Enterobacteriaceae Updated Microbiology PDF

THE ENTERBACTERI ACEAE Hannah Reilly MLS PA(ASCP) CHARACTERISTICS OF THE ENTERBACTERIACEAE GROUP Gram negative rods Facultative anaerobes Glucose fermenters Oxidase negative Nitrate positive – Reduce nitrate to nitrite WHERE DO THEY LIVE? Intestines of man and animals...

THE ENTERBACTERI ACEAE Hannah Reilly MLS PA(ASCP) CHARACTERISTICS OF THE ENTERBACTERIACEAE GROUP Gram negative rods Facultative anaerobes Glucose fermenters Oxidase negative Nitrate positive – Reduce nitrate to nitrite WHERE DO THEY LIVE? Intestines of man and animals –Thus called “enterics” or “coliforms” Soil and water COMMON GENERA Escherichia Klebsiella Enterobacter Citrobacter Salmonella Serratia Shigella Proteus Yersinia Providencia Edwardsiella Morganella ISOLATION OF ENTERBACTERIACEAE All grow well and quickly in lab in room air and 35°-37°C Sheep Blood agar Most look alike but can see swarming of Proteus Selective and differential agars MacConkey and EMB Primary classification as LF, NLF XLD, HE, SS (stool only) for pathogens DISEASE PROCESS: OPPORTUNISTIC INFECTIONS Normally found in the GI tract. Will cause disease when they have the opportunity to invade another area of the body. Can be infected by their own organism or passed from person to person in a nosocomial setting Examples of opportunistic infections: UTIs, wound infections, pneumonia, bacteremia Examples of nosocomial transmission: Catheters, Hospital devices that invade the patient, Contaminated IV fluids, & Unclean hands  COMMON OPPORTUNISTIC PATHOGENS E coli Proteus Klebsiella Providencia Enterobacter Morganella Citrobacter Serratia  PRIMARY PATHOGENS NOT normal flora. When in the body they will cause disease. Almost all are transmitted by fecal-oral route – Contaminated food and water, unclean hands Examples of primary pathogens: Salmonella, Shigella, E. coli gastroenteritis, Yersinia pestis CLASSIFICA TION CLASSIFICATION OF ENTEROBACTERIACEA E Huge group of members Divided into tribes of similar members, then further subdivided into genus and species Classification based on similarities in DNA DETERMINATION OF SUBSPECIES Based on ID of surface antigens-found on bacterial cell O or somatic antigen: heat-stable cell wall antigen H or flagellar antigen: heat-sensitive flagellar antigen (can destroy by boiling sample) K or capsular antigen: also heat-sensitive – Vi is a capsular antigen used in Salmonella typing NOMENCLATURE OF SUBSPECIES To denote serotype, a nomenclature system is used – Example: E. coli O157:H7 O (somatic antigen) is type 157 H (flagellar) is type 7 BIOCHEMICAL TESTING Identify species by using a series of biochemical tests which determine each individual’s species pattern of: – Carbohydrate fermentation – Presence of other enzymes such as urease and lysine decarboxylase Classically performed in test tubes Now are miniaturized and automated Principles guiding the biochemical reactions are the same for both classic and automated identification SEROLOGICAL CONFIRMATION Certain Enterobacteriaceae must be serotyped after biochemical ID to: – Make a definitive ID – Epidemiological purposes Organisms that are serotyped include: – Salmonella – Shigella – E. coli O157:H7 THE TRIBES ESCHERICIAE, EDWARDSIELLA, SALMONELLA CITROBACTEREAE, KLEBSIELLEAE, PROTEAE, YERSINIAE, TRIBE ESCHERICHIAE Genera includes: – Escherichiae Species E. coli – Shigella E. COLI CLINICAL SIGNIFICANCE Most commonly isolated bacteria in the clinical lab Usually normal flora in the GI tract, causes infection elsewhere Opportunistic pathogen Most common cause of UTI Also causes wound infections, neonatal meningitis, & septicemia TOXIGENIC STRAINS Have toxin genes (phage-borne) that allow them to cause diarrhea, intestine damage Different levels of pathogenicity: – Enterotoxigenic just causes fluid loss – Enteroinvasive invades the mucosa – Enterohemorrhagic can cause cell death and organ failure TOXIGENIC E. COLI Enterotoxigenic – Traveler’s diarrhea. “Montezuma’s Revenge” when visiting developing countries. – Toxin action is similar to cholera toxin – Caused by ingestion of contaminated food or water Entero- invasive/enteropathogenic – Causes dysentery especially in children. – Have fever, mucosal invasion, WBCs in stool – Will have bloody, mucous stools Enterohemorrhagic – Shigella-like verotoxin (shigatoxin) – E.coli 0157:H7 is an example – Toxin kills cells Enteropathogenic – Neonatal diarrhea/meningitis – Daycare center outbreaks