 sorbitol negative
decarboxylase test (arginine, lysine, ornithine)
ID GN enteric, NF, oxidase+, GNR, based on prod. of arginine dihydrolase and lysine and ornithine decarboxylase
peptones/yeast extract supply nitrogenous nutrients
pyridoxal enzyme for aa decarboxylase
dextrose is fermentable carbo.
bromcresol purple and cresol red pH indicators
arginine, lysine, ornithine are added 10g/L to detect enzyme for these- if ferment dextrose lower pH change color, purple to yellow, acidic cond. stimulates decarboxylase- if ferment appropriate enzyme aa degraded yielding an amine (A= citrulline, L= cadaverine, O= putrescine,) elevates pH changing color back to purple, if not stays yellow
must add oil
Shigella spp.
cause severe GI sympotoms Shigella A (dysenteriae) DIRTY Shigella B (flexneri) FINGERS Shigella C (boydii) BRINGS Shigella D (sonnei) SHIGELLA
Shigella spp. morph.
GNR on GS med. grey on BA NLF on MAC non-reactive non-motile sonnei late lactose fermenter ONPG +
ONPG test
O-nitrophenyl-beta-D- galactopyranoside
tests directly for beta galactosidase
positive in late lactose fermenters- turns yellow for +
bacteria not have permease (LF do have) which allows lactose to enter bact. cell wall where it is broken down into glucose & galactose by beta galactosidase to be metabolized
Edwardsiella tarda
carride by turtles/reptiles causes diarrhea, wound infec., bacteremia NLF lysine+ H2S + indole + motile urea = citrate = MIO +/+/+
Indole test
indole (benzyle pyrrole) metab. degradation prod. of aa tryptophan
bact. w/ tryptophanase hydrolyze and deaminiate tryptophan into indole, pyruvic acid, and ammonia. red color cmplx when indole rcts w/ p-dimethylaminobenzaldehyde
Kovac’s reagent (HCl, p-dimethylaminobenzaldehyde, amyl alcohol)
1.innoculate tube (w/ 18-24hr culture) incubate 35*C for 24-48hrs
2.remvoe 2mL to new tube. add .5mL or 10drops Kovacs reagent. shake. apperance of red ring on surface w/in 30 sec is postive for indole prod.
spot indole test
indole combines w/ p-deimethylaminocinnamaldehyde (DMACA) @ acidic pH to prod. blue/ blue-green color
- saturate filter paper w/ 2 drops indole spot reagent (DMACA) or drop on cotton swab
- smear w/ 18-24hr culture on BA. observe for blue/blue-green color w/in 1-3 mins
Salmonella spp.
acute gastroenteritis and typhoid fever (S. typhi) GNR motile H2S + lysine + NLF indole = VP= PDA= urease= API for speciation
famous Salmonella carrier
typhoid Mary
cooked at hospital
NY
Citrobactereae
related to Salmonella C.freundii H2S + looks like Salmonella C.koseri H2S = citrate+ MR+ LF sometimes NLF VP= ONPG+ can mimic Salmonella on Hektoen agar
Klebsiellae spp.
Klebsiella pneumoniae
Klebsiella oxytoca
Klebsiella aerogenes
Klebsielleae morph
fat shorter GNR
muccoid (capsule)
LF
Klebsiella tests
non motile except K.aerogenes (part of SKY grp)
LF
VP + (part of KES grp)
urea+
K.pneumoniae MIO =/=/=, VP+, bulls eye LF
K.oxytoca MIO =/+/=, VP+
K.aerogenes MIO +/=/+, VP + urea =
Enterobacter (in Klebsielleae tribe)
LF Enterobacter cloacae Enterobacter sakazakii mucoid citrate + MR= VP + motile MIO +/=/+
Serratia marcescens (in Klebsielleae tribe)
NLF pigmented (pink-orange-red) [false LF+ don't misread] motile VP+ gelatin + arabinose =
Hafnia alvei (Klebsielleae tribe)
only in immuno.
ID w/ automation
NLF
Proteeae tribe
Proteus
Providencia
Morganella morganii
Proteus spp.
swarm Proteus mirabilis: indole =, ornithinine + Proteus vulgaris: indole +, ornithine = can be Salmonella mimic on Hektoen agar H2S+ PDA+
Providencia spp.
Providencia retferii
Providencia stuartii
PDA +
Morganella morganii tests
MIO +/+/+
PDA +
Yersinia enterocolitica
non motile 37*C motile 25*C ornithine + ONPG + urea + causes GI tract infection rxns similar to S.sonnei
Yersinia pestis
very non reactive MIO =/=/= urea = ONPG + causes plague from rodents/prairie dogs
CIN agar
differential & selective
isolate Yersinia entercolitica
GN & GP inhibited by crystal violet, Na deoxycholate and antimicrobials: Cefsuldoin, Irgasan, and Novobiocin
incubate @ room temp
colonies w/ dark red center surrounded by transparent border are possible Y. entercolitica
KIA test
1:10 glucose;lactose
H2S ferrous sulfate and Na thiosulfate
peptones
AK/AK (RED/RED): no fermentation
AK/A (RED/YELLOW): only glucose fermented
A/A (YELLOW/YELLOW): both glucose & lactose fermented
BLACK DEEP: H2S prod. (requires acid environment)
black deep w/ red (Salmonella)
black deep w/ yellow (Citrobacter)
Methyl Red/ Voges Proskauer
positive for one or the other
MR: prod. strong acids thru acid fermentation; pH - red color
VP: detects acetoin, end prod. of butylene glycol pthwy; add 40% KOH, alpha naphthol - red color
Citrate test
detects ability to use citrate as a sole source of energy
bromothymol blue in presence of alkaline metabolic prod.
green===blue +
Urease test
use Christensen’s agar
Urea, dextrose, phenol red
urea breakdown==ammonia===pink+
for fungus too
Phenylalanine Deaminase (PDA)
prod. phenyl-pyruvic acid and ammonia
add FeCl3 === green color +
Lysine iron agar tube (LIA)
deter. if can decarboxylate lysine and form H2S
charac. of Salmonella
Ferric ammonium citrate and Na thiosulfate for H2S prod.
bromocresol purple pH indicator
glucose (dextrose); no lactose
lysine to detect decarboxylating lysine
LYSINE DECARBOXYLATION DETECT. IN BUTT:
purple slant/ purple butt: positive [or black butt]
purple slant/ yellow butt: negative fermentation of glucose only
LYSINE DEAMINATION DETCT. ON SLANT:
positive: red slant
negative: slant purple
H2S PROD.:
postivie: black ppt
negative: no black color
gas: bubbles/cracks in medium
Hugh Leifson OF medium
peptone decreased to reduce formation of alkaline amines that neutralize small amounts of weak acids formed
carbo increased to increase amount of acid that forms
agar [ ] decreased making it semi solid to permit acid that form on surface to penetrate thru media making pH change easier to see
bromothymol blue pH indicator (yellow-acid blue-alkaline)
open tube to see if oxidizer
Triple Sugar Agar
1% lactose 1% sucrose 0.1% glucose Peptones Ferrous sulfate Screens for Salmonella and Shigella Sucorse for other enterics not Sal & Shig Ak/A Shigella Ak/A H2S Salmonella
MIO
Low level agar
Pancreatic digest of casein, gelatin, yeast extract, bromocresol purple
Glucose ferm but no aa decarboxylated yellow
Aa decarboxylated purple
Ferment glucose==yellow==aa decarboxylated==purple
Overlay w/oil
Add indole reagent if positive red ring around top
