Dr.Babasaheb N. Kumbhar M.V.Sc ENTEROBACTERIACEAE

Enterobacteriaceae Habitat digestive tube(colon) of human and animals 150 Species Facultative anaerobes Diarrheal illnesses 3 million death/year 4 billion infectoions/ worldwide

Enterobacteriaceae Classification – more than15 different genera Escherichia Shigella Edwardsiella Salmonella Citrobacter Klebsiella Enterobacter Hafnia Serratia

Enterobacteriaceae Proteus Providencia Morganella Yersinia Erwinia Pectinobacterium

Family Enterobacteriaceae (nonmotile) True pathogen Certain E.coli strains can be considered true pathogens

Morphology and General Characteristics Are facultative anaerobes Gram-negative, non-sporing, rod shaped bacteria If motile, motility is by peritrichous flagella (Except Shigella & Klebsiella) Many are normal inhabitants of the intestinal tract of man and other animals Some are enteric pathogens and others are urinary or respiratory tract pathogens Differentiation is based on biochemical reactions and differences in antigenic structure

Morphology and Physiology Short gram-negative rods. Facultative anaerobes. Grow readily and rapidly on simple media. K. pneumoniae Klebsiella spp. have large capsule (form large and very mucoid colonies); those of Enterobacter have smaller capsule; the others produce diffusible slime layers (form circular, convex and smooth colonies).

ENTEROBACTERIACEAE PHYSIOLOGY Glucose is fermented with strong acid formation and often gas Reduce nitrates to nitrite Do not liquify alginate Oxidase negative Basis for speciation within a family - differences in carbohydrate they ferment - variations in end-product production - variation in substrate utilization

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

Incidence of Enterobacteriaceae Associated with Bacteremia

CULTURAL CHARACTERISTICS On non differential or nonselective media blood agar or infusion agar - no species distinction - appear as moist, smooth, gray colonies Selective media -To isolate Shigella and salmonella from fecal matter Differential media – selectively inhibit gram-positive organisms and to separate enterics in broad categories

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

Urinary Tract Infection, Pneumonia Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus mirabilis Pneumonia: Enterobacter spp., Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis Wound Infection: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis Bacteremia: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis Intestinal Infection Shigella sonnei (serogroup D) Salmonella serotype enteritidis Salmonella serotype typhimurium Shigella flexneri (serogroup B) Escherichia coli O157:H7 Yersinia enterocolitica

MacConkey (MAC) Agar

Eosin Methylene Blue (EMB) Agar (Levine)

Antigenic Structure –Most are motile by peritrichous flagella --H antigens. –Capsule – K antigen ( Vi for Salmonella). –Cell envelope (wall) –LPS (endotoxin) – O antigen. O antigen. –various outer membrane proteins. membrane proteins. –Pili - various antigen types, some encoded by plasmids types, some encoded by plasmids

Pathogenesis and Immunity Common virulence factors Type III secretion systems: possessed by some Enterobacteriaceae pathogens, e.g., E. coli, Yersinia, Salmonella, and Shigella; facilitate transport of bacterial virulence factors directly into host cells. Endotoxin (Lipid A of LPS) Capsule Antigenic phase variation Acquisition of growth factors (e.g. Fe) Resistance to serum killing Antimicrobial resistance

HE Agar: Growth of Enteric Pathogens and Commensals

Escherichia coli Toxins: two types of enterotoxin; Shiga-type toxin; Enteroaggregative ST-like toxin; Hemolysins; Endotoxin Virulence factors that protect the bacteria from host defenses: Capsule/Iron capturing ability (enterochelin)

E. coli May be hemolytic on CBA – more common in pathogenic strains KEY tests for the normal strain: TSI is A/A + gas LIA K/K Urea – Indole + Citrate – Motility + There is an inactive biotype that is anaerogenic, lactose –, and nonmotile.

Enteropathogenic E. coli  fever  infant diarrhea  vomiting  nausea  non-bloody stools  Destruction of surface microvilli loose attachment mediated by bundle forming pili (Bfp);  Stimulation of intracellular calcium level;  rearrangement of intracellular actin, Enterotoxigenic E. coli A watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days. Travellers diarrhea and diarrhea in children in developing countries Transmission is via contaminated food or water.

