Prevalence and molecular characterization of sorbitol fermenting and non-fermenting Escherichia coli O157:H7+/H7 isolated from cattle at slaughterhouse and slaughterhouse wastewater

https://doi.org/10.1016/j.ijfoodmicro.2014.01.002Get rights and content

Highlights

  • E. coli O157:H7 was examined in cattle and slaughterhouse wastewater samples.

  • Effect of season, age, gender and breed on prevalence was determined.

  • Major virulence genes and gene variants were assessed by PCR.

  • Sorbitol fermenting E. coli O157:H7 was isolated and characterized.

  • Wastewater is a significant source of E. coli O157:H7 environmental contamination.

Abstract

The prevalence and seasonal distribution of E. coli O157:H7+/H7 in an array of aged cattle at slaughter and its dissemination with slaughterhouse wastewater over a two year period in Turkey were investigated. For this purpose, a total of 720 samples (240 rectoanal mucosal swap [RAMS], 240 carcass sponge and 240 bile samples) of 240 cattle categorized according to age, gender, breed and sampling site were collected along with additional 24 wastewater samples and were subjected to immunomagnetic separation based cultivation technique to efficiently isolate E. coli O157 from the background flora. Identification (rfbEO157, fliCh7), detection of major virulence factors (stx1, stx2, eaeA, hly, lpfA1-3 and espA), intimin variants (eae-α1, eae-α2, eae-β, eae-β1, eae-β2, eae-γ1 and eae-γ2/θ) and shiga toxin variants (stx1c, stx1d, stx2c, stx2d, stx2e, stx2f and stx2g) of all the isolates were assessed by PCR. From 10 (4.2%) of RAMS and 11 (4.6%) of carcass sponge samples and 5 (20.8%) of slaughterhouse wastewater samples, a total of 102 colonies (99 sorbitol negative and 3 sorbitol positive) were isolated. Overall, 17 (7.1%) and 15 (6.3%) of 240 sampled cattle were shown to harbor E. coli O157 and E. coli O157:H7, respectively either in their RAMS or carcass sponge samples analyzed. Statistically significant differences between categories; season, age, gender and breed of cattle were not observed (p > 0.05). None of the isolated E. coli O157:H7+/H7 strains harbored any of the investigated intimin types other than eaeγ1 or shiga toxin variants stx1d, stx2e, stx2f or stx2g while all were lpfA1-3+ except 5 E. coli O157:H7 strains. Intimin variant eaeγ1 and shiga toxin 1 variant stx1c were detected from all of the eaeA+ (97/102, 95.1%) and stx1+ (32/102, 31.3%) strains, respectively while from stx2+ (80/102, 78.4%) isolates, both stx2c (68/80, 85.0%) and stx2d (12/80, 15.0%) variants were determined. In the last decade, prevalence of E. coli O157:H7 has an increasing trend in cattle. Slaughterhouses are the significant sources of environmental contamination with E. coli O157:H7. Isolation and molecular characterization of sorbitol fermenting E. coli O157:H7 are a novel finding and may lead to a revision of reference isolation procedure of E. coli O157:H7 in future.

Introduction

Escherichia coli O157:H7 has emerged as a pathogen of significant public health concern worldwide, and it is recognized as the major etiologic agent of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. Primarily cattle and other ruminants can carry the agent in their gastrointestinal tracts as asymptomatic reservoirs while feces and hide are the major sources of bacterial carcass contamination at slaughterhouse (Elder et al., 2000, Gun et al., 2003, McEvoy et al., 2000). Epidemiological studies indicated that the transmission of E. coli O157:H7 occurs through consumption of contaminated raw or undercooked meats of especially bovine origin such as minced meat and related products (Meng et al., 2001). In 2011–2012 alone, 1.4 million tons of bovine meat originating from culture, crossbred and native cattle were produced in Turkey (TSI, 2013), but the public health risk arising from E. coli O157:H7 contamination is fairly unclear.

