Escherichia coli Terry Arthur Research Microbiologist Meat Safety - - PowerPoint PPT Presentation
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 An equal opportunity approval to the provider and
An equal opportunity provider and employer Use of product names by USDA implies no approval to the exclusion of others that may also be suitable
Shiga-like toxin (SLTEC) Vero toxin (VTEC) Shiga toxin (STEC)
Nomenclature
dysenteriae
an E. coli strain
Enterohemorrhagic E. coli (EHEC) Cause disease in humans
Lipopolysaccharide (LPS) Flagella = H antigen = O antigen H1-H56 O1-O185
O157:H7 O111:H8 O26:H11
Non-O157 STEC – emerging?
O26 strains, not O157. (Konowalchuk et al. 1977)
from the early 1970’s. (Bettelheim et al. 1982)
caused by STEC O111, but no isolates were screened for Shiga toxin carriage. (Johnson et al. 1996)
(Riley et al. 1983)
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.
non-O157) – (O26, O45, O103, O111, O121, and O145)
A
O157:H7 and O157:NM, which are common causes of outbreaks and HUS in most countries.
B
Associated with outbreaks and HUS, but less frequently than seropathotype A (CDC Top-6).
C
Associated with sporadic HUS but not epidemics.
D
Associated with diarrhea but not with outbreaks or HUS.
E
Multiple STEC serotypes that have never been associated with human disease and appear to be linked exclusively to animal infections in an agricultural setting.
CDC Top Six non-O157 STEC Serotypes
O26 O45 O103 O111 O121 O145 :H11 or NM :H2 or NM :H2, H11, H25 or NM :H8 or NM :H19 or H7 :NM 22% 7% 12% 16% 9% 5% 71%
The cause of 71% of non-O157 diseases in the US
Brooks et al, 2005, JID 192:1422.
Shiga toxins – two types: stx1 and stx2
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?
Virulence of
Determinants of infection
“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
Virulence of
Determinants of infection
Dose Immune status
http://bites.ksu.edu/nonO157outbreaks
Country Prevalence Method Reference United States 5.9% colony hybridization Cray et al. 1996 19% vero cell assay Wells et al. 1991 Argentina 37% vero cell assay Blanco et al. 1997 Canada 38.2% vero cell assay Van Donkersgoed et al. 1999 France 70% PCR Pradel et al. 2000 Japan 78.9% PCR Shinagawa et al. 2000 100% nested PCR Kobayashi et al. 2001
Prevalence of non-O157 STEC in Cattle “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
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.
bovine human
factors and are potentially less virulent.
O157 STEC (van Dieman et al. I&I 2005)
– 4-day old calves inoculated with 1010 CFU
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
KG = Will swab sampling of RAJ be useful for detecting non-O157 STEC? Naturally colonized dairy cattle shown to excrete non-O157 STEC (O113, O22) for >6 months (Monrath et al. 2011) Persistent shedder – yes, Super shedder - ?
Barkocy-Gallagher et al., 2003.
Arthur et al., 2002; Barkocy-Gallagher et al., 2003; Bosilevac et al., 2007.
Hides Pre-evis Final Trim % stx positive 91.7 96.5 16.2 30.0 % STEC isolate 56.6 58.0 8.9 5.7 % Top 6 ND 11.1 0.0 0.0
Prevalence of Non-O157 contamination of post-intervention beef cattle carcasses
U.S.1 France2 Hong Kong3 Total Samples 326 851 986
1Arthur et al. 2002. 2Rogerie et al. 2001. 3Leung et al. 2001.
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)
stx1 152 135 17 stx2 93 78 15 stx1, stx2 15 15 stx1, eae 2 2 stx1, hlyA 8 3 5 stx2, hlyA 19 17 2 stx1, stx2, hlyA 31 23 8 stx1, stx2, eae 1 1 stx1, eae, hlyA 8 6 2 stx2, eae, hlyA 20 20 STEC virulence factors # of Isolates Preevis Post stx1, stx2, eae, hlyA 12 10 2 Total 361 310 51
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, 2011.
n 220 223 256 487 % stx gene 30 9.7 28 30 % STEC isolated 3.6 1.8 15.6 5.7 % Potential EHEC isolated 0.9 2.3 1.0 Australia Uruguay New Zealand USA
Bosilevac et al., 2007.
Prevalence of STEC in beef 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) 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.
Ogura et al. PNAS 2009
EHEC strains carry large virulence plasmid (pEHEC)
EHEC Commensal EAEC ExPEC EPEC Shigella Ogura et al. PNAS 2009
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?
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. 2000.
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. 2002.
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
1 2 3 4 O157 O26 O103 O111 O145 1.2 a 1.5 a 1.0 a 1.1 a 1.5 a Log reduction
STEC Pool 1 STEC Pool 2
1 2 3 4 O157 O45 O121 0.9 a 1.1 a 0.9 a
200 ppm, pH = 2.8 (Inspexx™)
http://www.amif.org/ht/d/sp/i/26883/pid/26883#Ecoli
Family: Enterobacteriaceae Gram negative Rod-shaped Motile Ferment: several sugars including sorbitol b-glucuronidase positive
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 10-100 organisms
Family: Enterobacteriaceae Gram negative Rod-shaped Motile: flagella Ferment: several sugars including sorbitol b-glucuronidase positive
Non-O157 STEC
Produce Shiga toxins
Non-O157 STEC Detection
Detection has focused on identification of strains carrying the Shiga toxin genes or expressing the toxin proteins PCR EIA Colony hybridization Vero cell assay
O26:H11 stx eae O36:H20 eae O26:H5 O13:H8 stx Presumptive positive Presumptive positive
grounda beef groundb beef trimb stx 24.3% 5.5% 15% stx and eae 10.1% 1.5% 4.7%
aBosilevac and Koohmaraie, 2011 bHill et al. 2011
strains
– Dr. Jim Bono, USMARC
– Specific for target strains – Rapid – Not inhibitory to any of the target strains – Dr. Mick Bosilevac, USMARC
colonization?
interventions? Answered