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This study was designed to identify Enterococcus faecalis from clinical mastitis of cattle and determine their antimicrobial resistance and virulence determinants to evaluate their potential public health significance. A total of 105 composite milk samples (80 from cattle with clinical mastitis and 25 from apparently healthy cattle) were analyzed. E. faecalis were isolated by culturing on enterococcal selective media and identified by PCR and sequencing. Antimicrobial resistance phenotype was elucidated by the disc diffusion method, and MIC was determined by broth microdilution method according to CLSI guidelines. Detection of antimicrobial resistance and virulence genes was done by PCR. E. faecalis were isolated from 11.25% (9/80) of the clinical mastitis and 4% (1/25) of the apparently healthy cattle milk samples. The disc diffusion test revealed 40% isolates as resistant to tetracycline and azithromycin, respectively. Among them, 20% (2/10) of isolates showed resistance to both tetracycline and azithromycin. Tetracycline-resistant isolates showed MIC ranging from ≥64 to >128 μg/ml and carried tetracycline-resistant genes tetK, tetL, and tetM in 25%, 25%, and 50% of the resistant isolates, respectively. On the other hand, all the isolates were sensitive to amoxicillin, ampicillin, bacitracin, chloramphenicol, gentamicin, penicillin, and vancomycin. In addition, the isolates carried at least one of the nine virulence genes screened with pil having the highest frequency, followed by fsrB, fsrC, ace, sprE, gelE, and agg genes. Positive correlations were evident between ace, fsrC, gelE, and sprE genes that are associated with the attachment and biofilm formation in E. faecalis. E. faecalis isolated in this study carried antibiotic resistance and virulence determinants which explain their competence to be potential human pathogens.

Streptococci are the major etiology in mastitis, a cause of huge economic losses in the dairy industries. Streptococcus (S.) agalactiae, S. dysagalactiae and S. uberis are mostly encountered in bovine mastitis; however, data on the diversity and characteristics of Streptococcus in clinical mastitis of cattle in Bangladesh is lacking. Thus, the present study was aimed to determine the diversity and antimicrobial resistance pattern of Streptococcus spp. isolated from clinical mastitis of cattle reared in Bangladesh. A total of 105 milk samples comprising eighty (80) from cattle with clinical mastitis (CCM) and twenty-five (25) from apparently healthy cattle (AHC) in four prominent dairy farms and one dairy community were purposively collected and examined in this study. Milk samples were enriched in Luria Bertani broth (LB) and Streptococcus spp. was isolated on Modified Edwards Medium and identified by 16S rRNA gene sequencing. Among eighty (80) clinical samples, eighteen (18) were positive for Streptococcus spp. while none of the milk from AHC revealed Streptococcus by cultural and molecular examination. Sequencing and phylogenetic analysis identified 55.6%, 33.3%, 5.6% and 5.6% of the Streptococcus isolates as S. uberis, S. agalactiae, S. hyovaginalis and S. urinalis, respectively. Antibiotic sensitivity testing with antimicrobials commonly used to treat clinical mastitis revealed 100%, 100%, and 30% of the S. agalactiae, S. hyovaginalis, and S. uberis as multidrug-resistant, respectively. Molecular characterization through whole genome sequencing of five (5) S. uberis isolates identified at least two novel ST types of S. uberis circulating in the study areas with one ST (4/5 isolates) clustered with the isolates from China, India and Thailand, and the other (1/5) with UK, Ireland and Australia. Pan-genome analysis and phylogeny of the core genome sequences also clustered the isolates into two sub-clusters, indicating the presence of at least two different subtypes of S. uberis in the study area. On virulence profiling, all the isolates of this study were found to harbor at least 35 virulence and putative virulence genes probably associated with intramammary infection (IMI) indicating all the S. uberis isolated in this study as potential pathogen. From the overall findings it was evident that Strepococcus occurring in bovine mastitis are diverse and S. uberis genome carries an array of putative virulence factors which need to be investigated genotypically and phenotypically to identify a specific trait or determinant governing the virulence and fitness of this bacterium. Moreover, Streptococcus isolated in this study carried multidrug resistance which needs careful consideration during the selection of a treatment regimen for mastitis.

IntroductionStreptococci are the major etiology in mastitis in dairy cattle, a cause of huge economic losses in the dairy industries. This study was aimed to determine the diversity of Streptococcus spp. isolated from clinical mastitis of cattle reared in Bangladesh.MethodsA total of 843 lactating cattle reared in four prominent dairy farms and one dairy community were purposively included in this study where 80 cattle were positive to clinical mastitis (CM) based on gross changes in the udder (redness, swelling, and sensitive udder) and/or milk (flakes and/or clots). Milk samples were collected from all the eighty cattle with clinical mastitis (CCM) and twenty five apparently healthy cattle (AHC). Samples were enriched in Luria Bertani broth (LB) and one hundred microliter of the enrichment culture was spread onto selective media for the isolation of Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli and Corynebacterium spp., the major pathogen associated with mastitis. Isolates recovered from culture were further confirmed by species specific PCR.Results and DiscussionOut of 105 samples examined 56.2% (59/105), 17.14% (18/105), 9.52% (10/105) and 22.9% (24/105) samples were positive for Staphylococcus, Streptococcus, Enterococcus faecalis and E. coli, respectively. This study was then directed to the determination of diversity of Streptococcus spp. through the sequencing of 16S rRNA. A total of eighteen of the samples from CCM (22.5%) but none from the AHC were positive for Streptococcus spp. by cultural and molecular examination. Sequencing and phylogenetic analysis of 16S rRNA identified 55.6, 33.3, 5.6 and 5.6% of the Streptococcus isolates as Streptococcus uberis, Streptococcus agalactiae, Streptococcus hyovaginalis and Streptococcus urinalis, respectively. Considering the high prevalence and worldwide increasing trend of S. uberis in mastitis, in-depth molecular characterization of S. uberis was performed through whole genome sequencing. Five of the S. uberis strain isolated in this study were subjected to WGS and on analysis two novel ST types of S. uberis were identified, indicating the presence of at least two different genotypes of S. uberis in the study areas. On virulence profiling, all the isolates harbored at least 35 virulence and putative virulence genes probably associated with intramammary infection (IMI) indicating all the S. uberis isolated in this study are potential mastitis pathogen. Overall findings suggest that Streptococcus encountered in bovine mastitis is diverse and S. uberis might be predominantly associated with CM in the study areas. The S. uberis genome carries an array of putative virulence factors that need to be investigated genotypically and phenotypically to identify a specific trait governing the virulence and fitness of this bacterium. Moreover, the genomic information could be used for the development of new genomic tools for virulence gene profiling of S. uberis.