microbiology, clinical

Enterobacteriaceae 21

EnterobacteriaceaeEnterobacteriaceae76,000,000 cases of foodborne disease in U.S.

yearly

Enterobacteriaceae account for over 7 million cases of diarrheal disease yearly

Account for 80% of clinically significant gram-negative isolates and

~50% of all clinically significant isolates.

Classification: >25 different genera, Classification: >25 different genera, 110 species110 species

– Escherichia– Shigella– Edwardsiella– Salmonella– Citrobacter– Klebsiella– Enterobacter– Hafnia– Serratia

– Proteus– Providencia– Morganella– Yersinia

General Characteristics of Enteric Gram-General Characteristics of Enteric Gram-Negative RodsNegative Rods

• GNR or coccobacilli• Facultative anaerobes• Motile species have peritrichous flagella• Typical colony on nonselective medium is

gray, circular, convex, and smooth with definite edges

• Most are nonhemolytic

Enterobacteriaceae: Motility (peritrichous flagella)

Non-motile species: Klebsiella, Shigella, Yersinia

E. coli


•Transduction and conjugation readily occurs

•High antigenic variability•Can be resistant to multiple antibiotics

(unpredictable antibiogram)


•Three characteristics in common– Ferment glucose with production of acid– Reduce nitrates to nitrites– Oxidase negative (except for Plesiomonas)

Selective and Differential MediaSelective and Differential Media

•Selective:– Incorporation of dyes and bile salts to

inhibit G+ organisms – May suppress growth of non-pathogenic

species.•Differential

– Based on lactose fermentation and/or H2S production.

Selective and Differential MediaSelective and Differential Media

• EMB• MAC• XLD• HE• SS• BIS

EMB

HEK

HEK agar

E. coli orangeSalmonella sp. black

Biochemical IdentificationBiochemical Identification

•Useful Biochemical Tests– TSI– Carbohydrate fermentation tests– Enzyme activity– End products from substrates


• Most often used tests include:– Oxidase– Indole– MRVP– Citrate– Motility– Phenylalanine deaminase– Decarboxylases/dihydrolase


• Most often used tests include:– Gelatin hydrolysis– Urease– H2S– Fermentation of glucose, lactose, and other

carbohydrates

Serological IdentificationSerological Identification

• Location of Antigens

Enterobacteriaceae: AntigensEnterobacteriaceae: Antigens

• Antigenic structure: differences genus/species.1. O antigens –heat stable polysaccharide (galactose-

mannose-rhamnose)2. Flagellar H antigens –heat labile (flagellin)3. Envelope or capsule K antigens – over surface of O

antigen and may block agglutination by O specific antisera. – Boiling for 15 min will destroy the K antigen– The K antigen is called the Vi (virulence) antigen

Agglutination Test (Typing) For Presence Of Antigens

Serological IdentificationSerological Identification

•Antigenic formula– Always listed in the same sequence– Sequence is O, K, H– Example: Salmonella typhi

Has O antigens 9 and 12; Vi antigen; phase 1 flagellar antigen d; and no phase 2 flagellar antigen

Antigenic formula is 9, 12 (Vi): d:-

Gram-negative StructureGram-negative Structure

Enterobacteriaceae: VirulenceEnterobacteriaceae: Virulence

• Endotoxin•Capsule•Antigenic variation• Type III secretion systems• Sequestration of Growth Factors

– Binding of growth factors in vivo.

• Serum Resistance•Antimicrobial Resistance

EndotoxinEndotoxin

• Lysis of organism releases endotoxin.• Lipid A released.• Fever,

leukopenia,hypotension/shock, tissue necrosis

• Activation of alternative complement cascade

• Disseminate Intravascular Coagulation

EnterotoxinsEnterotoxins• Cause changes in permeability of small intestinal that leads to diarrhea.

• Many enteric pathogens produce A-B enterotoxins.

• Food-poisoning bacteria often produce cytotoxins or superantigens.

