Shiga toxin-producing Escherichia coli

Transcription

1 Shiga toxin-producing Escherichia coli Terry Arthur Research Microbiologist Meat Safety and Quality Research Unit U.S. Meat Animal Research Center Use of product names by USDA implies no approval to the exclusion of others that may also be suitable An equal opportunity provider and employer

2 Outline -Background -Non-O157 STEC prevalence pre- and post-harvest -Non-O157 STEC response to interventions -Detection difficulties -Knowledge gaps

3 Nomenclature - Shiga toxin initially discovered as a product of Shigella dysenteriae - Antibodies against Shiga toxin shown to inhibit cytotoxicity of an E. coli strain - E. coli strain discovered to be toxic to vero cells Shiga-like toxin (SLTEC) Vero toxin (VTEC) Shiga toxin (STEC) Cause disease in humans Enterohemorrhagic E. coli (EHEC)

4 Lipopolysaccharide (LPS) = O antigen O1-O185 E. coli serotyping O157:H7 O111:H8 O26:H11 Y Y Flagella = H antigen H1-H56

5 Non-O157 STEC emerging? Initial STEC isolation from clinical cases were O111 and O26 strains, not O157. (Konowalchuk et al. 1977) Retrospective analysis found STEC O20 isolates dating from the early 1970 s. (Bettelheim et al. 1982) A 1954 disease outbreak in New England thought to be caused by STEC O111, but no isolates were screened for Shiga toxin carriage. (Johnson et al. 1996) O157:H7 not associated with human disease until (Riley et al. 1983) Retrospective search identified O157:H7 isolates in CDC repository from (Wachsmuth et al. 1997)

6 Non-O157 STEC Estimated to cause one-half of the clinical EHEC cases. Over 200 STEC serotypes have been isolated from cattle. KG = The proportion of non-o157 STEC able to cause disease in humans is unknown.

7 STEC emphasis progression Initially O157:H7 Then O157:H7 and non-o157 Now O157, Top 6 non-o157, & the rest of the non-o157 (non-top 6 non-o157) (O26, O45, O103, O111, O121, and O145) Breadth of STEC strains viewed as a continuum.

8 To assess the clinical and public health risks associated with non-o157 STEC a seropathotype classification defined. A B C D E O157:H7 and O157:NM, which are common causes of outbreaks and HUS in most countries. Associated with outbreaks and HUS, but less frequently than seropathotype A (CDC Top-6). Associated with sporadic HUS but not epidemics. Associated with diarrhea but not with outbreaks or HUS. Multiple STEC serotypes that have never been associated with human disease and appear to be linked exclusively to animal infections in an agricultural setting.

9 CDC Top Six non-o157 STEC Serotypes 22% 7% 12% 16% 9% 5% 71% O26 O45 O103 O111 O121 O145 :H11 or NM :H2 or NM :H2, H11, H25 or NM :H8 or NM :H19 or H7 :NM The cause of 71% of non-o157 diseases in the US Brooks et al, 2005, JID 192:1422.

10 STEC virulence factors Shiga toxins two types: stx1 and stx2 - ribosome inactivating proteins Intimin (eae) attachment to epithelial cells tir/espe - translocated intimin receptor espa and espb required for intimate attachment/attaching and effacing (AE) lesions characteristic of STEC infection hlya - po157 enterohemolysin releasing hemoglobin from red blood cells katp - po157 catalase peroxidase that defends the cell against oxidative damage etpd - po157, encodes part of a type II secretory pathway transporting proteins across the outer membrane lpf - chromosomal long polar fimbriae espp - po157 extracellular serine protease autotransporter saa - po113 STEC agglutinating adhesion suba subtilase cytotoxin serine protease KG = What are essential virulence components?

11 Determinants of infection Virulence of organism E. coli + stx E. coli O157:H7 Interestingly, different virulence gene profiles were detected within strains from the same serotype, for example, O26:H11 (10 isolates) displayed 3 different virulence profiles: stx1/stx2/stx2d/eaea/hlya/tir/lpfao113/espp/toxb/iha (7 isolates), stx1/eaea/hlya/tir/lpfao113/espp/toxb/iha (2 isolates) and stx1/eaea/hlya/lpfao113/espp/toxb/iha (1 isolate). Monaghan et al., 2011

12 Determinants of infection Virulence of organism E. coli + stx E. coli O157:H7 Dose Immune status of individual

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14 Outbreaks Since 1994, there have been approximately 31 non- O157 STEC outbreaks in the U.S. From 2000 to 2006 the CDC reported an average of 40 E. coli O157:H7 outbreaks per year.

