Bovine mastitis, a major disease in dairy farms, causes significant economic losses due to cow culling, milk discard and treatment costs (Shaheen et al., Reference Shaheen, Ha and Su2015). While developed countries have reduced their prevalence to below 10% through technological advancements (Francoz et al., Reference Francoz, Bergeron, Nadeau and Beauchamp2012), developing nations like Brazil, Colombia and China report rates ranging from 34.4% to 92% (Keefe et al., Reference Keefe, Marquez, Ceballos-Marquez, Jaramillo M and Toro2010; Bi et al., Reference Bi, Wang, Qin, Vallverdú, García, Sun, Li and Cao2016; Carvalho-Castro et al., Reference Carvalho-Castro, Silva, Paiva, Custódio, Moreira, Mian, Prado, Chalfun-Junior and Costa2017). Streptococcus agalactiae is a primary pathogen, necessitating stringent sanitary and disinfection measures for prevention. Although antibiotics are the main treatment, their overuse has led to antimicrobial resistance (AMR), a global concern (Cheng et al., Reference Cheng, Qu, Barkema, Nobrega, Gao, Liu, De Buck, Kastelic, Sun and Han2019). Monitoring AMR is critical for guiding treatment and drug selection (Kaczorek et al., Reference Kaczorek, Małaczewska, Wójcik, Rękawek and Siwicki2017), especially as S. agalactiae poses zoonotic and foodborne transmission risks (Botelho et al., Reference Botelho, Ferreira, Fracalanzza, Teixeira and Pinto2018). Shandong, Hebei and Inner Mongolia, key dairy-producing regions in China, contribute over 2 million tons of milk annually (Lin et al., Reference Lin, Chen and Liu2019). This study analyses AMR profiles of S. agalactiae isolates from these regions, hypothesizing regional resistance differences due to varying antibiotic usage practices, to improve dairy production and mastitis treatment.
Materials and methods
Isolating S. agalactiae in clinical bovine mastitis
Clinical mastitis was identified by redness, swelling and abnormal milk changes. Samples were collected after cleaning the udder and discarding initial milk, with pathogens isolated within 24 h. A total of 128 S. agalactiae strains (83 from Shandong, 26 from Hebei and 19 from Inner Mongolia) were stored for early mastitis detection, as shown in Supplementary Table 1.
Broth microdilution assay
Twelve antibiotics used in the field of human medicine and veterinary medicine were selected in this micro broth dilution test, including penicillin, cefalexin, ceftiofur, cefquinome, oxacillin, clindamycin, tetracycline, enrofloxacin, amoxicillin, daptomycin, erythromycin and vancomycin. The criteria were based on CLSI (Clinical Laboratory Standards Institute, 2020, 30th edition), with Streptococcus pneumonia (ATCC 49619) and S. agalactiae (ATCC 13813) as quality control strains.
Statistical analysis
Data analysis and statistical software were Excel 2019 and SPSS 26.0. Fisher's exact test was used to analyse the difference in drug resistance between different provinces and farms. The difference in minimum inhibitory concentrations (MIC) between provinces was tested by an independent sample Kruskal–Wallis test.
Results and discussion
All isolates were sensitive to penicillin, ceftiofur, amoxicillin, cefquinome and vancomycin. High sensitivity was also noted for cefalexin (98.4%), enrofloxacin (98.4%), oxacillin (96.1%), erythromycin (91.4%) and clindamycin (89.3%). However, only 19.3% and 0.7% of isolates were sensitive to tetracycline and daptomycin, respectively (Supplementary Table 2). These findings indicate that while most antibiotics remain effective against S. agalactiae, significant resistance to tetracycline and daptomycin poses a challenge, particularly in regions where these antibiotics are heavily used. Daptomycin, although rarely used in veterinary medicine, exhibited a high level of resistance in this study. This resistance may be mediated through horizontal gene transfer, although the precise mechanisms require additional study. Tetracycline resistance, linked to its widespread use as a growth promoter, is mediated by ribosomal protection genes (tetM, tetO and tetS) and efflux pump genes (tetL and tetK) (Gao et al., Reference Gao, Yu, Luo, He, Hou, Zhang, Li, Su and Han2012; Kaczorek et al., Reference Kaczorek, Małaczewska, Wójcik, Rękawek and Siwicki2017; Tomazi et al., Reference Tomazi, de Souza Filho Af, Heinemann and Dos Santos2018).
