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Practices of Molecular Epidemiology Lecture 8: Pathovar vs non-pathovar: Part Intestinal and extra-intestinal pathogenic Escherichia coli National Institute of Health January 17, 2017 Infectious disease epidemiological problems addressed by molecular biology techniques (2016)               Tracking strains across time and geography Distinguishing endemic from epidemic disease occurrence Stratification of data to refine study designs Distinguishing pathovars vs commensal flora or saprophytes Identifying new modes of transmission Studying microorganisms associated with healthcare or institutional infections Surveillance and monitoring response to intervention Characterizing population distribution and determinants of distribution of parasitic organisms Identifying genetic basis for disease transmission Validating microdiversity genotyping methods applied to epidemiology Virus quasispecies population structure analysis Identifying direction and chain of transmission Identifying hidden social networks and transmission links Analyzing microbiomes to study non-infectious disease epidemiology Pathovar vs non-pathovar: definition  Pathovar (pathotype): a pathogenic variant of an organism that causes disease in a host with no recognizable underlying medical condition (immunosuppression, chronic disease, medications)  Non-Pathovar: a nonpathogenic variant of an organism not associated with any disease syndrome elicited by the usual route of infection of its pathogenic variant Pathovars categories  Organisms belonging to the same species that occur as commensals in a host  Organisms that cause disease at the same site where their corresponding commensal strains reside  Organisms that cause disease when they breach a sterile site from a nonsterile niche  Organisms in the environment that cause disease in human hosts under special circumstances (saprophytes) Examples of pathovars  Organisms that cause disease at the same site where their corresponding commensal strains reside:  Escherichia coli  Helicobacter pylori  Organisms that cause disease when they breach a sterile site from a nonsterile niche:  Escherichia coli and other Gram negative bacteria  Staphylococcus aureus  Pneumococcus Examples of pathovars—cont  Organisms that occur in the environment that cause disease in human hosts under special circumstances:  Pseudomonas aeruginosa  Klebsiella pneumoniae  Acinetobacter baumanii  Other health-care associated Gram-negative bacterial spp Escherichia coli newscenter.lbl.gov Theodor Escherich, 1885 • Commensal E coli • Intestinal pathogenic E coli (IPEC) • Extraintestinal pathogenic E coli (ExPEC) E coli pathogens  Intestinal pathogenic E coli (IPEC):  Associated with diarrhea, hemolytic-uremic syndrome  Extraintestinal E coli pathogens (ExPEC):  Associated with urinary tract infection (UTI), blood stream infection (BSI), meningitis, wound infection newscenter.lbl.gov IPEC: Definite enemy—epidemiologic evidence Cause outbreaks and epidemics!  EHEC, EaggEC-EHEC,EPEC Cause foodborne illnesses!  EHEC, EIEC, EaggEC-EHEC, STEC Transmitted by person-to-person  EHEC, EIEC, ETEC, EPEC, EaggEC Transmitted by animal-to-person  EHEC, STEC ExPEC: Enemy??  Extraintestinal E coli pathogens (ExPEC): Urinary tract infection (UTI) Blood stream infection (BSI) Meningitis Wound infection newscenter.lbl.gov Enterohemorrhagic E coli  Pathogenesis:  Shares several virulence factors with EPEC  intimin, Tir, secretion apparatus proteins  In addition, expresses Shiga-like toxins (SLTI, SLTII) or verotoxins (VTI, VTII), encoded by a converting phage  The most common serotype O157:H7 derived from an EPEC strain belonging to serotype O55:H7 Enteroaggregative E coli  Secretory diarrhea; strongly associated with persistent diarrhea  Worldwide occurrence  Most recently found to be important cause of traveler’s diarrhea  Reservoir: ?  