Enterobacteriaceae

Dr Ekta Chourasia Lecturer, Microbiology

General Features of Enterobacteria

Present in large intestine Gram negative bacteria Aerobic or facultative anaerobic Motile by peritrichate flagella or non motile Grow on ordinary media ( non fastidious ) Ferments glucose with acid & gas or only acid Reduce nitrates to nitrites Catalase + ve & oxidase -ve Dr Ekta  Page 2

Classification of Enterobacteriaceae

 1.

2.

3.

Based on lactose fermentation

– oldest method : Lactose fermenters e.g. Escherichia, Klebsiella.

Late lactose fermenters e.g. Shigella sonnei Non lactose fermenters e.g Salmonella, Shigella Commensal intestinal bacteria: LF Intestinal pathogens: NLF Dr Ekta  Page 3

Classification of Enterobacteriaceae

 1.

2.

Modern taxonomy

– group together bacteria that possess: Common morphological and biochemical properties Similar DNA base compositions  Family – Tribe / Group - Genera Dr Ekta  Page 4

Enterobacteriaceae (Tribes & Genera )

CDC 1989

Tribe 1 Eshcherichieae

Escherichia Shigella

Tribe 2 Edwardsielleae Edwardsiella Tribe 3 Salmonelleae

Salmonella

Tribe 4 Citrobactereae Citrobacter Tribe 5 Klebsielleae Serratia Hafnia Tribe 6 Proteeae Providentia Tribe 7 Yersinieae Yersinia Tribe 8 Erwinieae Erwinia

Klebsiella

Enterobacter Pantoea

Proteus

Morganella Dr Ekta  Page 5

Escherichia coli

    Named after

Escherich

, first to describe colon bacillus Normal flora of the human & animal intestine.

Remains viable in the feces for few days.

Detection of E.coli in the drinking water – indicates recent pollution with human or animal feces.

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Antigenic Structure of Gram –ve Bacteria

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2.

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Three antigens – serotyping of E.coli

H – flagellar antigen O – somatic antigen K – capsular antigen Majority do not possess K Ag.

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Virulence Factors

- Two types of virulence factors: Surface Ags & Toxins 1.

-

Surface Antigens

LPS surface O Ag – endotoxic activity, protects from phagocytosis and bactericidal effects of complement Envelope or K Ag – protects against phagocytosis and antibacterial factors inserum Fimbriae – colonisation factors, found in strains causing diarrhoea and urinary tract infections Dr Ekta  Page 8

Virulence Factors

2.

Toxins (Exotoxins)

– two types Enterotoxins – pathogenesis of diarrhoea - 3 types : LT (heat labile toxin), ST (heat stable toxin) & VT (verocytotoxin or shiga- like toxin) Hemolysins – may be nephrotoxic Dr Ekta  Page 9

Heat Labile Toxin (LT)

 Resembles cholera toxin in its structure, function and mode of action  Complex of polypeptide subunits.

 LT: one subunit of A (action- enzymic), five subunits of B (binding) Dr Ekta  Page 10

Heat Labile Toxin (LT)

Gut lumen

Escherichia coli / Vibrio cholerae

Intestinal epithelial cell Dr Ekta  Page 11

E.coli toxins

Heat Labile Toxin (LT) Activates Adenyl cyclase Heat Stable Toxin (ST) Activates guanyl cyclase increased production of cAMP Increased production of cGMP Increased secretion of Na, Cl and water from the cell Inhibition of ionic uptake in intestinal cells Osmotic loss of water from cells Dr Ekta  Page 12

Pathogenicities/ Clinical Infections

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3.

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Urinary tract infection Diarrhoea Pyogenic infections Wound infection, especially after surgery of lower intestinal tract.

Peritonitis.

Biliary tract infection.

Neonatal meningitis .

Septicemia – can lead to fatal conditions like Septic shock Systemic Inflammatory Response Syndrome Dr Ekta  Page 13

Lab Diagnosis of UTI

Specimens Urine Mid stream urine (MSU) Catheter specimen urine (CSU) Supra pubic aspiration (SPA) Microscopy Wet mount Gram stain Urine Culture Pus cells / hpf Bacteria / crystals/ casts Gram negative bacteria (1bacteria / oil field is significant) Kass semi-qauntative method Standard loop technique To know significant bacteriuria Dr Ekta  Page 14

Lab Diagnosis of E. coli UTI

Significant bacteriuria

Culture

> 10 5 organism / ml of MSU BA / MAC : LF (flat)

Identification tests

I M Vi C test : + + - -

TSI agar AST Acid, no gas Dr Ekta  Page 15

Diarrheagenic E.coli

 Enteropathogenic E.coli (EPEC)  Enterotoxigenic E.coli (ETEC)  Enteroinvasive E.coli (EIEC)  Enterohemorrhagic E.coli (EHEC) or Verotoxigenic E.coli (VTEC)  Enteroaggregative E.coli (EAEC) : “stacked brick” appearance.

