Enterobacteriaceae General Features of Enterobacteria Present in large

Enterobacteriaceae

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

Classification of Enterobacteriaceae § Based on lactose fermentation – oldest method : 1. Lactose fermenters e. g. Escherichia, Klebsiella. 2. Late lactose fermenters e. g. Shigella sonnei 3. Non lactose fermenters e. g Salmonella, Shigella - Commensal intestinal bacteria: Coliform bacilli LF - Intestinal pathogens: NLF - Paracolon bacilli

Classification of Enterobacteriaceae § Modern taxonomy – group together bacteria that possess: 1. Common morphological and biochemical properties 2. Similar DNA base compositions § Family – Tribe / Group - Genera

Enterobacteriaceae (Tribes & Genera) CDC 1989 Tribe 1 Eshcherichieae Tribe 2 Klebsielleae Tribe 3 Proteeae Klebsiella Escherichia Proteus Enterobacter Shigella Providentia Serratia Edwardsiella Morganella Hafnia Salmonella Citrobacter Tribe 4 Erwinieae Erwinia Tribe 5 Yersinieae Yersinia

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.

Morphology & Culture Morphology - Gram negative straight rod, occasinally capsulated - Motile by peritrichate flagella Cultural characters: - Facultative anaerobe, non fastidious - Colonies are smooth, translucent, - BA: haemolytic colonies - MA: lactose fermenting pink colonies.

Antigenic Structure of Gram –ve Bacteria § Three antigens – serotyping of E. coli 1. H – flagellar antigen 2. O – somatic antigen 3. K – capsular antigen Majority do not possess K Ag.

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

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

Heat Labile Toxin (LT) § Resembles cholera toxin in its structure, function and mode of action § Complex of polypeptide subunits. § one subunit of A (action- enzymic), § five subunits of B (binding)

Heat Labile Toxin (LT) Escherichia coli / Vibrio cholerae Gut lumen Intestinal epithelial cell

E. coli toxins Heat Labile Toxin (LT) Heat Stable Toxin (ST) Activates Adenyl cyclase Activates guanyl cyclase increased production of c. AMP Increased secretion of Na, Cl and water from the cell Increased production of c. GMP Inhibition of ionic uptake in intestinal cells Osmotic loss of water from cells

Verocytotoxin (VT) § So named because of cytotoxic effect on Vero cell lines § Similar to Shigella dysenteriae type 1 toxin in its physical, antigenic and biological properties § VT gene is phage coded.

Pathogenicity/ Clinical Infection 1. Urinary tract infection 2. Diarrhoea 3. Pyogenic infections - Wound infection, especially after surgery of lower intestinal tract. - Peritonitis. - Biliary tract infection. - Neonatal meningitis. 4. Septicemia – can lead to fatal conditions like - Septic shock - Systemic Inflammatory Response Syndrome

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

Lab Diagnosis of E. coli UTI Significant bacteriuria > 105 organism / ml of MSU Culture BA / MAC : LF (flat) Identification tests I M Vi C test: + + - TSI agar AST Acid, no gas

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)

Enteropathogenic E. coli (EPEC) § Infantile diarrhea § Institutional outbreaks § Noninvasive, nontoxigenic § Pathogenesis – adhesion via fimbriae, to the enterocyte membrane & 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

Enterotoxigenic E. coli (ETEC) § Traveller’s diarrhea: From developed to endemic areas § Resembles cholera § Noninvasive, toxigenic § Pathogenesis – production of plasmid coded toxins (LT/ ST) along with colonisation factor Ags (CFA) § 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

Demonstration of ETEC toxins Assay In vivo tests Ligated rabbit illeal loop Infant rabbit bowel LT ST + + + Infant mouse intragastric Adult rabbit skin (Vascular permeability factor) In vitro tests Tissue culture tests Rounding of Y 1 mouse adrenal cells ELISA Passive agglutination tests + + -

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. § Lab Diagnosis: 1. Sereny test- instillation of suspension of freshly isolated EIEC or Shigella in the eyes of guinea pig – mucopurulent conjunctivitis and severe keratitis 2. Penetration of He. La or Hep 2 cells in tissue culture

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 § Clinical features - Mild diarrhea (bloody) to fatal complications (esp. in young children and elderly): 1. Hemorrhagic colitis – destruction of mucosa followed by hemorrhage.

Enterohemorrhagic E. coli (EHEC) 2. Hemolytic Uremic syndrome – triad of acute renal failure, hemolytic anemia and thrombocytopenia. § Serotype O 157: H 7 is most commonly involved. § Outbreaks of food poisonings (fast foods, contaminated hamburgers)

Enterohemorrhagic E. coli (EHEC) § Lab Diagnosis: 1. Demonstration of bacilli or VT in feces or in culture 2. Sorbitol Mac. Conkey agar for O 157: H 7 – does not ferment sorbitol unlike other E. coli 3. Cytotoxic effects on Vero or He. La cells 4. DNA probes to detect toxins

Enteroaggregative E. coli (EAEC) § Persistent diarrhea in children in developing countries. § Aggregate to give a “Stacked brick appearance” on Hep 2 cells or glass (due to fimbria) § Pathogenesis – shortening of villi, mucus biofilm, heat stable cytotoxin (hemorrhagic necrosis and edema)

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 Pneumonia, Urinary tract infections Atrophic rhinits K. rhinoscleromatis Rhinoscleroma

Pathogenicity of Klebsiella pneumoniae § Pulmonary infections - Pneumonia (lobar): 1. High fatality 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 – 1. Meningitis & enteritis in infants 2. UTI 3. Septicemia § An important cause of nosocomial infections.

Lab Diagnosis - Klebsiella Specimens Urine, sputum, nasal secretions / swab, blood Culture BA / MAC : LF (mucoid) Identification tests I M Vi C test: - - + + TSI agar Urease Acid with gas Positive

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, P vulgaris UTI 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

Lab Diagnosis - Proteus Specimens Urine, sputum, wound swab Culture BA: swarming MAC : NLF, fishy/ seminal smell Identification tests Indole: PM - / PV + TSI agar K / A (H 2 S) Urease +

Enterobacter, Serratia, Citrobacter § Moist environments in hospitals – common reservoirs. § Pathogenicity – - UTI, - Wound & respiratory infections in hospitalized patients, - Outbreaks in ICUs, burn units & other special units