B.A.Fontanilla, MD Department of Microbiology and Parasitology ENTEROBACTERIACEAE B.A.Fontanilla, MD Department of Microbiology and Parasitology

GENERAL CHARACTERISTICS Taxonomy Morphology Physiology Antigenic Structure Determinants of Pathogenicity Clinical Infection

ENTEROBACTERIACEAE Large number of closely related species - found in soil, water, decaying matter - found in large intestines of human, animals and insects ----”enterics” or “enteric bacilli” - includes causative agents of gastrointestinal diseases - causative agents of nosocomial infections

ENTEROBACTERIAECEAE TAXONOMY Cedecea Citrobacter Edwardsiella Enterobacter Escherichia Ewingella Hafnia Klebsiella Kluyvera Morganella Proteus Providencia Rhanella Salmonella Serratia Shigella Yersinia Enteric group

ENTEROBACTERIACEAE MORPHOLOGY Small 0.5 x 3.0 µ Gram-negative non-spore-forming bacilli May be motile or non-motile If motile – peritrichous flagella nonmotile – Shigella and Klebsiella

ENTEROBACTERIACEAE Biochemically diverse Facultative organisms When grown in anaerobic or low O2 - ferment carbohydrates When grown in sufficient O2 - utilize the TCA cycle and the electron transport system for energy production

ENTEROBACTERIACEAE PHYSIOLOGY Ferment glucose 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 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

ENTEROBACTERIACEAE ANTIGENIC STRUCTURE In serologic typing, antigenic structures are used for classification and epidemiologic studies - Capsular (K) antigens - Flagellar (H) antigens - Somatic (O) antigens

ENTEROBACTERIACEAE ANTIGENIC STRUCTURE Capsular (K) antigens - Klebsiella species has a well defined polysaccharide capsule - in other genera, amorphous slime layer surrounding the bacterial cell - in E.coli – proteins and form fimbriae not capsules - Vi antigen of Salmonella typhi

ENTEROBACTERIACEAE ANTIGENIC STRUCTURE Flagellar antigens - proteins -antigenic variation is due fo differences in amino acid sequences -antigenic typing of Salmonella is based on serologic typing of flagellar antigens

ENTEROBACTERIACEAE ANTIGENIC STRUCTURE Somatic antigens - O antigens is the most external part of the cell wall lipopolysaccharide (LPS) - may enhance the establishment of the organism in the host.

ENTEROBACTERIACEAE ANTIGENIC STRUCTURE Flagellar (H) antigens - proteins - antigenic variation of various flagellar types due to differences in amino acid sequences - serologic typing of flagellar antigens – basis for antigenic typing of Salmonella typhi

ENTEROBACTERIACEAE DETERMINANTS OF PATHOGENICITY 1. ENDOTOXIN - LPS (review structure p. 26 Jawetz) - resides in the lipid A portion of the LPS - produces, fever, fatal shock, leukocytic alterations, regression of tumors, alteration in host response to infection - pooling of blood in the microcirculation - causing cellular hypoxia and metabolic failure due to inadequacy of blood in vital organs

ENTEROBACTERIACEAE DETERMINANT OF PATHOGENICITY 2. ENTEROTOXIN -Toxins that affect the small intestines - transduction of fluid in the lumen –diarrhea 3. SHIGA TOXINS AND SHIGALIKE TOXINS (Verotoxins) – actions on Vero (African green monkey) tissue culture cells - Shigella – toxin that interferes with protein synthesis of cells - E.coli – hemolytic diarrhea

ENTEROBACTERIACEAE DETERMINANTS OF PATHOGENICITY 4. COLONIZATION FACTORS - cellular surface factors: capsule – Klebsiella pneumoniae “Vi” antigen – S. typhi 5. Other Factors - ability to penetrate epithelial lining – E.coli - Shigella - Salmonella

ESCHERICHIA

ESCHERICHIA Includes 6 species 5 species associated with human disease Escherichia blattae Escherichia coli Escherichia fergusonii Escherichia hermanii Escherichia vulneris

ESCHERICHIA COLI Grows well on commonly used media On enteric isolation media – lactose fermenting colonies On blood agar – β-hemolytic (assoc with UTI) Majority – non-pigmented, motile Produce lysine decarboxylase, use acetate as carbon source, hydrolysis of tryptophan to indole

ESCHERICHIA COLI Serologic typing is based on the determination of the O antigen type, the H antigen type and when applicable the K antigen type. - 164 O antigens - 100 K antigens - 50 H antigens Example: Serotype O157:H7 – hemorrhagic colitis Serotype O124:H30 –enteroinvasive; bacillary dysentery

ESCHERICHIA COLI DETERMINANTS OF PATHOGENICITY Surface Factors a. K1 capsule – E.coli with K1 capsule cause neonatal meningitis b. O antigen c. S fimbriae 2. Enterotoxins –produces watery diarrhea caused by the outpouring of fluids and electrolytes - plasmid mediated

