ENTEROBACTERIACEAE

Characteristics of the Enterobacteriaceae Gram-negative bacilli Ferment glucose Reduce nitrates to nitrites Oxidase negative Catalase positive (except Sh.disentry type 1) All except Klebsiella, Shigella and Yersinia are motile by peritrichous flagella

Grow readily on MacConkey (MAC) agar Eosin methylene blue (EMB) agar Grow readily at 35oC except Yersinia (25o-30oC) Do not form spores Natural Habitat: Environment (soil, water, and plants) Intestines of humans and animals

Enterobacteriaceae Opportunistic pathogens Escherichia coli Meningitis Opportunistic pathogens Escherichia coli Klebsiella pneumoniae Enterobacter aerogenes Serratia marcescens Proteus spp. Providencia spp. Citrobacter spp. Pneumonia Sepsis Diarrhea UTI Obligate pathogens Salmonella spp. Shigella spp. Yersinia spp. Some E. coli strains

Classification of Enterobacteriaceae: - Based on lactose fermentation LF LLF NLF

Classification of Enterobacteriaceae: Very large number of organisms in Family Enterobacteriaceae, species are grouped into - TRIBES Tribe 1 – Escherichieae Tribe 2 – Klebsiellae Tribe 3: Proteae Tribe 4 : Yersiieae Tribe 5 : Erwinieae Within each Tribe, species are further subgrouped under genera

Major Genera of family Enterobacteriaceae: Escherichia Shigella Salmonella Yersinia Klebsiella Proteus

Antigenic Factors of Enterobactericeae: Ability to colonize, adhere, produce various toxins and invade tissues Some possess plasmids that may mediate resistance to antibiotics Imp antigens used to identify the organisms: O antigen – somatic, cell wall, heat-stable Ag H antigen – flagellar, heat labile Ag K antigen – capsular, heat-labile Ag

Clinical Significance of Enterics: Ubiquitous in nature Except for Salmonella, Shigella and Yersinia, most are present in the intestinal tract of animals and humans as commensal flora; “fecal coliforms” Some live in water, soil and sewage

Clinical Significance of Enterics: Based on clinical infections produced, enterics are divided into two categories: Opportunistic pathogens – normally part of the usual intestinal flora, that may produce infection outside the intestine Primary intestinal pathogens – Salmonella, Shigella, and Yersinia sp.

Enterobacteriaceae: Modes of Infection Contaminated food and water (Salmonella spp., Shigella spp., Yersinia enterocolitica, Escherichia coli O157:H7) 2. Endogenous infection (urinary tract infection, primary bacterial peritonitis, abdominal abscess) 3. Abnormal host colonization (nosocomial pneumonia) Transfer between debilitated patients 4. Insect (flea) vector: unique for Yersinia pestis

Urinary tract infection: Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus mirabilis Pneumonia: Klebsiella pneumoniae; Enterobacter spp., Wound Infection: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis 4. Bacteremia: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis

Enterobacteriaceae: Intestinal Infection Escherichia coli Shigella Salmonella Yersinia enterocolitica

Identification of Enterobacteriaceae: IMViC Test Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests: The IMViC series of reactions allows for the differentiation of the various members of family Enterobacteriaceae.

A) Indole –Positive, B) MR –Positive C) VP- Positive, D) Citrate- Negative

Methyl Red-Voges Proskauer (MR-VP) Tests Principle Glucose Acidic pathway Or Neutral pathway Acety methyl carbinol (ACETOIN) Mixed acids  pH less than 4.4 Barrit’s A Barrit;s B Methyl Red indicator VP positive Klebsiella MR positive E. coli Red color Pink color

MR/VP test Voges-Proskauer test Methyl Red test Pink: Positive VP (Klebsiella) Red: Positive MR (E. coli) Yellow or orange: Negative MR (Klebsiella) No pink: Negative VP (E. coli)

Result Example Result Reaction on TSI H2S Slant color Butt color Non fermenter e.g. Pseudomonas Alk/Alk/- (No action on sugars) Negative Red LNF e.g. Shigella A/Alk/- (Glucose fermented without H2S) Yellow e.g. Salmonella & Proteus A/Alk/+ (Glucose fermented with H2S) Positive black in butt LF e.g. E. coli, Klebsiella, Enterobacter A/A/- (three sugars are fermented)

TSI Reactions of the Enterobacteriaceae A/A + g = acid/acid plus gas (CO2) A/A = acid/acid A/A + g, H2S = acid/acid plus gas, H2S Alk/A = alkaline/acid Alk/A + g = alkaline/acid plus gas Alk/A + g, H2S = alkaline/acid plus gas, H2S Alk/A + g, H2S (w) = alkaline/acid plus gas, H2S (weak)

A/A + g Escherichia coli Klebsiella A/A + gas, H2S Citrobacter freundii Proteus vulgaris Non-lactose, sucrose fermenter

Alk/A Alk/A + g Alk/A + g, H2S Shigella Providencia Salmonella serotype Paratyphi A Alk/A + g, H2S Salmonella Proteus mirabilis Edwardsiella tarda

