Introduction: Streptococcus agalactiae is a highly contagious pathogen that causes bovine mastitis, leading to significant economic losses. This study aimed to (1) identify and characterize S. agalactiae strains responsible for bovine mastitis by examining their phenotypic and genotypic characteristics in Thai dairy-intensive farming areas and (2) determine their susceptibility profiles to antimicrobial agents.Material and methods: In total, 100 S. agalactiae isolates obtained from clinical and subclinical mastitis cases from 13 dairy herds located in the central region of Thailand were examined. To confirm the identity of the bacterial pathogens, conventional microbiological procedures recommended by the National Mastitis Council (NMC) and the VITEK^® 2 system were employed.Results: All 100 isolates were successfully identified as S. agalactiae using the NMC procedure, whereas 94 isolates were identified as S. agalactiae using the VITEK^® 2 system. Finally, the S. agalactiae-specific gene dlt S was identified in all the examined isolates using polymerase chain reaction. Capsular polysaccharide (CPS) typing revealed that all strains belonged to CPS type Ia. Multilocus sequence typing identified 33 selected isolates as sequence type 103. Random amplified polymorphic DNA (RAPD) typing yielded 43 RAPD types, with 6 RAPD clusters identified. These results demonstrated a high level of genetic diversity among S. agalactiae within the studied herds. RAPD analysis suggested that specific S. agalactiae strains could persist in dairy farms for 2–12 months. Furthermore, antimicrobial susceptibility testing was performed using the broth microdilution method. Most strains demonstrated susceptibility to ampicillin, penicillin, penicillin/novobiocin, cephalothin, oxacillin, ceftiofur, and erythromycin.Discussion: This study revealed the phenotypic and genotypic characteristics of S. agalactiae isolates responsible for bovine mastitis in the central region of Thailand. The rapid identification of S. agalactiae and application of molecular typing methods can provide valuable epidemiological information regarding S. agalactiae causing mastitis in dairy farms. The antimicrobial susceptibility of S. agalactiae indicates that antimicrobial treatment for control and eradication could be a successful protocol. Our findings revealed that a single clonal strain of S. agalactiae affected the 13 studied farms. Further research is needed to explore the feasibility of vaccine development and application.

Staphylococcus aureus and S. epidermidis are the major teat skin bacteria and lead to severe bovine mastitis. Teat antiseptic is an important tool for controlling intramammary infection. The antibacterial activity of lactic acid (LA) against one reference strain of S. aureus ATCC 25923 and two field strains including S. aureus and S. epidermidis was investigated using the broth microdilution method. Its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 0.5% for strains belonging to both species. An antiseptic preparation containing 5% LA with modified rice gel (LA-RG) was successfully prepared. Rheological behavior of LA-RG was found to be a pseudoplastic flow with thixotropy with viscosity of approximately 0.007 Pas. LA-RG exhibited a sufficient adhesive property in the rolling ball test with a length of 9.67 ± 0.04 cm. Killing kinetic studies of LA-RG showed that the killing rate of LA-RG was significantly faster than that of LA. After 32 min of exposure to LA-RG, approximately 86% and 60% of S. aureus and S. epidermidis were reduced, respectively. Abnormal bacterial cell surface after exposure to LA-RG was observed by scanning electron microscopy. It is concluded that LA-RG is a promising preparation as an alternative product for preventing mastitis in dairy cattle.