High infectious inoculum: 109-10  Pathogenesis:  attachment mediated by a 65-Mda plasmid  plasmid contains a homolog of the ST enterotoxin (EAST1)  AggR; AAF/I Diffuse adherent E coli  Comprised of different subgroups, some of which may indeed be pathogenic; volunteer studies have not demonstrated disease production  Secretory diarrhea in children  Reservoir: ?  Pathogenesis: ? Diffuse-adherent E coli (DAEC) German E coli (STEC) O104:H4 outbreak, May-June, 2011 German E coli outbreak, May-July, 2011  May 1-July 4, 2011  3816 cases  845 hemolytic-uremic syndrome (HUS) cases  54 deaths  15 other countries  Cucumbers from Spain? No  ? Sprouts—June  ? Sprouts –June No  ? Sprouts—June 10 Yes (Frank et al, NEJM, 2011) E coli 104:H4  Enteroaggregative E coli producing shigatoxin (EaggEC/STEC)  Rare  Unusual clinical presentation:  High proportion of HUS (~25%)  >80% of HUS occurred in adults (mostly women)  Late June, 2011: 10 new cases in Bordeaux, France, case in Sweden in non-travelers (O104:H4, genetically similar to he German strain), traced to bean sprouts, ? imported from England Where did E coli O104:H4 come from?  Animal reservoir?  Genetic alteration from a strain of enteroaggregative E coli that gained a stx gene?  Expect to see other E coli strains in the future that cause hemorrhagic colitis and HUS?  Spread by globalization of food trade? Detection of E coli associated with diarrhea  Serologic—serotype association with pathogenic group  Bioassays  Suckling mouse assay (ST)  Rabbit ileal loop assay (LT)  Vero cell cytotoxicity (shigatoxin—EHEC, STEC)  Tissue culture association assays (EPEC, EIEC, DAEC, EaggEC)  Sereny test (EIEC)  Gene probes, PCR  ETEC (ST, LT)  EPEC (bfp, eae, EAF plasmid)  EHEC (stx1, stx2, fliC, chuA)  EIEC (ipaC, ipaH) Partial list of serogroups characteristics of diarrheagenic E coli groups (enterotoxigenic E coli and enteropathogenic E coli) Category Serogroup H antigen Category Serogroup H antigen ETEC 06 H16 EPEC 055 H6, NM 08 H9 086 H34, NM 011 H27 0111 H2, H12 015 H11 0119 H6, NM 020 NM 0125ac H21 025 H42, NM 0126 H27, NM 027 H7 0127 H6, NM 078 H11, H12 0128 H2, H12 0128 H7 0142 H6 Detection of EHEC/STEC  Most strains of E coli O157:H7 are sorbitol-negative in the first 24 hrs  MacConkey agar’s lactose replaced with sorbitol (SMAC)  Test SMAC-negative colonies for agglutination in antiO157 antiserum  Test strain for anti-H7 antibody (usually reference lab)  Test for stx1 or stx2 gene  Other tests specific for E coli O157:H7 Gene targets for DNA-probe or PCR-based detection of diarrheagenic E coli E coli group EPEC ETEC, ST producer ETEC, LT producer EIEC STEC EHEC EAEC Gene target bfpA, eae, EAF plasmid ST gene LT gene ipaC, ipaH stx1, stx2 stx1, stx2, fliC, hly, chuA EcoR1-PstI plasmid frag Pathovar PCR ETEC (LT) EPEC EIEC ETEC (ST) RSS-PCR of E coli strains (Kimura R et al, Appl Env Micfrobiol, 2000) 10 Comparing Salmonella and E coli by RSS-PCR ... Tir (translocated intimin receptor); Tir involved in pedestal formation  Exports virulence determinants (Tir) via Type III secretion system; (Esp) Enteropathogenic E coli  Pathogenesis:  Causes... of microvilli leads to malabsorption and net fluid output —diarrhea Bundle-forming pili (BFP), EPEC (from J Giron) Localized adherence (LA) of EPEC on HeLa c Enterotoxigenic E coli overview ... Staphylococcus aureus  Pneumococcus Examples of pathovars—cont  Organisms that occur in the environment that cause disease in human hosts under special circumstances:  Pseudomonas aeruginosa