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Tvorko M. S. Enterobacteriaceae Escherichia Shigella Edwardsiella Salmonella Citrobacter Enterobacter Serratia Providencia Yersinia Klebsiella Hafnia Proteus Morganella Erwinia Escherichia coli Characteristics • Discovered in 19th century by Bavarian pediatrician Theodor Escherich • Gram negative eubacteria • Facultative anaerobe • Usually motile • Universal inhabitant of human (mammalian) colon Escherichia coli Found in the intestines of humans and animals, this bacterium is usually harmless, but some strains can cause food poisoning and more serious illnesses. Most outbreaks involve contaminated beef that was not cooked thoroughly. The strain known as O157:H7 is considered a potential biological weapon. E. coli Lives in Colon of Healthy People (member of commensal flora) Includes E. coli Scanning electron micrograph Cultivation. Colonies of E. coli on meat-peptone agar Colonies of E. coli on Endo's medium Colonies of E. coli on Ploskirev's medium Colonies of E. coli on blood agar Escherichia coli is highly motile and will show turbidity throughout the tube. Fermentative properties. “+” -ve test “—” -ve test Positive (left) reactions of isolates E. coli in glucose fermentation broth. Note the formation of acid (yellow color) and gas. Observe the bubble in the Durham tube. Indole reaction A. Salmonella B. E. coli is the positive microbe. A B E. coli can reduce nitrate to nitrite. Note the bubble formation. Catalase positive Antigenic structure. The antigenic structure of E. coli is characterized by variability and marked individuality. Along with the H- and O-antigens, the presence of other antigens has been shown in some strains, i.e. the surface somatic (membranous, capsular) K-antigens which contain the thermolabile L- and B-antigens and the thermostable Aand M-antigens. On the basis of antigenic structure an antigenic formula is derived which fully reflects the antigenic properties of the strain For example, one of the most widely spread serotypes is designated 0111 : K58 : H2. Enteropathogenic E. coli (EPEC) • First E. coli pathotype described • UK pediatrician John Bray, 1945 • Causes potentially fatal infant diarrhea in developing areas • Contract organism by ingestion of contaminated water, food or fomites EPEC Colonization and Lesion Development • patchy colonization of the small intestine • Generates unique histopathology termed “attaching and effacing” lesion • destroys microvilli • Expresses numerous toxins/effectors that manipulate host cell systems to serve the pathogen • EPEC, like other pathogenic E. coli strains, is a master cell biologist Toxin production. a gluco-lipo-protein complex with which their toxic, antigenic, and immunogenic properties endotoxins thermolabile neurotropic exotoxins haemotoxins pyrogenic substances, proteinases, deoxyribonucleases, urease, phosphatase hyaluronidase aminoacid decarboxylases Pathogenic E. coli Virulence Mechanisms Generalities • Virulence systems frequently encoded on mobile genetic elements • successful combinations reflected in pathotypes • Like other mucosal pathogens, pathogenic E. coli use multi-step strategy to infect host • attachment • evasion of host defenses • multiplication • damage host • Pathogenic E. coli often colonize host niches not normally inhabited by E. coli EPEC A/E Lesion and Pedestal Formation Fig. 1. Scanning electron micrograph showing microcolonies of EPEC displaying the characteristic localized adherence pattern of adherence to HEp-2 cells. Fig. 2. High power scanning electron micrograph of EPEC displaying localized adherence to HEp-2 cells. Note the elongated microvilli to which the bacteria appear to attach. Enterohaemorrhagic E. coli (EHEC) • First described in 1982 • Causes bloody diarrhea (haemorrhagic colitis), non-bloody diarrhea and haemolytic uremic syndrome (HUS) • ~5% of EHEC infections result in HUS; predominantly in children <5 years old and elderly • ~5% of HUS cases are fatal • Contract organism by ingesting contaminated food • common inhabitant of bovine gut • low infectious dose for humans (<100 organisms) • organism may be resistant to stomach acid EHEC Virulence Factors • O157:H7 serotype dominant in North America, UK, Japan • O26 and O111 serogroups more prominent in other countries • Evolved from LEE containing EPEC serogroup O55 • Like EPEC, generates A/E lesion • Colonizes the large intestine • Expanded repertoire of adhesion factors • Encodes toxins/effectors in EPEC LEE Additional virulence factors (plasmid and chromosomal) • RTX toxin similar to haemolysin • StcE activates host Complement cascade • Stx (Shiga) toxin Stx (Shiga) Toxin • Toxin encoded on bacteriophage • >200 E. coli serotypes encode Stx • Shiga toxin E. coli (STEC) • most do not encode LEE (not virulent) • A/B toxin • pentameric B subunits bind holotoxin to host cell surface • A subunit cleaves host ribosomal RNA arresting protein synthesis and cell death (apoptosis) • Stx produced in colon travels through bloodstream to kidney • Damages renal endothelium and induces inflammation that may lead to acute renal failure and death Shiga toxin • Distinguishing virulence factor • Subunit toxin: A: acts at ribosomal level, inhibits protein synthesis B: binds glycolipid receptor in mammalian cells (renal endothelium) • Stx1, Stx2 – Stx2 variants: 2c,2d,2e,2f HUS: Stx (Shiga) Toxin Activity Direct and indirect contact transmission EHEC Ecology and Transmission Has HACCP led to a reduction in human incidence? Excretion Re-colonization Death Environment Direct contact transmission Indirect contact (environmental) transmission B.Enterotoxigenic E coli ( ETEC) – Common cause for travelers diarrhoea, and watery diarrhoea in children. • Colonisation factor facilitates the attachment to the intestinal epithelium. – Some ETEC produces heat labile exotoxin LT and heat stable or either of the toxins • • LT has two sub units A B Action -Activate Adenylate cyclase Increase local CAMP Intense, prolonged hypersecretion of water , Lumen of gut fill with water Hypermobility and diarrhoea results. LT is antigenic and cross reacts with the enterotoxin of Vibrio cholerae. Some ETEC produces heat stable enterotoxin STa/b STa activates guanylyl cyclase. STb activates cyclic nucleotides. Releases water Enteroinvasive E coli (EIEC) • Produces disease similar to shigellosis. •In adults this has been isolated with Shigella •Commonly affect children in developing countries, and travelers. •Disease is due to invasion into mucosal cells of the intestine multiply inside the cells and destruction /inflammation/ulceration diarrhoea with blood EIEC are nonlactose fermenter, or late lactose fermenter and non motile. E. coli Enteroinvasive E. coli • member normal gut flora • Agent of bacillary dysentery like disease Normal Colon Dysenteric Colon • healthy GI tract • diseased GI tract • Fever • Severe abdominal pain • Bloody discharge with mucous E. Enteroaggregative E coli (EAEC) • Produce acute/chronic diarrhoea in persons in developing countries. • Sepsis When normal host defense is poor sepsis can happen. • Common in new born babies whose IgM level is low. Treatment of E.coli related diarrhoea 1st Line • Nitrofurantoin • Nalidixic acid • Norfloxacin ABST’ SHOULD BE DONE • Ampicillin • Cotrimoxazole 2nd line • Ciprofloxacin/Ceftriaxone/Cefuroxime Gentamicin Salmonella •Salmonella enterica –one species, ~2000 serovars –Non standard nomenclature •S. enterica serovar Typhimurium •or S. typhimurium •rod-shaped, non-spore-forming Gramnegative bacterium •belongs to the family Enterobacteriaceae –close relative of E. coli •Motile by peritrichous flagella (H antigen). –nonmotile exceptions: S. gallinarum and S. pullorum Salmonella infections in humans • Enteric fever – typhoid and paratyphoid fevers – typhi, paratyphi A, B, C – systemic infection – infects only humans – GI symptoms may not be evident • Salmonella gastroenteritis – non-typhi serovars – zoonosis: predominantly food-borne – can be complicated by septicaemia • more common with some serovars, e.g. S. dublin (15% mortality rate when septicemic in the elderly) • Metastatic disease, e.g. osteomyelitis 10 most frequently isolated Salmonella strains causing human disease – – – – – – – – – – S typhimurium (22.1%) S enteritidis (26.1%) S enteritidis heidelberg (4.8%) Salmonella enteritidis newport (4.3%) Salmonella hadar (2.7%) Salmonella enteritidis agona (2.0%) Salmonella enteritidis montevideo (1.7%) Salmonella oranienburg (1.6%) Salmonella muenchen (1.5%) Salmonella enteritidis thompson (1.5%) Salmonella typhi Scanning electron micrograph Gram’s staining Cultivation. Colonies of S. paratyphi on Ploskirev's medium Colonies of S. typhus on Ploskirev's medium Colonies of Salmonella on Mac-Conkey medium Colonies of Salmonella on CLED medium Colonies of S. typhus on on bismuth-sulphite agar Fermentative properties. Antigenic Structure • Kauffmann-White antigenic scheme – agglutination reactions with specific antisera against Salmonella antigens • O antigens – characteristic sequence of