L9Enterobacteriaceae

Lecture prepared by Dr. M.Watts

Objectives

� students will become familiar with

� a range of tests designed to identify

Enterobacteriaceae

� a range of diseases caused by these

organisms

� Know where they fit into the classification

schemes

Medically important bacteria:So far.. Gram +

� Gram-positive cocci

� Micrococaceae� Streptococcacaea

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

� Anaerobic � Actinomyces sp.� Clostridium spp

Now: Gram Negative

Organisms

� Cocci (only a few)

� Aerobic

� Neisseria

� Moraxella

� Anaerobic

� Veillonella sp.

� Rods (several hundred)

� Aerobic

� Anaerobic

� Facultative/fermentative

GNRs: Facultative/fermentative

� Enterobacteriaceae

� Fermenting glucose and lactose

� 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 pestis

� Others (non-enteric) misc GNBs� Pseudomonas spp.� Aeromonas sp. � Plesiomonas shigelloides� Vibrio cholerae� Vibrio parahaemolyticus� Vibrio vulnificus

sugar fermented is hugely important:

Enterobacteriaceae

What are they?

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

What are they?

� Gram negative rods grouped together on strong phenotypic grounds

� Most confirmed by molecular methods

� Currently, they are classed into 31 genera and over 100 species.

Where do you find them?

� 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

Are they medically important? YES!

� Cause a range of infections

� Enteric: Salmonella,

Shigella, E. coli.

� UTI: E. coli.

� Nosocomial:

� Septicaemia,

� Pneumonia

� Wound infection

� Have the opportunity

� reasonably resistant to antibiotics

�have the necessary virulence attributes

E. coliClinical aspects:

� normal flora of GIT � produces vitamin K in the large intestine

� most commonly isolated pathogen in hospitalized patients� UTI, neonatal meningitis,

gastroenteritis, wound infections, pneumonia, septicaemia.

� LPS (endotoxin) is released when the cell dies� Treating infections with antibiotics may

place the patient in severe shock

� some strains have acquired additional genetic information from plasmids, transposons, and phages which allows them to be pathogenic

E.coli: a highly variable organism

� 5 classes cause diarrhoeal diseases

� ETEC (Enterotoxigenic)

� EPEC (Enteropathogenic)

� EIEC (Enteroinvasive)

� EHEC 0157

(Enterohaemorrhagic)

� EaggEC

EHEC 0157(Enterohaemorrhagic)

How can variants of the same organism be so different?

Salmonella

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

http://video.google.com/videosearch?q=whale&emb=0#emb=0&q=beached%20whale%20new%20zealand&src=2

Salmonella nomenclature controversial

� 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

� paratyphi

� gallinarium

� pullorum

� Subgroup 2 : salamae

� Subgroup3a: arizonae

� Subgroup3b: diarizonae

� Subgroup 4: houtenae

� Subgroup 5: bongori

� Subgroup 6: indica

Eg Salmonella enterica serovars (e.g.,

Enteritidis, Typhi, Typhimurium)

Ie Salmonella ser. Typhimurium (not

italicized)

Some books:

S. Typhimurium

S. Typhi

Why the problem?

� three kinds of major antigens

� Somatic (O) or Cell Wall Antigens67 used for serological identification.

� Surface (capsular) Antigens Vi antigen well known

� Flagellar (H) AntigensAntiflagellar antibodies can immobilize bacteria with corresponding H antigens.

So many surface antigens

Clinical Aspects:

� two distinct diseases

� enteric fever (typhoid),

� bacterial invasion of the bloodstream (S. typhi)

� acute gastroenteritis

� foodborn Salmonella: (S.typhimurium, S.enteritidis)

� chicken and eggs reservoir.

� implicated in more than 50,000 cases of bacterial food poisoning in the United States every year

� S. typhi only carried by humans

� Role of carriers

� About 5% of patients clinically cured from typhoid remain carriers for months or even years.

� Antibiotics are usually ineffective on Salmonellacarriage because the site of carriage (gall bladder) may not allow penetration by the antibiotic.

Outbreak of salmonella

� http://video.google.com/videoplay?docid=623

8435932617357831&q=&hl=en

Shigella� Severe diarrhea accompanied by fever

and also invasive

� reservoir human only� small inoculum (10 to 200 organisms) is

sufficient to cause infection.

� Shiga toxin A:B, enters cell disrupts protein synthesis

� Outbreaks in daycare, nursing homes

� spread� Four “ f’s “ faeces food flies fingers

� four species � Serotype A- S. dysenteriae� Serotype B- S. flexneri� Serotype C- S. boydii (rare)� Serotype D- S. sonnei (most

common)

KLEBSIELLA

� Klebsiella pneumoniae

� Common cause of nosocomial pneumonia and UTI(second only to E. coli)

� Produces a heat-stable enterotoxin

� contain resistance plasmids (R-plasmids)

� can be transferred to other enteric bacteria (not necessarily of the same species)

P. mirabilis

• Proteus: urinary

pathogens

• Can form stones

• caused by infection of the urine with urea-

splitting bacteria.

