slides student l9[1]pathology

8/13/2019 Slides Student L9[1]Pathology

1/80

L9Enterobacteriaceae

Lecture prepared by Dr. M.Watts


2/80

Objectives

.students will become familiar with.a range of tests designed to identifyEnterobacteriaceae.a range of diseases caused by theseorganisms.Know where they fit into the classificationschemes


3/80

.Gram-positive cocci.Micrococaceae.StreptococcacaeaMedically important bacteria:So far.. Gram +

.Gram-positive rods.Aerobic.Bacillus spp..Lactobacillus sp..Corynebacterium spp.Listeria spp..Erysipelothrix rhusiopathiae

.Nocardia sp.

.Streptomyces spp..Anaerobic.Actinomyces sp..Clostridium spp


4/80

Now: Gram NegativeOrganisms

.Cocci (only a few).Aerobic.Neisseria.Moraxella.Anaerobic.Veillonella sp..Rods (severalhundred).Aerobic.Anaerobic

.Facultative/fermentative


5/80

GNRs: Facultative/

.Enterobacteriaceae.Fermenting glucose andlactose.Citrobacter sp..Enterobacter sp..Escherichia coli.Klebsiella pneumoniae.Fermenting glucose but NOTlactose.Proteus sp..Salmonella enteriditis

.Salmonella typhi

.Shigella sp..Serratia marcescens.Yersinia enterocolitica.Yersinia pestisfermentative

.Others (non-enteric) miscGNBs.Pseudomonas spp..Aeromonas sp..Plesiomonas shigelloides.Vibrio cholerae.Vibrio parahaemolyticus

.Vibrio vulnificussugar fermented is hugely important:


6/80

Enterobacteriaceae

What are they?Where do you find them?Are they medically important?Which are most commonly encountered?How to recognise and identify


7/80

What are they?

.Gram negative rods grouped together on strongphenotypic grounds.Most confirmed by molecular methods.Currently, they are classed into 31 genera and over100 species.


8/80

Where do you findthem?

.Primarily the bowel.1012 per gram of faeces.large range of animals, both warm and cold blooded.Also on some plants and in the soil..wherever you find animal faeces.Prefer moist environments


9/80

Are they medically important?YES!Have the opportunityAre they medically important?YES!Have the opportunity.Cause a range ofinfections.Enteric: Salmonella,Shigella, E. coli..UTI: E. coli..Nosocomial:.Septicaemia,.Pneumonia.Wound infection.

reasonably resistant toantibioticshave the necessary virulence attributes


10/80

.normal flora of GIT.produces vitamin K in the large intestine.most commonly isolated pathogen inhospitalized patients.UTI, neonatal meningitis,gastroenteritis, wound infections,pneumonia, septicaemia..LPS (endotoxin) is released when thecell dies.Treating infections with antibiotics mayplace the patient in severe shock.some strains have acquired additionalgenetic information from plasmids,transposons, and phages whichallows them to be pathogenicE. coli

Clinical aspects:


11/80

E.coli: a highly variable organism

.5 classes causediarrhoeal diseases.ETEC (Enterotoxigenic).EPEC (Enteropathogenic).EIEC (Enteroinvasive).EHEC 0157(Enterohaemorrhagic).EaggEC


12/80

EHEC0157(Enterohaemorrhagic)


13/80

How canvariants of thesame organismbe so different?


14/80

http://video.google.com/videoplay?docid=6673841890662551798

Salmonella

http://video.google.com/videosearch?q=whale&emb=0#emb=0&q=beached%20whale%20new%20zealand&src=


15/80

Salmonella nomenclaturecontroversial

.original taxonomy based on clinical considerations,.e.g., Salmonella typhi, Salmonella cholerae-suis, Salmonella abortus-ovis,.Now known that all Salmonella serovars form a single species (Salmonella enterica)composed of seven subgroups.Subgroup 1 contains most common serotypes.typhi.cholerae-suis Eg Salmonella enterica serovars (e.g.,.paratyphiEnteritidis, Typhi, Typhimurium)

.

gallinarium.pullorumIe Salmonella ser. Typhimurium (not

.Subgroup 2 : salamaeitalicized)

.Subgroup3a: arizonae.Subgroup3b: diarizonae

Some books:

.Subgroup 4: houtenae.Subgroup 5: bongoriS. Typhimurium.Subgroup 6: indicaS. Typhi


16/80

Why the problem?