15 Outline -Background -Non-O157 STEC prevalence pre- and post-harvest

16 Prevalence of non-o157 STEC in Cattle Country Prevalence Method Reference United States 5.9% colony hybridization Cray et al % vero cell assay Wells et al Argentina 37% vero cell assay Blanco et al Canada 38.2% vero cell assay Van Donkersgoed et al France 70% PCR Pradel et al Japan 78.9% PCR Shinagawa et al % nested PCR Kobayashi et al Of the 1800 samples analysed, 40% (480/1200) of faecal and 27% (162/600) of soil samples were stx1 and/or stx2 positive. STEC were cultured from 1.9% (23/1200) of faecal and 0.7% (4/600) of soil samples Monaghan et al., 2011

17 Prevalence of STEC in Cattle Calves appear to be more susceptible to STEC colonization than older cows. STEC strains harboring stx1 are more commonly isolated from cattle than those harboring stx2. Bovine-related STEC isolates lack accessory virulence factors intimin and hemolysin EHEC serotypes (O157:H7, O111:H8, and O26:H11) are infrequently isolated form cattle when using unbiased methods.

18 bovine human - Majority of bovine STEC strains lack accessory virulence factors and are potentially less virulent. - Cannot distinguish between virulent and nonvirulent STEC.

19 Colonization Tissue tropism difference reported for O157 and non- O157 STEC (van Dieman et al. I&I 2005) 4-day old calves inoculated with CFU O26 = spiral colon O157 = distal colon/raj Later work with tissue explants on 6 wk old calves determines similar binding patterns. (Girard et al. AEM 2007) O26 and O111 can bind at RAJ O157 can adhere and induce A/E lesions at intestinal sites other than the terminal rectum

20 Colonization Naturally colonized dairy cattle shown to excrete non-o157 STEC (O113, O22) for >6 months (Monrath et al. 2011) Persistent shedder yes, Super shedder -? KG = Will swab sampling of RAJ be useful for detecting non-o157 STEC?

21 Prevalence of STEC by Sample Type Barkocy-Gallagher et al., 2003.

22 What do we know from the processing plant studies regarding the Top6 STEC Hides Pre-evis Final Trim % stx positive % STEC isolate % Top 6 ND Arthur et al., 2002; Barkocy-Gallagher et al., 2003; Bosilevac et al., 2007.

23 Prevalence of Non-O157 contamination of post-intervention beef cattle carcasses U.S. 1 France 2 Hong Kong 3 Total Samples Total non-o157 STEC positive 27 (8.3) 16 (1.9) 17 (1.7) PCR positive for stx genes 43 (13.4) 3 91 (10.7) 112 (11.4) 1 Arthur et al Rogerie et al Leung et al

24 STEC virulence factors # of Isolates Preevis Post stx stx stx1, stx stx1, eae stx1, hlya stx2, hlya stx1, stx2, hlya stx1, stx2, eae stx1, eae, hlya stx2, eae, hlya stx1, stx2, eae, hlya Total

25 Prevalence of STEC in beef Number (%) of ground beef samples Total stx positive STEC confirmation Potential EHEC 4,133 (100) 1,006 (24.3) 300 (7.3) 10 (0.24) Bosilevac and Koohmaraie, Australia New Zealand Uruguay USA n % stx gene % STEC isolated % Potential EHEC isolated Bosilevac et al.,

26 Prevalence of STEC in beef 16.8% retail meat samples positive for stx genes (n= 296, Samadpour et al. 2002) However, it is important to note that mere possession of the Stx toxin gene is not sufficient to convert a nonpathogenic E. coli strain into a pathogen. Although the prevalence of STEC in retail ground beef is much higher than that of E. coli O157:H7, which is a member of the group, it does not follow that the public health impact of the group is higher by the same magnitude. (Samadpour et al. 2002)