Some Organisms that produce A/B toxins: V. cholera, E. coli, Salmonella,

C. tetani, C. botulinum, C. diphtheriae

Enteric PathogensEnteric Pathogens

Salmonella

SalmonellaSalmonella

•General characteristics– O, H, and Vi antigens– Seven groups based on DNA-DNA

hybridization– Group I with more then 1400 serotypes

cause most human infections


•General characteristics– Motile– Usually do not ferment lactose or sucrose– Most found in intestine of humans and

animals– Infections associated with consumption of

fecal contaminated food and water


•Usual specimens isolated from– Stool– Blood– Urine– Bone marrow

Salmonella: culture mediaSalmonella: culture media

•To detect nonlactose fermenters EMB, MAC

–To detect H2S production SS, HE, XLD

–Enrichment medium-fecal pathogens GN broth, Selenite F, Tetrathionate broth

XLD Salmonella and flora onMacConkey agar

Salmonella: Peritrichous flagella Gram Stain

XLDSalmonella

produces black centers on

XLD and HEK


•Final Identification– Biochemical Tests

H2S and lysine decarboxylase positive• Indole and ONPG negative• S. typhi is differentiated from other Salmonella by a

negative citrate and ornithine decarboxylase test • Salmonella is PYR negative while Citrobacter is PYR

positive


•Final Identification– Agglutination Tests

Detect O antigens (A, B, C1, C2, D or E)

SchottmuelleriHirschfeldii


•Pathogenesis and Infectious Diseases Typhoid fever (S. typhi) Bacteremia Enterocolitis

ShigellaShigella

• General characteristics– 4 serogroups

A (S. dysenteriae, 12 serotypes) B (S. flexneri, 6 serotypes) C (S. boydii, 23 serotypes) D (S. sonnei, 1 serotype)

– Fecal-Oral Transmission and contaminated water– Found in intestines of humans and some

nonhuman primates

ShigellaShigella

•Cultural, biochemical and other characteristics– Nonmotile– Do not ferment lactose– Ferment glucose and other carbohydrates

but no gas production

Shigella

Shigella on MacConkey

Species SerotypesS. dysenteriae A

A1 S. flexneri B S. boydii C

C1 C2

S. sonnei D

Shigella Serotypes

Shigella is relatively biochemically inert

ShigellaShigella

• Pathogenesis and Infectious Disease– Shigellosis also known as bacillary dysentery– Intense inflammatory response– Endotoxin– Shiga-toxin– Enterotoxin– Neurotoxin– Highly infectious

Stool from patient with shigellosis. Shigella cause bacillary dysentery that is characterized by the appearance of blood and pus in the stool. Note the presence of both red and white blood cells.

Edwardsiella tardaEdwardsiella tarda

• General characteristics– Primarily found in freshwater fish– Other hosts include turtles, lizards, snakes,

alligators, crayfish, birds and mammals– Also isolated from lakes, rivers, and sewage

water– Important cause of gastrointestinal distress

(diarrheal illness). Also known to cause wound infections.

Edwardsiella tardaEdwardsiella tarda

• Cultural, biochemical and other characteristics– Motile GNR– H2S positive– Ferments Glucose with gas production– Does not ferment lactose, sucrose or most other

carbohydrates

YersiniaYersinia

•Three important species:– Yersinia pestis –plague– Yersinis enterocolitica – enteric– Yersinia pseudotuberculosis – enteric

– Plague: urban and sylvatic zoonosis

Yersinia pestisYersinia pestis

•Non-motile•Bipolar staining• Slow growth of small colonies on ordinary

culture media – it grows better at lower temperature (25-300 C)

Y. pestisY. pestis

• Bubonic Plague:– Transmitted by fleas from rodent

1. Engulfed by fixed macrophages in lymph node2. Enlarged lymph nodes in the groin and armpit

(buboes). 3. Bacteria go from lymph nodes to blood. 4. Lysis of the bacteria releases LPS, septic shock. 5. Black death- Subcutaneous hemorrhages, LPS

causing DIC.