.three kinds of majorantigens.Somatic (O) or Cell WallAntigens67 used for serologicalidentification.

.Surface (capsular)AntigensVi antigen well known.Flagellar (H) AntigensAntiflagellar antibodies canimmobilize bacteria withcorresponding H antigens.

So many surface antigens


17/80

Clinical Aspects:

.two distinct diseases.enteric fever (typhoid),.bacterial invasion of thebloodstream (S. typhi).acute gastroenteritis.foodborn Salmonella:(S.typhimurium, S.enteritidis).chicken and eggs reservoir..implicated in more than50,000 cases of bacterialfood poisoning in the UnitedStates every year

.S. typhi only carried by humans

.Role of carriers.About 5% of patients clinicallycured from typhoid remaincarriers for months or evenyears..Antibiotics are usuallyineffective on Salmonellacarriage because the site of

carriage (gall bladder) may notallow penetration by theantibiotic.


18/80

Outbreak of salmonella

.http://video.google.com/videoplay?docid=6238435932617357831&q=&hl=en


19/80

.Severe diarrhea accompanied by feverand also invasive.reservoir human only.small inoculum (10 to 200 organisms) issufficient to cause infection..Shiga toxin A:B, enters cell disruptsprotein synthesis.Outbreaks in daycare, nursinghomes.spread.Four fs faeces food flies fingers.four species.Serotype A-S. dysenteriae.

Serotype B-S. flexneri.Serotype C-S. boydii (rare).Serotype D-S. sonnei (mostcommon)Shigella


20/80

KLEBSIELLA

.Klebsiella pneumoniae.Common cause ofnosocomial pneumonia andUTI(second only to E. coli).Produces a heat-stableenterotoxin.contain resistanceplasmids (R-plasmids).can be transferred toother enteric bacteria(not necessarily of thesame species)


21/80

P. mirabilisProteus: urinarypathogensCan form stonescaused by infection ofthe urine with urea-splitting bacteria.Staghorn calculus

Kidney


22/80

Yersinia pestis Yersinia pestisYersinia pestis rodent pathogen,The flea draws viable Y. pestis organisms into its intestinal tract.These organisms multiply in the flea.humans an accidental host


23/80

Los Angeles Times Los Angeles Times


24/80

Others.

.NF gut can all cause opportunistic infections.EDWARDSIELLA.E. tarda known to cause gastroenteritis and wound infections.CITROBACTER.C. freundii is suspected to cause diarrhea.C. diversus has been linked to a few cases of meningitis in newborns..ENTEROBACTER.E. aerogenes and E. cloacae are sometimes associated with UT & RT infections.SERRATIA.Serratia marcescens is considered a harmful human pathogen.

known to cause urinary tract infections, wound infections, and pneumonia..also have many antibiotic resistance properties


25/80

Identification

Uses biochemistry of bacteriaThink about what they do andwhere they live


26/80

12 most commongenera

.Often collectively.called coliforms..

.Escherichia coli: type.species.

ESCHERICHIASHIGELLAEDWARDSIELLASALMONELLACITROBACTERKLEBSIELLA

ENTEROBACTERSERRATIAPROTEUSMORGANELLAPROVIDENCIAYERSINIA


27/80

Which tests aremost useful?

.Diagnostic microbiology.Most useful tests for quick ID.Empirical:.Ist stage tests


28/80

Identification:GNR

E.coli, a vibrio & apseudomonadWhich is which??


