ISSN: 0973-7510
E-ISSN: 2581-690X
Foodborne infections are major concerns for public health, and are occurring due to microbial contamination of meat. E. coli blotches on surfaces of the meat especially pork and beef. Meat, as a source of protein, and provides energy to our body pork meat has inhabited as special place in the diet of majority of Thai-Population for a wide variety of reasons such as tradition and availability. This study was aimed to evaluate the protein patterns among E. coli strains from pork meat samples of thalath Ongkharak market, Nakhonnayok, Thailand. To evaluate the similarity or intra-specific polymorphism degrees based on whole-cell protein fingerprinting, plasmid profiles and antibiotic resistance patterns. It was determined that the SDS-PAGE method may provide better criteria than plasmid and antimicrobial susceptibility for the taxonomic and epidemiological studies of E. coli isolates. Molecular characterization of isolates of E. coli was carried out by whole cell protein analysis by SDS-PAGE. Whole cell protein analysis of isolates carried out by SDS-PAGE, and were observed visually and compared among samples.
Pork Meat, E. coli, Prevalence, Chloramphenicol, protein, and fingerprinting.
Globally, foodborne pathogens are the origin of diseases and deaths. To prevent the foodborne pathogens billions of dollars spending1. E. coli, is one of the common origin for foodborne diseases in mammals. Resistant strains of E. coli infect at all the age groups and acid resistance, wide range of infections are the severe consequences2. Severity of the infections depends on the host susceptibility, virulence of the E. coli, and dose. Prevalence of resistant strains of E. coli results mild and severe bloody diarrhea, hemorrhagic colitis, and or hemolytic uremic syndrome, which lead to kidney failure2, 3. Livestock animals are the primary reservoirs of E. coli and meat products are determined ass major root cause of foodborne transmission2,4. Carcass contamination occurs through skin-to-carcass or fecal-to-carcass transfer of the pathogen during slaughter process at processing plants5,6,7. These are the major concerns for human infection. However, during the processing of meat direct, indirect and or cross-contamination may occur. Antimicrobials are using mainly reduce pathogen shedding8,9 and washing of skin and carcass10.
For the first time, by using genetic fingerprinting techniques, Kudva et al.11 found multiple strains of E. coli O157:H7 in a single flock of sheep and showed that a single animal shed multiple strains simultaneously and that strains shed by individuals changed over time. Escherichia coli O157:H7 has been isolated from animal drinking water, animal feed, flies, and a pigeon at dairy farms in Wisconsin12. The majority of isolates collected at these farms had the same genetic fingerprint. Although various methods are available for genetic characterization of bacterial isolates, random amplification of polymorphic DNA (RAPD) has been used successfully in the past for E. coli O157:H713, 14,15 and is less costly and time-consuming than other methods. As a source of animal protein, goat meat has for long occupied a special place in the diet for a variety of reasons including taste preference, prestige, religion, tradition and availability, in almost all the communities of the country with the nutritional aspects being included more recently. Meat was the first important food that met up the hunger of ancient people living in cave16. It plays a very vital role in keeping the human body strong in order to provide energy and health17. But, microorganisms present in meat may be harmful for human and may cause spoilage and may be used as indicator organisms. Many researchers have isolated and identified heterogeneous types of microflora from fresh meat.
Protein profiling by SDS-PAGE is a reliable and reproducible molecular technique that has been used by many workers to type various microorganisms of epidemiological interest. This technique was utilized for differentiating the pathogenic and the non-pathogenic strains. Plasmid analysis has also proved a useful method for differentiating bacterial isolates18, 19. The number and size of the plasmids present is used as the basis for strain identification. This strain typing technique has been used successfully for analysis of outbreaks of nosocomial infections20 and community-acquired infections21 caused by a variety of species of Gram negative rods. In the present study aimed to investigate the whole cell protein patterns for characterizing and differentiating E. coli isolates from meat samples
A total number of 74 pork meat samples were collected equally from slaughter yards and meat stalls. After collection, bacteriological analysis of the samples was performed to asses the selected microbial attributes in goat meat cuts of different sources by using MacConkey (MC) agar or nutrient agar medium to find out the sanitary quality and identification of bacterial strains in goat meat. Bacteria were cultured on these media in aerobic conditions at 37°C for 24h22 and colonies were identified by standard biochemical tests.
Whole cell protein analysis
The total protein samples were extracted as described by the Kishore et al.23 (1996). Total protein analysis was carried out by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described previously. Each run included marker proteins of known molecular weight (Fermentas). The gel was stained overnight with Coomassie Brilliant Blue G-250 according to Bushuk24 and Demiralp25.