27 Outline -Background -Non-O157 STEC prevalence pre- and post-harvest -Non-O157 STEC response to interventions

28 Genome comparison EHEC strains carry large virulence plasmid (pehec) Ogura et al. PNAS 2009

29 Genome comparison EHEC Commensal EAEC ExPEC EPEC Shigella Ogura et al. PNAS 2009

30 Genome comparison These results indicate that the whole-gene repertoires of the EHECs are more similar to each other than to any of the other strains. Ogura et al. PNAS 2009 KG = What are the selective pressures and mechanisms driving the development of EHEC? Does parallel evolution have implications for non-virulence-related attributes? O104 outbreak horizontal transmission

31 Previous conclusions Similarly, E. coli O111:H8 and E. coli O26:H11 associated with beef surfaces were reduced by the interventions to approximately the same extent as E. coli O157:H7. Based on these findings, interventions used by the meat industry to reduce Salmonella spp. and E. coli O157:H7 appear to be effective against Salmonella Typhimurium DT 104 and non-o157:h7 STEC. Cutter and Rivera- Betancourt J. Food Prot When the E. coli isolates were grouped as O157 or non-o157 strains, there was no statistical difference between the groups in their sensitivities to D-lactate. However, the non-o157 strains were more susceptible to L-lactate than were the O157strains. Leitsch and Stewart Appl. Environ. Microbiol This indicates that where a process (based on low ph or heat) is validated to ensure absence of E. coli O157:H7, the same conditions should also be effective against E. coli O26. Duffy et al. Int. J. Food Microbiol

32 Log reduction Effect of Peroxyacetic Acid (POAA) on Reduction of STEC Strains a 0.9 a 1.1 a a 1.2 a 1.5 a 1.0 a 1.1 a 0 0 O157 O45 O121 O157 O26 O103 O111 O145 STEC Pool 1 STEC Pool ppm, ph = 2.8 (Inspexx ) Dr. Nor Kalchayanand unpublished

33 Outline -Background -Non-O157 STEC prevalence pre- and post-harvest -Non-O157 STEC response to interventions -Detection difficulties

34 Family: Enterobacteriaceae Gram negative Rod-shaped Motile Ferment: several sugars including sorbitol b-glucuronidase positive E. coli

35 E. coli O157 Family: Enterobacteriaceae Gram negative Rod-shaped Motile: flagella Ferment: several sugars not sorbitol b-glucuronidase negative Resistant to tellurite, novobiocin, and vancomycin Produce Shiga toxins Infectious dose organisms

36 Non-O157 STEC Family: Enterobacteriaceae Gram negative Rod-shaped Motile: flagella Ferment: several sugars including sorbitol b-glucuronidase positive Produce Shiga toxins

37 Non-O157 STEC Detection -Sampling is the same as for O157 -Enrichment is the same as for O157 -No universal immunomagnetic separation method available -Cannot use sorbitol-based detection -Cannot use b-glucuronidase-based detection Detection has focused on identification of strains carrying the Shiga toxin genes or expressing the toxin proteins PCR Colony hybridization EIA Vero cell assay

38 Testing Detection methods for O157:H7 should not be confused with non-o157 methods Sensitivity is comparable, specificity is not Non-O157 STEC confirmation is as much by chance as by design

39 O36:H20 eae O26:H11 stx eae O13:H8 stx O26:H5 Presumptive positive Presumptive positive

40 Screening of commercial beef trim for STEC ground a beef ground b beef trim b stx 24.3% 5.5% 15% stx and eae 10.1% 1.5% 4.7% a Bosilevac and Koohmaraie, 2011 b Hill et al. 2011

41 Detection needs Genetic markers specific for top6 or potential EHEC strains Dr. Jim Bono, USMARC Confirmation method that is: Specific for target strains Rapid Not inhibitory to any of the target strains Dr. Mick Bosilevac, USMARC

42 Outline -Background -Non-O157 STEC prevalence pre- and post-harvest -Non-O157 STEC response to interventions -Detection difficulties -Knowledge gaps

43 Knowledge Gaps What are the selective pressures and mechanisms driving the development of EHEC? Does parallel evolution have implications for non-virulence-related attributes? O104 outbreak horizontal transmission What are the determinants of STEC risk? Need genome sequences from across the STEC continuum. Do non-o157 STEC colonize the RAJ? Will aid in preharvest intervention design. Do non-o157 STEC attain super shedding levels through natural colonization? How do non-o157 STEC respond to current and emerging pre-harvest interventions? Answered

44 Knowledge Gaps Do EHEC specific targets exist for detection? We must move detection methodology forward.

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