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Genotyping and Antimicrobial Susceptibility Profiling of Streptococcus uberis Isolated from a Clinical Bovine Mastitis Outbreak in a Dairy Farm.

ABSTRACT: Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of β-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

SUBMITTER: Monistero V

PROVIDER: S-EPMC8229259 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionStreptococcus 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 methodsIn 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.ResultsAll 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.DiscussionThis 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.

Project description:Intramammary infections are a major problem for dairy sheep farms, and Streptococcus uberis is one of the main etiological agents of ovine mastitis. Surveys on antimicrobial resistance are still limited in sheep and characterization of isolates is important for acquiring information on resistance and for optimizing therapy. In this study, a sampling of 124 S. uberis isolates collected in Sardinia (Italy) from sheep milk was analyzed by multilocus-sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) for genetic relatedness. All isolates were also subjected to antimicrobial susceptibility analysis by the disk diffusion test using a panel of 14 antimicrobials. Resistance genes were detected by PCR assays. MLST analysis revealed that the isolates were grouped into 86 sequence types (STs), of which 73 were new genotypes, indicating a highly diverse population of S. uberis. The most frequently detected lineage was the clonal complex (CC)143, although representing only 13.7% of all characterized isolates. A high level of heterogeneity was also observed among the SmaI PFGE profiles, with 121 unique patterns. Almost all (96.8%) isolates were resistant to at least one antimicrobial, while all exhibited phenotypic susceptibility to oxacillin, amoxicillin-clavulanic acid and ceftiofur. Of the antimicrobials tested, the highest resistance rate was found against streptomycin (93.5%), kanamycin (79.8%) and gentamicin (64.5%), followed by novobiocin (25%) and tetracycline-TE (19.3%). Seventy-four (59.7%) isolates were simultaneously resistant to all aminoglycosides tested. Seventeen isolates (13.7%) exhibited multidrug resistance. All aminoglycosides-resistant isolates were PCR negative for aad-6 and aphA-3' genes. Among the TE-resistant isolates, the tetM gene was predominant, indicating that the resistance mechanism is mainly mediated by the protection of ribosomes and not through the efflux pump. Three isolates were resistant to erythromycin, and two of them harbored the ermB gene. This is the first study reporting a detailed characterization of the S. uberis strains circulating in Sardinian sheep. Further investigations will be needed to understand the relationships between S. uberis genotypes, mastitis severity, and intra-mammary infection dynamics in the flock, as well as to monitor the evolution of antimicrobial resistance.

Project description:β-hydroxybutyrate (BHB) has been associated with disease incidence in early lactation dairy cattle, but such associations do not demonstrate causation. Therefore, the objective of this study was to examine the effects of BHB during an intramammary Streptococcus uberis challenge. A secondary objective was to elucidate the mechanisms behind BHB effects on cytokine transcript abundance using the RAW 264.7 cell line. Late lactation multiparous dairy cows (n = 12) were continuously infused intravenously with either BHB to induce hyperketonemia (target concentration: 1.8 mM) or with saline (CON) for 72 h during a S. uberis intramammary challenge. Body temperature, dry matter intake (DMI), milk production, and milk S. uberis cfu were measured daily until one week post-challenge. Blood samples were collected during infusion to assess changes in metabolism (glucose, insulin, glucagon, NEFA, and cortisol) and systemic inflammation (IL-1β and SAA). Mammary biopsies were conducted at 72 h post-challenge to assess transcript abundance of inflammation-associated genes. BHB-infused cows exhibited a delayed febrile response, noted by a lesser vaginal temperature during the final day of infusion, followed by a greater vaginal temperature 6 d post-challenge. Consequently, BHB-infused cows had greater S. uberis cfu on d 4, 6, and 7 as compared to CON. Accordingly, BHB-infused cows consumed less DM, produced less milk, had reduced blood glucose, and had increased cortisol concentrations, however, no effects were seen on other systemic parameters or transcript abundance of inflammation-related genes in mammary tissue. To elucidate mechanisms behind the impaired immune defenses, RAW 264.7 cells were transfected with a GPR109A siRNA for 24 h and then treated with or without 1.8 mM BHB and challenged or left unchallenged with S. uberis for an additional 3 h. Transfection with siRNA reduced Gpr109a by 75%. Although BHB treatment did not significantly increase Il10, GPR109A knockdown as compared to the scrambled control reduced Il10 by 90% in S. uberis challenged macrophages treated with BHB, suggesting that macrophage immune responses to S. uberis can be altered via a GPR109A-dependent mechanism. Taken together, these data suggest that BHB altered the immune response promoting tolerance toward S. uberis rather than resistance.

Dataset Information

Genotyping and Antimicrobial Susceptibility Profiling of Streptococcus uberis Isolated from a Clinical Bovine Mastitis Outbreak in a Dairy Farm.

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