Molecular characteristics and antibiotic susceptibility profiles of Mycoplasma bovis associated with mastitis on dairy farms in China

Abstract

Mycoplasma bovis (M. bovis) is regarded as the most prevalent mycoplasma species causing bovine mastitis worldwide. This study was conducted with the objectives to: (1) estimate M. bovis prevalence in samples from clinical mastitis and bulk tank milk; (2) assess genetic diversity and population structure of isolates; and (3) determine antibiotic susceptibility of isolates to nine antimicrobials. Milk samples (n = 476), including 450 clinical mastitis and 26 bulk tank milk samples from 23 farms (each with >1000 lactating cows) in 10 provinces of China were collected between May 2018 and September 2019. M. bovis cultured from milk samples were analyzed by multi-locus sequence typing. Minimum inhibitory concentrations of all isolates to nine antimicrobials were determined. Differences in minimum inhibitory concentration values were assessed by Kruskal-Wallis test with Bonferroni correction. The positive proportions of M. bovis in clinical mastitis samples and bulk tank milk samples were 39/450 (8.7%) and 11/26 (42.3%), respectively. Based on multi-locus sequence typing, the 50 isolates were identified as three sequence types, including sequence type 10 and two novel sequence types (newly registered as sequence type 172 and sequence type 173). The most prevalent type, sequence type 172 (31/50, 62.0%), had allelic profile 4, 3, 2, 3, 5, 7, 4. In addition, sequence type 10 with allelic profile 4, 3, 2, 3, 5, 3, 4 had a mid-range prevalence (11/50, 22.0%), whereas sequence type 173 with allelic profile 10, 3, 6, 13, 21, 6, 10 was the least prevalent (8/50, 16.0%). Both sequence type 10 and sequence type 172 were clustered in Clonal Complex 3, with isolates from the USA. M. bovis isolates in this study uniformly had low level minimum inhibitory concentrations to enrofloxacin and tiamulin. Overall variances among isolates were significant (Kruskal-Wallis test) for clindamycin (P = 0.006), erythromycin (P = 0.012) and tylosin (P =0.004). Relative to the sequence type 10 group, there were higher minimum inhibitory concentrations levels for the sequence type 173 group (H = -19.795, P = 0.003, for clindamycin; H = -19.574, P = 0.003, for erythromycin; and H = -18.881, P = 0.003, for tylosin) by post-hoc comparisons using pairwise comparisons of mean ranks following Kruskal-Wallis test with Bonferroni correction. Hence, increasing antimicrobial resistance may have contributed to emergence of novel sequence types. These data provided a baseline for elucidating genetic diversity and antibiotic susceptibility profiles of M. bovis in the main dairy-farming provinces in China.