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ENTEROBACTERIACEAE Dr. Rouchelle Dept of Microbiology Characteristics of the Enterobacteriaceae • Gram-negative bacilli Four major features: 1. Ferment glucose 2. Reduce nitrates to nitrites 3. Oxidase negative 4. All except Klebsiella, Shigella and Yersinia are motile by peritrichous flagella • Grow readily on – MacConkey (MAC) agar – Eosin methylene blue (EMB) agar • Grow readily at 35oC except Yersinia (25o-30oC) • Do not form spores • Natural Habitat: – Environment (soil, water, and plants) – Intestines of humans and animals Classification of Enterobacteriaceae: • Very large number of organisms in Family Enterobacteriaceae, species are grouped into - TRIBES • • • • • Tribe 1 – Escherichieae Tribe 2 – Klebsiellae Tribe 3: Proteae Tribe 4 : Yersiieae Tribe 5 : Erwinieae • Within each Tribe, species are further subgrouped under genera Major Genera of family Enterobacteriaceae: 1. Escherichia 2. Shigella 3. Salmonella 4. Yersinia 5. Klebsiella 6. Proteus Antigenic Factors of Enterobactericeae: • Ability to colonize, adhere, produce various toxins and invade tissues • Some possess plasmids that may mediate resistance to antibiotics • Imp antigens used to identify the organisms: – O antigen – somatic, cell wall, heat-stable Ag – H antigen – flagellar, heat labile Ag – K antigen – capsular, heat-labile Ag Clinical Significance of Enterics: • Ubiquitous in nature • Except for Salmonella, Shigella and Yersinia, most are present in the intestinal tract of animals and humans as commensal flora; “fecal coliforms” • Some live in water, soil and sewage Clinical Significance of Enterics: • Based on clinical infections produced, enterics are divided into two categories: 1. Opportunistic pathogens – normally part of the usual intestinal flora, that may produce infection outside the intestine 2. Primary intestinal pathogens – Salmonella, Shigella, and Yersinia sp. Enterobacteriaceae: Modes of Infection 1. Contaminated food and water (Salmonella spp., Shigella spp., Yersinia enterocolitica, Escherichia coli O157:H7) 2. Endogenous infection (urinary tract infection, primary bacterial peritonitis, abdominal abscess) 3. Abnormal host colonization (nosocomial pneumonia) 4. Transfer between debilitated patients 5. Insect (flea) vector: unique for Yersinia pestis 1. Urinary tract infection: Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus mirabilis 2. Pneumonia: Klebsiella pneumoniae; Enterobacter spp., 3. Wound Infection: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis 4. Bacteremia: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis Enterobacteriaceae: Intestinal Infection • Escherichia coli • Shigella • Salmonella • Yersinia enterocolitica Identification of Enterobacteriaceae: • IMViC Test • Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests: – The IMViC series of reactions allows for the differentiation of the various members of family Enterobacteriaceae. A) Indole –Positive, B) MR –Positive C) VP- Positive, D) Citrate- Negative Methyl Red-Voges Proskauer (MR-VP) Tests Principle Glucose Acidic pathway Or Acety methyl carbinol (ACETOIN) Mixed acids pH less than 4.4 Barrit’s A Barrit;s B Methyl Red indicator Red color Neutral pathway MR positive E. coli VP positive Klebsiella Pink color MR/VP test Methyl Red test Red: Positive MR (E. coli) Yellow or orange: Negative MR (Klebsiella) Voges-Proskauer test Pink: Positive VP (Klebsiella) No pink: Negative VP (E. coli) Result Example Reaction on TSI Butt color Red Slant color Red H2 S Negative Negative Yellow Yellow Yellow Red Red Yellow Positive black in butt Negative Result Alk/Alk/(No action on sugars) A/Alk/(Glucose fermented without H2S) A/Alk/+ (Glucose fermented with H2S) A/A/(three sugars are fermented) Non fermenter e.g. Pseudomonas LNF e.g. Shigella LNF e.g. Salmonella & Proteus LF e.g. E. coli, Klebsiella, Enterobacter TSI Reactions of the Enterobacteriaceae • • • • • • • A/A + g = acid/acid plus gas (CO2) A/A = acid/acid A/A + g, H2S = acid/acid plus gas, H2S Alk/A = alkaline/acid Alk/A + g = alkaline/acid plus gas Alk/A + g, H2S = alkaline/acid plus gas, H2S Alk/A + g, H2S (w) = alkaline/acid plus gas, H2S (weak) A/A + g • Escherichia coli • Klebsiella A/A + gas, H2S • Citrobacter freundii • Proteus vulgaris • Non-lactose, sucrose fermenter Alk/A – Shigella – Providencia Alk/A + g – Salmonella serotype Paratyphi A Alk/A + g, H2S – Salmonella – Proteus mirabilis – Edwardsiella tarda Lactose fermenting colonies Non lactose fermenting colonies Escherichia coli • • • • Most significant species in the genus Important potential pathogen in humans Common commensal in the intestine Pink (lactose positive) colony with surrounding pink area on MacConkey – Ferments glucose, lactose, trehalose, & xylose – Usually motile – Positive indole and methyl red tests – Does NOT produce H2S – Simmons citrate negative – Voges-Proskauer test negative Infections caused by E.coli: – Meningitis, gastrointestinal, urinary tract, wound, and bacteremia – Gastrointestinal Infections : 1. Enteropathogenic (EPEC) – Primarily in infants and children; – Outbreaks in hospital nurseries and day care centers – Stool has mucous but not blood – Identified by serotyping Enteropathogenic E.coli Destruction of surface microvilli • Fever Gut lumen • Diarrhea • Vomiting • Nausea • Non-bloody stools 25 Enterotoxigenic E. coli • Diarrhea resembling cholera • Travelers diarrhea 26 Enterotoxigenic E. coli • Heat labile toxin – Like cholera toxin – Adenyl cyclase activated – Cyclic AMP – Secretion water/ions • Heat stable toxin – Guanylate cyclase activated – cyclic GMP – uptake water/ions 27 Enteroinvasive E. coli (EIEC ) •Dysentery - Resembles shigellosis Enterohemorrhagic E. coli • Usually O157:H7 Flagella Transmission electron micrograph 29 O157:H7 transmitted- through meat products or sewage-contaminated vegetables • Hemorrhagic diarrhea – Bloody, copious diarrhea – Few leukocytes – Afebrile • Hemolytic-uremic syndrome (HUS) – Hemolytic anemia – Thrombocytopenia (low platelets) – Kidney failure 30 Enterohemorrhagic E. coli • Vero toxin- “shiga-like” • Hemolysins Enteroaggregative (EaggEC) Cause diarrhea by adhering to the mucosal surface of the intestine; watery diarrhea; symptoms may persist for over two weeks 31 Urinary Tract Infections • E. coli is most common cause of UTI and pyelo-nephritis in humans • Usually originate in the large intestine • Able to adhere to epithelial cells in the urinary tract Septicemia & Meningitis • Most common causes of septicemia and meningitis among neonates • Acquired in the birth canal before or during delivery • E. coli also causes bacteremia in adults, secondary to genitourinary tract infection or a gastrointestinal source Klebsiella, Enterobacter • Usually found in intestinal tract • Wide variety of infections, primarily pneumonia, wound, and UTI • General characteristics: – Non-motile – Simmons citrate positive – H2S negative, Weakly urease positive – MR negative; VP positive • Usually found in Gastro-intestinal tract • K. pneumoniae is most commonly isolated species • Possesses a polysaccharide capsule - which protects against phagocytosis and antibiotics • Makes the colonies moist and mucoid – Frequent cause of nosocomial pneumonia – Significant biochemical reactions • Lactose positive • Most are urease positive • Non-motile • Nosocomial pneumonia: Spread by health care personnel and equipment • Frequently caused by K. pneumoniae • Often seen in middle-aged males who abuse alcohol • Difficult to diagnose due to commensals in sputum Proteus, Morganella & Providencia species • All are normal intestinal flora • Opportunistic pathogens • Deaminate phenylalanine • Non lactose fermenters Proteus species • P. mirabilis and P. vulgaris are widely recognized human pathogens • Isolated from urine, wounds, and ear and bacteremic infections • Both produce swarming colonies and have a distinctive “burned chocolate” odor • Both are strongly urease positive • Both are phenylalanine deaminase positive • Exhibits characteristic “swarming” Laboratory Diagnosis of Enterics Specimen collection: • Specimens collected and transported in Cary-Blair, Amies, or Stuart media Isolation and Identification – Site of origin must be considered – Enterics from sterile body sites are highly significant – Routinely cultured from stool Media for Isolation and Identification of Enterics: – Blood agar and a selective/differential medium such as MacConkey – On MacConkey, lactose positive are pink; lactose negative are clear and colorless – For stool, highly selective media, such as Hektoen Enteric (HE), XLD, or SS is used along with MacConkey agar • Identification: All enterics are Oxidase negative Ferment glucose Reduce nitrates to nitrites SHIGELLA & SHIGELLOSIS Characteristics of Shigella: – – – – – – Short gram negative bacilli Non-motile Do not produce gas from glucose Fragile organisms Non-lactose fermenting Resistant to bile salts CLASSIFICATION: • Shigellae are classified into 4 subgroups (A, B, C, D) based on biochemical and serological specificity • Subgroup A – Mannitol fermentation negative • Subgroup B, C, D – Mannitol positive SERO GROUP ORGANISM REACTIONS DISTRIBUTION A S. dysenteriae (Sh.shigae) 12 serotypes -Catalase negative - mannitol negative Developing Countries Africa, Latin America, Asia B S.flexneri 6 serotypes Few biotypes form acid & gas Common in developing countries C S.boydii 18 serotypes Bacillary dysentery Indian subcontinent D S.sonnei Late lactose fermenter Developed countries Clinical Infections – Dysentery (bloody stools, mucous, and numerous WBC) – S. sonnei and S. flexneri common – Humans are only known reservoir – Oral-fecal transmission – Low infectious dose (102-104 CFU) • Incubation period = 1-3 days • Watery diarrhea with fever; changing to dysentery • Outbreaks in daycare centers, nurseries, institutions Pathogenesis of Shigellosis (2 stages) Early stage: • Watery diarrhea - Enterotoxic activity of Shiga toxin • Following ingestion and noninvasive colonization, multiplication, and production of enterotoxin in the small intestine Second stage: • Adherence and tissue invasion of large intestine symptoms of dysentery • Cytotoxic activity of Shiga toxin increases severity of dysentry Virulence attributable to: 1. Invasiveness • Attachment (adherence) and internalization • Large multi-gene virulence plasmid regulated by multiple chromosomal genes 2. Exotoxin (Shiga toxin) 3. Intracellular survival & multiplication Invasiveness in Shigella-Associated Dysentery • Penetrate through colonic mucosa, invade and multiply in the colonic epithelium but do not typically invade beyond the epithelium into the lamina propria • Preferentially attach to and invade into M cells in Peyer’s patches (lymphoid tissue, i.e., lymphatic system) of small intestine Invasiveness in Shigella-Associated Dysentery M cells - transport foreign antigens from the intestine to underlying macrophages, Shigella can lyse the phagocytic vacuole (phagosome) and replicate in the cytoplasm Note: This contrasts with Salmonella which multiplies in the phagocytic vacuole Actin filaments propel the bacteria through the cytoplasm and into adjacent epithelial cells with cell-tocell passage, thereby effectively avoiding antibodymediated humoral immunity (similar to Listeria monocytogenes) In Shigella pathogenesis Toxins produced by Shigella: 1. Endotoxin 2. Exotoxin: acts as Enterotoxin & Neurotoxin 3. Shiga toxin: (Verocytotoxin) produced by Sh.dysentriae type 1. • Made of 2 