Streptococcus agalactiae is a contagious pathogen that causes bovine mastitis. The ability of S. agalactiae to cause widespread mastitis relies on bacterial virulence factors. In this study, we detected 10 virulence determinants associated with mastitis pathogenicity using conventional PCR. The antimicrobial susceptibility of 100 S. agalactiae isolates from 13 Thai dairy herds was assessed using the Kirby–Bauer disk diffusion susceptibility test. All strains had at least three virulence factors responsible for invasion, adhesion, and infection (fbsB, bibA, and cfb, respectively). The predominant virulent profile of S. agalactiae strains revealed the presence of fbsA, fbsB, bibA, cfb, and cyl (n = 96). Most strains were sensitive to penicillin, ampicillin, amoxicillin-clavulanic acid, cefotaxime, ceftiofur, erythromycin, sulfamethoxazole-trimethoprim, and vancomycin. However, all strains were resistant to aminoglycosides, including kanamycin and gentamicin attributed to the unnecessary antimicrobial use. Furthermore, we identified seven multidrug resistant (MDR) S. agalactiae strains among four dairy herds, of which, two were vancomycin resistant. Our study provides profiles for virulence factors and antimicrobial susceptibility, which are beneficial for the clinical monitoring, prevention, and control of bovine mastitis in dairy cattle in Thailand. Moreover, we emphasize the need for awareness regarding the judicious use of antimicrobials on dairy farms.

The instability of the protein expression in Pichia pastoris strains has been an issue for various peptide productions. Some modifications to the traditional fermentation process could potentially solve the problem. Here, we consider a four-stage fermentation process to express the CAP2 (cell-penetrating antimicrobial peptide 2) candidate in P. pastoris KM71H, a slow methanol utilization strain. During the fermentation process, CAP2 productivity is limited (6.15 ± 0.21 mg/L·h) by the low overall methanol consumption (approximately 645 g), which is mainly the result of the slow methanol utilization of the P. pastoris KM71H. To overcome this limitation, we increased the cell concentration two-fold prior to the induction stage. A fed-batch process with exponential and dissolved oxygen tension (DOT) stat feeding strategies was deployed to control the glycerol feed, resulting in an increase in cell concentration and enhancement of the volumetric methanol consumption rate. The improved fermentation process increased the overall methanol consumption (approximately 1070 g) and the CAP2 productivity (13.59 ± 0.24 mg/L·h) by 1.66 and 2.21 times, respectively. In addition, the CAP3 (cell-penetrating antimicrobial peptide 3) candidate could also be produced using this improved fermentation process at a high yield of 3.96 ± 0.02 g/L without any further optimization. Note that there was no oxygen limitation during the improved fermentation process operating at high cell density. This could be due to the controlled substrate addition via the DOT stat system.

Antimicrobial resistance in bovine mastitis pathogens poses interconnected threats to dairy productivity and public health; however, longitudinal evidence on the association between veterinary oversight and lower resistance to critically important antimicrobials in tropical smallholder systems remains limited. This study analyzed 1347 quarter milk samples from 47 dairy farms in Saraburi Province, Thailand, submitted consecutively over five years (2020–2025), to evaluate whether participation in a veterinary-led herd health (HH) program was independently associated with reduced vancomycin resistance and multidrug resistance (MDR). Bacterial identification was conducted using standard methods, and susceptibility was determined using the Kirby–Bauer disk diffusion method. Among 1069 culture-positive samples, Streptococcus spp. (25.3%) and coagulase-negative staphylococci (16.8%) predominated. Vancomycin resistance declined from 15.9% to 5.4% between early (2020–2022) and recent (2023–2025) periods. Of the 686 isolates tested against ≥3 antibiotic classes, 61 (8.9%) were MDR; vancomycin resistance co-occurred in 34.4% of these samples. Repeated logistic regression identified non-HH farm status (OR = 4.035; p = 0.027) and early-year groups (OR = 4.611; p = 0.002) as independent risk factors for vancomycin resistance. As HH participation and year group showed a significant interaction, their joint effect for MDR was modeled: compared with non-HH farms in the early period (reference), all other combinations—including HH farms in 2023, HH farms in 2020, and non-HH farms in 2023—were associated with significantly lower odds of MDR. These findings provide the first evidence from Thailand that structured veterinary herd health programs are independently associated with lower critically important antimicrobial resistance, supporting their integration into a One-Health stewardship policy.