Enterobacteriaceae Classification of E.coli, Klebsiella, Proteus and Pseudomonas PDF

ENTEROBACTERIACEAE (Lactose Fermenters & Proteu) Pseudomonas By: Dr. Haidy Samir Mohamed khalil Professor of Medical Microbiology & Immunology Faculty of Medicine Helwan University  Objectives Explain the characters and the classificatio...

ENTEROBACTERIACEAE (Lactose Fermenters & Proteu) Pseudomonas By: Dr. Haidy Samir Mohamed khalil Professor of Medical Microbiology & Immunology Faculty of Medicine Helwan University  Objectives Explain the characters and the classification of the family Enterobacteriaceae Identify & explain the morphology, culture character, pathogenesis, clinical picture, diagnosis, treatment of E.coli and Klebsiella Identify & explain the morphology, culture character, pathogenesis, clinical picture, diagnosis, treatment of Proteus Enumerate the most important characters of Providencia, Morganella Identify & explain the morphology, culture character, pathogenesis, clinical picture, diagnosis, treatment of Pseudomonas  Belong to which family ENTEROBACTERIACEAE  What is the characters of this family 1. G –ve bacilli 2. Non sporing 3. Aerobe facultative anaerobe 4. Ferment glucose 5. Oxidase –ve 6. Catalase +ve 7. Reduce nitrate to nitrite  ENTEROBACTERIACEAE Members of the family Enterobacteriaceae are gram negative bacilli Found primarily in the colon of humans and other animals Many as part of the normal flora They cause a variety of diseases with different pathogenic mechanisms  Commercially prepared biochemical test kits (API 20E) are used for the differentiation of the species of Enterobacteriaceae The family includes many genera e.g. Escherichia, Shigella, Salmonella, Klebsiella, Enterobacter, Serratia, Citrobacter and Proteus  They have certain features in common: 1- They are facultative anaerobes 2- They all ferment glucose (fermentation of other sugars varies) 3- They are oxidase negative 4- They reduce nitrates to nitrites as part of their energy-generating processes  The pathogenic genera are the Shigella, Salmonella, and some strains of the Escherichia The opportunistic pathogens are Klebsiella, Enterobacter, Serratia, Citrobacter, Proteus and Escherichia  They are classified according to What  According to their effect on lactose, Enterobacteriaceae are divided to: 1- The lactose fermenters : Escherichia, Klebsiella and Enterobacter. They are collectively called coliforms. 2- The lactose non-fermenters: Salmonella, Shigella, and Proteus 3- Late lactose fermenters: Sh.sonnei , Citrobacter & Provedentia MacConkey’s agar Blood agar Lactose fermenter Lactose non-fermenter  Escherichia coli These are normal inhabitants of the intestine of man and animals. However, some can cause disease in man.  Refer to the practical Morphology: - Gram negative bacilli - motile - some strains are capsulated  Refer to the practical Cultural characters: -Facultative anaerobes, grow on simple media. -On MacConkey's medium, they produce rose-pink colonies due to lactose fermentation. -E. coli strains causing urinary tract infections produce haemolysis on blood agar.  Refer to the practical Biochemical activity: IMVC ++ -- Ferment glucose, lactose, maltose, mannite, sucrose and salicin with production of acid and gas. Indole positive V.P. negative M.R. positive Citrate Urease and H2S negative  Serological characters: O (somatic) H (flagellar) antigens Many pathogenic E. coli possess K (capsular) antigen  The enteropathogenic E. coli possess O antigens with numerical designations e.g. 26, 55, 111,119. Serotype 0157:H7 cause haemorrhagic colitis.  Virulence factors: 1. Pili or colonization factors 2. K or capsular polysaccharide antigen 3. Endotoxin 4. Two exotoxins (enterotoxins) 5. Verotoxin or Shiga toxin  Virulence factors: 1- Pili or colonization factors: Enable the organism to adhere to mucosal cells Genes for pili are carried on plasmids  Virulence factors: 2- K or capsular polysaccharide antigen: interferes with phagocytosis  Virulence factors: 3- Endotoxin: It is the lipopolysaccharide (LPS) that causes endotoxic manifestations that may be associated with E. coli infections  Virulence factors: 4- Two exotoxins (enterotoxins): One is heat labile (LT) and the second is heat stable (ST) They are produced by enterotoxigenic strains of E. coli They are genetically determined by a plasmid  Virulence factors: 5- Verotoxin or Shiga toxin : It is produced by enterohaemorrhagic E. coli  Diseases caused by E. coli: 1- Urinary tract infections 2- Hospital acquired infections 3- Neonatal meningitis 4- Pneumonia, sepsis, bacteraemia and endotoxic shock 5- Intestinal diseases  Diseases caused by E. coli: 1- Urinary tract infections: E. coli causes 90% of community acquired urinary tract infections (UTI) Certain strains known as uropathogenic E. coli colonize the vagina and periurethral region from where they ascend to the bladder or kidney causing cystitis or pyelonephritis  Cont. These strains possess pili with adhesive proteins that bind to specific receptors on the urinary tract epithelium They also possess K antigens and exotoxins (haemolysins)  Any instructions to the mothers of the female girls ? 