Lactose fermenting colonies Non lactose fermenting colonies

Escherichia coli Most significant species in the genus Important potential pathogen in humans Common commensal in the intestine Pink (lactose positive) colony with surrounding pink area on MacConkey

Ferments glucose, lactose, trehalose, & xylose Usually motile Positive indole and methyl red tests Does NOT produce H2S Simmons citrate negative Voges-Proskauer test negative

Pathogenesis and clinical diseases Sepsis In people with inadequate host defenses, e.g. the newborns. Usually originates from UT or GI infections. Some infections may be endogenous. Meningitis E. coli (particularly K1 strains) and S. agalactiae are the leading causes of meningitis in infants. Bacteremia

Urinary tract infection E. coli is the most common cause of urinary tract infection. Community- vs. hospital-acquired UT infection Most infections originate from colon; the bacteria contaminate the urethra, ascend into the bladder, and may migrate into the kidney or prostate. Symptoms: urinary frequency, dysuria, hematuria, and pyuria. Can result in bacteremia and sepsis. Uropathogenic E. coli strains produce P (Pyelonephritis-associated) pili, which is associated with renal colonization and may induce protective immunity, and hemolysin HlyA.

Infections caused by E.coli: Meningitis, gastrointestinal, urinary tract, wound, and bacteremia Gastrointestinal Infections: - diarrhoegenic E. coli Enteropathogenic (EPEC) Enterotoxigenic E. coli (ETEC) Enteroinvasive E. coli (EIEC ) Enterohemorrhagic E. coli (EHEC) Enteroaggregative E. coli (EAEC)

Enteropathogenic (EPEC) Primarily in infants and children; Outbreaks in hospital nurseries and day care centers Stool has mucous but not blood Identified by serotyping

Enteropathogenic E.coli Destruction of surface microvilli Fever Diarrhea Vomiting Nausea Non-bloody stools Gut lumen 31

Enterotoxigenic E. coli Diarrhea resembling cholera Travelers diarrhea 32

Enterotoxigenic E. coli Heat labile toxin Like cholera toxin Adenyl cyclase activated Cyclic AMP Secretion water/ions Heat stable toxin Guanylate cyclase activated cyclic GMP uptake water/ions 33

Enteroinvasive E. coli (EIEC ) Dysentery - Resembles shigellosis

Enterohemorrhagic E. coli Usually O157:H7 Transmission electron micrograph Flagella 35

Bloody, copious diarrhea Few leukocytes Afebrile O157:H7 transmitted- through meat products or sewage-contaminated vegetables Hemorrhagic diarrhea Bloody, copious diarrhea Few leukocytes Afebrile Hemolytic-uremic syndrome (HUS) Hemolytic anemia Thrombocytopenia (low platelets) Kidney failure 36

Vero toxin- “shiga-like” Enterohemorrhagic E. coli Vero toxin- “shiga-like” Hemolysins Enteroaggregative (EaggEC) Cause diarrhea by adhering to the mucosal surface of the intestine; watery diarrhea; symptoms may persist for over two weeks 37

Urinary Tract Infections E. coli is most common cause of UTI and pyelo-nephritis in humans Usually originate in the large intestine Able to adhere to epithelial cells in the urinary tract

Septicemia & Meningitis Most common causes of septicemia and meningitis among neonates Acquired in the birth canal before or during delivery E. coli also causes bacteremia in adults, secondary to genitourinary tract infection or a gastrointestinal source

Klebsiella, Enterobacter Usually found in intestinal tract Wide variety of infections, primarily pneumonia, wound, and UTI General characteristics: Non-motile Simmons citrate positive H2S negative, Weakly urease positive MR negative; VP positive

Usually found in Gastro-intestinal tract K. pneumoniae is most commonly isolated species Possesses a polysaccharide capsule - which protects against phagocytosis and antibiotics Makes the colonies moist and mucoid Frequent cause of nosocomial pneumonia

Significant biochemical reactions Lactose positive Most are urease positive Non-motile

Nosocomial pneumonia: Spread by health care personnel and equipment Frequently caused by K. pneumoniae Often seen in middle-aged males who abuse alcohol Difficult to diagnose due to commensals in sputum

Proteus, Morganella & Providencia species All are normal intestinal flora Opportunistic pathogens Deaminate phenylalanine Non lactose fermenters

Proteus species P. mirabilis and P. vulgaris are widely recognized human pathogens Isolated from urine, wounds, and ear and bacteremic infections Both produce swarming colonies and have a distinctive “burned chocolate” odor Both are strongly urease positive Both are phenylalanine deaminase positive

Exhibits characteristic “swarming”

Laboratory Diagnosis of Enterics Specimen collection: Specimens collected and transported in Cary-Blair, Amies, or Stuart media Isolation and Identification Site of origin must be considered Enterics from sterile body sites are highly significant Routinely cultured from stool

Media for Isolation and Identification of Enterics: Blood agar and a selective/differential medium such as MacConkey On MacConkey, lactose positive are pink; lactose negative are clear and colorless

For stool, highly selective media, such as Hektoen Enteric (HE), XLD, or SS is used along with MacConkey agar Identification: All enterics are Oxidase negative Ferment glucose Reduce nitrates to nitrites