 Diffusely adherent E.coli (DAEC) Dr Ekta  Page 16

Enteropathogenic E.coli (EPEC)

 Infantile diarrhea  Institutional outbreaks  Noninvasive, nontoxigenic  Pathogenesis – adhesion via fimbria, disruption of brush border microvilli  Clinical features – fever, diarrhea, vomiting, nausea, non bloody stools  Lab Diagnosis – testing colonies grown on BA/ MA with EPEC O antisera Dr Ekta  Page 17

Enterotoxigenic E.coli (ETEC)

 Traveller’s diarrhea  Resembles cholera  Noninvasive, toxigenic  Pathogenesis – production of plasmid coded toxins (LT/ ST)  Clinical features - Diarrhea, vomiting and abdominal pain  Lab Diagnosis – demonstration of enterotoxin by in vitro or in vivo methods, detection of LT/ St by gene probes Dr Ekta  Page 18

Enteroinvasive E.coli (EIEC)

    Bloody diarrhea (dysentery), resembles Shigella dysentery Passage of blood, mucus & leucocytes in stool Pathogenesis - Invades epithelial cells by endocytosis and can spread laterally to adjacent cells, causes tissue destruction, necrosis and ulceration. 1.

2.

Lab Diagnosis : Sereny test- instillation of suspension of freshly isolated EIEC or Shigella in the eyes of guinea pig – mucopurulent conjunctivitis and severe keratitis Penetration of HeLa or Hep2 cells in tissue culture Dr Ekta  Page 19

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Enterohemorrhagic E.coli (EHEC)

   Produces verocytotoxin (VT), a shiga-like toxin (SLT); hence also known as Verocytotoxigenic E.coli (VTEC) Pathogenesis – EHEC attaches to the colonic mucosa and releases VT. VT targets vascular endothelial cells, inhibits protein synthesis - cytotoxicity 1.

2.

Clinical features - Mild diarrhea (bloody) to fatal complications (esp. in young children and elderly): Hemorrhagic colitis – destruction of mucosa followed by hemorrhage.

Hemolytic Uremic syndrome – triad of acute renal failure, hemolytic anemia and thrombocytopenia.

  Serotype O157: H7 is most commonly involved.

Outbreaks of food poisonings (fast foods, contaminated hamburgers) Dr Ekta  Page 21

Enterohemorrhagic E.coli (EHEC)

 1.

2.

3.

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Lab Diagnosis: Demonstration of bacilli or VT in feces or in culture Sorbitol MacConkey agar for O157:H7 – does not ferment sorbitol unlike other E.coli

Cytotoxic effects on Vero or HeLa cells DNA probes to detect toxins Dr Ekta  Page 22

Enteroaggregative E.coli (EAEC)

 Persistent diarrhea in children in developing countries.  Aggregate to give a “Stacked brick appearance” or glass (due to fimbria) on Hep2 cells  Pathogenesis – shortening of villi, mucus biofilm, heat stable cytotoxin (hemorrhagic necrosis and edema) Dr Ekta  Page 23

Epidemiology & Treatment

Epidemiology

 EPEC & ETEC - most important causes of diarrhea globally  EHEC – in developed countries.

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2.

Treatment

Based on symptoms: Primary treatment – fluid replacement Secondary treatment – antibiotics in severe cases with systemic involvement Dr Ekta  Page 24

Klebsiella

Normal

gut flora in the intestine Gram negative coccobacilli (short & plump) Capsulated, non-motile , Mucoid LF colonies on MAC

Species

K. pneumoniae K. oxytoca K. ozaenae K. rhinoscleromatis Pneumonia, Urinary tract infections Atrophic rhinits Rhinoscleroma Dr Ekta  Page 25

Pathogenicities of Klebsiella pneumoniae

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Pulmonary infections - Pneumonia (lobar): High fatality 1.

2.

3.

2.

In middle aged or older persons with medical problems like DM, alcoholism, chronic bronchopulmonary disease 3.

Extensive necrosis & hemorrhage resulting in thick, mucoid, brick red sputum “currant jelly like” Extrapulmonary infections – Meningitis & enteritis in infants UTI Septicemia An important cause of nosocomial infections .

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Lab Diagnosis - Klebsiella

Specimens

Culture Urine, sputum, nasal secretions / swab, blood

BA / MAC : LF (mucoid)

Identification tests

I M Vi C test : - - + +

TSI agar Urease Acid with gas

Positive

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Proteus

Normal gut flora in the intestine Gram negative bacilli , pleomorphic Motile, Non lactose fermenter NLF on MAC Swarms on BA, Urease +, H 2 S + Species P mirabilis UTI P vulgaris Pneumonia Wound infections Urease converts urea to NH 4 & CO 2 causing alkalinization of urine leading to renal calculi (stones) Proteus antigens are used in the Weil - Felix test to diagnose Rickettsial diseases Dr Ekta  Page 28

Lab Diagnosis - Proteus

Specimens

Culture Urine, sputum, wound swab

BA: swarming MAC : NLF,

fishy/ seminal smell Identification tests Indole: PM - / PV + TSI agar K / A (H 2 S) Urease + Dr Ekta  Page 29

Enterobacter, Serratia, Citrobacter

 Moist environments in hospitals – common reservoirs.

 Pathogenicities – UTI, Wound & respiratory infections in hospitalized patients, Outbreaks in ICUs, burn units & other special units Dr Ekta  Page 30