Buboes and pneumoniaBuboes and pneumonia

Y. pestisY. pestis

•Pneumonic Plague:– Bacteria reach the lungs – Ingested by lung macrophages

transmitted directly via aerosol.

Yersinia PestisYersinia Pestis Bipolar Staining Bipolar Staining

Yersinia pestisYersinia pestis

•TSI- K/A no gas•Urea (–)•Direct fluorescent antibody test•New DNA probe test

Other pathogenic Yersinia speciesOther pathogenic Yersinia species::

• Ingestion contaminated food/H2O.

– Y. enterocolitica-watery diarrhea – Y. pseudotuberculosis

Fever and abdominal pain.

Detection of Y. enterocoliticaDetection of Y. enterocolitica

•Cefsulodin-irgasan-novobiocin agar (CIN) – Selective media

Mannitol and phenol red plus antibiotics The media is incubated at 250C.•Pink bulls-eye colonies.•Mannitol fermentation

Yersinia spp.

Y. ent. are motile, growth and produce biochemical reactions faster at 25°C (2 left tubes) and non-motile at 35°C ( 2 right tubes)

CINMAC

UREABile Esculin

Bull-eye colonies

Enteric OpportunistsEnteric Opportunists

EscherichiaEscherichia coli coli

– Normal inhabitant of G.I. tract. – Various forms of gastroenteritis.– Cause of UTI, septicemia,and neonatal

meningitis.– May have capsule.– ID: Biochemistry reactions

Most are motile.Indole positive

Escherichia coli

•Epidemiology– Epidemics occur from:

Person-to-person spreadContaminated food and waterUnpasteurized milk and juices

– Humans, domestic and wild animals-sources of pathogens

E. coliE. coli

•May be hemolytic on BAP – more common in pathogenic strains

•KEY tests :– TSI is A/A + gas– Indole +– Motility +

•There is an inactive biotype that is anaerogenic, lactose –, and nonmotile.

E. coli: E. coli: Clinical SignificanceClinical Significance

•Neonatal meningitis – is the leading cause of neonatal meningitis and septicemia with a high mortality rate.– strains with K1 capsular antigen.

In premature babies and newborns < 3 months, causes are: group B streptococciEscherichia coliListeria monocytogenes

Older children are more commonly affected by: Neisseria meningitidis (meningococcus) and Streptococcus pneumoniae (serotypes 6, 9, 14, 18 and 23)Haemophilus influenzae type B

In adults, Neisseria meningitidis and Strep pneumoniae together cause 80% of bacterial meningitis cases.

Risk of infection with Listeria monocytogenes is increased in persons over 50 years old.

Meningitis

E. coliE. coli: : Clinical SignificanceClinical Significance

• Leading cause urinary tract infections.• New evidence in women who suffer from

recurrent UTIs suggests that this is due to the formation of pod-like E. coli biofilms inside bladder epithelial cells.– Bacteria living on the edges of the biofilms may

break off leading to repeated infection.

Pod-like BiofilmPod-like Biofilm

Escherichia coli Gastroenteritis

• Pathogenesis– 2 important virulence factors

EnterotoxinAdherence (small intestine)

• Symptoms– Depends on virulence of strain– Symptoms range from vomiting and diarrhea to

severe cramps and bloody diarrhea– Fever not prominent– Recovery usually within 10 days

• Gastroenteritis:– enterotoxigenic E. coli (ETEC) – enteroinvasive E. coli (EIEC) – enteropathogenic E. coli (EPEC) – enteroaggregative E. coli (EAEC)– enterohemorrhagic E. coli (EHEC)

E. coli: Clinical Significance

E coli 0157: H7E coli 0157: H7

• Enterohemorrhagic: – Hemorrhagic diarrhea

(young children and elderly) – Hemolytic Uremic Syndrome

•Cause: – undercooked, contaminated ground beef,

unpasteurized milk, contaminated water and vegetables.