Fourteen strains (10.9%) exhibited multidrug resistance, with the most common resistance pattern being clindamycin–tetracycline–daptomycin–erythromycin (7%, n = 9). Inner Mongolia had the highest multidrug resistance rate (42.1%, 8/19), while no multidrug resistance was observed in herds J (Shandong) and K (Hebei) (Supplementary Table 3). The higher prevalence of multidrug resistance in Inner Mongolia may reflect regional differences in antibiotic usage practices, particularly the overuse of certain antibiotics in livestock management. Due to the absence of direct evidence (e.g. veterinary prescription records and antimicrobial sales data) and the limited sample size, these findings remain speculative. Further studies are warranted to elucidate the underlying mechanisms. Resistance genes for lincosamides, often located on plasmids, require continuous monitoring to prevent their spread (Bozdogan et al., Reference Bozdogan, Berrezouga, Kuo, Yurek, Farley, Stockman and Leclercq1999). The low overall prevalence of multidrug resistance (10.9%) may be attributed to sampling from large-scale farms with standardized practices (Tian et al., Reference Tian, Zheng, Han, Ho, Wang, Wang, Wang, Li, Liu and Yu2019), which typically have stricter antibiotic use protocols compared to smaller, less regulated farms.
Inner Mongolia isolates showed significantly higher resistance to oxacillin, clindamycin and erythromycin compared to Shandong (P < 0.05). In contrast, Shandong isolates were more resistant to tetracycline than those from Hebei and Inner Mongolia (P < 0.01). Inner Mongolia also exhibited higher tetracycline resistance than Hebei (P < 0.01) (Table 1). Additionally, MIC values for most antibiotics varied significantly among provinces, with Inner Mongolia consistently showing higher values, indicating greater resistance (Supplementary Table 4). These regional variations in resistance patterns and MIC values likely reflect differences in antibiotic usage and management practices across provinces. The higher resistance levels in Inner Mongolia may be linked to the region's intensive livestock farming practices and less stringent antibiotic stewardship compared to Shandong and Hebei (Tian et al., Reference Tian, Zheng, Han, Ho, Wang, Wang, Wang, Li, Liu and Yu2019; Deng et al., Reference Deng, Liu, Wang, Yang, Xu, Wang, Dai, Xiao, Gu, Zhang and Qu2023). These findings underscore the urgent need for rational antibiotic use and standardized practices to curb the evolution of resistance and ensure public health safety.
Antimicrobial resistance differences among isolates distributed in different provincesa
Table 1 Long description
The table reports counts of sensitive and resistant isolates for seven antibiotics across three provinces, with sample sizes of 83 in Shandong, 26 in Hebei, and 19 in Inner Mongolia. Cefalexin shows near-universal sensitivity in all provinces, with only one resistant isolate in Shandong and none in Hebei or Inner Mongolia. Oxacillin and clindamycin resistance differs by province: Shandong has no oxacillin resistance and very low clindamycin resistance, while Inner Mongolia has the most resistance for both. Tetracycline resistance is extremely high in Shandong, low in Hebei, and intermediate in Inner Mongolia, and these provincial differences are marked as statistically meaningful. Enrofloxacin remains mostly sensitive in all provinces, with small numbers of resistant isolates and no clear evidence of a provincial difference. Daptomycin is largely resistant in Shandong but fully sensitive in Hebei and Inner Mongolia, yet the overall test does not indicate a clear difference by province. Erythromycin resistance varies by province, with Inner Mongolia highest and Shandong lowest, and the overall comparison indicates a statistically meaningful difference; letter markers indicate which provinces differ from each other.
S: sensitive; R: resistance.
a A–C means within three major provinces with different superscript differ (P < 0.05).
b Bold font means a statistically significant difference (P < 0.05).
Bovine mastitis, affecting one-third of 220 million dairy cows globally, incurs significant economic losses, with incidence rates ranging from 46% to 70% (Gussmann et al., Reference Gussmann, Denwood, Kirkeby, Farre and Halasa2019). Streptococcus agalactiae is a key pathogen in bovine mastitis, and the overuse of antibiotics has exacerbated AMR, posing public health risks (Yang et al., Reference Yang, Liu, Ding, Yi, Ma, Fan and Lu2017; Awandkar et al., Reference Awandkar, Kulkarni and Khode2021). The findings of this study highlight the importance of region-specific interventions to address antibiotic overuse and promote standardized practices. Rational antibiotic use is crucial for mitigating the spread of AMR and safeguarding both animal and public health. The higher resistance and elevated MIC values observed in Inner Mongolia isolates emphasize the need for targeted efforts to improve antibiotic stewardship in regions with intensive livestock farming.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029925101283.
Acknowledgements
This study was financially supported by Yunnan Fundamental Research Projects (grant no. 202301AS070081) and Rural Vitalization Science and Technology Project – Gejiu Cattle Industry Science and Technology Mission in Yunnan Province (grant no. 202304BI090007).
