Full Length Research Paper
ABSTRACT
The current work aimed to determine the prevalence of Escherichia coli in fresh ground beef purchased from butchers' shops in Suez Governorate, Egypt, and the antibiotics susceptibility pattern of the isolated bacteria. E. coli was isolated and detected on tryptone bile glucuronide agar (TBGA) plates as chromogenic selective medium for this species. The sensitivity and resistance of the isolated bacteria to antibiotics were performed according to the National Committee for Clinical Laboratory Standards guidelines (NCCLS). A total of 299 bacterial isolates were recovered from 130 ground beef and E. coli had the highest frequency of occurrence (81.5%). The isolated enteric bacteria were identified phenotypically and genotypically as Serratia marcescens, E. coli, Enterobacter cloacae and Klebsiella pneumoniae and deposited in the GenBank nucleotide sequence database under accession numbers KU237235, KU237236, KU237237 and KU237238, respectively. Antibiotic susceptibility test showed that the four isolated species were susceptible to norfloxacin, pefloxacin, kanamycin and ceftriaxone, and resistant to clindamycin and the other tested antibiotics showed different susceptibility pattern with each tested species. Precautions and strict hygienic measures should be taken during the processing stages of ground beef in order to avoid contamination by enteric bacteria.
Key words: Contamination, Enterobacteriaceae, genotype, resistance, susceptibility, meat.
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
The appearance of these different coloured colonies besides the E. coli colonies might be due to different intracellular enzymes by these bacteria or different reactions between the components of the medium and intracellular metabolites. Generally, this chromogenic medium may be suitable for better detection of these four groups of isolated bacteria. Isolation and enumeration of E. coli is used as reliable indicator of fecal contamination and probability of toxigenic microorganisms’ presence in this food. Group B which represents E. coli had the highest frequency of occurrence and represented by 81.5% recovered from 106 samples out of 130 samples, while groups A, C and D is represented by 5.4, 43 and 66.9%, respectively (Table 1). The results are almost in line with that of Greeson et al. (2013) who studied the prevalence of Enterobacteriaceae in 36 samples of meat and reported that E. coli was the most frequent contaminant and its prevalence was 72.2%. E. coli is known as a fecal contamination indicator in foods due to its presence in the intestinal tract.
The gastrointestinal tract and the hands of personnel were recorded as major transferors of Klebsiella spp. and E. coli (Gundogan and Yakar, 2007). On the other hand, the current study results are in contrast with those of Mohammed et al. (2014) who recorded low frequency (15.89%) of E. coli isolated from 384 meat samples. Also, a similar study on isolation of E. coli from retailed meat was performed by Nossair et al. (2014) who isolated different members of Enterobacteriaceae from 50 samples of retailed meat collected from buffaloes and it was found that different species of bacteria were isolated at different rates, where E. coli is the highest isolated bacterium (40%) followed by K. pneumoniae. The obtained results showed that the examined ground beef of cow origin were highly contaminated with E. coli and other enteric bacteria which showed fecal contamination potential for severe hazard (Mohammed et al., 2014). The isolated bacteria were members of the intestinal flora of human and animals and many of them might lead to food deterioration and toxicities (Gundogan and Yakar, 2007; Haryani et al., 2007).
These results emphasized the role played by meat in transmission of E. coli that could constitute public hazard and food poisoning outbreaks (Reuben and Gyar, 2015; Kabiru et al., 2015). Contamination of both carcasses and the environment by E. coli from the intestinal contents of cattle during slaughter is one of the most significant risk factors in transmission to humans (Koohmaraie et al., 2005; Bosilevac et al., 2009). Moreover, Enterobacteriaceae contaminating ground beef in butchers' shops may originate from human carriers (workers) who handle and prepare the meat during cutting and grinding. Also, infected rodents that may be present in the butchers' shops or slaughterhouse could represent a neglected nsource of contamination by E. coli and other coliforms bacteria (Okonko et al., 2010). The four isolated coloured bacterial groups were characterized morphologically, biochemically and genotypically.