Enterohemorrhagic E. coliEnterohemorrhagic E. coli

• Shiga-type toxin –(verotoxin) •Produced by EHEC •Cytotoxic, enterotoxic, neurotoxic, and

diarrhea,ulceration of G.I. tract. – Two types

Shiga-like toxin 1 /shiga-like toxin 2. Inhibit protein synthesis by cleaving a 28S

rRNA that’s part of the 60S subunit

•Diagnosis: – Slow fermentation of sorbitol – Chromagar– MUG negative

(methylumbelliferyl-beta-D-glucuronide)

– Toxin production– Latex agglutination tests (typing) – Characteristic biochemical codes.

E coli 0157: H7E coli 0157: H7

Spot Indole

Typical E. coli O157:H7 latex agglutination result.

E. Coli 0157 Identification

KlebsiellaKlebsiella

• General characteristics– Produce a large capsule– Nonmotile

• K. pneumoniae:– Colonies are glistening, large, and mucoid– Ferment lactose and nonmotile– Ferment glucose with gas– Positive citrate and lysine decarboxylase tests

KlebsiellaKlebsiella

•Pathogenesis and Infectious Disease– K. pneumoniae is a significant cause of

respiratory (especially pneumonia) and urinary tract diseases (opportunist).

– K. ozaenae and K. rhinoscleromatis are associated with chronic inflammatory diseases of the upper respiratory tract

– K. granulomatis is the causative agent of granuloma inguinale.

Klebsiella

EnterobacterEnterobacter• NF of GI tract• TSI and urea -variable results• Citrate +• Motile+• Clinical significance

Nosocomial infections Bacteremia in burn patients Antimicrobial resistance E. aerogenes, E. cloacae

Enterobacter aerogenes growing on MacConkey (left) and blood agar (right).

– TSI A/A or K/A; (+/- gas) (does not ferment lactose)

– Citrate (+), VP and lysine/ornithine+– Motility (+)– Urea (+/-)– RT, UT and varied infections– Is resistant to many antimicrobics– Red colonies (S. marcescens)

SerratiaSerratia

Serratia

Proteus-Providencia-MorganellaProteus-Providencia-Morganella

•General characteristics– Major species:

Proteus (P. mirabilis and P. vulgaris) Providencia (P. rettgeri – most common;

P.alcalifaciens, and P. stuartii) Morganella morganii

– In this group, P. mirabilis is most frequent isolate from clinical specimens

Proteus, Providencia, and MorganellaProteus, Providencia, and Morganella

• NF of the GI tract (except Providencia).

• All motile, with Proteus swarming

• PDA (+)

• Lysine deamination (+)

• Urea (+) Proteus and P. rettgeri

• TSI variable, H2S (+)

• UTI, kidney stones (Proteus), RTI, septicemia, wounds

Proteus

Proteus-Providencia-MorganellaProteus-Providencia-Morganella

•P. mirabilis is indole negative and ampicillin sensitive

•P. vulgaris is indole positive

– TSI- K/A or A/A (+ gas and H2S)– Urea usually (+)– Motility (+)– PYR (+)– Citrate (+)– Opportunists -urinary tract, wounds,

respiratory tract , osteomyelitis, endocarditis, and meningitis( C. koseri).

Citrobacter

HafniaHafnia

• Cultural, biochemical and other characteristics– Motile– Usually indole and urease negative– VP, lysine and ONPG positive– Ferment glucose with gas production– Often seen as a cause of pediatric gastroenteritis– Rare respiratory tract infections, urinary tract infections,

liver abscesses, appendicitis, and postoperative endophthalmitis

– Biliary abscesses

• Isolated from freshwater, freshwater fish, and shellfish– Also from many types of animals including cattle,

goats, swine, cats, dogs, monkeys, vultures, snakes, and toads.

•Gastroenteritis, followed by septicemia•Plesiomonas is oxidase positive. Plesiomonas is

negative for DNAse; distinguishes it from Aeromonas sp.

Plesiomonas shigelloidesPlesiomonas shigelloides

microbiology, clinical

Documents

o antigens

surface of o antigen

antigen phase

flagellar antigen d

rhamnoseflagellar h

o specific antisera

differential mediaselective

commonferment glucose