2- E. coli is an important cause of hospital acquired infections including UTI, which is associated with urinary catheterization and drug resistant strains 3- Neonatal meningitis: E. coli causes 40% of neonatal meningitis followed by group B streptococci. Such strains usually possess capsular antigen Kl. 4- Pneumonia, sepsis, bacteraemia and endotoxic shock: may follow any E. coli infection specially in immunocompromised hosts or neonates. 5- Intestinal diseases: Diarrhoeagenic strains of E. coli cause diarrhoea through different mechanisms: a- Enterotoxigenic E. coli (ETEC) b- Enteropathogenic E. coli (EPEC) c- Enteroinvasive E. coli (EIEC) d- Enterohaemorrhagic E. coli (EHEC) e- Enteroaggregative E. coli (EAEC)  a- Enterotoxigenic E. coli (ETEC) It causes "travellers' diarrhoea" and infantile diarrhoea. The diarrhoea is watery and ranges from mild to severe (cholera-like) and may be fatal. The organism adheres to intestinal epithelium via the pili or colonization factors. Then they liberate LT and ST enterotoxins. LT causes the watery diarrhoea by stimulating adenylate cyclase activity in cells of the small intestine resulting in increase in the concentration of cAMP, which causes excretion of the chloride ion, inhibition of sodium ion absorption and significant fluid and electrolyte loss into the lumen of the gut. ST activates guanylate cyclase in enteric epithelial cells and stimulates fluid secretion.  b- Enteropathogenic E. coli (EPEC) It is an important cause of diarrhoea in infants. Certain serotypes e.g. O55, Om,026 previously caused outbreaks of neonatal diarrhoea in nurseries. They act mainly by adhering tightly to intestinal mucosa resulting in loss of microvilli and cupping of cells around the bacteria. Thus, preventing the normal functions of absorption and secretion. Resulting in sever watery diarrhoea, vomiting and fever. It is usually self limited.  c- Enteroinvasive E. coli (EIEC): These cause dysentery-like diarrhoea through invasion of intestinal epithelial cells. Like shigella, EIEC are non-lactose or late lactose fermenters and are non-motile.  d- Enterohaemorrhagic E. coli (EHEC): These belong mainly to serotype 0157:H7. They produce a toxin known as "verotoxin" (so called because it is toxic to Vero cells (monkey kidney cells in tissue culture). It is also called shiga toxin as it is similar to those produced by Shigella species. These strains are associated with outbreaks of haemorrhagic colitis, which is a severe form of bloody diarrhoea that mainly follows ingestion of undercooked hamburger at fast-food restaurants.  Some patients may end up with a life-threatening complication called haemolytic uraemic syndrome (haemolytic anaemia, thrombocytopenia and acute renal failure). Treatment of the diarrhoea with antibiotics increases the risk of developing this syndrome by increasing the amount of verotoxin produced by the organism.  e- Enteroaggregative E. coli (EAEC) These exhibit a specific pattern of aggregative adherence to the mucosa in patches. They cause acute and persistent diarrhoea in children and in HIV patients. EAEC produce ST-like toxin and a haemolysin. The available methods used for their identification are DNA probes and HEp-2 cell cultures to determine the aggregative phenotype.  Diagnosis: The specimen e.g. urine, pus, stools, CSF...etc are cultured on different media. Lactose fermenting pink colonies on MacConkey are further identified by their morphology and biochemical reactions. (Refer to the practical )  In case of diarrhoea, isolated E. coli should be further tested serologically and virulence proved: 1- Serotyping by slide agglutination for EPEC and EHEC strains. 2- When EHEC infection is suspected; rapid diagnostic methods are used to detect the verotoxin by ELISA, or to detect the organism by immunofluorescence in stools. 3- Various in vivo assays, tissue cultures, immmuno assays, DNA probes and PCR may be used for detection of toxin production or its gene, mainly in reference laboratories.  Treatment Treatment of E. coli infections depend on the site and on the sensitivity pattern of the isolated organism. Diarrhoeal diseases usually do not require treatment. However, the duration of the diarrhoea can be shortened by antibiotics. Rehydration is essential.  ESBL ?  What are the indicators of the faecal pollution of the water  Indicators of faecal pollution of water: There are some organisms that normally occur in the stools and if isolated from a water sample, this means that the water is contaminated with stools. These organisms are; E. coli, Enterococcus faecalis and CI. perfringens.  KLEBSIELLA Klebsiella pneumoniae are normal inhabitants of the intestine and respiratory tract. They are saprophytes in soil and water. Some may cause diseases in man, which are mainly nosocomial due to multi-drug resistant strains. There are 77 serotypes based on capsular polysaccharide, which is the most important virulence factor.  Refer to the practical Morphology: Gram negative, non-motile, capsulated bacilli. Cultural characters: They give pink colonies on MacConkey. Colonies are mucoid due to the production of abundant extracellular slime. Biochemical activities: They ferment glucose, lactose, maltose, mannite, sucrose and salicin with production of acid and gas. They are indole negative, V.P. positive, M.R. negative and citrate positive.  Diseases caused by Klebsiella: K. pneumoniae causes ülobar pneumonia üurinary tract infection üsepticaemia ü neonatal meningitis. üIt is a common cause of hospital acquired infections. It is highly pathogenic to mice and causes their death within 24-48 hrs when injected intra-peritoneally. Capsulated organisms can be seen in smears from tissues stained by gram.  