Antibiotic resistance of Escherichia coli and Campylobacter jejuni and multilocus sequence typing of pathogenic Escherichia coli isolated from selected orang Asli and Malay villages in Malaysia

Campylobacter jejuni and Escherichia coli are prominent bacterial causes of human gastroenteritis in the developing countries and the emergence of antibiotic resistance of these bacteria has been widely reported to be on the increase, particularly because of the increase in the number of resistant C. jejuni and E. coli isolated from human infections. Poultry, in particular chickens and wild birds are reported to be frequently infected with C. jejuni and E. coli. Wild birds or chickens harboring resistant C. jejuni and E. coli can transfer the genes to microorganisms in their gastrointestinal tracts. A number of studies in the country had shown the occurrence of C. jejuni and E. coli in chickens and chicken meat. Wild birds were observed in abundance at residential areas, market places and farms. It was reported that wild birds harbor and may transmit C. jejuni and E. coli to residential areas and farms environment. However, very few studies had been carried out on antibiotic resistance of C. jejuni and E. coli from wild birds, chickens and humans and their environment in Malaysia. Also, very few studies had been conducted to detect the pathogenic E. coli genes and no study had been conducted to detect the sequence typing (STs) of pathogenic E. coli from different sources in Malaysia. Thus, the objectives of this study were, (i) to determine the presence of C. jejuni and E. coli in wild birds, chickens, environment and human at selected villages in Perak and Kedah, (ii) to determine the antibiotic resistance profiles among C. jejuni and E. coli isolates, (iii) to detect pathogenic E. coli (EPEC, EIEC) among E. coli isolated, (iv) to characterize E. coli using Multilocus Sequence Typing (MLST) assay, and (v) to determine the interrelatedness among the pathogenic E. coli sequence types (STs) from different sources. A total of three Orang Asli villages were selected in Perak and three Malay villages in Kota Setar, Kedah. Ninety-nine (99) human stool, cloacal swabs from 113 chickens 78 from wild birds were collected and environmental samples included flies (85) water (100) and soil (88). Campylobacter jejuni was isolated from 24(21.2%) chickens and 19(22.4%) flies. A total of 92(92.9%) humans were found positive for E. coli while no human samples were positive for C. jejuni. Fifty-eight (74.4%) of the birds and 84(74.3%) of the chickens were positive for E. coli. Eight-four (74.3%) of the chickens were positive for E. coli. Out of 273 environmental samples, 220 (80.6%) showed positive for E. coli mainly isolated from flies, 77(35%) and water, 74 (33.6%). The C. jejuni and E. coli isolates were subjected to antibiotic susceptibility test using disc diffusion method. The isolates were tested against 10 antibiotics, namely ampicillin-sulfbactam, tetracycline, gentamicin, erythromycin, ciprofloxacin, nalidixic acid, enrofloxacin, sulfamethoxazole-trimethoprim, cefpodoxime, streptomycin. The C. jejuni isolates from chickens showed resistance to 1-7 antibiotics. The highest resistance was to nalidixic acid (40.9%) while all isolates were found sensitive to gentamicin. Flies isolates showed resistance to 2-7 antibiotics. The highest resistance was to cefpodoxime (65%) and isolates were sensitive to gentamicin and erythromycin. The E. coli isolates from humans showed resistance to 1-9 antibiotics and the highest resistance was to erythromycin (100%). Chicken isolates showed resistance to 2-9 antibiotics with highest resistance was to erythromycin (98.8%). Escherichia coli isolates from wild birds showed resistance to 1-8 antibiotics. Hundred percent (100%) of the wild birds isolates were found resistant to erythromycin followed by tetracycline (87.3%). Flies isolates showed resistance to 2- 9 antibiotics. The highest resistance was to erythromycin (97%) followed by tetracycline (57.5%). The isolates from water showed resistance to 3-10 antibiotics. Hundred percent (100%) of the water isolates were found resistant to erythromycin. Soil isolates showed resistance to 1-10 antibiotics. The highest resistance was to erythromycin (100%) followed by tetracycline (60.3%). The occurrence of enteropathogenic E. coli (EPEC) and enteroinvasive (EIEC) was through the detection of eaeA and bfpA, and ial genes respectively. Thirty-eight (45.2%) of the chicken isolates were positive for eaeA gene only, six (7.1%) and two (2.4%) were positive for EPEC and EIEC respectively. Seven (12.1%) of the wild bird isolates were positive for eaeA gene only. Sixteen (29.6%) of the environmental isolates were positive for eaeA gene, EPEC was detected in isolates from water (1.9%) and EIEC was detected in isolates from soil (1.9%). The typing of pathogenic E. coli isolates and interrelatedness among the isolates was done using Multilocus sequence typing (MLST) and eBURST analyses. Multilocus sequence typing (MLST) analysis revealed twenty sequence types (STs) from 42 pathogenic E. coli. Seven wild bird isolates revealed six STs namely ST 2705, ST 2491, ST 484, ST 2253, ST 453 and ST295. Thirteen STs were revealed from 19 chicken isolates namely ST 5686, ST 155, ST 10, ST 181, ST 3856, ST 1141, ST 202, ST 542, ST 295, ST 188, ST 453, ST 206 and ST 48 and one undefined ST. Three flies isolates revealed three STs namely ST 295, ST 155 and ST 181. Six water isolates revealed two STs namely ST 162, ST 69 and one undefined ST. Five STs were revealed from seven soil isolates namely ST 5686, ST 181, ST 46, ST 2419, ST 202 and two undefined STs. From this study, the overlapping of similarities shown among several of the pathogenic E. coli sequence types (STs) isolated from the wild birds and chickens with the environmental isolates in the villages could imply that these animals are main sources of the pathogenic E. coli in the villages. Also, the isolates from wild birds in several villages showed high multidrug resistance (MDR) and the overlapping of similarities shown between several of the pathogenic E. coli STs isolated from the wild birds (ST 295, ST 2491 and ST 453) with the chicken and environmental isolates in several villages could possibly showed that these birds had been the main sources of the antibiotic resistant E. coli and pathogenic E. coli. The presence of high MDR in C. jejuni and E. coli could compromise treatment in humans and in particular, if the bacteria is resistant to the drugs of choice and alternative drugs for treatment and therefore poses a significant public health risk. The chickens and flies could play a role in the dispersal and spread of or may had acquire antibiotic resistant C. jejuni in the villages environment. Thus, the presence of C. jejuni in chickens and flies, and pathogenic E. coli in wild birds, chickens and the environment (flies, water, soil) may cause health hazard to human upon exposure to the organisms.

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