Out of 74 samples 46 (60.3%) were detected as positive by disc diffusion method, 60.9% of isolates were resistant to tetracycline, ampicillin (45.66%), chloramphenicol (32.61%) and streptomycin (39.1%) respectively. However, 39.1%, 28.26%, 28.26% for tetracycline, ampicillin, chloramphenicol and streptomycin respectively. notwithstanding, 26.08%, 23.91%, 32.61% were sensitive to tetracycline, ampicillin, chloramphenicol and streptomycin respectively (table.1). The highest prevalence of antimicrobial resistant strains in the study area shows the high concern for the community health. Environmental sampling would be helpful in this regard, to check whether it has any impact on the persistence and dissemination of resistant strains. High prevalence of resistant strains indicating the detrimental and devastating effects of excess usage of antimicrobials.
Table (1):
Prevalence of antimicrobial resistance in E. coli isolates of pork meat.
Meat sample |
Tetracycline (%) |
Ampicillin (%) |
Chloramphenicol (%) |
Streptomycin (%) |
|---|---|---|---|---|
Pork (n=46) |
||||
Sensitive |
0 |
12(26.08) |
11(23.91) |
15(32.61) |
Resistance |
28(60.9) |
21(45.66) |
15(32.61) |
18(39.1) |
Intermediate |
18(39.1) |
13(28.26) |
20(43.47) |
13(28.26) |
Fig. 1: Extraction of whole cell protein of E. coli resistant strains by PAGE.Whole-cell protein profiles of meat E. coli
Lane: 1: SDS-PAGE protein profiles of E. coli isolates
from fresh pork meat sample.
Lane: 2: SDS-PAGE protein profiles of E. coli isolates
from frozen pork meat sample.
Lane: 3: SDS-PAGE protein profiles of E. coli isolates
from pork meat sample.
Lane: M: 1Kb DNA marker
Whole-cell protein profiles of meat E. coli isolates obtained by SDS-PAGE were inspected visually and compared among samples. The protein profiles of all E. coli isolates exhibited different banding patterns. The genetic distance of the strains based on whole protein profiles of E. coli isolates was detected. SDS-PAGE is used in studies to discriminate the bacterial strains. Plasmid analysis is also used method for differentiation among some bacterial strains26, 27. This study showed that antibiotic susceptibility, plasmid DNA and SDS-PAGE analysis of whole cell proteins have a discriminatory power to distinguish the E. coli strain. E. coli strains are routinely exposed to a wide range of antimicrobial agents. E. coli also has a very wide natural distribution28, 29 and a propensity for plasmid carriage29. Resistance to various antibiotics is relatively common in clinical pathogens in Turkey and also common in E. coli strains30, 31, 32 and it is frequently plasmid-mediated33.
The high prevalence of resistance to the various class of antimicrobials in the clinical and animal origin indicates further investigations to confirm. However, it is impossible to mimic epidemiologic association between the observed high resistance to the antimicrobials. This may depend on frequency of utilization of these antimicrobials in the study area, and it demonstrate that antimicrobials could be the most commonly used in Thailand to treat human and livestock animals. Hence, need further studies to elucidate the devastating effects of antimicrobials.
Resistance in E. coli was observed at high prevalence from the pork samples, collected from raw meat sellers. It indicates transmission of the pathogen by the animal to human. This investigation indicates that the high prevalence of E. coli in pork meat could be due to current sanitary systems at retail shops and processing units. Further epidemiological investigations needed to initiate on pork production and processing continuum are recommended to further substantiate the findings of the study.
Plasmids were screened to determine their antibiotic resistance profiles. In this study, plasmids were screened to determine their antibiotic resistance profiles. It was observed that there was no close relation between plasmid occurrence and multiple antimicrobial resistance in isolates. This could be due to isolates has resistance without plasmids. SDS-PAGE analysis was the most efficient method for characterizing E. coli species used in this study, because these species showed differences in their electrophoretic protein patterns.
- Havelaar AH, et al. World Health Organization global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Med. 2015; 12(12): e1001923.
- Ferens WA, Hovde CJ. Escherichia coli O157:H7: animal reservoir and sources of human infection. Foodborne Pathog Dis. 2011; 8(4):465–87.
- Smith JL, Fratamico PM, Gunther NWT. Shiga toxin-producing Escherichia coli. Adv Appl Microbiol. 2014; 86:145–97.
- Croxen MA, et al. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013; 26(4):822–80.
- Arthur TM, et al. Super shedding of Escherichia coli O157:H7 by cattle and the impact on beef carcass contamination. Meat Sci. 2010; 86(1):32–7.
- Brichta-Harhay DM, et al. Salmonella and Escherichia coli O157:H7 contamination on hides and carcasses of cull cattle presented for slaughter in the United States: an evaluation of prevalence and bacterial loads by immunomagnetic separation and direct plating methods. Appl Environ Microbiol. 2008; 74(20):6289–97.
- Elder RO, et al. Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing. Proc Natl Acad Sci U S A. 2000; 97(7): pp 2999–3003.
- Arthur TM, et al. Evaluation of a direct-fed microbial product effect on the prevalence and load of Escherichia coli O157:H7 in feedlot cattle. J Food Prot. 2010; 73(2):366–71.
- Smith DR. Cattle production systems: ecology of existing and emerging Escherichia coli types related to foodborne illness. Annu Rev Anim Biosci. 2014; 2:445–68.