Age, gender, breed, weaning period, housing, transportation, feed composition of the cattle, season and strain specificity of E. coli O157:H7 were all identified as risk factors for carriage and infection of cattle (Callaway et al., 2009, Kulow et al., 2012). In previous studies, unweaned calves (< 4–8 months) generally showed higher prevalence than adult cattle (> 24 months) while adult cattle showed lower prevalence of E. coli O157:H7 than young stock (12–24 months) as well. Higher prevalence was also observed in female than male cattle (Albihn et al., 2003, Nielsen et al., 2002, Rugbjerg et al., 2003, Yilmaz et al., 2002, Van Donkersgoed et al., 1999). Some of the previous works had speculated the possible influence of genetic difference of cattle on E. coli O157:H7 prevalence (Rugbjerg et al., 2003, Widiasih et al., 2003) while a more recent work showed the genetic and physiological influence of the animal in persistence of the pathogen in cattle (Jeon et al., 2013). In general the prevalence of E. coli O157:H7 is higher during summer and autumn than winter (Hussein and Bollinger, 2005) consequently resulting in higher rates of human infections in summer and autumn (Vugia et al., 2007).

Shiga toxins and intimin are considered as the most critical virulence factors of E. coli O157:H7, involved in development of HUS and attaching-effacing (AE) lesions, respectively (Gyles, 2007). Accumulated reports showed differences in clinical outcomes (Friedrich et al., 2002) and toxicities of shiga toxin variants on different cell cultures (Lefebvre et al., 2009) while intimin variants were shown to affect tissue tropism and consequently the colonization site (Fitzhenry et al., 2002, Mundy et al., 2007). Furthermore, additional factors such as; EspA (E. coli secreted protein A) and Lpf (Long polar fimbria) were determined to be important in tropism, attachment, persistence and virulence (Abe et al., 1998, Farfan and Torres, 2011, Torres et al., 2009).

By the present study, we aimed to provide scientific data on the influence of season, age, gender, breed and sampling site on the prevalence of E. coli O157:H7+/H7 in an array of aged cattle at slaughter over a two years period in Kirikkale, Turkey. Additionally, prevalence and seasonal distribution of slaughterhouse wastewater contamination were assessed for estimation of environmental contamination and the E. coli O157:H7 risk that might arise for public health. Furthermore, major and contributor virulence factors of sorbitol fermenting and non-fermenting Escherichia coli O157:H7 isolates were investigated.

Section snippets

Sampling design and sample collection

Between July 2011 and June 2013, within a two-year period, 744 cattle and wastewater samples (240 rectoanal mucosal swap, 240 carcass sponge, 240 bile and 24 wastewater samples) were analyzed for the detection of E. coli O157 by immunomagnetic separation (IMS) based cultivation technique, which enables efficient isolation of the pathogen from the background flora, and PCR. For this purpose, during 24 months period, weekly visits to slaughterhouse were done to achieve a total of 31 samples per

Isolation and identification of E. coli O157:H7 from cattle and wastewater samples

A total of 744 samples including, 240 of each RAMS, carcass sponge and bile samples belonging to 240 cattle and 24 slaughterhouse wastewater samples were analyzed for the detection of E. coli O157:H7 over a two year period. From 10 (4.2%) of 240 RAMS, 11 (4.6%) of 240 carcass sponges and 5 (20.8%) of 24 slaughterhouse wastewater samples, a total of 102 colonies (99 sorbitol negative and 3 sorbitol positive) were isolated as E. coli O157 by IMS based cultivation technique and latex

Discussion

In this study, over a two year period, we attempted determining E. coli O157:H7 prevalence at the slaughterhouse and slaughterhouse wastewater, its seasonal distribution and the effects of age, breed and gender of cattle on its distribution. Furthermore, pathogenic potential of the isolated strains was investigated by determination of the major virulence genes. In the last decade, limited number of studies on the prevalence of E. coli O157:H7 in cattle at abattoir environment have been reported

Acknowledgments

This work was supported by TUBITAK (The Scientific and Technological Research Council of Turkey) grant no. 110R013. We are grateful to J. Osek and C. Garcia-Aljaro for kindly supplying the reference strains.

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