29/80

OF test: most are fermenters

A BWhich is fermenter? Which is oxider?


30/80

Next?

Have GNR fermentative :

???? how to differentiate Enterobacteriaceafrom the others

The OXIDASE test

.Oxidase neg:enterobacteriacea.Oxidase pos: = theothers.Vibrios.Aeromonads.Pseudomonads (aerobic).

Will do these next lecture


31/80

Nitrate reduction Nitrate reduction.+ve for Enterobacteriaceae.Detects whether bacteria uses nitrate (NO3)as electron acceptor (ie).Nitrate reduced to nitrite (or othercompounds) via nitrate reductase.NO3 ----> NO2 ----> NH3 or N2.Grow organism in nitrate broth: test forend products.reagents: a-naphthylamine and sulfanilic acid.Formation of red color after addition of the


32/80

Nitrate reduction

Which are +ve?


33/80

Another example: Following incubationand addition of nitrate reagents:

34Which are positive??


34/80

Now which are positive? (Following additionof Zn dust)

What about 1?


35/80

differentiating pathogens from non-pathogens:ability to ferment lactose

0%95%0 -1%5%0 1%2%98%0%98%2%Citrobacter freundii (50%)


36/80

Commonly used lactose containingselective and differential mediaCommonly used lactose containingselective and differential media.MacConkey.XLD.EMB.KIA.TSI


37/80


38/80

Reactions on Mac


39/80

A ABC DHave a guess?


40/80

XLD

.selective and differential medium designedfor the isolation of Gram-negative entericpathogens from clinical specimens..contains xylose, lysine, sodiumdesoxycholate, sodium thiosulfate and ferricammonium citrate.


41/80

Principle:

.Differentiation of Shigella and Salmonella from nonpathogenic bacteriais accomplished by three reactions:.1) xylose fermentation,.2) lysine decarboxylation, and.3) hydrogen sulfide production..Xylose: enterics (except Shigella) ferment xylose rapidly..Salmonella rapidly exhaust xylose and decarboxylate lysine, and revertto alkaline conditions (simulates the Shigella reaction)..Lactose and sucrose, added in excess, prevent lactose fermenters fromsimilarly reverting..The production of hydrogen sulfide under alkaline conditions results inthe formation of colonies with black centers, whereas, under acidicconditions, this black precipitation is inhibited


42/80

Salmonella on XLD agar: lysine positive, Salmonella on XLD agar: lysine positive,xylose fermented, positive H2S E. coli on XLD agar: lactose and sucrose(colonies with black centers) fermented, lysine and H2S negative.

Proteus on XLD agar:swarming inhibited, xylose only Shigella on XLD agar:fermented, lysine negative. non-fermentive, no H2S


43/80

Reactions on XLD Reactions on XLD


44/80

EMB:

.Peptones 10.0;.di-potassium hydrogen phosphate 2.0;.lactose 5.0;.sucrose 5.0;.eosin Y;.methylene blue 0.07; agar-agar 13.5.


45/80

KIA formula

.Meat extract...........................................................................3,00.Yeast extract..........................................................................3,00.Peptone.................................................................................20,00.Lactose..................................................................................10,00.Sodium chloride..................................................................... 5,00.Dextrose................................................................................ 1,00.Ammonium Ferrous citrate......................................................0,50

.Sodium thiosulfate..............................................................

..... 0,50

.Phenol red.............................................................................0,03.Agar.......................................................................................15,00.Final pH 7,4 0,2


46/80

KIA:allows fordeterminationof:

.1. Fermentation of glucose, lactose.2. Production of gas during fermentation.3. Production of H2S from the sulfur source.Phenol red is used as PH indicator: yellow in acid,red in alkaline


47/80

KIAinterpretationLactoseglucoseKIAinterpretationLactoseglucoseWhy includeglucose?

nonfermenters such as Pseudomonas and others K/KSalmonella, Edwardsiella, Shigella K/AE. coli, Yersinia, Aeromonas, Vibrio A/ASOME COMMON SUGAR REACTIONS IN KIATHIS MEDIUM IS RUN ON GRAM -RODS ONLY!


