BackgroundThe human and animal intestinal tract harbors a complex community of microbes which enables bacteria to inherit antibiotic resistance genes. The aims of this study were to investigate clonality, antimicrobial resistance, prevalence and gene cassette arrays of class I and II integrons among commensal Escherichia coli from human and animals.MethodsA total of 200 E. coli isolates from human, chicken, cattle, and sheep were isolated followed by phenotypic antibiotic susceptibility testing and detection of class I and II integrons gene cassettes arrays. The clonal relationship of the isolates were analyzed by (GTG)5-PCR.ResultsOf 200 isolates, 136 isolates were multi drug resistance (MDR) including 47, 40, 31 and 18 isolates from chicken, human, cattle and sheep, respectively. Class I integron was detected in 50, 38, 6 and 16 %, while class II was detected in 26, 8, 0 and 4 % of chicken, human, cattle and sheep isolates, respectively. Variable regions were amplified and sequenced. Cassette arrays in class I integrons were: dfrA1, dfrA5, dfrA7, dfrA12, aadA1, dfrA17 aadA1, aadA22, aadB–aadA2 and dfrA12–orfF–aadA2, and for class II, dfrA1-sat-aadA1, and sat-sat1-aadA1 were detected. Six class I and three class II positive strains did not produce any amplicons for variable region. Integron-positive isolates showed higher rate of resistance to streptomycin and trimethoprim–sulphamethoxazole, especially in chicken isolates which were fed antibiotics. Low similarity and great genetic diversity of class I and II integrons carrying isolates indicated no clonal relation.ConclusionsIntegrons encoding for antibiotic resistance are significantly present among non-pathogenic commensal E. coli, especially from the hosts medicated by antibiotics. Uncontrolled use of antibiotics will increase the numbers of multiple drug resistant isolates and integrons prevalence.
The aim of this study was to analyze the enterobacterial repetitive intergenic consensus (ERIC)-types, phylo-groups and antimicrobial resistance (AMR) patterns of Escherichia coli and to investigate if these approaches are suitable for microbial source tracking (MST). E. coli strains were isolated from cattle faeces and Karaj River. For genetic diversity, AMR profile, and phylo-grouping, we applied ERIC-PCR, disk diffusion, and multiplex-PCR, respectively. Fifty isolates from each sample group were used in the study. ERIC fingerprinting produced ten different bands, demonstrating 64 unique and 36 repetitive profiles. Six isolates from the river showed the same ERIC pattern of the cattle, of which four expressed the same AMR profile. E. coli isolates from water were represented in A, B1, C, and D phylo-groups. Phylo-groups A, B1, and E were more prevalent in the cattle isolates and B2 was absent in both sources. Three of the water isolates with the same ERIC-type and AMR to cattle isolates showed the same phylo-groups. Genetic characteristics, AMR, and phylo-groups of the isolates from the river are diverse and complex. For accurate MST, complementary approaches should be applied together and a comprehensive library should be provided.
Microbial water-borne diseases still affect developing countries and are major water quality concerns throughout the world. Routine culture-based methods of identifying bacterial pathogens in water sources are laborious and time-consuming. Recently, the use of molecular techniques such as the polymerase chain reaction (PCR) has provided rapid and highly promising detection methods. In this study, we developed two multiplex PCR assays for simultaneous detection of six water-borne bacteria. Two triplex PCR protocols were developed to detect six target genes. The first protocol targets uidA (Escherichia coli), int (Shigella spp.), and gyrB (Pseudomonas aeruginosa) genes, while invA (Salmonella spp.), ompW (Vibrio cholera), and lacZ (coliforms) were amplified by the second protocol. Specificity testing was carried out for 12 reference strains. Furthermore, the applicability of the multiplex PCR assays for detection of these bacteria was investigated for 52 surface water samples. The results indicated that all primer pairs showed specificities only for their corresponding target organisms. The detection sensitivity of both multiplex PCR assays was 3 × 102 − 3 × 103 colony forming units. The developed assays represent simple and efficient diagnostic procedures for co-detection of water-borne bacteria and have the potential to provide earlier warnings of possible public health threats and more accurate surveillance of these organisms.