K. rhinoscleromatis causes ürhinoscleroma which is a granulomatous lesion in the nose and throat. K. ozaenae is assosiated with atrophic rhinitis. K. oxytoca causes hospital acquired infections.  Enterobacter. Citrobacter and Serratia (Read only) They are found in soil, water and stools. They may cause urinary tract, wound and blood stream infections in hospitalized and immunocompromised patients, especially those under invasive procedures such as, respiratory intubations, intravenous and urinary catheters. They are gram negative bacilli, motile and are differentiated by their biochemical reactions.  Proteus species Found in soil and water, and are normal inhabitants of the intestine of man They cause infections only when they leave the intestine The genus includes two important species; P. vulgaris and P. mirabilis  The Proteus cause: Urinary tract infections (UTI) -most commonly by P. mirabilis Wound infections Otitis media Pneumonia Meningitis Bacteremia What is your  Infections may be hospital or community acquired Proteus infections are resistant to antibiotics Antibiotic sensitivity tests should be done before giving treatment  Morphology (Refer to the practical) Gram negative bacilli Very pleomorphic Highly motile with …………… Non capsulated Non sporing  How we can test the motility? Refer to the practical section  Cultural characters Facultative anaerobes Due to their high motility, they give colonies which swarm in successive waves over the surface of nutrient agar On MacConkey, they produce pale non-lactose fermenting colonies Smell : ………………………..  Biochemical activities (Refer to the practical) They are phenylalanine deaminase positive Urease positive: which differentiate them from Salmonella and Shigella They produce H2S which blackens the butt of triple sugar iron (TSI) agar Sugar fermentation : ……………………….  Diagnosis Refer to the practical section  Treatment According to the results of antibiotic sensitivity tests However, most strains are sensitive to aminoglycosides and trimethoprim-sulfamethoxazole Cephalosporins are used for resistant strains  Morganella morganii & Providencia rettgeri (Read only) Were found to be different from Proteus by molecular DNA studies and were placed in separate genera They are similar to Proteus in several characters: üThey are gram negative ü non-lactose fermenters üphenylalanine deaminase and urease positive However, they are differentiated by other biochemical properties  They are found in soil and water They are normal inhabitants of the intestine of man They cause UTI and occasionally other infections They are emerging as important agents of nosocomial infections They are resistant to antibiotics Pseudomonas  It causes: Urinary tract infection Wound infection Otitis externa Corneal ulcers in contact lens users Pneumonia Sepsis with ecthyma gangrenosum in the skin Osteomyelitis & endocarditis in intravenous drug users  Infections are severe and occur in hospitalized and compromised hosts: ü neutropenic patients ü burned patients ü cystic fibrosis patients  Morphology (Refer to the practical) Gram negative motile bacilli  Cultural characters (Refer to the practical) Aerobe Grows on nutrient agar leading to greenish coloration of the medium due to its diffusible exopigment which consists of pyocyanin (blue) and pyoverdine (yellow- green fluorescent) Cultures have a sweet grape-like odour Some strains hemolyze blood  Pathogenesis: They are invasive and toxigenic due to several virulence factors: 1. Pili 2. Endotoxin 3. Enzymes (elastase and protease), that facilitate invasion. 4. Exotoxin A: very similar to diphtheria toxin in its action and causes tissue necrosis  Biochemical activities (Refer to the practical) Oxidase positive Does not ferment any sugar Acid is produced from glucose by oxidation only  Strains isolated from cystic fibrosis possess an exopolysaccharide (glycocalyx) which mediate adherence of the organism to mucous membranes and allows it to live in a biofilm away from antibodies and phagocytosis  P. aeruginosa causes 10-20% of hospital acquired infections due to: 1- Their ability to grow in aqueous solutions that favors their persistence in hospital environment contaminating üRespiratory equipment üAnesthesia equipment üI.V. fluids 2- Their remarkable ability to withstand disinfectants. They were found growing in hexachlorophene-containing soap solutions, antiseptics and detergents. 3- The presence of the compromised patients in hospitals e.g. those with extensive burns, chronic respiratory diseases, cystic fibrosis and UTI in those with indwelling catheters  Diagnosis of infections: Refer to the practical section 1- Specimen 2- Smears 3- Cultures 4- Bacteriophage and pyocin typing are used for epidemiologic purposes (tracing the source of infection).  Treatment The organism is resistant to many antibiotics Antibiotic sensitivity tests should be done to find the proper antibiotic. Penicillin -ticarcillin or piperacillin- plus an aminoglycoside e.g. gentamycin are commonly used, as well as, imipenem, ciprofloxacin and ceftazidime.  Some organisms are like Jokers they can cause any type of infection, can you enumerate some of them? Time for ………