L9 Enterobacteriaceae

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

Rods (several hundred)

Aerobic Anaerobic Facultative/fermentative

Anaerobic
Veillonella sp.

GNRs: Facultative/ fermentative

Enterobacteriaceae
Fermenting glucose and lactose Citrobacter sp. Enterobacter sp. Escherichia coli Klebsiella pneumoniae Fermenting glucose but NOT lactose 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. coli Clinical 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?

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=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

Eg Salmonella enterica serovars (e.g., Enteritidis, Typhi, Typhimurium) Ie Salmonella ser. Typhimurium (not italicized) Some books: S. Typhimurium S. Typhi

Subgroup 2 : salamae Subgroup3a: arizonae Subgroup3b: diarizonae Subgroup 4: houtenae Subgroup 5: bongori Subgroup 6: indica

Why the problem?

three kinds of major antigens Somatic (O) or Cell Wall Antigens 67 used for serological identification. Surface (capsular) Antigens Vi antigen well known Flagellar (H) Antigens Antiflagellar antibodies can immobilize bacteria with corresponding H antigens.

So many surface antigens

Clinical Aspects:
two distinct diseases enteric fever (typhoid), 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 Salmonella carriage because the site of carriage (gall bladder) may not allow penetration by the antibiotic.

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

Outbreak of salmonella
http://video.google.com/videoplay?docid=623 8435932617357831&q=&hl=en

Severe diarrhea accompanied by fever and also invasive

Shigella

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 fs 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 ureasplitting bacteria.

Staghorn calculus Kidney

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?

Next? Have GNR fermentative : ???? how to differentiate Enterobacteriacea

from the others

The OXIDASE test

Oxidase neg: enterobacteriacea Oxidase pos: = the others Vibrios Aeromonads Pseudomonads (aerobic) Will do these next lecture

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

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

Another example: Following incubation and addition of nitrate reagents:

3 Which are positive??

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

What about 1?

differentiating pathogens from non-pathogens: ability to ferment lactose

95% 0% 0 1% 98% 5% 0 - 1% 98% 0% 2% 2%

Citrobacter freundii (50%)

Commonly used lactose containing selective and differential media

MacConkey XLD EMB KIA TSI

MacConkey Agar selective 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 fermenters are colorless.

Reactions on Mac

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

Salmonella on XLD agar: lysine positive, xylose fermented, positive H2S (colonies with black centers)

E. coli coli XLD agar: lactose and sucrose E. on on XLD agar: lactose fermented, lysine and H2S negative. and sucrose fermented, lysine and H2S negative.

Proteus on XLD agar: swarming inhibited, xylose only fermented, lysine negative.

Shigella on XLD agar: non-fermentive, no H2S

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

KIA: allows for determination of:

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 interpretation

Lactose glucose

Why include glucose?

A/A K/A K/K A/K

SOME COMMON SUGAR REACTIONS IN KIA E. coli, Yersinia, Aeromonas, Vibrio Salmonella, Edwardsiella, Shigella nonfermenters such as Pseudomonas and others erroneously inoculated a Gram + or some other weird thing

THIS MEDIUM IS RUN ON GRAM - RODS ONLY!

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

K.pneumoniae E.coli

tests the ability of organism to split indole from tryptophan (ie have tryptophanase) Indole reagents: Kovac's reagent: Pdimethylaminobenzaldehyde in alcohol Positive reaction: formation of red color at the interface of the broth and reagent
tryptone broth

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
peptone broth

MR-VP media buffered-dextrose 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 Enterobacter produce more neutral products from glucose (acetoin) pH rises above 6.2. The reagents: alpha-napthol and 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 bgalactosidase True NLFs lack both of these enzymes Late LFs produce b-galactosidase but lack permease (eg Shigella 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 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 CHOs 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

BUTconsider: Biochemical variability from tables

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

Probability and identification

ONPG Result in your test E. coli Shigella Sorbitol Indole

+
95 5

+
95 30

+
98 50

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)
MOT + 1 0 5 MR 4 VP + 1 XYL + 0 1 ARA 0 LDC 0 CIT 0 4 H2S 4 IND +

514 is unique to those results.

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