Kidney

Staghorn calculus

Yersinia pestis

•Yersinia pestis rodent pathogen,

•The flea draws viable Y. pestis organisms into its intestinal tract.

•These organisms multiply in the flea.

•humans an accidental host

Los Angeles Times

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

Identification

Uses biochemistry of bacteria

Think about what they do and where they live

12 most common genera

� Often collectively called “coliforms”.

� Escherichia coli: type species

� ESCHERICHIA � SHIGELLA� EDWARDSIELLA� SALMONELLA� CITROBACTER� KLEBSIELLA� ENTEROBACTER� SERRATIA� PROTEUS� MORGANELLA� PROVIDENCIA� YERSINIA

Which tests are most useful?

� Diagnostic microbiology

� Most useful tests for quick ID

� Empirical:

� Ist stage tests

Identification:GNR

E.coli, a vibrio & a pseudomonad

Which is which??

OF test: most are fermenters

Which is fermenter? Which is oxider?

A B

Next?

Have GNR fermentative :

???? how to differentiate Enterobacteriacea

from ‘the others’

� Oxidase neg: enterobacteriacea

� Oxidase pos: = ‘the

others’

� Vibrios

� Aeromonads

� Pseudomonads (aerobic)

� Will do these next lecture

The OXIDASE test

Nitrate reduction

� +ve for Enterobacteriaceae

� Detects whether bacteria uses nitrate (NO3)as electron acceptor (ie)

� Nitrate reduced to nitrite (or other compounds) via nitrate reductase

� NO3 ----> NO2 ----> NH3 or N2

� Grow organism in nitrate broth: test for end products� reagents: a-naphthylamine and sulfanilic acid

� Formation of red color after addition of the

Nitrate reduction

Medium undefined: contains large amounts of nitrate (KNO3). Which are +ve?

Another example: Following incubation and addition of nitrate reagents:

3 4

Which are positive??

Now which are positive? (Following addition of Zn dust)

What about 1?

differentiating pathogens from non-pathogens: ability to ferment lactose

0%95%

0 - 1%

5%

0 – 1%

2%

98%

0%

98%

2%

Citrobacter freundii (50%)

Commonly used lactose containing selective and differential media

� MacConkey

� XLD

� EMB

� KIA

� TSI

MacConkey Agarselective and

differential

contains� lactose, � bile salts, � neutral red (and crystal violet).

� selective media � Gram-positive organisms are inhibited by the bile salts and the

crystal violet.

� Differential� When bacteria ferment lactose and produce enough acid products

to reduce the pH below 6.8, the neutral red turns from colorless to red.

� Thus, MacConkey is a differential media on which lactose fermenting colonies appear red (or pink). Nonlactose fermentersare colorless.

Reactions on Mac

A B

C D

Have a guess?

XLD

� selective and differential medium designed for the isolation of Gram-negative enteric pathogens from clinical specimens.

� contains xylose, lysine, sodium desoxycholate, sodium thiosulfate and ferric ammonium citrate.

Principle:

� Differentiation of Shigella and Salmonella from nonpathogenic bacteria is 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 revert to alkaline conditions (simulates the Shigella reaction).

� Lactose and sucrose, added in excess, prevent lactose fermenters from similarly reverting.

� The production of hydrogen sulfide under alkaline conditions results in the formation of colonies with black centers, whereas, under acidic conditions, this black precipitation is inhibited

E. coli on XLD agar: lactose

and sucrose fermented,

lysine and H2S negative.

E. coli on XLD agar: lactose and sucrose

fermented, lysine and H2S negative.

Shigella on XLD agar:

non-fermentive, no H2S

Salmonella on XLD agar: lysine positive,

xylose fermented, positive H2S

(colonies with black centers)

Proteus on XLD agar:

swarming inhibited, xylose only

fermented, lysine negative.

Reactions on XLD

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.

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

� 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

KIA:allows for determination of:

KIA interpretation

Lactose

glucose

erroneously inoculated a Gram + or some other weird thingA/K

nonfermenters such as Pseudomonas and othersK/K

Salmonella, Edwardsiella, ShigellaK/A

E. coli, Yersinia, Aeromonas, VibrioA/A

SOME COMMON SUGAR REACTIONS IN KIA

THIS MEDIUM IS RUN ON GRAM - RODS ONLY!

Why include

glucose?

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)

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 change from red to yellow.

Amino acid decarboxylation

� Decarboxylation only takes place in an acid environment

� & needs to be anaerobic

� Net reaction is alkaline

� How can you be sure decarboxylation has occurred?

� 2 tubes inoculated:

� one without amino acid but

both with glucose (for……….?)

� sterile mineral oil overlay

� Tube without aa is control

(goes yellow)

Typical reactions: which set is +ve?

Motility test

� A non-motile organism will

have a clearly defined edge

as it grows on the stab line

� Motile organisms will be

turbid throughout the tube

or have fuzzy, diffuse

growth at the edges.

