Drug Susceptibility Test and Analysis of Quinolone Resistance Genes in Mycoplasma hyopneumoniae Vaccine Strains and Field Isolates from China

Abstract

Background

Enrofloxacin is a commonly used animal-specific drug in veterinary clinics. However, this drug has no epidemiological cutoff values (ECVs/ECOFFs) for Mycoplasma hyopneumoniae in CLSI and EUCAST. Defining the epidemiological cutoff values (ECOFFs) of enrofloxacine to Mycoplasma hyopneumoniae (M. hyo) can inform an early detection of bacterial resistance to better manage the resistance prevention and also help in establishing drug resistance breakpoints;.

Methods

We determined the susceptibility breakpoint of M. hyo to enrofloxacin by the American Clinical and Laboratory Standards Institute (CLSI) standard method based on the PCR of vaccine strains and wild strains drug resistance genes;.

Results

Eighty strains of M.hyo isolated in Tibet were moderately sensitive (S) to tetracycline, florfenicol, spiramycin, erythromycin thiocyanate, tilmicosin, tiamulin, lincomycin, clindamycin, ofloxacin, enrofloxacin, gentamicin, amikacin, with MICs below 0.5 μg/mL. For vaccine 168L, RM48, and J strains, the susceptibility to the same antibacterial drugs was lower compared to the Tibetan isolates. The resistance of J strain to erythromycin thiocyanate was confirmed. Gene point mutation was confirmed in Quinolone Resistance Determining Regions (QRDR) of HNSH strain Topoisomerase IV subunit A, this finding is compared with the sequencing results of 168L strain reference sequence (Accession number: CP003131). Arg-Lys amino acid mutation (G921A and G1179A) was confirmed for the increase of MIC value involved in M.hyo to enrofloxacine;.

Conclusion

The cut-off value of M.hyo to enrofloxacin was set as 1 μg/mLby ECOFFinder XL 2010 V2.1.

Keywords:

• Mycoplasma hyopneumoniae
• drug resistance
• Tibetan pigs
• MIC
• enrofloxacin

Data Sharing Statement

The datasets generated and/or analysed during the current study are available in the GenBank repository,(https://www.ncbi.nlm.nih.gov/nuccore/CP003131.1/), (https://www.ncbi.nlm.nih.gov/nuccore/CP002274.1/).

Institutional Review Board Statement

The animal study protocol was approved by the Animal Ethics and Welfare Committee (AEWC) of Xinyang Agriculture and Forestry University (AEWC −2021011803 and January 17, 2021).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no conflict of interest.

Additional information

Funding

This research was funded by The Natural Science Foundation of Henan Province OF Department of Science and Technology of Henan Province, grant number 212300410385; the Foundation of Central Laboratory of Xinyang Agriculture and Forestry University (FCL202013, Qiu Gang), the Key Scientific Research Project of Colleges and Universities of Henan Province (21A230016, Qiu Gang), and the Key Scientific Research Project of Colleges and Universities of Henan Province (21B230009, Rui Yapei). The APC was funded by The Natural Science Foundation of Henan Province, the Foundation of Central Laboratory of Xinyang Agriculture and Forestry University, and the Key Scientific Research Project of Colleges and Universities of Henan Province.