ESCHERICHIA COLI DETERMINANTS OF PATHOGENICITY 2. Enterotoxin – produces watery diarrhea - plasmid mediated a. LT enterotoxin - similar to enterotoxin of Vibrio cholera - stimulates adenylate cyclase in the epithelial cells of the small intestines, increasing perrmeability of the intestinal lining, resulting to loss of fluids and electrolytes

ESCHERICHIA COLI DETERMINANTS OF PATHOGENICITY 2. Enterotoxin b. ST enterotoxin – ST producing E.coli do not cause diarrhea Verotoxins (Shigalike Toxins) - associated with 3 human syndrome: - diarrhea, hemorrhagic colitis, hemolytic uremic syndrome - inhibit protein synthesis similar to Shigatoxin

ESCHERICHIA COLI DETERMINANT OF PATHOGENICITY 4. Other factors a. Enteroinvasiveness – strains have large plasmids that encode for O antigens b. Hemolytic – strains are nephropathogenic

ESCHERICHIA COLI CLINICAL MANIFESTATION Pulmonary infections – nosocomial pneumonia - most patients are 50 yrs or oldeer - with underlying chronic disease - main source: endogenous aspiration of oral secretions containing E.coli 2. Neonatal meningitis – with subsequent neurologic or developmental abnormalities

ESCHERICHIA COLI CLINICAL MANIFESTATION 3. Wound infections – especially occurring in the abdomen 4. Sepsis – can invade the bloodstream from any of the primary infection sites

ESCHERICHIA COLI CLINICAL MANIFESTATIONS 5. Diarrheal disease a. Enteropathogenic E.coli (EPEC) - cause of infantile diarrhea - adhere to mucosal cells of small bowell - loss of microvilli - produce watery diarrhea

ESCHERICHIA COLI CLINICAL MANIFESTATION 5. Diarrheal disease b. Enterotoxigenic E.coli – common cause of travelers diarrhea - some strains produce LT, plasmid mediated - toxin activates adenylyl cyclase - intense and prolonged hypersecretion of water and chlorides and inhibits reabsorption of sodium

ESCHERICHIA COLI CLINICAL MANIFESTATION 5. Diarrheal disease c. Enterohemorrhagic E. coli (EHEC) - produces verotoxin - associated with hemorrhagic colitis and hemolytic uremic syndrome - serotype O157:H7 – most common

ESCHERICHIA COLI CLINICAL MANIFESTATION 5. Diarrheal diseases d. Enteroinvasive E. coli (EIEC) - cause bacillary dysentery in all age groups - disease is very similar to shigellosis - occurs most commonly in children - invade intestinal mucosal cells

ESCHERICHIA COLI CLINICAL MANIFESTATION 5. Diarrheal diseases f. Enteroaggregative E.coli (EAEC) - causes acute and chronic diarrhea in persons in developing countries - produce ST-like toxins

To be continued….

KLEBSIELLA

KLEBSIELLA TAXONOMY Klebsiella pneumoniae (Friedlander’s bacillus) Klebsiella oxytoca – Klebsiella ozeana – Klebsiella rhinoscleromatis

KLEBSIELLA BIOCHEMICAL AND CULTURAL CHARACTERISTICS Appear as lactose fermenting colonies on differential enteric media Non-motile Large capsule – colonies appear large, moist and mucoid

KLEBSIELLA ANTIGENIC STRUCTURE Possess O and K antigens K antigens are most useful in serologic typing

KLEBSIELLA DETERMINANTS OF PATHOGENICITY Capsule – resist phagocytosis - encapsulated strains are more virulent 2. Endotoxin 3. Enterotoxin – isolated in patients with tropical sprue - similar to E.coli ST and LT - plasmid mediated

KLEBSIELLA CLINICAL INFECTION Klebsiella pneumoniae - cause primary community-acquired pneumonia - typical patient: middle or older aged with underlying medical problems – alcoholism, chronic bronchopulmonary disease, diabetes mellitus - most patients – thick, non-putrid bloody sputum – necrosis and abscess formation

KLEBSIELLA CLINICAL INFECTION Klebsiella pneumonia - can cause urinary tract infection -wound infections, bacteremia, meningitis 2. Klebsiella oxytoca – causes chronic atrophic rhinitis – fetid odor 3. Klebsiella rhinoscleromatis – infects nose and pharynx; produces granulomatous inflammation

ENTEROBACTER

ENTEROBACTER Enterobacter cloaca Enterobacter aerogenes Non motile Isolated less frequently than Klebsiella and E.coli - Capable of infecting any tissue in the body - Frequently associated with urinary tract infection

ENTEROBACTER Most infections occur in patients with underlying problems Usually nosocomial Among elderly Risks for development of Enterobacter bacteremia - long hospitalization, placement of intravenous catheters, respiratory colonization, use of antibiotics