subunits: A & B • Subunit B binds to intestinal cells • Subunit A inhibits protein synthesis Shiga toxin (3 effects) 1. Enterotoxic 2. Cytotoxic 3. Inhibits protein synthesis Enterotoxic Effect: – Adheres to small intestine receptors – Blocks uptake of electrolytes, glucose, and amino acids from the intestinal lumen 57 Shigellosis: • Incubation: 1 to 2 days (up to 7 days) • Duration: 4 days to 2 weeks (shedding!) Clinical Signs: • Mild watery diarrhea • Severe dysentery • Abdominal pain, • Fever • Bloody stools with mucus, tenesmus • Dehydration, acidosis and death – in young, old, immunocompromised & malnourished Complications of Shigellosis • • • • • • Intestinal perforation Septicemia Toxic megacolon Seizures Reactive Arthritis HUS (S. dysenteriae) Laboratory diagnosis: 1. Specimen collection: • Stool • Rectal swabs Transport medium used- Sach’s buffered glycerol saline 2. Microscopy (wet mount) • Fecal leucocytes are found in large No. (>50 cells/HPF) in Shigellosis *Clumps of fresh leucocytes, RBCs & Macrophages typical of Shigellosis STOOL IN SHIGELLOSIS 3. Inoculation of primary isolation media • MA- convex, colorless NLF colonies • XLD- red, smooth colonies • SSA- colourless, transluscent colonies • Selinite F broth – enrichment medium Confirmtion is by : 4. Biochemical identification 5. Serological identification – Polyvalent antisera for Group A, B, C and D serogroups – slide agglutination test Treatment: • Proper hydration - generally a self-limiting disease • Sever disease – treated with antibiotics (children) • Nalidixic acid , Norfloxacin - used • Effective antibiotic treatment – Reduces duration of illness from 5-7 days to 3 days – Reduces period of Shigella excretion after symptoms subside. • Antimicrobial resistance a worldwide problem Prevention of Shigellosis: • Proper sanitation, cooking and storage of food • Identification of carriers, especially food handlers SALMONELLA Genus: SALMONELLA Clinically important species • S. typhi • S. paratyphi A, B, C • Other Salmonella species Diseases caused: • Enteric fever • Gastroenteritis • Septicemia Normal habitat: • Intestines of animals - especially pigs, cattle, rodents, shellfish (contaminated meat and fish) • S.typhi and S. paratyphi - found only in humans • Excreted in faeces & urine of patients , and present in the gall bladder of long term carriers. • Mode of Infection - By ingestion organism in food/water or contaminated hands. • S. typhi causes typhoid – spread mainly by water • S. paratyphi by food. General Characteristics of Salmonella: Coliform bacilli (Enteric rods) Motile gram-negative facultative anaerobes Non-lactose fermenting H2S producing (except S.paratyphi A & S.cholersuis)) Biochemical reactions: • Salmonellae ferment glucose, Mannitol and maltose – producing acid and gas. • ( S.typhi does not produce gas) • Utilise citrate (except S.typhi & S.paratyphi A) • Indole negative • Do not ferment lactose • Most produce H2S in TSI agar. (except S.paratyphi A & S.cholersuis) Antigenic structure: • Salmonellae possess 3 types of antigens based on which they are classified 1. Flagellar antigen ‘H’ 2. Somatic antigen ‘O’ 3. Surface antigen ‘Vi’ Flagellar antigen ‘H’ • Present on the flagella • Heat labile protein • Destroyed by boiling, alcohol & acids • Strongly immunogenic • When mixed with antisera, H agglutination occursproducing large , loose. Fluffy clumps • Flagellar antigens present in 2 phases – phase 1 & phase 2 Somatic antigen ‘O’ • • • • LPS – part of the cell wall Heat stable Resistant to boiling, alcohol & acids Less immunogenic than H antigen – Titre of ‘O’ Ab in serum after infection / immunisation. • When mixed with antisera- agglutination produces compact, chalky, granular clumps. Surface antigen ‘Vi’: • Surface antigen, Interferes with agglutination of freshly isolated strains • Poorly immunogenic - Induces production of low titres of Ab following infection- not useful for diagnosis • Total absence of Vi antibody in a typhoid case- poor prognosis • Persistence of Vi Ab indicates- CARRIER STATE Classification: • The Kaufmann-White system used to classify Salmonella based on identifying the O and H antigens by agglutination • The detection of Vi antigen is also used in the detection of Salmonella typhi and other species. 