ENTEROHEMORR HAGIC E. COLI Transmitted through undercooked beef, raw milk, or raw fruits/veggies contaminated with fecal matter Children and the elderly are especially vulnerable to severe disease Causes bloody diarrhea Can complicate into Hemolytic Uremic Syndrome (HUS) which can lead to multiple organ failure and death Special media used to isolate E. coli O157:H7: – Sorbitol-MacConkey (SMAC) – Is sorbitol negative so colonies will be clear on media – Have to confirm with serotyping MUG test can also be used for identifying – Detects an enzyme beta-D-glucuronidase – O157:H7 is negative IDENTIFYING ENTEROHEMORRHAG IC E. COLI -Colorless colonies on SMAC -Pink colonies on MAC -Biochemical ID as E. coli -MUG negative -Serotyping for subspecies – usually sent out to reference lab -Can also do toxin assay (EIA) from GN broth KEY BIOCHEMICAL REACTIONS TSI: A/A NOTE: Some strains of E. coli are lactose Indole: + negative! So may be an NLF on MAC plate VP: - Citrate: - A few strains can appear lactose (–) but Motility: + are ONPG + H2S: - Lysine: + A quick presumptive ID of E. coli can be Ornithine: + made from the following results: ONPG: + – Beta-hemolytic on BAP Urea: - – Flat LF PAD: - – Indole + SHIGELLA CLINICAL SIGNIFICANCE ALWAYS considered a pathogen – A primary pathogen Transmitted fecal-oral route – By ingesting food or water contaminated with infected feces – Causes varying degrees of diarrhea Is exclusively a human GI pathogen Very infectious! – Ingestion of 2 µg/ml ceftazidime < 22 mm ceftazidime > 2 µg/ml aztreonam < 27 mm aztreonam > 2 µg/ml cefotaxime < 27 mm cefotaxime > 2 µg/ml ceftriaxone < 25 mm ceftriaxone > 2 µg/ml CONFIRMING ESBL PRODUCTION CLSI recommends performing phenotypic confirmation of potential ESBL- producing isolates of K. pneumoniae, K. oxytoca, or E. coli by testing both cefotaxime and ceftazidime, alone and in combination with clavulanic acid Testing can be performed by the broth microdilution method or by disk diffusion. – For MIC testing, a decrease of > 3 doubling dilutions in an MIC for either cefotaxime or ceftazidime tested in combination with 4 µg/ml clavulanic acid, versus its MIC when tested alone, confirms an ESBL-producing organism. – For disk diffusion testing, a > 5 mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid versus its zone when tested alone confirms an ESBL-producing organism. CONFIRMATIO N OF ESBL Ceftazidime and cefotaxime 30 µg each will be used alone and in combination with 10 µg of clavulanic acid in the phenotypic confirmatory disk diffusion tests (PCDDT A) and B (PCDDT B) respectively. Individual discs are placed at least 3cm center to center apart on a MH plate. An increase in zone diameter of either ceftazidime or cefotaxime by ≥ 5mm with clavulanic acid versus its diameter when tested alone is considered as ESBL positive REPORTING ESBL POSITIVE ORGANISMS All penicillins, cephalosporins, and aztreonam should be reported as resistant. Cephamycins (cefotetan and cefoxitin) should be reported according to their routine test results. If an isolate is not confirmed as an ESBL-producer, current recommendations suggest reporting results as for routine testing. Do not change interpretations of penicillins, cephalosporins, and aztreonam for isolates not confirmed as ESBLs. ALL positive ESBLs need to be reported to the physician and the infection control department. WHAT IS CARBAPENEM RESISTANT ENTERBACTERIACEAE (CRE)? CRE are Enterobacteriaceae that are non-susceptible to carbapenem antibiotics In general, these carbapenem resistance mechanisms include: – The production of carbapenemases (called CP-CRE), enzymes that break down carbapenems and related antimicrobials making them ineffective – This includes enzymes like Klebsiella pneumoniae carbapenemase (KPC) – The combination of mechanisms other than carbapenemase production (called non-CP-CRE) – Most commonly the production of beta-lactamases (e.g., AmpC) in combination with alterations in the bacteria’s cell membrane (e.g., porin mutations). CON’T….. Some Enterobacteriaceae (e.g., Proteus spp., Morganella spp., Providencia spp.) have intrinsic elevated MICs to imipenem and therefore results for meropenem, doripenem, and ertapenem should be used for these organisms to determine if these organisms meet the CRE definition. Imipenem resistance alone (i.e., without resistance to at least one other carbapenem) does not allow these organisms to meet the CRE surveillance definition. MODIFIED HODGE TEST