ENTEROBACTER Enterobacter cloaca Enterobacter aerogenes

ENTEROBACTER BIOCHEMICAL AND CULTURAL CHARACTERISTICS Motile Grows on media used for the isolation of enterics Rapid lactose fermenters and produce pigmented colonies

ENTEROBACTER ANTIGENIC STRUCTURE - Antigenic subgrouping not as developed as E.coli and Klebsiella

ENTEROBACTER CLINICAL INFECTION Isolated less frequently than E. coli and Klebsiella Capable of infecting any tissue Most frequently associated with urinary tract infection Most infections occur in patients with underlying problems – nosocomial Risks: long term hospitalization, plac ement of intravenous catheters, respiratory colonization; prior use of antibiotics,

SERRATIA

SERRATIA Serratia marcescens Serratia liquifaciens Can be differentiated from other members of the Enterobacteriaceae by: - ability to produce extracellular deoxyribonuclease (Dnase), lipase and gelatinase - resistance to colistin and cephalosporin

SERRATIA O and A antigens are important epidemiologic markers All Serratia infections – associated with underlying disease, changing physiologic patterns, immunosuppressive therapy or mechanical manipulations - 90% are hospital acquired: UTI, wound infections, pneumonia, septicemia

PROTEUS

PROTEUS Proteus mirabilis Proteus vulgaris

PROTEUS CULTURAL CHARACTERISTICS Produce a translucent sheet of growth on non-selective media such as blood agar - swarming

PROTEUS BIOCHEMICAL CHARACTERISTICS - Distinguished from other enterics – produce phenylalanine deaminase - All species produce urease: urea = ammonia + CO2 Proteus mirabilis does not hydrolyze tryptophan to indole – basis of grouping into indole-positive and indole-negative

PROTEUS ANTIGENIC STRUCTURE All members possess O, H and K antigens certain P. vulgaris strains share antigens with Rickettsia – used as -antigens for the detection of rickettsial antibodies in Weil_Felix test ( OX-19, OX-K, OX-2)

PROTEUS CLINICAL INFECTION Proteus mirabilis -2nd leading cause of community acquired UTI - major cause of nosocomial infection - urease formation causes urine to become alkaline – stone formation - rapid motility – invasion of urinary tract

PROVIDENCIA

PROVIDENCIA Providencia rettgeri – previously Proteus Providencia alcalifaciens Providencia stuartii Members of the normal intestinal flora All cause urinary tract infections Often resistant to antimicrobial therapy

CITROBACTER

CITROBACTER Citrobacter freundii – isolated from patients with diarrhea Citrobacter diversus- neonatal meningitis and brain abscesses

TO BE CONTINUED…..

SHIGELLA

SHIGELLA TAXONOMY Shigella – genetically indistinguishable from E.coli - divided into 4 serogroups given species name serogroup A – Shigella dysenteriae serogroup B – Shigella flexneri serogroup C – Shigella boydii serogroup D – Shigella sonnei

SHIGELLA BIOCHEMICAL PROPERTIES AND CULTURAL CHARACTERISTICS Factors that distinguish from Salmonella Appear as non-lactose fermenting colonies Non-motile Do not produce H2S Do not produce gas from glucose (except S. flexneri)

SHIGELLA RESISTANCE TO PHYSICAL AND CHEMICAL AGENTS Less resistant than most enterics to physical and chemical agents Susceptible to most common disinfectants Can tolerate low temperatures if adequate moisture is present Can survive for more than 6 months in water and room temperature

SHIGELLA ANTIGENIC STRUCTURE -Shigella are divided into 4 major O antigenic groups – A, B, C, D subgrouping – based on minor O antigens ex: 12 serologic types of group A 6 serologic types of group B 18 serologic types of group C - No H antigens - nonmotile

SHIGELLA DETERMINANTS OF PATHOGENESIS 1. Surface properties – due to O antigens Survive the passage through upper GIT, 2. Invasiveness - attach to colonic cells and penetrate the epithelial cells by induced phagocytosis, escape from phagocytic vacuole, multiply and spread inside the cytoplasm and passage to adjacent cells.

SHIGELLA DETERMINANTS OF PATHOGENICITY 3. Toxins -Shiga toxin interferes with protein synthesis - inactivates the 60S ribosomal unit bacillary dysentery is a 2-stage disease Shigella multiply in a noninvasive manner in the jejunum- and produce the toxin which is taken up by small bowel receptors- result in an activated secretory process second phase – involve the large intestines

SHIGELLA TOXINS Endotoxin - from the LPS - causes irritation of the bowel wall 2. Shigella dysenteriae exotoxin - neurotoxic –meningisimus, coma - enterotoxic – produces diarrhea

SHIGELLA PATHOGENESIS Spectrum of disease asymptomatic infection to severe bacillary dysentery with high fever, chills, convulsions, abdominal cramps, tenesmus and frequent bloody stools Organisms rarely penetrate the intestinal wall and spread to other parts of the body For children and elderly - dehydration

SHIGELLA DIAGNOSTIC LABORATORY TESTS