2nd bacteremia (mononuclear phagocytes ) liver、spleen、gall、 Bone Marrow early stage (1-3W) Bacilli. In gall Bac. In Bac. In feces feces peyer's patches & mesenteric lymph nodes Enterorrha gia,intestin al perforation LN Proliferate,swell necrosis defervescence stage (3-4w) 1st bacteremia (Incubation stage) 10-14d S.Typhi eliminated convalvescence stage (4-5w) Pathogenecity S. Typhi causes : Typhoid fever. Symptoms – Fever with low pulse rate, Headache, Spleenomegaly Mental confusion, Rashes [rose spots] on light colored skin, In uncomplicated typhoid, total WCC is low with relative lymhocytosis and anaemia. S. Paratyphi A&B • Paratyphoid or enteric fever - milder than typhoid fever. • Diarrhea and vomitting Other Salmonella species cause: • Food poisoning [enterocolitis] • Food poisoning strains can also cause bacteremia, inflammation of the gall bladder, ostitis and occasionally abscesses. Laboratory diagnosis of enteric fever: • Specimens: Blood, Bone marrow, Urine, Stools, Intestinal contents by enterotube • Commonly Blood, urine & feces for culture. • Blood Culture– positive in 75-90% of patients during first 10 days of infection • Faeces – S.typhi can be isolated from 80% of patients in the 3rd week. Microscopy: • Gram negative motile bacilli • Faeces may contain blood and few pus cells Culture • Enrichment & selective media for enrichment of Salmonella from faeces –Selenite F broth. • Differential media is XLD and BSA • XLD-pink colonies with black centers • BSA- black colonies with silver metallic sheen. XLD Agar XLD - Red pink black centered colonies Salmonella BSA- Black colonies with silver metallic sheen. Biochemical reactions TSI • K/Ag+ Salmonella species • K/A+ (H2S) Salmonella typhi Lysine Salmonella typhi Salmonella species + Shigella - Indole - Mannitol + + - O Antigens • Cell wall heat stable antigens. • Groups are designed A- Z • Medically important Salmonella belong to groups A to G. • Each group has a group factor, which is an O antigen common to all members of the group and not possessed by Salmonella belonging to other groups. H Antigens • Flagella – heat stable antigens • Serotyped by their H antigens • Many Salmonella are diphasic, i.e. occur in 2 antigen forms referred to as phase l and phase ll. • Phase l antigens are given in alphabetical letters and phase ll are either numbered or given a letter if known to occur in both phases. Vi Antigen: • Surface or capsule antigen can be found in S. typhi, S. paratyphi C and a few other Salmonella. • Associated with virulence and can be detected using Vi antiserum. • It can interfere with O antigen testing • Saline suspension of organism should be heated in water bath for 20 mins and after being allowed to cool, the bacterial cells should be retested with the O antiserum. Grouping and serotyping of Salmonella • Specific O, H and Vi antisera to identify the S. typhi. • The following sera are required to identify S. typhi: Salmonella O antiserum Factor 9 [Group D] Salmonella H antiserum Salmonella Vi antiserum Widal Test • This tests for presence of O & H antibodies in the patients serum. • Useful test when culturing facilities are not available. Buffy coat cultures • A low yield is related to low numbers of Salmonella (<15 organisms per milliliter) in infected patients and/or to recent antibiotic treatment. • Centrifugation to isolate and culture the buffy coat, which contains abundant blood