48/80

KIA interpretation

.If the butt of tube is yellow (A): glucose fermenter.If the slant of tube is yellow (A): lactose fermenter.If the slant is red (K): non-lactose fermenter.Black ppt: H2S prodn.Gas: gaps in agar.A/A, K/A and K/K.Could you get A/K??????.(If the butt of tube is red (K): non-glucose fermenter)


49/80

Carbohydrate fermentation

.Single CHO.Test Result.1.Control Negative.2. S. aureus ?.3. P. vulgaris ??.4. P.aeruginosa.5. E. coli ??+ve Acid prodn: color changefrom red to yellow

.


50/80

Amino acid decarboxylation

.Decarboxylation only takesplace in an acid environment.& needs to be anaerobic.Net reaction is alkaline.How can you be suredecarboxylation has occurred?.2 tubes inoculated:.one without amino acid butboth with glucose (for.?.sterile mineral oil overlay.Tube without aa is control(goes yellow)


51/80

Typical reactions: which set is+ve?


52/80

Motility test

.A non-motile organism willhave a clearly defined edgeas it grows on the stab line.Motile organisms will beturbid throughout the tubeor have fuzzy, diffusegrowth at the edges..Some organisms are somotile that the entire tubebecomes very turbid(cloudy).


53/80

Indole-methyl red-VP-citrateIMViC tests (4 tests)

.used to differentiate Enterobacter andKlebsiella from E.coli.three sets of media are inoculated:.indole test (tryptone broth).MR-VP broth,.Simmons citrate medium


54/80

The Indole Test

.tests the ability of organism tosplit indole from tryptophan(ie have tryptophanase).Indole reagents:.Kovac's reagent: Pdimethylaminobenzaldehydein alcohol.Positive reaction: formation ofred color at the interface of thebroth and reagentE.coliK.pneumoniaetryptone broth


55/80

MR-VP

.All enterics oxidize glucose forenergy.end products vary depending onbacterial enzymes.MR and VP tests are used todetermine what end productsresult.MR-VP media buffered-dextrosepeptone broth.tests are read from a singleinoculated tube of MR-VP broth..After 24-48 hours of incubationthe MR-VP broth is split into twotubes.


56/80

Methyl red:

.It tests the ability of organismto produce and maintainstrong, mixed acid frombuffered-dextrose peptonebroth.E. coli is one of the bacteriathat produces acids, causingthe pH to drop below 4.4..When the pH indicator methylred is added to this acidicbroth it will be cherry red (apositive MR test)


57/80

Vogues Proskauer

.Klebsiella and Enterobacterproduce more neutral productsfrom glucose (acetoin).pH rises above 6.2..The reagents: alpha-naptholand potassium hydroxide..If acetoin is present reagentsturn a pink-burgundy color (apositive VP test)..color may take 20 to 30minutes to develop.


58/80

The Citrate Test: Citrate usealkalinises the medium

.Simmon's medium.Typical Composition (g/liter)Ammonium dihydrogen phosphate 1.0;

.di-potassium hydrogen phosphate 1.0;.sodium chloride 5.0;.sodium citrate 2.0; sole C and energy source.magnesium sulfate 0.2;.bromothymol blue 0.08;.pH of 6.9: agar-agar 13.0..

yellow at acidic pH's (around 6), and blueat more alkaline pH's (around 7.6)..positive citrate: blue.Enterobacter and Klebsiella + E.coli


59/80

Why does citrate use alkalinisethe medium??

Bacteria that use citrate also utilize the ammonium saltas a nitrogen source and create ammonia as a result.


