Characterization and resistant determinants linked to mobile elements of ESBL-producing and mcr-1-positive Escherichia coli recovered from the chicken origin.

The spread of antimicrobial resistance (AMR) in Escherichia coli is a complex process linked with various mobile genetic elements (MGEs) like plasmids, transposons, and integrons. This study aimed to determine the co-occurrence of ESBL and mcr-1 and their physical linkage with MGEs in E. coli. E. coli strains of chicken origin were obtained from different commercial farms of eastern China from 2010 to 2011. Antimicrobial sensitivity testing, identification of different antibiotic-resistant genes (ARGs), and prevalence and evidence involvement of integrons, ISEcp1, ISCR1, and ISApl1, were determined. A multiplex PCR was used to detect virulence genes and the phylogenetic clustering of isolates. Conjugation experiments, plasmid replicon typing were performed to know the transferability of ARGs and MGEs. A total of 83.33% of isolates were found to be multidrug-resistant (MDR). The incidence rate of blaCTX-M, blaSHV,blaTEM, and mcr-1 was found to be 30%, 10.95%, 8.09%, and 36.66%, respectively. The most prevalent combination was noticed for mcr-1 and blaCTX-M 73%, whereas the most prominent blaCTX-M alleles found, were blaCTX-M-55 46%, followed by blaCTX-M-14 31%, and blaCTX-M-15 13%. The frequency of ISEcp1, ISCR1, ISApl1, and int1 was 27.77%, 53.70%, 51.85%, and 70.37% respectively. Most ß-lactamases, especially blaCTX-M, blaSHV, and blaTEM, were associated with ISEcp1, ISCR1, and Integron 1, whereas the ISAPl1-mcr-1 segment was observed in mcr-1-positive E. coli isolates. Phylogrouping revealed that group A was the most predominant phylotype, whereas the common virulence genes detected in these isolates were EHEC, EAEC, and EPEC. Conjugation assay also indicated that multiple genetic elements were involved; common plasmids identified were FIB 61.11%, followed by IncHI2 48.14%, and FrepB 33.33%. Propagation of such MDR strains carrying multiple resistance elements among the bacterial population is a threat of worry.

Sequence Duplication Within pmrB Gene Contribute to High-Level Colistin Resistance in Avian Pathogenic Escherichia coli.

Beyond the emergence of plasmid-encoded mechanisms, mutation within the pmrAB genes remains one of the primary colistin resistance mechanisms in Escherichia coli. However, the mechanisms of high-level colistin resistance (HLCR) have not been elucidated. In this study, we evaluated the HLCR mechanisms in five colistin-susceptible Avian pathogenic Escherichia coli (APEC) isolates after colistin exposure. Three PmrB substitutions (G19R, L167P, V88E) and two PmrB sequence duplication (PmrB-sd) mutations (68-77dup and 94-156dup) were detected. Chromosomal replacement and deletion mutagenesis revealed the two PmrB-sd mutations contribute to, but are not fully responsible for, HLCR in APEC strains. Quantitative reverse transcription/polymerase chain reaction (qRT-PCR) revealed that the PmrB-sd induction mutants showed an increased pmrAB transcript level and the PmrB-sd reversion mutants exhibited a reduction of pmrAB expression. All five induction mutants exhibited decreased minimum inhibitory concentrations to florfenicol and tetracycline. In addition, four mutants (G19R, L167P, V88E, and 94-156dup) and two mutants (68-77dup and 94-156dup) also displayed increased sensitivity to ceftiofur and gentamicin, respectively. Zeta potential measurement of the induction mutants showed that there was less negative charge on the cell surface compared with its parental strains in the absence of colistin. The induction mutants also showed an increase of lag time and decrease of fitness. In summary, the identification of novel PmrB-sd mutations contributing to HLCR is helpful to broaden the knowledge of colistin resistance. Attention should be paid to the use of colistin for the treatment of infections caused by APEC strains.

High incidence of multidrug-resistant Escherichia coli coharboring mcr-1 and bla CTX-M-15 recovered from pigs.

PURPOSE: The coexistence of mobile colistin (COL)-resistant gene mcr-1 with extended-spectrum beta-lactamase (ESBL) gene in Escherichia coli has become a serious threat globally. The aim of this study was to investigate the increasing resistance to COL and in particular its coexistence with ESBL-producing E. coli recovered from pig farms in China. MATERIALS AND METHODS: E. coli were isolated from 14 pig farms in Jiangsu China. Susceptibility testing was identified by micro-dilution method. PCR assay and nucleotide sequencing were used to detect COL-resistant genes, mcr-1 to -5, as well as ESBL genes, bla CTX-M, bla SHV and bla TEM. Conjugation experiment, plasmid replicon typing of the multidrug resistance (MDR), S1-PFGE and DNA southern hybridization were performed to study the transferability of these genes. RESULTS: Overall, 275 E. coli isolates were recovered from a total of 432 cloacal and nasal swabs. More than 90% of the isolates were MDR, of which 70.18% were resistant to COL. Of these 275 isolates, mcr-1 was identified as the most predominant gene carried by 71.63% (197/275) of isolates, 39.59% (78/197) of the isolates were harboring both mcr-1 and ESBL genes (bla CTX-M, bla SHV and bla TEM). ESBL genotyping showed that bla CTX-M was the most predominant ESBL (68.49%) followed by bla SHV (16.4%) and bla TEM (15%). Sequencing revealed that the most common variants of bla CTX-M identified were, bla CTX-M-15 (69%), bla CTX-M-55 (29%) and bla CTX-M-1 (1.8%). IncHI2, IncFIB, IncFIC, IncN and IncX4 were found to be the most common Inc-types found both in donors and in transconjugants and were associated with the transfer of the mcr-1 and ESBL encoding genes. Six strains carried a total of five different plasmids: approximately 97-, 130-, 160-, 227- and 242-kb plasmids. CONCLUSION: The coexistence of the mcr-1- and bla CTX-M-15-carrying isolates displaying high MDR, recovered from E. coli of pig origin, is a major concern for both humans and veterinary medicine.