- Koohmaraie M, et al. Post-harvest interventions to reduce/eliminate pathogens in beef. Meat Sci. 2005, 71(1):79–91.
- Kudva, I. T., P. G. Hatfield, and C. J. Hovde. Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep. J. Clin. Microbiol. 1997; 35:892-899.
- Shere, J. A., K. J. Bartlett, and C. W. Kaspar. Longitudinal study of Escherichia coli O157:H7 dissemination on four dairy farms in Wisconsin. Appl. Environ. Microbiol. 1998; 64:1390–1399.
- Birch, M., D. W. Denning, and D. Law. Rapid genotyping of Escherichia coli O157 isolates by random amplification of polymorphic DNA. Eur.J. Clin. Microbiol. Infect. Dis. 1996; 15:297–302.
- Pacheco, A. B. F., B. E. C. Guth, K. C. C. Sores, D. F. de Almeida, andL. C. S. Ferreira. Clonal relationships among Escherichia coli serogroupO6 isolates based on RAPD. FEMS Microbiol. Lett. 1971; 48:255–260.
- Wang, G, T. S. Whittam, C. M. Berg, and D. E. Berg. RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains. Nucleic Acids Res. 1993; 21: 5930–5933.
- Johanson L, Underdal B, Grosland K, Whelehan OP and Roberts TA. A survey of the hygienic quality of beef and pork carcasses in Norway. Acta Veterinaria Scandinavica 1983; 24: 11-13.
- Rahman MM. Fundamentals of Meat Hygiene. ‘Bismillah’ Farming and Frozen Meat Ltd. Dhaka, Bangladesh. 2000; 76-101.
- Waschmut IK, Griffin PM and Wells JG. Escherichia coli O157:H7, a cause of hemorrhagic colitis and hemolytic uremic syndrome. Acta Paediatr Jpn. 1991; 33: 603-612.
- Dorn CR, Silapanuntakul R, Angrick EJ and ShipmanLD. Plasmid analysis and epidemiology of Salmonella enteritis’s infection in three commercial layer flocks. Avian Dis. 1992; 36: 844-851.
- Schaberg DR, Tompkins LS and Falkow S. Use of agarose gel electrophoresis of plasmid deoxyribonucleic acid to fingerprint gram-negative bacilli. J Clin Microbiol. 1981; 13: 1105-1110.
- Fornasini M, Reeves RR, Murray BE, Morrow AL andPickering LK. Trimethoprim-resistant Escherichia coli in households of children attending day care centers. J Infect Dis. 1992; 166: 326-330.
- Forbes BA, Sham DF, Weissfeld AS, Trevino Enterobacteriaceae. Baily and Scott’s Diagnostic Microbiology. Mosby New York, 1998; 509-526.
- Kishore L, Natarajan K and Babu LR. Total soluble protein and membrane lipopolysaccharide profiles in differentiating Rhizobium isolates. Microbios. 1996; 86: 143-156.
- Bushuk W, Hay RL, Larsen NG, Sara RG, Simmons LD and Sutton KH. Effect of mechanical dough development on the extractability of wheat storage proteins from bread dough. Cereal Chem. 1977; 74: 389-395.
- Demiralp H, Çelik S and Köksel H. Effects of oxidizing agents and defatting on the electrophoretic patterns of flour proteins during dough mixing. EurFood Res Technol. 2000; 211: 322-325.
- Tanner ACR, Listgarten MA and Ebersole JL. Bacteroides forsythus sp. nov., a slow-growing, fusiform Bacteroides sp. from the human oral cavity. Int J Syst Bacteriol. 1986; 36: 213-221.
- Wallia TM, Williamson T, Kaiser A and Tewari R. Usefulness of protein patterns and epidemiology of Salmonella enteritis infection in three commercial layer flocks. Eur J Clin Microbiol Infect Dis. 1988; 7: 248-255.
- Selander RK and Levin BR. Genetic diversity and structure in Escherichia coli. Science. 1980; 210: 545–547.
- Sherley M, Gordon DM and Collignon PJ. Species differences in plasmid carriage in the Enterobacteriaceae. Plasmid. 2000; 349: 79–85.
- Elçi S, Özerden AN and Gül K. ‹drar örneklerinden izole edilen E. coli sufllar›n›n baz› kinolonlaraduyarl›l›klar›. ANKEM Derg. 1998; 12: 86-90.
- Tekerekolu MS, Durmaz B, Sönmez E, Korolu M andfiahin K. Uriner sistem infeksiyonlar›n›n tedavisindekullan›lan antibiyotiklere in vitro dirençdurumu. Infeksiyon Derg. 1998; 12: 375.
- Ozden M, Kalkan A, Demirda K, and Ozdarendeli. A ciprofloxacin and cotrimoksazole resistance and extended spectrum beta-lactamaseproduction in E. coli strains isolated from urinarytract infections. Int J Antimicrob Agents. 2003; 21: 492-493.
- Neu HC. The crisis in antibiotic resistance. Science.257: 1064–1072, 1992.
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