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PH 152 Medical and Public Health Microbiology INTRODUCTION TO ENTEROBACTERIACEAE December 3, 2010 Sylvia Nachura, Gino Lacsamana

Objective To discuss the family Enterobacteriaceae based on the following general characteristics: virulence factors diseases produced host response laboratory identification treatment prevention and control epidemiologic features

true pathogenic nonenteric o Yersinia pestis

Introduction also called coliforms large, heterogeneous group of gram-negative rods natural habitat: intestinal tract of man and animals, soil and water, decaying matter some members are part of the normal intestinal flora (ex. E.coli), while others are regular human pathogens (ex. Salmonella and Shigella)

Scientific Classification Kingdom Bacteria Phylum Proteobacteria Class Gamma Proteobacteria Order Enterobacteriales Family Enterobacteriaceae Genera coliforms: lactose-fermenting, normal flora, opportunistic o Escherichia coli o Klebsiella o Enterobacter o Hafnia o Serratia o Citrobacter Non-coliforms in normal flora: lactose-negative, opportunistic o Proteus o Morganella o Providencia o Edwardsiella Groupings of Enterobacteriaceae Intestinal pathogens: Salmonella, Shigella, Yersinia Opportunistic pathogens: other genera

*more than 50 genera have been defined, but only 20-25 species are clinically significant motile with peritrichous flagella or nonmotile (Klebsiella and Shigellae) presence of capsule or slime layer in some fimbriae or pili is present in most species facultative anaerobes to aerobes

Biochemical characteristics ferment a wide range of carbohydrates o ferment glucose often with the presence of gas catalase positive oxidase negative reduce nitrate to nitrite

Colony characteristics form moist, smooth, gray colonies in non-differential or nonselective media

Bacteriocins virus-like bacterial substances act against strains of the same or closely related species its production is controlled by plasmids bacteriocin-producing strains are resistant to their own bacteriocin E. coli: colicins Serratia: marcescens Pseudomonas: pyocins

*at present, the distinction is less clear cut due to genetic similarities among different genera Important species true pathogenic enterics o Salmonella typhi o Salmonella cholerae-suis o Salmonella enteritidis o Shigella dysenteriae o Shigella flexneri o Shigella boydii o Shigella sonnei

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Antigenic Structure: 1. O antigen found in the most external part of the cell wall: Lipopolysaccharide (LPS) somatic or cell wall antigen made up of repeating polysaccharide units heat and alcohol resistant (heat-stable) appears to be involved in attachment to the host and protection against the complement system detected by bacterial agglutination antibodies against it are predominantly IgM a single organism can carry many O antigens, and some O antigens are associated with specific diseases implicated in endotoxic shock

3. Shiga toxins and Shiga-like toxins (Exotoxins) shiga toxins: interfere with protein synthesis in cells of mammals shigalike toxins of E. coli called verotoxins cause hemolytic diarrhea and hemolytic uremic syndrome

4. Colonization factors capsule: prevent phagocytosis Vi antigen: may have protective functions (prevention of intracellular destruction) fimbriae: attachment to target tissues O antigen: may bind organism to receptor sites 5. Invasive factors invasins facilitate invasion of target cells

2. K antigen capsule or slime layer polysaccharide or protein includes Vi antigen of Salmonella typhi external to O on some Enterobacteriaceae may interfere with agglutination by O antisera may be associated with virulence o K1 antigen in E. coli: neonatal meningitis o K antigens of E. coli attach to epithelial cells before gastrointestinal and urinary tract infections o K antigen of Klebsiella for respiratory tract infections and UTI

6. Serum resistance protection against the complement system due to a lipopolysaccharide in the outer layer of the cell that prevents initiation of the complement system

7. Cumulation of Fe2+ active transport of Fe2+ by siderophores depletes iron in the body

8. Others tissue penetration o Shigellae and E.coli: penetrates epithelium of the small intestine o Salmonellae: penetrates the epithelium of the large intestine resistance genes o plasmid- borne, gives drug resistance

3. H antigen flagellar antigen denatured or removed by heat or alcohol (heat labile) preserved by formalin agglutinate with anti-H antibodies: IgG a single amino acid sequence in the flagellar protein (flagellin) indicates the form of the antigen present in one or both of two forms in serotype: Phase 1 (designated by lower case letters) and/or Phase 2 (designated by arabic numerals) o Phase variation: process by which an organism changes phases may interfere with agglutination by anti-O antibody -

Clinical Infections bacteremia urinary tract infection wound infections pneumonia meningitis gastrointestinal disorders opportunistic infections o occur outside the intestine o require the alteration of host by mechanical, physiologic, or infectious processes

Virulence factors 1. Endotoxin (LPS) toxic because of the lipid A portion of the molecule incorporated in the cell wall can cause fever, fatal shock, leukocytic alterations, regression of tumors, alterations in host response to infection and other metabolic changes

