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Infectious disease epidemiological problems addressed by molecular biology techniques 2016  Tracking strains across time and geography  Distinguishing endemic from epidemic disease occ

National Institute of Health

January 17, 2017

Lecture 8: Pathovar vs non-pathovar: Part 1

Intestinal and extra-intestinal pathogenic Escherichia coli

Practices of Molecular Epidemiology

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.

 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:

Examples of pathovars—cont.

 Organisms that occur in the environment that cause

disease in human hosts under special circumstances:

Escherichia coli

• Commensal E coli

• Intestinal pathogenic E coli (IPEC)

• Extraintestinal pathogenic E coli (ExPEC)

Theodor Escherich, 1885

newscenter.lbl.gov

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!

 Cause foodborne illnesses!

 Transmitted by person-to-person

 Transmitted by animal-to-person

ExPEC: Enemy??

 Extraintestinal E coli pathogens (ExPEC):

 Urinary tract infection (UTI)

 Blood stream infection (BSI)

 Meningitis

 Wound infection

newscenter.lbl.gov

IPECs: E coli organisms associated with diarrhea diseases

2011

Part 1: Intestinal pathogenic E coli

IPECs: E coli organisms associated with diarrhea diseases

 Enteropathogenic E coli (EPEC)

 Enterotoxigenic E coli (ETEC)

 Enteroinvasive E coli (EIEC)

 Shigatoxin producing E coli (STEC)

 Enterohemorrhagic E coli (EHEC)

 Other Shigatoxin producing E coli (VTEC, STEC)

 Entero-aggregative E coli (EAEC)

 Diffuse-adherent E coli (DAEC)

 Enteroaggragative-EHEC (EAEHEC)

2012

Story of E coli

 1885-described by Theodor Escherich

 1945-Bray, showed E coli O111to be associated with an

outbreak in a pediatric clinic.

 1950s- >13 serogroups associated with outbreaks

 By 1955: term, “enteropathogenic E coli” came to be used to

refer to those strains associated with infantile diarrhea.

 1956: De et al (India) found some strains of E coli to be

toxigenic in rabbit ileal loop assay.

 By 1970: Some strains of E coli produced LT and ST toxins.

Story of E coli—cont.

 1960s: Some E coli strains recognized to cause diarrhea indistinguishable from that

caused by Shigella; they were also positive by Sereny test (Japan) These came to be called

enteroinvasive E coli (EIEC)

 1970s: Volunteer studies conclusively showed that nontoxigenic, noninvasive E coli

obtained from diarrhea outbreaks could cause diarrhea in adults (Levine et al); these came to be called EPEC.

 1980s: Some strains of E coli produced shiga-like toxins; eventually these came to be

called shigatoxin-producing E coli (STEC)

 1980s: Several outbreaks of bloody diarrhea (hemorrhagic colitis) were found to be

associated with E coli O157:H7; enterohemorrhagic E coli (EHEC) identified; later

shown to be part of STEC

 2011: Enteroaggregative-shigatoxin E coli (Eagg/STEC) O104:H4 outbreak in Europe

Enteropathogenic E coli (EPEC)

 Prolonged, loose stool, watery diarrhea, low-grade fever, sometimes leads to persistent diarrhea

 Major cause of diarrhea among infants in urban centers of developing countries; high mortality

 Associated with hospitals

 Frequently drug resistant

 Reservoir: human host, possibly animals

 Transmission: person-to-person

 Infectious inoculum: 107-8

Enteropathogenic E coli

 Pathogenesis:

 Causes “attaching and effacing” (A/E) lesion; genes responsible for the A/E lesion

located in a 35-kb region called locus of enterocyte effacement (LEE), pathogenicity island

of EPEC

 Stages of infection

 attachment: mediated by bundle-forming pili (BFP); BFP encoded by a plasmid

 signal transduction: cytoskeletal rearrangement, pedestal formation, microvilli

effacement

 intimate attachment, mediated by a protein intimin encoded by eae gene

 intimin binds to a receptor in the host cell called Tir (translocated intimin receptor); Tir

involved in pedestal formation

 Exports virulence determinants (Tir) via Type III secretion system; (Esp)

Enteropathogenic E coli

 Pathogenesis:

 Causes “attaching and effacing” (A/E) lesion; genes responsible for the

A/E lesion located in a 35-kb region called locus of enterocyte effacement (LEE), pathogenicity island of EPEC

Loss 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

 Secretory diarrhea watery diarrhea, low-grade fever

 Worldwide occurrence; most common cause of traveler's diarrhea; morbidity greater than that due to cholera

because of higher prevalence; children under 2-3 yrs of age experience 2-3 episodes/yr

 Reservoir: ? Animals, ?environment, humans

 Transmission: person-to-person; water, foods

 Most common cause of traveler’s diarrhea

 Infectious inoculum: 108

Enterotoxigenic E coli

 Pathogenesis:

 Colonization factor antigens (CFAs): fimbrial structures, encoded by plasmids

 Same plasmids encode enterotoxins LT, ST

 Heat-labile (LT) toxin

 LT resembles CT; activates adenylate cyclase

 Heat-stable (ST) toxin (STa or STI; STb or STII)

 Sta activates guanylate cyclase, which causes elevation in cyclic GMP; effect is reversible

 May also activate protein kinase C

Enteroinvasive E coli overview

 Invasive (inflammatory) diarrhea, resembling shigellosis

 Worldwide occurrence

 Reservoir: humans

 Infectious inoculum: similar to Shigella, 10-100

 Invasiveness mediated by a 140-MDa plasmid containing genes identical to those in Shigella responsible for cell invasion

Implicated: Daikon sprouts

Itoh, Y et al, Appl Environ Microbiol, 1998

Contaminated

thrombocytopenic purpura (TTP) in adults

 Predominantly associated with cattle (beef, milk), and water or produce contaminated with cattle feces (apple juice, lettuce, sprouts, spinach, etc)

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

 attachment mediated by a 65-Mda plasmid

 plasmid contains a homolog of the ST enterotoxin (EAST1)

 AggR; AAF/I

Diffuse adherent E coli

may indeed be pathogenic; volunteer studies have not demonstrated disease production

Diffuse-adherent E coli (DAEC)

German E coli (STEC) O104:H4 outbreak, May-June, 2011

German E coli outbreak, May-July, 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, 1 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?

enteroaggregative E coli that gained a stx gene?

 Expect to see other E coli strains in the future that

cause hemorrhagic colitis and HUS?

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)

anti- 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

RSS-PCR of E coli strains

(Kimura R et al, Appl Env Micfrobiol, 2000)

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Comparing Salmonella and E coli by RSS-PCR

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