� Some organisms are so

motile that the entire tube

becomes very turbid

(cloudy).

Indole-methyl red-VP-citrate

IMViC tests (4 tests)

� used to differentiate Enterobacter and Klebsiella from E.coli

� three sets of media are inoculated:

� indole test (tryptone broth)

� MR-VP broth,

� Simmons citrate medium

The Indole Test

� tests the ability of organism to split indole from tryptophan (ie have tryptophanase)

� Indole reagents:

� Kovac's reagent: P-dimethylaminobenzaldehydein alcohol

� Positive reaction: formation of red color at the interface of the broth and reagent

tryptone broth

E.coli

K.pneumoniae

MR-VP

� All enterics oxidize glucose for energy

� end products vary depending on bacterial enzymes

� MR and VP tests are used to determine what end products result

� MR-VP media buffered-dextrose peptone broth

� tests are read from a single inoculated tube of MR-VP broth.

� After 24-48 hours of incubation the MR-VP broth is split into two tubes.

Methyl red:

� It tests the ability of organism to produce and maintain strong, mixed acid from buffered-dextrose peptone broth

� E. coli is one of the bacteria that produces acids, causing the pH to drop below 4.4.

� When the pH indicator methyl red is added to this acidic broth it will be cherry red (a positive MR test)

Vogues Proskauer

� Klebsiella and Enterobacterproduce more neutral products from glucose (acetoin)

� pH rises above 6.2.

� The reagents: alpha-naptholand potassium hydroxide.

� If acetoin is present reagents turn a pink-burgundy color (a positive VP test).

� color may take 20 to 30 minutes to develop.

The Citrate Test: Citrate use

alkalinises 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 blue at more alkaline pH's (around 7.6).

� positive citrate: blue � Enterobacter and Klebsiella + E.coli -

Why does citrate use alkalinise the medium??

Bacteria that use citrate also utilize the ammonium salt

as a nitrogen source and create ammonia as a result.

IMViC Interpretation

� E.coli gives ++--

� Enterobacter and Klebsiella give the reverse:

--++

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 ) Artificial substrate

� turns yellow in the presence of beta-galactosidase.

http://vcell.ndsu.nodak.edu/animations/lacOperon/index.htm

ONPG test: detects the presence of β-

galactosidase

� organism needs to be growing in/on any medium with lactose (to induce the production of galactosidase)

� Pipette 0.5ml of the saline into a sterile tube.

� Inoculate with the bacterium and add the ONPG disc in a sterile manner (forceps dipped in alcohol and flamed) to the tube.

� Incubate at 37C for 4 hours.

� INTERPRETATION:� any shade of yellow + for galactosidase

enzyme.

� Precautions� A heavy inoculum is necessary to obtain

a high concentration of enzyme.

Urease test

� Some bacteria produce urease:

� Urease splits urea into ammonia and carbon dioxide

� Organisms that produce urease will turn hot pink due to ammonia prodn

Urease +ve Proteus

SERRATIA

�characteristic red pigment.

Summary: Recognition and Identification

� Is it a “coliform” or 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 or E.coli 0157?)

� Requires a “battery” (10-20) of biochemical tests

� Usually commercially packaged

� Many of these are broadly discriminatory.

� A range of CHO’s added for more fine discrimination.

Characteristics shared by all

Enterobacteriaceae

� Gram negative rods

� oxidase negative

� All can ferment glucose

� Facultative anaerobes

� reduce nitrate to nitrite

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

BUT………consider: Biochemical variability from tables

--57--Pr. mirabilis

-vvvvSalmonella spp.

9483-7795E. coli

-++++K. pneumoniae

6+62-95C.freundii

INDARACITLDCONPG

Probability and identification

50305Shigella

989595E. coli

+++Result in your test

IndoleSorbitolONPG

Could be either E.coli or Shigella, but E. coli more

probable. More tests in your panel = better

probability.

API strips are therefore very useful!!!!!

The use of codes and code books

� Do tests in “set” order

� group the results in sets of three.

� Assign 0 for a (-) and 1, 2 or 4 for a (+).

� - - - = 0, + - - =1, - + - = 2, + + - = 3

� - - + = 4, + - + = 5, - + + = 6, + + + =7

� ( note that if 1, 2, 3 then + + - =3 and - - + = 3)

� 514 is unique to those results.

415

400001401

+----++-+

INDH2SCITLDCARAXYLVPMRMOT

API profile result sheet

Spot the deliberate mistake!!!!!!

Mechanics of identification

� Do the 10 or 20 tests.

� + - - + + - + + + - - + + + - - + +

� Number?

� 137436

� Now compare your results with a table of expected

results for over 100 different organisms.

� This could be a table of 30 x 100!

Automation

� Many systems now available.

� organism in diluent.

� aspirates into “black box”

� Distributes inoculum into wells with dehydrated substrates and incubates.

� Instrument reads results with spectro.

� Determines + or -, and compares with database.

� Prints out name of organism.

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

Compose a flow charts from major tests

Enterobacteriaceae flow chart

End of lecture

� Review questions