repeating polysaccharide units in LPS. • H antigens – flagellar antigens (protein) and may occur in one of two phase variations. • Vi antigen – a capsular polysaccharide homopolymer of N-acetyl galactosamineuronic acid Toxin production. S. typhi contains gluco-lipo-protein complexes. The endotoxin is obtained by extracting the bacterial emulsion with trichloracetic acid. This endotoxin is thermostable, surviving a temperature of 120° C for 30 minutes, and is characterized by a highly specific precipitin reaction and pronounced toxic and antigenic properties. Investigations have shown the presence of exotoxic substances in S. typhi which are inactivated by light, air, and heat (80° C), as well as enterotropic toxin phosphatase, and pyrogenic substances. Infectious dose • typically about 1,000,000 bacteria • much lower if the stomach pH is raised • much lower if the vehicle for infection is chocolate – protects the bacteria in their passage through the stomach – an infectious dose of about 100 bacteria Clinical Features Enteric Fever • incubation period 10 to 14 days • septicaemic illness – myalgia and headache – fever – splenomegaly – leukopenia – abdominal pain – Rose spots (macular rash on abdomen) • 10% fatal • positive blood, urine, and stool cultures • Sequelae: intestinal haemorrhage and perforation Pathogenesis and diseases in man. • The causative agent is primarily located in the intestinal tract. Infection takes place through the mouth (digestive stage). • Salmonellae penetrate into the intestine, during which inflammatory processes develop in the isolated follicles and Peyer's patches (invasive stage). Salmonellae enter the blood (bacteriemia stage). Here they are partially destroyed by the bactericidal substances contained in the blood, with endotoxin formation. • During bacteraemia typhoid salmonellae invade the patient's body, penetrating into the lymph nodes, spleen, bone marrow, liver, and other organs (parenchymal diffusion stage). During the second week of the disease endotoxins accumulate in Peyer's patches, are absorbed by the blood, and cause intoxication. The general clinical picture of the disease is characterized by status typhosus, disturbances of thermoregulation, activity of the central and vegetative nervous systems, cardiovascular activity, etc. •On the third week of the disease a large number of typhoid bacteria enter the intestine from the bile duct. This results in the development of hyperergia and ulcerative processes. (excretory and allergic stage). Clinical recovery (recovery stage) does not coincide with the elimination of the pathogenic bacteria from the body. The majority of convalescents become carriers during the first weeks following recovery, and 3-5 per cent of the cases continue to excrete the organisms for many months and years after the attack and, sometimes, for life. Epidemiology carrier states • carrier state may last from many weeks to years with faecal shedding – convalescent carrier • chronic carrier – ~3% of persons infected with S. typhi – ~0.1% of those infected with non-typhoidal salmonellae • potential for cross-contamination of foods by the infected handler – “Typhoid Mary” Mallone – but more common in textbooks than in real life Typhoid Mary's real name was Mary Mallon. Irish immigrant who made her living as a cook. Mallon was the first person found to be a "healthy carrier" of typhoid fever in the United States. She herself was not sick – but over 30% of the bacteria in her feces were S. typhi Mallon is attributed with infecting 47 people with typhoid fever, three of whom died. Laboratory Diagnosis • Isolated from stool, blood and urine in enteric fever (blood cultures need to be taken!) Isolation of haemoculture. • Isolated from stool in gastroenteritis • Appears as a non-lactose fermenter – on MacConkey agar or similar selective agar Blood (5 - 10 ml) BHI Incubate 37 0C Subculture (18-24hrs/every 2nd day for 7days) MacConkey Stools and urine first should be cultured in enrichment media Laboratory Diagnosis • Biochemical tests and serological tests must be done in parallel – Some other bacteria, e.g. Citrobacter, may have similar serological profiles The isolated culture is identified by inoculation into a series of differential media and by the agglutination reaction. The latter is performed by the glass-slide method using monoreceptor sera or by the test-tube method using purified specific sera LABORATORY DIAGNOSIS 1. Isolation of the bacteria 2. Demonstration of antibodies 3. Demonstration of