Molecular Characterization And Mechanism Of Dissemination Of CTX-M-Type Extended-Spectrum-β-Lactamase Genes In Multidrug-resistant Isolates Of Escherichia Coli And Proteus Mirabilis From Poultry

With the widespread use of antimicrobial agents for prevention and therapy in infected animals, the rapid dissemination of antimicrobial resistance, especially multidrug-resistance (MDR), is increasing among poultry-original Enterobacteriaceae in the worldwide. Extended-Spectrum-β-Lactamase (ESBLs)-producing E. coli have increased in prevalence and diversity through the last decade, largely due to the emergence of CTX-M beta-lactamases. CTX-M producing E. coli have been found in humans, food producing animals, companion animals, wild animals and the environment. There are many ways for bacteria to obtain multidrug-resistance, example for Conjugative transfer, transposition,IS elements and mobile DNA elements encoding site-specific gene-integration functions (integrons). The purpose of this study was to characterize the molecular characterisation and mechanism of dissemination of CTX-M genes in Multidrug-resistant isolates of Escherichiu coli (E. coli) and Proteus mirabilis(p. mirabilis) from Poultry.The Multidrug-resistant isolates were examined by isolation and identification, antimicrobial susceptibility testing, confirmation of ESBL and AmpC, resistant genes screening, plasmid analysis, conjugation experiments, and the plasmid incompatibility groups in parental strain and its transconjugants was investigated by PCR-based replicon typing. The multiplex PCR was carried to determine whether this strain were commensal (A and B1) or were associated with phylogroups exhibiting extraintestinal virulence (B2and D). Multilocus sequence typing (MLST) was used to study the genetic relatedness of a collection of isolates of E. coli. PCR mapping and gene cloning were performed to investigate the genetic environment of blaCTX-M and rmtB, respectively.1. Forty-three isolates of E. coli and twenty-one isolates of p. mirabilis were identified with Vitek-32system (BioMerieux, France), which were collected from the dead poultry from different geographic regions. The results of ESBL and AmpC confirmation showed that19isolates of E. coli produced the ESBLs,1isolate of E. coli produced the AmpCs,10isolates of p. mirabilis produced the ESBLs,6isolate of p. mirabilis produced the AmpCs. The ESBL-producing isolates of E. coli and the isolates of p. mirabilis showed different patterns of antibiotic resistance based on their resistant phenotype by antimicrobial susceptibility testing. Most of these isolates were resistant to ampicillin, ceftriaxone, cefotaxime, kanamycin. Moreover,4isolates of E. coli also exhibited a high level resistance to aminoglycosides including kanamycin, amikacin, gentamicin. In the present study, Phylogenetic analysis indicated that the4isolates of E. coli belonged to group D, the other belonged to group A. The results of MLST revealed that20isolates of E. coli belonged to11kinds(ST23, ST93, ST117, ST155, ST156, ST162, ST224, ST410, ST539, ST602, ST2847). MLST analysis showed a high diversity of the isolates.2.15transconjugants from20E. coli isolates were successfully obtained by conjugation experiments.BlaTEM-1genes were detected in16of the43E. coli isolates (37%). BlaTEM-M genes were detected in19of the43E. coli isolates (44%). The most common CTX-M types were blaCTX-M-65(n=9),blaCTX-M-14(n=5), followed by blaCTX-M-55,blaCTX-M-3,blaCTX-M-24,blaCTX-M-27.BlaOXA-1was detected in5isolates. blacny-2was detected in one isolate.qnrS1gene was detected in1of the43E. coli isolates (2.3%). BlaTEM-1genes were detected in10of the21p. mirabilis isolates (48%). blaCTX-M genes were detected in10of the21p. mirabilis isolates (48%). The most common CTX-M types were blaCTX-M-14(n=6), blaCTX-M-65(n=4). blacmy-2was detected in6isolates (29%). qnrD gene was detected in3of the21E. coli isolates (14%). BlaOXA-1was detected in6isolates, and BlaOXA-10was detected one same isolate simultaneously. BlaSHV was not detected in all isolates.3. The genetic environments of blaCTX-M genes were shown specific for the subtype of the genes.A mobile genetic element ISEcpl (in some cases truncated or inserted by IS26, IS10sequences) were detected upstream of blaCTX-M-3, blaCTX-M-55, and blaCRX-M-14,blaCTX-M-24, blaCTX-M-27, blaCTX-M-65genes. Importantly, for the first time, a new arrangement, ISEcp1Δ-IS1294-ΔISEcpl-blaCTX-M-55-ORF477was identified, in which the ISEcpl element was disrupted by another insertion sequence, IS1294. The nucleotide sequence of the genetic environment of blaCTX-M-55was deposited in GenBank under the accession no. JN977127. A special characteristic was the sequence between ISEcp1and blaCTX-M gene:48bp for blaCTX-M-55and45bp for blCTX-M-55;42bp for CTX-M-9group genes. Nucleotide sequence analysis revealed that orf477was located downstream of blaCTX-M-3and blaCTX-M-55, and insertion sequence IS903(intact or truncated) was detected downstream of CTX-M-9group gene. It was also shown that blaCTX-M genes were mostly located on conjugative plasmids belonging mainly to the incompatibility groups IncI1and IncF, while conjugative plasmids, which harboring blacmy-2, belonged IncA/C.4. The rmtB gene was detected in4of the43E. coli isolates (9%), in4of the20beta-lactamase-producing E. coli isolates(20%). It is worth noting that CTX-M-producing isolates of E. coli coproduced16S rRNA methylase, and cmy-2-producing isolate of E. coli coproduced16S rRNA methylase, and this poses a severe threat to the clinical use of β-lactam antibiotics and aminoglycosides in human and veterinary medicine. The blaCTX-M-55and rmtB genes were found to be present in the separate plasmids belong to the IncI1and IncN, respectively. These antibiotic resistant plasmids could be transferred to the recipient strain alone or together. The results also showed that the rmtB gene was carried by Tn3transposonIn conclusion, CTX-M-producing E. coli have increased in prevalence and diversity in poultry. The genetic environments of blacCTX-M genes were shown specific for the subtype of the genes.Nucleotide sequence analysis revealed that a mobile genetic element ISEcp1were detected upstream of blaCTX-M, insertion sequence IS903(intact or truncated) was detected downstream of CTX-M-9group gene, and orf477was located downstream of CTX-M-1group gene. The blaCTX-M-55and rmtB genes were detected firstly in the same strain of E. coli C21isolated from a chicken in China, and were found to be present in separate plasmids belonging to the Incl1and IncN families, respectively.Both plasmids harboring these genes were conjugative. This study also described, for the first time, a new arrangement,ISEcp1Δ-IS1294-AISEcp1-blaCTX-M-55-ORF477, in which the ISEcpl element was disrupted by another insertion sequence, the IS1294element. Conjugative plasmid, transposition and IS elements may contribute to the rapid dissemination of antibiotic resistance genes, and this is of great significance to human and veterinary medicine.