E.coli-Enteroinvasive (EIEC) The organism attaches to the intestinal mucosa via pili Outer membrane proteins are involved in direct penetration, invasion of the intestinal cells, and destruction of the intestinal mucosa. There is lateral movement of the organism from one cell to adjacent cells. Symptoms- fever,severe abdominal cramps, malaise, and watery diarrhea followed by scanty stools containing blood, mucous,& pus. resembles shigellosis E.coli-c. Enteropathogenic (EPEC) Malaise and low grade fever diarrhea, vomiting, nausea, non- bloody stools Bundle forming pili are involved in attachment to the intestinal mucosa. This leads to changes in signal transduction in the cells, effacement of the microvilli, and to intimate attachment via a non-fimbrial adhesion called intimin. This is a problem mainly in hospitalized infants and in day care centers.

Enterohemorrhagic (EHEC) Enterohemorrhagic (EHEC) Hemorrhagic – bloody, copious diarrhea – few leukocytes – afebrile hemolytic-uremic syndrome – hemolytic anemia – thrombocytopenia (low platelets) – kidney failure Usually O157:H7 Transmission electron micrograph

Enteroaggregative E. coli a cause of persistent, watery diarrhea with vomiting and dehydration in infants. That is autoagglutination in a ‘stacked brick’ arrangement. the bacteria adheres to the intestinal mucosa and elaborates enterotoxins (enteroaggregative heat-stable toxin, EAST). The result is mucosal damage, secretion of large amounts of mucus, and a secretory diarrhea.

Shigella

Shigella species Shigella Contains four species that differ antigenically and, to a lesser extent, biochemically. S. dysenteriae (Group A) S. flexneri (Group B) S. boydii (Group C) S. sonnei (Group D) bacillary dysentery, shigellosis, bloody feces, intestinal pain, pus Biochemistry TSI K/A with NO gas LIA K/A Urea – Motility - All ferment mannitol except S. dysenteriae S. sonnei may show delayed lactose fermentation

Shiga toxin 1. Chromosomally encoded 2. Neurotoxic 3. Enterotoxic 4. Cytotoxic Enterotoxicity Enterotoxicity can make the disease clinically diarrhea appear as a diarrhea. toxin inhibits protein synthesis The toxin inhibits protein synthesis (acting on the 60S ribosome and lysing 28S rRNA).

Clinical significance man only "reservoir" man only "reservoir" mostly young children mostly young children – fecal to oral contact – children to adults transmitted by adult food handlers transmitted by adult food handlers – unwashed hands

Diagnosis of Shigella infection Specimen: stool. Culture and Identification Quick immunological methods: 1. Immunofluorescent “ball” test; 2. Coagglutination.

XLD Agar: Growth of Shigella and Proteus

Only a few types that are commonly associated with characteristic human diseases - 1. S. enteritidis 2. S. cholerae-suis 3. S. typhi Salmonella several syndromes including gastroenteritis, enteric (typhoid) fever or septicemia

Salmonella Biochemistry TSI K/A + gas and H 2 S: S. typhi produces only a small amount of H 2 S and no gas, and S. paratyphi A produces no H 2 S LIA K/K with H 2 S with S. paratyphi A giving K/A results Urea – Motility + Citrate +/- Indole - Virulence factors Endotoxin – may play a role in intracellular survival Capsule (for S. typhi and some strains of S. paratyphi) Adhesions – both fimbrial and non-fimbrial

Salmonella typhi The organism is transmitted from: 1. a human reservoir 1. a human reservoir 2. in the water supply (if poor sanitary conditions) 2. in the water supply (if poor sanitary conditions) 3. in contaminated food 3. in contaminated food The antigenic structures of salmonellae used in serologic typing

Virulence factors Endotoxin – may play a role in intracellular survival Capsule (for S. typhi and some strains of S. paratyphi) Adhesions – both fimbrial and non-fimbrial Type III secretion systems and effector molecules – 2 different systems may be found: –One type is involved in promoting entry into intestinal epithelial cells –The other type is involved in the ability of Salmonella to survive inside macrophages Outer membrane proteins - involved in the ability of Salmonella to survive inside macrophages Flagella – help bacteria to move through intestinal mucous Enterotoxin - may be involved in gastroenteritis Iron capturing ability