mononuclear cells associated with the bacteria, decreases time to isolation Stools/intestinal secretions culture: • 40-50- % positivity in 2nd week, 80 % in 3rd wk Urine culture: • 25 % in second week • Excretion intermittent • Multiple cultures needed Bone marrow • In long standing enteric fever more success in bone marrow cultures than with blood especially if patient on antimicrobials • The test is extremely painful. Clinicians should try to establish the diagnosis with less traumatic means. Culture/blood/buffy coat/Bone marrow • Blood collected in tryptic soy broth Enrichment cultures of specimens like stool /intestinal contents • Stool/intestinal contents are put in selenite F broth / tetrathionate broth before being cultured on to selective /differential media Selective /differential media for the isolation of salmonellae from clinical samples including blood from enrichment/selective broths • Media of medium to high selectivity • Inhibit gram positive and commensals including Gram negative enterics – DCA – Brilliant green Agar – Bismuth sulfite Agar – XLD Final identification • By biochemical methods • Slide and tube agglutination tests with specific antiserum DNA testing: • Polymerase chain reaction assays for identifying S typhi are available Stools Microscopy/enteric fever • Macrophages • Blood • Stools microscopy in other salmonellosis • Pus cells and red cells Widal • • Detection of Abs against the organisms of enteric fever Antigens known • O antigens and H antigens • Available commercially • Qualitative • Quantitative • Qualitative slide method • End point agglutination Widal • Quantitative, agglutination test • Doubling dilutions of serum and addition of constant quantity of antigen • Incubation water bath • Reaction • Followed by settling time • Examine the tubes with a hand lens against a dark background • End point carpet formation • Granular agglutination • Floccular agglutination • Also look for clearing in supernatant fluid Carpet Larger flocules Widal interpretation • Active typhoid if titers above 1:180 or 1:200 against O or H or both agglutinins is present • High titer > 1:160 against O antigen suggestive of infection • High titer > 1:160 against H antigen alone suggests past infection Widal interpretation • In acute infection, O Ab appears first • H Ab appears slightly later but persists longer and can be used to distinguish between various types of enteric fever. Limitations of Widal test: • Titers against almost all the antigens raised – TAB Vaccination • Titers against o and H antigens may be raised in the following diseases/conditions • Other salmonellosis • Chronic liver disease • Immunological disorders – rheumatic fever – Nephrotic syndrome, Ulcerative colitis etc. Prevention and control • Prevent consumption of contaminated food and water • Food like ice creams – can be contaminated with nontyphoidal Salmonella • Boiling of milk before drinking • Periodic check up of food handlers • Exclude carriers Antimicrobial treatment • Chloramphenicol -emergence of resistant strains • Co-trimoxazole, Ampicillin • Amoxycillin • Ciprofloxacin. Ofloxacin • Ceftriaxone or some other third gen cephlosporins • Emergence of MDR Multidrug resistant strains • These bacteria are resistant to chloramphenicol, ampicillin, trimethoprim, sulfonamides, and tetracycline. • Like chloramphenicol resistance, resistance to ampicillin and trimethoprim is plasmid-encoded. • Drug of choice for MDR treatment: Fluroquinolones Chronic carrier state (stool & urinary carriers) A small percentage become chronic carriers and continue to have positive stool cultures for at least 1 year • 1-4% of untreated patients become chronic carriers • Bladder infection with Schistosoma haematobium predisposes to urinary carriage. • . Typhoid Mary • 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.