Urinary tract infections bacteria ascend the urethra to reach the bladder pili attach to receptors in cells which line the bladder the superficial layer of the epithelium is sloughed off newly exposed cells are penetrated by endocytosis signs and symptoms: o urinary frequency o dysuria: painful urination o hematuria: blood in urine o pyuria: pus in urine o flank pain (seen in upper tract infections)

2. Enterotoxins causes transduction of fluid into the lumen of the small intestines: leads to diarrhea Salmonella, Shigella, strains of E. coli, Klebsiella pneumoniae, Citrobacter freundii, and Enterobacter

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Sepsis: septic shock low blood pressure and reduced amount of blood reaching the vital organs signs and symptoms: o nausea o tachycardia o cold, clammy skin o weak pulse caused by lipid A which triggers the secretion of interleukins, tumor necrosis factors and other cytokines o pyrogenic: induce fever o activate complement cascade o promote intravascular blood coagulation o results in circulation failure, tissue damage, hypertension and shock

Motility: shown by inverted pine tree appearance Enterotube: commercial identification system

Treatment sulfonamides, ampicillin, cephalosporins, fluroquinolones, aminoglycosides multiple drug resistance is common destroyed by heat, germicides and disinfectants o bacteriocides: phenolics, formaldehyde, beta-gluteraldehyde, halogen compounds sensitive to drying Surgical correction (urinary tract obstruction, resection of bronchiectatic portion of lung)

Prevention and Control Proper waste disposal Safe drinking water Good personal hygiene Proper cooking of food

Laboratory Identification 1. Specimens urine blood pus spinal fluid sputum tissue rectal swab feces

2. Culture selective, differential media allows rapid preliminary identification Blood agar plate and other differential media o MacConkey agar: bile salts, crystal violet, lactose, neutral red dye o Eosin methylene blue (EMB) agar: bile salts, eosin, methylene blue o Hektoen enteric agar: bile salts, bromthymol blue, acid fuchsin

OTHER ENTEROBACTERIA KLEBSIELLA Has 5 species, namely: o K. pneumonia o K. oxytoca o K. planticola o K. terrigena o Klebsiella group 47 Exhibit mucoid growth With large polysaccharide capsules Are nonmotile

3. Smears could not serve for species identification large capsules are characteristic of Klebsiella

4. Biochemical tests Lactose utilization: shows the presence of two enzymes (permease and beta-galactosidase) Triple Sugar Iron Slant (lactose, glucose, sucrose and iron salts) IMViC (Indole, methyl-red, Voges-Proskauer, Citrate test) o Indole test: Kovac's reagent o Methyl red: methyl red indicator o Voges-Proskauer: Barritt's reagent o Citrate media: citrate and bromothymol blue Lysine and ornithine decarboxylase: bromocresol purple Urea media: 2% urea, phenol red indicator Phenylalanine deaminase: ferric chloride -

Klebsiella: Laboratory Identification Most are lactose-fermenting on enteric isolation media (except K. rhinoscleromatis and most strains of K. ozaenae) Produces -lactamase: inactivates ampicillin and carbenicillin

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Klebsiella: Antigenic Structure O and K antigens K antigens are the most useful in serologic typing Capsule: resistance to phagocytosis and killing power of the serum Endotoxin: cause opportunistic infections Enterotoxin: o produced by some strains of K. pneumoniae o Plasmid mediated o Isolated from patients with tropical sprue (malabsorption disease, with abnormal flattening of villi and inflammation of lining of large int.) Cytotoxin from some strains of K. oxytoca, but role is unknown

Pneumonia Enterogenic E. cloacae: tropical sprue E. gergorviae o Nosocomial UTI o Isolated from respiratory specimens and blood E. sakazakii o Neonatal meningitis, brain abscess, bacteremia o Isolated from wounds and respiratory Often infects patients with underlying problems High risk for development of infection: long-term hospitalization, use of intravenous catheters, respiratory colonization, prior use of antibiotics

Enterobacter: Antibiotic Resistance Resistance to ampicillin and first and second generation cephalosporins Because of chromosomal -lactamase: ampC

Klebsiella: Diseases Produced K. pneumoniae: o Pneumonia: production of thick, non-putrid bloody sputum o Urinary tract and wound infections o Bacteremia o Meningitis K. oxytoca = Resembles K. pneumoniae in terms of disease spectrum K. ozaenae = Chronic atrophic rhinitis: fetid odor K. rhinoscleromatis = Infection of pharynx and nose, causes granulomatous destruction

SERRATIA Widely distributed in soil and water Common opportunistic pathogen Major entities in nosocomial infections S. marcescens is the major cause of human disease S. liquefaciens: o previously under Enterobacter o with 4 different groups of organisms O and H antigens are useful epidemiologic markers

ENTEROBACTER Most medically important species: E. cloacae, E. aerogenes, E. agglomerans, E. gergoviae , E. sakazakii, E. taylorae, E. asburiae, E. hormaechii Habitat: soil and water, intestines of man and animals Motile (some species) Form mucoid colonies in enteric media

