Prevalence of E. coli O157:H7 in Raw Minced Beef at Slaughterhouses in Tripoli, Lebanon
Enterohemorrhagic Escherichia coli O157:H7 is a pathogenic strain associated with infections caused by consumption of undercooked raw meat. Sensitive and rapid detection methods for this pathogen are essential for the meat industry to ensure a safe meat supply. This study was conducted to determine the prevalence of E. coli O157:H7 in raw minced beef. A total of 80 raw minced meat samples were collected from eighty different slaughterhouses. Samples were investigated for the presence of E. coli O157:H7 by using VIDAS system followed by culture, biochemical characterization and PCR confirmation.It was observed that 18.8% (15/80) of samples were positive for E. coli O157:H7 by VIDAS, 33.33% (5/15) were identified as E. coli by conventional biochemical tests, while toxin genes amplification by PCR showed negative results for all samples 0%.
Introduction
Food-borne diseases represent a growing public health problem, both in developed and developing countries. The World Health Organization (WHO) has reported that in 2005, 1.8 million people died from diarrheal diseases worldwide. In industrialized countries, where food-borne surveillance systems have been implemented, the percentage of the population suffering from food-borne diseases each year has been reported to be up to 30% [1]. In the U.S. the Center for Disease Control and Prevention (CDC) estimates that each year, one of six Americans (48 million) get sick, 128,000 are hospitalized and 3,000 die of food-borne diseases [2]. In contrast, statistics for food- borne diseases in developing countries are difficult to estimate due to the poor or non-existent reporting systems, therefore reliable statistics on these diseases are not available thus making it difficult to estimate the magnitude of the problem however, the high prevalence of diarrheal diseases in many developing countries suggests major underlying food safety problems [1, 3].
Escherichia coli is a facultative anaerobic Gram- negative, rod-shaped bacterium, belonging to the family Enterobacteriaceae. It is commonly found as part of the normal facultative anaerobic microflora in the intestinal tract of humans and warm-blooded animals [4]. Five virulence groups of E. coli are recognized, according to their effect on certain cell cultures, serological groupings, and to the disease syndromes and characteristics: enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), and enterotoxigenic (ETEC) [5]. Unfortunately, some strains, including Escherichia coli O157:H7 carry traits that can cause serious diseases in humans. E. coli O157:H7 is a member of the group of pathogenic E. coli strains enterohaemorragic (EHEC), verotoxinproducing (VTEC), verocytotoxigenic or shiga-toxin producing organisms (STEC). E. coli O157:H7 are characterized by the production of Shiga-toxins 1 and/or Shiga toxin 2, an essential virulence factor in the production of at least one Shiga toxin [4]. Food, a major trade commodity, represents an important vehicle for food-borne pathogen infections. Raw food, of animal origin, has been linked with food- borne outbreaks, and it has been widely reported that livestock animals are an important source of food-borne pathogens and that poultry, swine and cattle among others, are a reservoir for pathogens such as Salmonella, Campylobacter, and E. coli O157:H7 as well as other STEC [5]. For this reason we used to conduct a study on raw beef to determine the prevalence of E. coli O157.We focused on the use of VIDAS®technique in order to detect E. coli O157 after one day enrichment and then confirmation of the results conventionally by culturing on MacConkey Sorbitol, followed by biochemical tests and finally real- time PCR confirmation. This is the first study in Lebanon, especially in Tripoli, on the detection of the prevalence of E. coli O157 by different method in raw meat.
Material and Methods
Sample Collection
Eighty (80) raw meat samples from beef (minced meat) were collected from different slaughterhouses covered all Tripoli regions .Samples were placed in separate sterile plastic bags to prevent spilling and cross contamination and were immediately placed into cooler boxes at 4±1 ̊ C and transported back to the laboratory. The samples were analyzed within two hours of collection. This study was realized in Health and Environmental Microbiology Laboratory – Lebanese University.
Detection of Pathogens Using Conventional Methods and VIDAS® Technique
Twenty-five gram of each sample (aliquots were taken from five locations in the package (four corners and the middle) to adequately represent the package) were placed in a sterile stomacher bag with 225 ml of Buffered Peptone Water (BPW), stomached for 2 minutes, and incubated at 7-24 hours at 41.5 ± 1°C. After incubation, the contents of the stomacher were mixed manually.
