Study On Faecal Microbiota Resistant Gene And Antibiotics Resistance Of ESBLs-producing Escherichia Coli Chicken Origin

Antibiotic resistance genes (ARGs) are emerging contaminants, posing a potentialworldwide human health risk. Animal husbandry is believed to be a major contributorto the increased environmental burden of ARGs. The abuse of antibiotics in animalhusbandry in China has led to the increase of drug-resistant bacteria. However,background data regarding the prevalence of farm related ARGs is still lacking.1. Metagenomic profiling of antibiotic resistance genes in gut microbiota of chickenThe objective of this study was to use Illumina Hiseq2000highthroughputsequencing to investigate the occurrence, diversity and abundance of antibioticresistance genes (ARGs) in gut microbiota of layer and broiler. Metagenomic analysisshowed that Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria dominatedin the gut microbiota of chicken. At genus level, Escherichia coli were predominant inthe chicken feaces and has relatively abundant plasmids. Genes coding for antibioticresistance were identified in both communities. BLAST analysis against AntibioticResistance Genes Database (ARDB) further revealed that layer and broilerfeaces contains195ARGs, covering the current known main antibiotictypes. Sequencing reads from layer and broiler feaces revealed differences in theabundance of ARGs. ARGs types from layer feaces was significantly higher than thatfrom broile rfeaces. Compared with broiler feaces, vancomycin resistance genes (van)were relatively richer in layer feaces. β-lactam resistance genes were highly rich in thechicken feaces, among which bl2e_cbla had the highest abundance in broilerfeaces, but was absent in layer feaces.2. Antibiotic susceptibility testing of E. coli isolated from chickenFood animals colonized with extended-spectrum β-lactamase (ESBLs)-producing E.coli have been considered potential sources of resistant E. coli, causing infection inthe community. This study collected195ESBLs-producing E. coli isolates from20chicken farms and3live bird markets located in Heilongjiang, Liaoning, Jilin, and Jiangsu provinces from February2011to October2013. Susceptibilities to antibiotics,including cefazolin, cefotaxime, ceftazidime, cefepime, aztreonam, ampicillin,piperacillin, amoxicillin-clavulanate, ampicillin-sulbactam, imipenem, meropenem,amikacin, gentamycin, trimethoprim-sulfamethoxazole, chloramphenicol,levofloxacin, and tetracycline were reported according to data obtained from the BDPhoenixTM-100System. ESBL producers were initially screened by the BDPhoenixTM-100System, and were further characterized by a phenotypic confirmatorytest using both cefotaxime and ceftazidime, alone and in combination with clavulanicacid according to CLSI recommendations. Antimicrobial susceptibility tests showedthat195ESBLs-producing E. coli isolates were resistant to ampicillin, piperacillin,and cefazolin, while97.9,80.0,56.4,34.3,13.8,4.1, and1.5%of isolates showedresistance to cefotaxime, cefepime, aztreonam, ampicillin-sulbactam, ceftazidime,amoxicillin-clavulanate, and piperacillin-tazobactam, respectively. It is worth notingthat some of these isolates were also resistant to other classes of antibiotics, especiallytetracycline (92.3%), trimethoprim-sulfamethoxazole (89.7%), ciprofloxacin (80.5%),chloramphenicol (76.9%), levofloxacin (72.8%), gentamicin (57.2%), and amikacin(11.7%). However,195isolates were susceptible to imipenem and meropenem. Inaddition,96.9%(189/195) of the ESBLs-producing E. coli isolates expressed a MDRphenotype.3. Detection of β-lactamase genesβ-lactamase genes, including blaTEM, blaCTX-M, blaSHV, blaOXA, blaMOX, blaCIT,blaDHA, blaACC, blaEBCand blaFOXwere detected and characterized, and thesusceptibilities of these strains to various antimicrobial agents were determined. Onehundred and eighty-nine (189/198,96.9%) isolates showed resistance to at least threedifferent classes of agent. One hundred and ninety-one of these isolates carried one ormore bla genes. blaCTX-M, blaTEM-1, blaOXA, blaSHV-5, and blaFOXwere identified in183,121,47,2, and1isolates, respectively. This is the first study to report the blaSHV-5gene in E. coli isolated from chickens in China.The most common blaCTX-Mgeneswere blaCTX-M-15(68strains), blaCTX-M-65(41strains), blaCTX-M-55(35strains),blaCTX-M-14(32strains), followed by blaCTX-M-3, blaCTX-M-13, blaCTX-M-79, andblaCTX-M-101, as well as the chimeric genes blaCTX-M-64, blaCTX-M-123, and blaCTX-M-132. When categorized by region, the most common blaCTX-Mtype was blaCTX-M-15(66/145strains) in Northeast China and blaCTX-M-55(25/50strains) in Jiangsu. Fifteenstrains (7.7%) co-harboring CTX-M-1group and CTX-M-9group genes weredetected in195ESBLs-producing strains.Analysis of the genetic structure up-and downstream of the blaCTX-Mtype genesshowed the ISEcp1-like elements were located upstream of162(81.8%) of198blaCTX-Mgenes. ISEcp1was observed42bp upstream of the start codon of CTX-M-9group genes, whereas the spacer region between the right inverted repeats andCTX-M-1group genes varied from45bp to127bp. Orf477and IS903were detecteddownstream of most CTX-M-1group (90.3%,112/124) and CTX-M-9group genes(75.7%,56/74). ISs might play important roles in acquisition and mobility of blaCTX-Mamong the bacterial species.4. Conjugation and plasmid analysisConjugation experiments were conducted with blaCTX-M-producing isolates by thefilter method, using an azide-resistant mutant of E. coli J53as the recipient strain.Transconjugants were selected on tryptose soya agar containing cefotaxime (4μg/ml)and sodium azide (200μg/ml). The results indicated that the blaCTX-Mgene from37blaCTX-M-producing strains could be transferred to recipient strains, suggesting thatmost of the blaCTX-M-type genes (CTX-M-1and CTX-M-9) were likely to be locatedon conjugative plasmids. In this study, chloramphenicol and tetracycline resistancewere co-transferred with CTX-M-1subgroup genes, while gentamicin, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline resistance were co-transferredwith CTX-M-9subgroup genes, suggesting that the corresponding resistancemechanisms were transferable and located close to the CTX-M-1and CTX-M-9subgroup genes. However, compared with the CTX-M-1subgroup producers, theCTX-M-9subgroup producers were more frequently resistant to these non-β-lactamantimicrobials. blaCTX-Mgenes are carried by many kinds of transferable anduntypeable plasmids.5. Molecular epidemiology typingOne hundred and ninety-five ESBL-producing E. coli were divided into differentphylogenetic groups, as described in the materials and methods section. Group A was dominant (35.4%,69/195), followed by group D (34.9%,68/195), group B2(15.4%,30/195), group B1(10.8%,21/195), and the not typeable group (3.5%,7/195). It isworth noting that in this study, the phylogenetic group analysis showed that group Awas dominant among the E. coli isolates from faeces, while group D was dominantamong the strains isolated from liver or meat. The chromosomal DNA of45isolateswas available for PFGE typing and the isolates displayed34different PFGE profiles.A majority of the E. coli isolates showed unique PFGE profiles and, as a result, areunlikely to be the cause of an epidemic. Among45E. coli isolates studied,28different multilocus sequence types (ST) were identified. Although ST131was notdetected, we found6isolates belong to ST117, of which5strains belong to group Dand1strain belong to B2group. These multi-drug resistant E. coli strains, which alsobelong to pathogenic bacteria, can cause more damage to animal production andpublic health.