Characterization of Virulence Factors and Antimicrobial Susceptibility of Streptococcus agalactiae Associated with Bovine Mastitis Cases in Thailand.

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.

Genotypic and antimicrobial susceptibility of Streptococcus agalactiae causing bovine mastitis in the central region of Thailand.

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.

Potential of Pm11 antimicrobial peptide against bovine mastitis pathogens.

OBJECTIVE: To investigate an alternative treatment for bovine mastitis by using Pm11 antimicrobial peptide. SAMPLE: 5 bovine mastitis pathogens that were previously isolated from cows affected by either clinical or subclinical mastitis. PROCEDURES: The current study introduces Pm11 antimicrobial peptide as an alternative treatment for bovine mastitis. The antibacterial activity of Pm11 was tested against Escherichia coli strain SCM1249, Klebsiella spp strain SCM1282, Staphylococcus aureus strain CM967, Streptococcus agalactiae strain SCM1084, and Streptococcus uberis strain SCM1310 using minimum bactericidal concentrations (MBCs) and time-kill kinetics. The pathogens' morphological changes were demonstrated using a scanning electron microscope (SEM). The cytotoxicity of Pm11 was assessed using the minimum hemolytic concentration assay. RESULTS: MBCs ranged from 2.5 to 10 µM and IC50 ranged from 0.32 to 2.07 µM. Time-kill kinetics at MBC demonstrated that Pm11 reduced viable cell counts of S agalactiae strain SCM1084 and S uberis strain SCM1310 from 105 to 0 CFU/mL within 1 h. E coli strain SCM1249 and S aureus strain CM967 were reduced from 105 to 0 CFU/mL within 4 h. The average Pm11-induced hemolytic activity was < 10% for all Pm11 concentrations tested except at the maximum concentration tested (160 µM: 10.19 ± 2.29%). Based on SEM, Pm11 induced morphological and cellular changes in S aureus and E coli. CLINICAL RELEVANCE: Pm11 antimicrobial peptide demonstrated in vitro antibacterial activity against the common bovine mastitis pathogens E coli, S aureus, S agalactiae, and S uberis, except Klebsiella spp, and should be further investigated in vivo.

Inhibitory Effects and Killing Kinetics of Lactic Acid Rice Gel Against Pathogenic Bacteria Causing Bovine Mastitis.

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.

The effect of a catastrophic flood disaster on livestock farming in Nakhon Sawan province, Thailand.

In 2011, a catastrophic flood disaster in Thailand affected not only humans but also took animal lives. Data on livestock losses, including death, loss, and decreased production, were collected in Nakhon Sawan province. The time-series map of the flooded area from August to December 2011 was available online from the Geo-informatics and Space Technology Development Agency. To evaluate the high-density areas of livestock loss, a spatial hot spot analysis was performed. The Getis-Ord Gi statistic with weighted zone of indifference and the Euclidean distance measurement were employed to identify spatial clusters of species that were affected by the flood. The results indicated that the majority of livestock losses were from poultry and swine farms. The density of poultry and swine loss was significantly different between sub-districts with clusters of high-density loss alongside the river, particularly in Chum Saeng and Kao Liew. Using spatial hot spot analysis as a tool to classify and rank the areas with high flood risks provides an informative outline for farmers to be aware of potential flood damage. To avoid unexpected loss from flooding, poultry and swine farms in risk areas should be properly managed, particularly during the flooding season between August and December.

Antimicrobial resistance and virulence genes in Salmonella enterica isolates from dairy cows.

One hundred sixty Salmonella enterica isolates from clinically healthy dairy cows were assayed for antibiotic susceptibilities, the presence of class 1 integrons, antimicrobial resistance genes and virulence genes, and conjugal transfer of antimicrobial resistance determinants. One hundred nine (68%) of the Salmonella isolates were resistant to at least 1 antibiotic, and 14 isolates (9%) were multiresistant. The most prevalent resistance observed was to streptomycin (64%). Class 1 integrons were detected in only two Salmonella isolates (serovar Singapore and Derby), and both integrons harbored the same cassette content aadA2. The Derby class 1 integrons were associated with Salmonella genomic island 1-A. Most commonly found resistance genes were strA and strB (9.2%). None of class 1 integrons were horizontally transferred, and the resistance genes were successfully transferred from six (5.5%) Salmonella strains. One hundred fifty-nine isolates (98.8%) were positive to the invasion gene invA, whereas the virulence plasmid-associated genes spvC and pefA were found in only two (1.3%) and one (0.6%) Salmonella isolates, respectively.

Role of the MexXY multidrug efflux pump in moderate aminoglycoside resistance in Pseudomonas aeruginosa isolates from Pseudomonas mastitis.

The contribution of the MexXY multidrug efflux system to aminoglycoside resistance was investigated in 18 clinical isolates of Pseudomonas aeruginosa obtained from dairy cows with Pseudomonas mastitis. All of the isolates expressed MexXY as determined by reverse transcription-PCR. The loss of mexXY resulted in increased susceptibility (two- to 16-fold decline in MIC) to aminoglycosides, confirming the contribution of this system in aminoglycoside resistance in these strains. As the impact of DeltamexXY varied, overexpression of MexXY alone is not sufficient for aminoglycoside resistance. Expression of mexXY also varied and did not strictly correlate with aminoglycoside insusceptibility. Transcription levels of mexY were independent on mutations in mexZ, suggesting the existence of additional regulatory mechanisms other than mexZ.