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.

Relationship between Penicillin-Binding Proteins Alterations and ß-Lactams Non-Susceptibility of Diseased Pig-Isolated Streptococcus suis.

Streptococcus suis is a zoonotic pathogen causing disease in both animals and humans, and the emergence of increasingly resistant bacteria to antimicrobial agents has become a significant challenge globally. The objective of this study was to investigate the genetic basis for declining susceptibility to penicillin and other ß-lactams among S. suis. Antimicrobial susceptibility testing and penicillin-binding proteins (PBP1a, PBP2a, PBP2b, and PBP2x) sequence analysis were performed on 225 S. suis isolated from diseased pigs. This study found that a growing trend of isolates displayed reduced susceptibility to ß-lactams including penicillin, ampicillin, amoxicillin/clavulanic acid, and cephalosporins. A total of 342 substitutions within the transpeptidase domain of four PBPs were identified, of which 18 substitutions were most statistically associated with reduced ß-lactams susceptibility. Almost all the S. suis isolates which exhibited penicillin-non-susceptible phenotype (71.9%) had single nucleotide polymorphisms, leading to alterations of PBP1a (P409T) and PBP2a (T584A and H588Y). The isolates may manifest a higher level of penicillin resistance by additional mutation of M341I in the 339STMK active site motif of PBP2x. The ampicillin-non-susceptible isolates shared the mutations in PBP1a (P409T) and PBP2a (T584A and H588Y) with additional alterations of PBP2b (T625R) and PBP2x (T467S). The substitutions, including PBP1a (M587S/T), PBP2a (M433T), PBP2b (I428L), and PBP2x (Q405E/K/L), appeared to play significant roles in mediating the reduction in amoxicillin/clavulanic acid susceptibility. Among the cephalosporins, specific mutations strongly associated with the decrease in cephalosporins susceptibility were observed for ceftiofur: PBP1a (S477D/G), PBP2a (E549Q and A568S), PBP2b (T625R), and PBP2x (Q453H). It is concluded that there was genetically widespread presence of PBPs substitutions associated with reduced susceptibility to ß-lactam antibiotics.

Antimicrobial Susceptibility of Streptococcus suis Isolated from Diseased Pigs in Thailand, 2018-2020.

Streptococcus suis is a porcine and zoonotic pathogen that causes severe systemic infection in humans and pigs. The treatment of S. suis infection relies on antibiotics; however, antimicrobial resistance (AMR) is an urgent global problem, pushing research attention on the surveillance of antibiotic-resistant S. suis to the fore. This study investigated the antimicrobial susceptibility of 246 S. suis strains isolated from diseased pigs in Thailand from 2018-2020. The major sources of S. suis strains were lung and brain tissues. PCR-based serotyping demonstrated that the most abundant serotype was serotype 2 or ½, followed by serotypes 29, 8, 9, and 21. To the best of our knowledge, this is the first report describing the distribution of AMR S. suis serotype 29 in diseased pigs. The antimicrobial susceptibility test was performed to determine the minimum inhibitory concentrations of 35 antimicrobial agents. The results showed that important antimicrobial agents for human use, amoxicillin/clavulanic acid, daptomycin, ertapenem, meropenem, and vancomycin, were the most effective drugs. However, a slight decrease in the number of S. suis strains susceptible to amoxicillin/clavulanic acid and vancomycin raised awareness of the AMR problem in the future. The data indicated a tendency of reduced efficacy of available veterinary medicines, including ampicillin, cefepime, cefotaxime, ceftiofur, ceftriaxone, chloramphenicol, florfenicol, gentamicin, penicillin, and tiamulin, for the treatment of S. suis infection, thus emphasizing the importance of the prudent use of antibiotics. The widespread of multidrug-resistant S. suis strains was identified in all serotypes and from different time periods and different regions of the country, confirming the emergence of the AMR problem in the diseased pig-isolated S. suis population.

Plasmid-mediated colistin resistance and ESBL production in Escherichia coli from clinically healthy and sick pigs.

This study aimed to determine the percentage of colistin resistant and ESBL-producing Escherichia coli from clinically sick and healthy pigs and understand the molecular mechanisms underlying colistin resistance and ESBL production. A total of 454 E. coli isolates from healthy pigs (n = 354; piglets, n = 83; fattening pigs, n = 142 and sows, n = 100) and sick pigs (n = 100) were examined for antimicrobial susceptibility, chromosomal and plasmid-mediated colistin resistance mechanisms and ESBL genes. The healthy (41%) and sick pig (73%) isolates were commonly resistant to colistin. Three mcr genes including mcr-1 (10.4%), mcr-2 (1.1%) and mcr-3 (45%) were detected, of which mcr-3 was most frequently detected in the healthy (33%) and sick pig (57%) isolates. Coexistence of mcr-1/mcr-3 and mcr-2/mcr-3 was observed in piglets (23%), fattening pig (3.5%) and sick pig (13%) isolates. Three amino acid substitutions including E106A and G144S in PmrA and V161G in PmrB were observed only in colistin-resistant isolates carrying mcr-3. The percentage of ESBL-producing E. coli was significantly higher in the sick pigs (44%) than the healthy pigs (19.2%) (P = 0.00). The blaCTX-M group was most prevalent (98.5%), of which blaCTX-M-14 (54.5%) and blaCTX-M-55 (42.9%) were predominant. The blaTEM-1 (68.8%) and blaCMY-2 (6.3%) genes were identified in ESBL-producers. All ESBL producers were multidrug resistant and the majority from piglets (97%), fattening pigs (77.3%) and sick pigs (82%) carried mcr gene (s). ESBL producers from piglets (n = 5) and sick pig (n = 1) simultaneously transferred blaTEM-1 (or blaCTX-M-55) and mcr-3 to Salmonella. In conclusion, pigs are important reservoirs of colistin-resistant E. coli that also produced ESBLs, highlighting the need for prudent and effective use of antimicrobials in pigs and other food-producing animals.

Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.

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.