Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (17): 3780-3788.doi: 10.3864/j.issn.0578-1752.2021.17.018

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Analysis of Plasmid-Mediated AmpC β-lactamases Gene and Plasmid in Poultry Proteus mirabilis Strains

ZHAO ShiYu1(),JIAO JiaJie1,DONG NingNing1,PAN YuanYue2,CUI MengMei1,PAN YuShan1()   

  1. 1College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002
    2College of Life Sciences, Henan Agricultural University, Zhengzhou 450002
  • Received:2020-08-28 Accepted:2021-01-25 Online:2021-09-01 Published:2021-09-09
  • Contact: YuShan PAN E-mail:zsmzhao@163.com;pylearn21@163.com

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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 blaCMY-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 blaCMY-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 blaCMY-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 blaCMY-2 gene was successfully transferred from P. mirabilis C12 to E. coli C600 recipient strain, however, conjugation experiments failed to obtain transconjugants for other blaCMY-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 blaCMY-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-blaCMY-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 blaCMY-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 blaCMY-2 gene in poultry Proteus mirabilis isolates. IncC plasmid was one of the predominant vehicles for the dissemination of multiple resistance genes, such as blaCMY-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, blaCMY-2, Tn1696 transposon

Table 1

Primers for AmpC β-lactamases genes"

引物名称 Primer name 引物方向（5′→3′） Primer sequence (5′→3′) 产物长度 Size (bp) 参考文献 Reference
MOX-F GCTGCTCAAGGAGCACAGGAT 520 [16]
MOX-R CACATTGACATAGGTGTGGTGC [16]
CIT-F TGGCCAGAACTGACAGGCAAA 462 [16]
CIT-R TTTCTCCTGAACGTGGCTGGC [16]
DHA-F AACTTTCACAGGTGTGCTGGGT 405 [16]
DHA-R CCGTACGCATACTGGCTTTGC [16]
ACC-F AACAGCCTCAGCAGCCGGTTA 346 [16]
ACC-R TTCGCCGCAATCATCCCTAGC [16]
EBC-F TCGGTAAAGCCGATGTTGCGG 302 [16]
EBC-R CTTCCACTGCGGCTGCCAGTT [16]
FOX-F AACATGGGGTATCAGGGAGATG 190 [16]
FOX-R CAAAGCGCGTAACCGGATTGG [16]
blaCMY-2-F ATGATGAAAAAATCGTTATGC 1146 本研究 This study
blaCMY-2-R TTATTGCAGCTTTTCAAGAATG 本研究 This study

Table 2

MICs of twelve antimicrobials against six blaCMY-2-carrying Proteus mirabilis strains and the transconjugant TC12 (µg·mL-1)"

抗菌药物
Antimicrobial agents
MICs (µg·mL-1)		 菌株 Strains
CY12 CY32 WS2 S31 S52 C12 TC12 E. coli C600 E. coli ATCC 25922
氨苄西林Ampicillin >256 >256 >256 >256 >256 >256 256 4 2
头孢他啶Ceftazidime 8 8 4 4 4 2 2 <0.125 <0.125
头孢噻肟Cefotaxime 4 1 2 2 2 4 4 <0.125 <0.125
头孢西丁Cefoxitin 32 32 64 32 32 32 32 2 2
环丙沙星Ciprofloxacin 4 4 1 8 8 4 <0.125 <0.125 <0.125
恩诺沙星Enrofloxacin 16 16 4 32 32 32 <0.125 <0.125 <0.125
卡那霉素Kanamycin 256 256 2 >256 128 128 64 4 4
阿米卡星Amikacin 4 2 0.5 8 4 1 1 <0.125 <0.125
庆大霉素Gentamycin 8 16 0.25 2 2 64 32 <0.125 <0.125
氟苯尼考Florfenicol >256 256 8 128 128 64 128 2 0.5
多西环素Doxycycline 128 64 128 64 64 64 64 0.25 <0.125
粘菌素Colistin >256 >256 >256 >256 >256 >256 <0.125 <0.125 <0.125

Fig. 1

Pulsed-Field Gel Electrophoresis of six Proteus mirabilis strains carrying blaCMY-2"

Fig. 2

Sequence comparisons of pC12 and other blaCMY-positive IncC plasmids pAR060302 (FJ621588), pSN254 (CP000604), pCVM22425 (CP009560), pSD_174 (JF267651), pUMNK88 (HQ023862), IncA/C-LS6 (JX442976) from P. mirabilis, E. coli, S. enterica, and K. pneumoniae Red arrows represent genes coding for antibiotic resistance or mercury resistance; yellow arrows represent genes coding for insertion sequence elements or transposase; cyan arrows represent genes coding for conjugal transfer; orange arrows represent genes coding for maintenance and stability; gray arrows represent genes coding for hypothetical proteins. Homologous segments generated by a BLASTn comparison (≥94% identity of nucleotide sequence) are shown as grey boxes"

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