XLD Agar: Appearance of Salmonella

Diagnosis A. Specimens A. Specimens a) Enteric fever: blood, bone marrow, stool, urine. a) Enteric fever: blood, bone marrow, stool, urine. b) Food poisoning: stool, vomitus, suspected food. b) Food poisoning: stool, vomitus, suspected food. c) Septicemia: blood. c) Septicemia: blood. B. Culture and identification B. Culture and identification C. Widal test C. Widal test

Klebsiella NF of GI tract, but potential pathogen in other areas TSI A/A + gas LIA K/K Urea + Citrate + MR-, VP+ Motility - Has both O and K antigens

Klebsiella Virulence factors Capsule Adhesions Iron capturing ability Clinical significance Causes pneumonia, mostly in immunocompromised hosts. Permanent lung damage is a frequent occurrence (rare in other types of bacterial pneumonia) A major cause of nosocomial infections such as septicemia and meningitis K.Pneumoniae On BA K.Pneumoniae on BA and MAC

Proteus General characteristics: “swarming” phenomenon on nonselective agar (P.vulgaris; P.mirabilis and P.myxofaciens) General characteristics: “swarming” phenomenon on nonselective agar (P.vulgaris; P.mirabilis and P.myxofaciens) P.vulgaris strains (OX-19, OX-K, OX-2)have common antigen with Rickettsia (Weil-Felix test). P.vulgaris strains (OX-19, OX-K, OX-2)have common antigen with Rickettsia (Weil-Felix test). urinary tract infections; food poisoning. urinary tract infections; food poisoning.

Proteus spp. Some enteric bacteria are motile. Klebsiella species are not motile, while Proteus species move very actively by means of peritrichous flagella, resulting in "swarming" on solid medium.swarming Some strains of E. coli produce hemolysis on blood plates.

CITROBACTER Resident of soil, water, stool C.freundii UTI and bacteremia

Serratia

Serratia A free-living saprophyte TSI A/A or K/A; +/- gas (does not ferment lactose) LIA usually K/K Citrate + Motility + Urea +/- Has been found in RT and UT infections Is resistant to many antimicrobics

Special Features: Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-). Positive test e.g. E. coli Negative test e.g. Klebsiella Indole test Indole test Positive Klebsiella, Enterobacter Negative E. coli Citrate Utilization Test Urease Test Positive test Negative test

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

EMBSS MacCon key O/F Nitrate reductase Oxidase Gram stain Metallic sheen LFLFO+/F++ve-ve -ve rod E. coli DarkLFLFO+/F++ve-ve -ve rods Citrobacter DarkLFLFO+/F++ve-ve Klebsiella DarkLFLFO+/F++ve-ve Enterobacter Colorless NLF/ H2S NLFO+/F++ve-ve -ve rods Salmonella ColorlessNLFNLFO+/F++ve-ve Shigella Colorless NLF/ H2S NLFO+/F++ve-ve -ve rods Proteus Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

MotilityUreaseCitrateVPMRIndoleTSI Motile-ve-ve-ve+ve+veA/A/- E. coli Motile-ve+ve-ve+ve+veA/A/- Citrobacter freundii Non motile +ve+ve+ve-ve-veA/A/- Klebsiella pneumoniae Motile+ve+ve+ve-ve-veA/A/- Enterobacter cloacae Motile-ve+ve-ve+ve-veA/Alk/+ Salmonella typhi Non motile -ve-ve-ve+ve-veA/Alk/- Shigella boydii MotileSwarwing+ve+ve-ve+ve-veA/Alk/+ Proteus mirabilis Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

Oxidase Test Positive Negative Pseudomonas Enterobacteriaceae Nitrate test: +ve further reduction to N 2 Growth on cetrimide agar: Pale colonies with green pigmentation MacConkey’s agar & TSI Lactose fermenter Pink colonies on MacConkey & acidic butt and slant on TSI colorless colonies on MacConkey & acidic butt alkaline slant onTSI Lactose non-fermenter IMV i C test & EMB IMV i C & black colonies with metalic shines on EMB E.coli IMV i C Klebsiella No H 2 S production (no blacking in TSI) H 2 S production (blacking in TSI) Shigella Urease production +ve Proteus -ve SS agar colorless colonies with black centers Salmonella O/F test: O + /F - Motility Not motile Motile Enterobacter

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