Citation of This ArticleKheiri R, Akhtari L: Clonal heterogeneity and efficacy of BOX and (GTG)5 fingerprinting methods for molecular typing of Escherichia coli isolated from chickens in IRI. Kafkas Univ Vet Fak Derg, 23 (2): 219-225, 2017. DOI: 10.9775/kvfd.2016 AbstractThis study evaluates the clonal heterogeneity and efficacy of BOX-PCR and (GTG)5-PCR for DNA-based typing of Escherichia coli strains isolated from feces of chickens in IRI. Fecal samples were collected from chicken husbandry followed by E. coli isolation through biochemical tests. Isolates were finger printed by BOX-A1 and (GTG)5 primers. Dendrograms were generated based on 80% similarity and Shannon-Weaver index was calculated. One hundred and six E. coli isolates were obtained from chicken's fecal sample. By (GTG)5 primer, of 106 isolates, two isolates were untypeable, while 104 isolates generated 100 unique, and 2 duplicate profiles. The dendrogram generated six clusters (G1-G6). With BOX-PCR, 106 E. coli isolates revealed 50 unique BOX profiles, in addition to 22 repetitive profiles, while 12 isolates were untypeable. Based on the bands and dendrogram, the 106 strains were grouped into six clusters (B1-B6). Shannon-Weaver index was 4.665 for (GTG)5-PCR and 0.281 for BOX-PCR. (GTG)5-PCR revealed complex clonal heterogeneity, more discriminatory power, less untypeable isolates, higher Shannon-Weaver index, and less isolates with the same profile in comparison to BOX-PCR. Although (GTG)5-PCR proved to be a powerful typing method, it is recommended to combine two or more different typing methods for higher discriminatory power. (GTG)5-PCR; BOX-PCR ile karşılaştırıldığında kompleks klonal heterojenite, daha fazla ayırt edici güç, daha az tiplendirilemeyen izolat, daha yüksek Shannon-Weaver endeksi ve daha az aynı profilli izolat elde edilmesini sağladı. (GTG)5-PCR daha güçlü tiplendirme metodu olmasına rağmen iki veya daha fazla tiplendirme metodunun birlikte kullanılması daha yüksek ayırıcı güç için önerilmektedir. Keywords: BOX-PCR, Chicken, Clonal heterogeneity, Escherichia coli, (GTG)5-PCR, Molecular typing BOX ve (GTG) 5 Parmak izi Metotlarının İran'da Tavuklardan İzole Edilen
It is believed that high antibiotic consumption and horizontal transfer of resistance genes are two major causes of antibiotic resistance in bacteria. To confirm or reject this belief, we studied the role of drug administration in antimicrobial resistance (AMR), prevalence of Class I and II integrons and integron-mediated resistance in Escherichia coli isolated from human and animal specimens. Recording demographic data, E. coli from different specimens including human, chicken, cattle and sheep was isolated followed by phenotypic antibiotic susceptibility testing and detection of Class I and II integrase genes. The correlation between integrons and resistance (P value) was evaluated using SPSS software. According to demographic records, chickens received the highest dose and variation of antibiotics. As expected, the most prevalent MDR strains and integrons were found in chicken strains. Chi square analysis showed a significant correlation between integrons and resistance pattern mostly in E. coli strains isolated from chicken rather than other specimens. Our survey confirmed that the use of antibiotics is strongly associated with the prevalence of antimicrobial resistance and integrons in commensal E. coli. Such results confirm that high doses of antibiotics and selection pressure may remove susceptible intestinal microorganisms followed by resistant ones. Keywords: Antimicrobial resistance, Cattle, Chicken, Escherichia coli, Integrons İnsan ve Hayvan Örneklerinden İzole Edilen Escherichia coli'nin İlaç Direnci ve İntegronların Yaygınlığında Antibiyotik Rolü ÖzetYüksek antibiyotik kullanımı ve dayanıklılık genlerinin yatay transferinin bakterilerde antibiyotik direncinin iki önemli nedeni olduğuna inanılmaktadır. Bu inancı onaylamak veya reddetmek için, insan ve hayvan örneklerinden izole edilen Escherichia coli'nin antimikrobiyel direnci (AMR), Sınıf I ve II integronları ve integron-aracılı direnç prevalansında ilaç uygulamasının rolünü araştırdık. Demografik verileri kaydetmek için; fenotipik antibiyotik duyarlılık testi ve Sınıf I ve II integraz genlerinin tespiti sonrası insan, tavuk, sığır ve koyun gibi farklı örneklerden E. coli izole edildi. İntegronlar ve direnç (p değeri) arasındaki korelasyon SPSS yazılımı kullanılarak değerlendirildi. Demografik kayıtlara göre, tavuklar en yüksek doz ve değişimli antibiyotik aldı. Beklenildiği gibi, en yaygın MDR suşları ve integronları tavuk suşlarında bulundu. Ki kare analizi, başka örnekler yerine çoğunlukla tavuktan izole edilen E. coli suşlarının integronları ve direnç modeli arasında anlamlı bir korelasyon gösterdi. Araştırmamız, antibiyotik kullanımının ortakçı E. coli'de antibiyotik direnci ve integronların yaygınlığı ile sıkı ilişkili olduğunu doğruladı. Bu sonuçlar, yüksek doz antibiyotik ve seçim baskısının dirençli olanların ardından duyarlı bağırsak mikroorganizmalarını ortadan kaldırabildiğini doğrulamaktadır.
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