Serratia: Laboratory Identification Can produce DNAse, lipase and gelatinase (except S. fonticola) Red pigment in 10% of the isolates = PRODIGIOSIN, usually found in S. marcescens

Enterobacter: Laboratory Identification Serratia: Clinical Significance Rapid lactose fermenters Form pigmented colonies Has ornithine decarboxylase (except E. agglomerans) Produce gas from glucose Citrate (+) Urinary tract infections, wound infections, pneumonia, septicemia, bacteremia , endocarditis Major causes: o Contaminated respiratory equipment o Poor catheterization techniques Disease associated with: o Underlying disease o Changing physiologic patterns o Immunosuppressive therapy o Mechanical manipulation Resistant to aminoglycosides and penicillins Treated with 3rd generation cephalosporins

Enterobacter: Antigenic Structure Not as developed as E. coli and K. pneumoniae Typing of E. cloacae: o 53 O antigens, 57 H antigens o 28 antigens o Cross react with antisera for Klebsiella capsules Typing of E. aerogenes: o use of Klebsiella capsular antisera

Enterobacter: Diseases Produced Urinary Tract Infections Wound and device infections (lethal when introduced in the bloodstream, as with contaminated IV fluids)

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PROTEUS Two medically important species: P. mirabilis, P. vulgaris Peritrichous flagella gives it rapid motility

Damage to renal epithelial cells Same laboratory characteristics as Proteus and Morganella, but is mainly UREASE (-) Invades burns, also causes UTI Often antimicrobial-resistant

Proteus: Laboratory Identification Motile at 37C Seen as swarming in nonselective media Urease (+) Phenylalanine deaminase (+) Produce H2S Slow to no lactose fermentation MORGANELLA deaminate phenylalanine motile grow on KCN medium ferment xylose urease-positive nosocomial pathogen produced morganii

Proteus: Antigenic Structure O, H and K antigens Used in diagnosis of rickettsial disease o Shared antigens with Rickettsia o Detection of rickettsial antibodies (Weil-Felix test)

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Morganella

CITROBACTER Medically important species: o C. freundii o C. diversus o C. amalonaticus (Levinea amalonatica) C. diversus (Levinea malonatica) C. koseri Formerly part of the Bethesda-Ballerup Group Was included in Salmonellae Typical pathogen, can infect any site in the body

Proteus: Diseases Produced Urinary tract infections Bacteremia Pneumonia Focal lesions Wound infections Septicemia ONLY PRODUCED WHEN BACTERIA LEAVE THE GIT

Citrobacter: Laboratory Identification Grows on enteric isolation media C. freundii: H2S production Weak urease Utilizes citrate No LDC (lysine decarboxylase) Very slow to no lactose fermentation

PROVIDENCIA Three medically important species: P. rettgeri, P. alcalifaciens, P. stuartii Part of normal intestinal flora Cause other infections occasionally Resistant to antimicrobial therapy Urinary tract infections of urease producing members Alkaline pH Formation of urinary calculi: precipitation of Ca and Mg salts

Citrobacter: Antigenic Structure O, H and K antigens C. freundii antigens o closely related to Salmonellae and Escherichia antigens o Some strains with Vi antigen of S. typhi outer membrane protein found in isolates from patients with neonatal meningitis without this protein strains are still virulent and can cause ventriculitis and brain abscesses

Citrobacter: Diseases Produced UTI, sepsis C. diversus: Neonatal meningitis, Brain abscesses, Neonatal septicemia

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SUMMARY OF LABORATORY IDENTIFICATION FOR ENTEROBACTERIACEAE

Laboratory Procedure MacConkey agar Levines EMB agar Hektoen enteric agar Lactose utilization TSI Indole test Methyl red test Voges-Proskauer test Citrate media LDC, ODC Urea Phenylalanine deaminase Motility

Results and Rationale (+) Colonies red to pink, differentiates rapid lactose fermenters from slow fermenters (+) Dark nucleus seen, metallic sheen on surface, differentiates lactose from nonlactose fermenters (+) Pink to orange (fermenter) or green to blue-green (nonfermenter) colonies Differentiates rapid from slow fermenters (+) A=acid, K=alkaline, black precipitate at bottom of tube, detection of sugar fermenters and gas producers (+) Bright red ring at surface of tube, detect ability of organism to cleave indole from tryptophan (+) Cherry red color, (-) yellow to orange, for genus differentiation (+) Pink to rosy red tinge in medium, (-) yellow color prevails, for genus differentiation (+) Medium has a royal blue color, detects citrate-utilizing enterobacteria (+) Bright lavender color, (-) yellow, for genus differentiation (+) Phenol red indicator turns bright pink, detect enterobacteria with urease (+) Olive green color of media, detects presence of this enzyme from Enterobacteria (+) Inverted pine tree appearance, detect motile enterobacteria

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