60/80

IMViC Interpretation

.E.coli gives ++-.Enterobacter and Klebsiella give the reverse:--++


61/80

ONPG test

.Principle.Lactose utilization requires 2 main enzymes,.permease and b-galactosidase.Enzymes ONLY induced in the presence of the lactose substrate(inducer).Need high lactose medium.Regular LFs produce both permease enzyme and b-galactosidase.True NLFs lack both of these enzymes.Late LFs produce b-galactosidase but lack permease (egShigella sonnei)

.ONPG: (Ortho-nitrophenyl-b-D-galactopyranoside ) Artificialsubstrate.turns yellow in the presence of beta-galactosidase.http://vcell.ndsu.nodak.edu/animations/lacOperon/index.htm


62/80

ONPG test: detects the presence of

.organism needs to be growing in/onany medium with lactose (to induce theproduction of galactosidase).Pipette 0.5ml of the saline into a steriletube..Inoculate with the bacterium and add theONPG disc in a sterile manner (forcepsdipped in alcohol and flamed) to the tube..Incubate at 37C for 4 hours..INTERPRETATION:.any shade of yellow + for galactosidaseenzyme..

Precautions.A heavy inoculum is necessary to obtaina high concentration of enzyme.


63/80

Urease test

.Some bacteriaproduce urease:.Urease splits ureainto ammonia andcarbon dioxide.Organisms thatproduce urease willturn hot pink due toammonia prodn


64/80

Urease +ve Proteus Urease +ve Proteus


65/80

SERRATIA

characteristic red pigment.


66/80

Summary: Recognitionand Identification

.Is it a coliformor another type of GNB?.Facultative, growth on Mac: then do oxidase (-).[OF (F) and NO3 reduction tests not usually needed].Which coliform is it?.Is it a major pathogen (salmonella or shigella orE.coli 0157?).Requires a battery(10-20) of biochemical tests.Usually commercially packaged.Many of these are broadly discriminatory..A range of CHOs added for more fine discrimination.


67/80

Characteristics shared by allEnterobacteriaceae

.Gram negative rods.oxidase negative.All can ferment glucose.Facultative anaerobes.reduce nitrate to nitrite


68/80

Then more tests to speciate

.Lactose fermentation.CHO fermentation.Decarboxylase reactions.H2S production.IMViC reactions:. I: indole. M: methyl red. V: Voges-Proskauer. C: citrate.And others


69/80

BUTconsider:Biochemical variability fromtables

ONPG LDC CIT ARA INDC.freundii 95 -62 + 6K. pneumoniae + + + + -E. coli 95 77 -83 94Salmonella spp. v v v v -Pr. mirabilis --57 --


70/80

Probability and identification Probability and identificationONPG Sorbitol IndoleResult inyour test+ + +E. coli 95 95 98Shigella 5 30 50

Could be either E.coli or Shigella, but E. coli moreprobable. More tests in your panel = betterprobability.


71/80

API strips are therefore very useful!!!!! API strips are therefore very useful!!!!!


72/80


73/80

API profile result sheet

Spot the deliberate mistake!!!!!!


74/80

Mechanics of identification

.Do the 10 or 20 tests..+--++-+++--+++--++.Number?.137436.Now compare your results with a table of expectedresults for over 100 different organisms..This could be a table of 30 x 100!


75/80

Automation

.Many systems now available..organism in diluent..aspirates into black box.Distributes inoculum into wellswith dehydrated substratesand incubates..Instrument reads results withspectro..Determines + or -, andcompares with database..Prints out name of organism.


76/80

Major ID tests

.GNB

.After growth on Mac, do oxidase test..If oxidase negative, do biochemical tests..Carbohydrate fermentation.KIA agar/TSI.IMViC.Urease.Decarboxylase reactions.ONPG.

H2S.Motility


77/80

Compose a flow chartsfrom major tests


78/80

Enterobacteriaceae flow chart Enterobacteriaceae flow chart


79/80

End of lecture

.Review questions


80/80

slides student l9[1]pathology

Documents