E. coli O157:H7 Preliminary Detection
An aliquot (0.5 ml) of BPW broth was placed into the sample well of the VIDAS®ECPT strip and heated for 5 ± 1 minutes using Heat and Go Operator. The strip was removed and left to cool for at least 10 minutes then placed in the VIDAS®instrument (Biomerieux-France) for 50 min with a VIDAS®ECPT solid phase receptacle (SPR). Results were analyzed according to the manufacturer’s instructions. Positive results were confirmed immediately by culturing onto Sorbitol MacConkey agar (SMAC). Sorbitol negative colonies were identified using several biochemical testssuch as urea, indole and culturing on Kligler Ion Agar (KIA) (Bio-RAD, France). To confirm the biochemical results, a RapID™ ONE System (Remel, USA) was used.
PCR Confirmation of E. coli O157:H7 Species
The Real-Tim PCR reactionwas used to detect the presence of genes encoding Shiga toxins (stx1 &stx2) and eae gene coding the intimin (specific adhesin for E. coli O157:H7) using Real-Time PCR in CFX96 thermocycler (Bio-Rad-Germany). The DNA extraction was performed using QIAamp DNA MiniKit (Qiagen-Germany). The foodproof® STEC Screening LyoKit (Biotecon-Germany) was used. The instructions, protocols and PCR program were followed as given by manufacturer instructions.
Results
E. coli O157:H7 Detection
VIDAS®ECPT analysis revealed that 15 out of 80 (18.75%) raw beef meat were positive for E. coli O157:H7. After biochemical screening for the 15 VIDAS® positive strains, 12 were positive to glucose, lactose, and indole whereas the urea was negative. The RapID™ ONE System (Remel-USA) results showed that only 5 strains of the 15 VIDAS® positive strains were identified as E. coli. The Real-time PCR results showed that all of the 15 VIDAS strains were not exhibit the stx1, stx2 and eae genes (Table 1).
| No. of positive | ||||||||
|---|---|---|---|---|---|---|---|---|
| Detection | ||||||||
| samples/No. of | Percentage | |||||||
| Methods | ||||||||
| sample tested | ||||||||
| VIDAS® | 15/80 | 18.75% | ||||||
| Culture and biochemical test | ||||||||
| 5/15 | 33.33% | |||||||
| Real-Time PCR | 0/5 | 0% |
Table1: Results of E. coli detection by different methods.
Discussion
Human infections of E. coli O157:H7 have mostly been recognized to be from food products with animal origin [6]. The current study demonstrated (0%) prevalence of contamination with E. coli O157:H7 organisms in raw minced beef according to the real-time PCR method. The absence of VTEC O157 contamination appears compatible with the low prevalence of this pathogen in stool samples and low human E. coli O157:H7 infection cases in Tripoli. The results of real-time PCR-based method were negative. However, other methods like VIDAS® system and conventional method gave positive results for E. coli O157:H7. The internal and positive control used in real- time PCR kit is often desired to rule out the possibility of false negatives. For this reason we gave our prevalence result (0%) depending on the real-time PCR analysis. The discrepancy between results issued from different methods could be explained. The E. coli O157:H7 is not present in the minced beef samples, but both conventional methods and VIDAS® technique yielded false positive results. Therefore, the specificity of VIDAS was affected by the presence of contaminating bacteria in the enrichment broth.For conventional method, the selective medium of E. coli O157:H7 was based on the sorbitol- fermenting activity. But since many bacteria other than E. coli O157:H7, like Shigella spp., Hafnia alvei and Edwardsiellatarda, formed colorless colonies on sorbitol MacConkey agar, false positive results were shown by conventional method. In comparison to other countries, the prevalence reported in this study is lower than that in previous studies. In Riyadh, Saudi Arabia, Ashgan et al., (2015) showed thatthe prevalence of E. coli O157H7in ground meat was (5%) [7]. Direct comparison of results is difficult due to differences in the study methodologies, such as sampling over a full year and the sampling of all regions in the country. In this study sampling occurred over 2 months only while in other studies it had occurred during 12 months-3 years, covering all the seasons.
In the study done by SimaHajian, et al. the highest prevalence of E. coli O157:H7 was found on meat sampled in fall and spring, which is in agreement with finding of previous studies on beef that reported peak prevalence occurs in early fall [8, 9]. Time of sampling has an effect on the results, thus we should take this into consideration.
Conclusion
The overall objective of this study was to determine the prevalence of E. coli O157:H7 in raw minced beef in Tripoli. The results showed that E. coli O157:H7 was absent in the raw minced beef samples.
Acknowledgment
The authors thank Mr. Taha ABDOU, Mrs. Mariam YEHIA and Mrs. Rayan SALMA for the technical contribution.
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