Abstract:

【Objective】 The aim of this study was to probe the genotype of AmpC β-lactamases gene and the complete nucleotide sequence of the conjugative plasmid carrying bla_CMY-2 in poultry P. mirabilis strains, so as to provide a theoretical basis for the prevent spreading of multidrug-resistant poultry P. mirabilis strains. 【Method】 Twenty-one P. mirabilis strains were characterized for the confirmation of AmpC β-lactamases genes by using three-dimensional test, polymerase chain reaction (PCR) amplification and sequencing. The bla_CMY-2-carrying P. mirabilis strains were further evaluated using pulse field gel electrophoresis (PFGE) and conjugation experiments. The complete nucleotide sequence of conjugative plasmid pC12 was determined by using high-throughput sequencing platform and compared with closely related plasmids. 【Result】 Six of twenty-one P. mirabilis strains produced AmpC enzymes, all of which carried the bla_CMY-2 gene and the detection rate was 28.6%. Antimicrobial susceptibility testing showed that six P. mirabilis strains exhibited high resistance to ampicillin, cefoxitin, doxycycline, florfenicol and colistin, but were susceptible to ceftazidime and amikacin. Conjugation assay revealed the bla_CMY-2 gene was successfully transferred from P. mirabilis C12 to E. coli C600 recipient strain, however, conjugation experiments failed to obtain transconjugants for other bla_CMY-2-bearing strains, despite repeated attempts. Three PFGE patterns of six P. mirabilis strains were determined. The findings demonstrated the vertical and horizontal dissemination of bla_CMY-2 gene in poultry P. mirabilis isolates. Sequence analysis revealed the P. mirabilis C12 harbored a conjugative plasmid, designated as pC12. pC12 was found to be a multi-drug resistant type 1b IncC plasmid with 161 319-bp size and an average GC content of 52.45%, and had at least 161 predicted open reading frames. The complete sequence of pC12 has been submitted to GenBank with the accession number MT320534. The pC12 harbored three antibiotic resistance regions: the first region, antibiotic resistance island ARI-B, carried floR, tet(A), strA, strB, and sul2 genes; the second region, ISEcp1-bla_CMY-2-blc-sugE, was a typical structure, and the ISEcp1 was truncated by IS10R; the third region, ARI-A, was a hybrid Tn1696tnp-pDUmer module. The ARI-A contained a sul1-containing class 1 integron with cassette array (aac(6')-Ib-cr|arr3|dfrA27|aadA16), and a mercury resistance cluster merEDBAPTR, and inserted into the plasmid backbone generating 5-bp direct repeats (TTGTA). 【Conclusion】 All the AmpC-producing P. mirabilis strains carried the bla_CMY-2 gene, and one of them harbored an epidemic type 1 IncC conjugative plasmid. Three PFGE patterns were identified. The findings demonstrated the vertical and horizontal dissemination of bla_CMY-2 gene in poultry Proteus mirabilis isolates. IncC plasmid was one of the predominant vehicles for the dissemination of multiple resistance genes, such as bla_CMY-2, tet (A), floR or class 1 integron cassette, which further increased the difficulty for the treatment of the infection caused by P. mirabilis. More attention should be paid on the epidemiology of IncC plasmid in pathogenic bacteria.

Key words: Proteus mirabilis, IncC plasmid, AmpC β-lactamases, bla_CMY-2, Tn1696 transposon