circulating antigens 4. Blood picture Isolation of bacteria Specimens - stools/urine/blood Blood culture - before giving chloramphenicol - repeat the culture - 80-90% +ve 1st week up to10days - +ve during relapses - can culture bone marrow +ve 1-2 days after drug therapy ? good - • When blood culturing is done there is diluting of the drug Stool and urine culture Stools - during the second and third week Urine - during the third and the fourth week, less frequently +ve than stools Repeated culture is necessary when blood culture is -ve Serological test Detection of antibodies to H and O antigens Widal test • Antibodies appear at the end of the first week • Rises during the 3rd week of the enteric fever • Two specimens are taken at interval of 7 to 10 days Interpretation of Widal test 1. Antibody appears at the end of 1st week/rises during 2nd and 3rd/steady at the 4th week then decline 2. Four fold rise of both H and O antibody in an interval of 7 - 10 days (between 1st and the 3rd week is highly significant) Commercial kits commonly used, e.g. API20 Phage typing done for epidemiological purposes E.g. to find source of outbreak Certain phage types predominate nationally S. typhimurium PT4 S. enteritidis DT109 Salmonella vaccines • Vaccination of travellers against typhoid recommended, but does not remove need for good hygiene • Three licensed vaccines – Traditional heat-killed • very reactogenic – Vi subunit vaccine – live oral vaccine, S. typhi Ty21A • Salmonellas can act as live attenuated carriers for other antigens – So far only experimental • No vaccines for gastroenteritis Enterocolitis • • • • • • Common manifestation of Salmonella Common pathogen is S typhimurium 8-48 hrs after ingestion nausea/ headache/ vomiting and profuse diarrhoea, low grade fever Resolution 2-3 days. Inflammatory lesions in bowel are present S. typhimurium in the mouse • S. typhimurium – causes gastroenteritis in humans – causes typhoid-like disease in mice • infection can be established orally or systemically • used as model of typhoid • primary mechanisms of pathogenesis – invasion of the intestine – survival and growth in macrophages Salmonella in eggs • various Salmonella serovars isolated from the outside of egg shells • S. enteritidis PT4 present inside the egg, in the yolk • vertical transmission – deposition of the organism in the yolk by an infected layer hen prior to shell deposition. Sources of in infection • • • • • contaminated water contaminated dairy products shellfish dried or frozen eggs meat and meat products of infected animals • Household pets Clinical features Gastroenteritis • incubation period depends on dose • symptoms usually begin within 6 to 48 hours – – – – – Nausea and Vomiting Diarrhoea Abdominal pain Myalgia and headache Fever • duration varies, usually 2 to 7 days • seldom fatal, except in elderly or immunocompromised Pathogenesis Gastroenteritis • Pathogenic salmonellae ingested in food survive passage through the gastric acid barrier • invade intestinal mucosa • invasion of epithelial cells stimulates the release of proinflammatory cytokines • induces an inflammatory reaction • causes diarrhoea and may lead to ulceration and destruction of the mucosa • Bacterimia rare • Blood culture -ve • Stools remain +ve for sometime after recovery Diagnosis 1.Faeces, vomitus, food stuffs (mainly a single source) 2. Convalescent serum often agglutinates the suspension of casual serotype Prevention and control •Sanitation Thorough cooking •Carriers should not handle food •Vaccination not successful long-term. Vibrio cholerae: 1-pure culture; 2- flagellate vibrios sacharose mannose arabinose Sheep erythrocyte hemolysis Lysis by specific O1 subgroup phages Agglutination by O1 cholera serum Sensitivity to polymixin B Vibrio cholerae biovar cholerae A A – – + + + Vibrio cholerae biovar El Tor A A – + + + – Vibrio cholerae biovar Proteus A A – + – – – Vibrio cholerae biovar albensis А – – – – – – Fermentation within 24 hrs Vibrio The golden rules for prevention of food poisoning 1- Choose foods processed for safety. 2- Cook food thoroughly. 3- Eat cooked food immediately. 4- Store cooked food carefully. 5- Reheat cooked food thoroughly. 6- Avoid contact between cooked and raw food. 7- Wash hands repeatedly. using lots of friction W—warm A—and S—soapy 8- Keep all kitchen surfaces meticulously clean. Wash dishes and utensils after contact with raw meat or eggs. 9- protect foods from insects, rodents and other animals. 10- use safe water. Keep hot food hot and cold food cold.