The morphological and biochemical characterization of the total recovered bacterial isolates belonging to the different four groups: A, B, C and D were performed by conventional methods. All the tested isolates belonging to the four groups were negative for Gram staining and have short rods shape. Also, the four groups were negative for oxidase and positive for glucose fermentation. On the other hand, the four groups differed in their result for lactose fermentation as the test was positive for the three groups: B, C and D, and was negative for group A. The results of the other biochemical tests showed more differences between the four bacterial groups in this investigation. Group A was characterized by red pigment production and was positive for VP, citrate and motility, whereas, was negative for methyl red, H2S, urease and indole. Group B was positive for methyl red, motility and indole but negative for VP, citrate, H2S and urease.
Group C was positive for VP and citrate but was negative for methyl red, H2S, urease, motility and indole. The fourth group showed positive result for each methyl red and citrate and gave result for VP, H2S, urease, motility and indole. The colonies colour differed on different culture media as presented in Table 3. Based on the morphological and biochemical characterizations, the isolated strains belonging to the four groups: A, B, C and D were related to Serratia marcescens, E. coli, Enterobacter cloacae, Klebsiella pneumoniae, respectively, according to the Bergey’s Manual of Determinative Bacteriology (Holt, 1994). The phenotypic-based identification was confirmed by genotypic identification. One isolate was selected from each of the four groups and their DNA were extracted, and a fragment of about 1500 bp from each one was amplified using the universal primers 16S F and 16S R (Figure 2). Comparison between 16S rRNA gene sequences of the tested isolates Ras1, Ras2, Ras3 and Ras4 belonging to groups A, B, C and D, respectively and 16S rRNA gene sequences on GenBank database as determined using Blast search analysis, were done.
Other tested antibiotics showed differential susceptibility. All the tested isolates of the four tested species were resistant to clindamycin. The tested isolates of S. marcescens were also resistant to cefuroxime sodium, cephalexin, cephalothin, streptomycin and rifampicin, whereas, were highly sensitive to norfloxacin, pefloxacin, kanamycin and ceftriaxone. The tested isolates of E. coli were highly sensitive to norfloxacin, pefloxacin, ceftriaxone and cephalothin but with moderate sensitivity to cefuroxime sodium, cephalexin and streptomycin and had low sensitivity to the others antibiotics. E. cloacae isolates were resistant for cephalexin, cephalothin and clindamycin, however, were highly sensitive to norfloxacin, kanamycin and ceftriaxone. On the other hand, K. pneumoniae was resistant to cephalothin, rifampicin in addition to clindamycin and was highly sensitive to norfloxacin, pefloxacin and ceftriaxone. Shiga toxin-producing E. coli isolated from samples of meat were multi-resistant, exhibiting resistance to ampicillin, ciprofloxacin, tetracycline, sulfamethoxazole-trimethoprim, gentamycin and streptomycin (Li et al., 2011).
In a study carried out by Kalmus et al. (2011) to evaluate antibiotic resistance of E. coli, ampicillin, streptomycin and tetracycline resistance were observed in 24.3, 15.6 and 13.5%, respectively, among the E. coli isolates. While examining the hygienic and sanitary quality of pasteurized cow's milk, E. coli was identified in 77.05% of the samples and the highest rates of resistance to antimicrobial agents were obtained for ampicillin (19.2%), cephalothin (18.9%) and tetracycline (17.1%) (Zanella et al., 2010). E. coli and other coliforms recovered from humans and animals had antibiotic resistance and several species were resistant to many antimicrobial agents commonly used in human and veterinary medicine (Greeson et al., 2013). The treatment of cattle with common antibiotics leads to increase in resistance and transfer of these resistant strains to human hosts (Rinsky et al., 2013; Barnett and Linder 2014; Cordoba et al., 2015).CONCLUSION
CONFLICT OF INTERESTS
ACKNOWLEDGEMENTS
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