In vitro immune responses of bovine mammary epithelial cells induced by Escherichia coli, with multidrug resistant extended-spectrum ß-lactamase, isolated from mastitic milk.

Bovine mastitis is an inflammatory condition of mammary glands causing huge economic losses for dairy industries. Infection with extended-spectrum ß-lactamase (ESBL)-producing sequence types (ST) 410-Escherichia coli (ESBL-ST410 E. coli) is considered a leading cause of bovine mastitis in China. However, pathogenic effects of these strains in an in vitro model, e.g. bovine mammary epithelial cells (bMECs), are unknown. Therefore, our objectives were to explore pathogenesis (adhesion and invasion, inflammation, oxidative stress and apoptosis) of ESBL-E. coli (highly prevalent in bovine mastitis) in bMECs. Non-pathogenic E. coli DH5α and a prototypical E. coli P4 were included as negative and positive controls, respectively. The bMECs were infected with our isolated ST410 strains, plus DH5α and P4, with assessment of the following end points: adhesive and invasive capabilities; lactate dehydrogenase (LDH) activities; inflammatory responses, including concentrations of interleukin-1ß (IL-1ß), IL-6, IL-10 and tumor necrosis factor-α; oxidative stress including intracellular reactive oxygen species production, malondialdehyde concentrations, activities of glutathione peroxidase and superoxide dismutase; and apoptosis. All ST410 strains had greater adhesive and invasive capabilities and increased LDH release, with varying degrees of inflammatory responses, oxidative stress and apoptosis compared to blank and DH5α groups, similar to P4-infected bMECs. In particular, ST410(4) was more likely than the other 3 isolates to adhere to and invade bMECs and increase LDH activities, cytokine release, oxidative stress and apoptosis. Thus, ST410 isolates had pathogenic manifestations of adhesive and invasive capabilities; furthermore, they induced inflammation, oxidative stress and apoptosis in bMECs. Finally, ST410(4) was the most pathogenic strain.

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

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.

Co-Occurrence of Plasmid-Mediated Colistin Resistance (mcr-1) and Extended-Spectrum ß-Lactamase Encoding Genes in Escherichia coli from Bovine Mastitic Milk in China.

Both mcr-1 phosphoethanolamine transferase enzymes and extended-spectrum ß-lactamases (ESBLs) are the main plasmid-mediated mechanisms of resistance to colistin and third-generation cephalosporins, respectively, and currently considered a major concern to humans and food animals. Prevalence of mcr-1 gene in Escherichia coli from dairy cattle has rarely been reported. Our objective was to determine prevalence and characteristics of mcr-1 carrying E. coli isolated from clinical mastitis cases in large dairy farms (>500 cows) in 16 provinces of China. A total of 249 E. coli was isolated from 2,038 mastitic milk samples. Among these isolates, 2.0% (n = 5) and 19.7% (n = 49) were colistin resistant mcr-1-positive and ESBL-producing isolates, respectively. All mcr-1-positive isolates that produced ESBLs also carried the blaCTX-M-15 gene and belonged to phylogroup-A. Most mcr-1 and blaCTX-M-15 genes were located on conjugative plasmids (IncP and IncF, respectively) that were successfully transferred to transconjugants in conjugation experiments. All mcr-1-positive E. coli isolates were multidrug resistant, exhibiting resistance to common antimicrobials. Multilocus sequence typing of these mcr-1-carrying E. coli isolates revealed four sequence types, reflecting substantial diversity. Multilocus sequence analysis detected evolutionary connection of mcr-1 carrying isolates with our recently reported ESBL-producing E. coli isolates, raising concerns regarding fast dissemination between bacteria. To our knowledge, this was the first nation-wide report describing isolates of E. coli from mastitic milk samples collected on large dairy farms in China, carrying mcr-1 and blaCTX-M-15 genes on conjugative plasmids. We concluded that dairy cattle are a potential source of mcr-1-carrying and ESBL-producing E. coli.