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Non-aureus staphylococci (NAS) represent a major etiological agent in dairy animal mastitis, yet their role and impact remain insufficiently studied. This study aimed to elucidate the genomic characteristics of a newly identified multidrug-resistant NAS strain, specifically Staphylococcus warneri G1M1F, isolated from murine feces in an experimental mastitis model. Surprisingly, NAS species accounted for 54.35 % of murine mastitis cases, with S. warneri being the most prevalent at 40.0 %. S. warneri G1M1F exhibited resistance to 10 major antibiotics. Whole-genome sequencing established a genetic connection between G1M1F and S. warneri strains isolated previously from various sources including mastitis milk in dairy animals, human feces and blood across diverse geographical regions. Genomic analysis of S. warneri G1M1F unveiled 34 antimicrobial resistance genes (ARGs), 30 virulence factor genes (VFGs), and 278 metabolic features. A significant portion of identified ARGs (64 %) conferred resistance through antibiotic efflux pumps, while VFGs primarily related to bacterial adherence and biofilm formation. Inoculation with G1M1F in mice resulted in pronounced inflammatory lesions in mammary and colon tissues, indicating pathogenic potential. Our findings highlight distinctive genomic traits in S. warneri G1M1F, signifying the emergence of a novel multidrug-resistant NAS variant. These insights contribute to understanding NAS-related mastitis pathophysiology and inform strategies for effective treatment in dairy animals., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: Staphylococcus spp. are the major causal agents of mastitis in dairy animals worldwide leading to profound economic losses and public health threats. Recently, Staphylococcus aureus has emerged as a multidrug resistant and zoonotic pathogen. This study aimed to characterize S. aureus in subclinical mastitis (SCM) milk samples of riverine buffaloes in Bangladesh through antibiogram and virulence gene(s) profiling, and 16S rRNA gene sequencing., Method: We characterized S. aureus in SCM milk samples (N = 500) of riverine buffaloes through antibiogram and virulence gene(s) profiling, and 16S rRNA gene sequencing., Results: Out of 500 milk samples tested, 188 (37.6%) were found positive for SCM. From 188 SCM samples, 291 isolates were obtained with a prevalence of S. aureus in 37.4% (109/291) isolates. Phylogenetic analysis revealed the evolutionary divergence of S. aureus isolates in bubaline SCM milk samples. The antibiogram profiling showed that about 96.0% S. aureus isolates were multidrug resistant (MDR). Notably, 29 and 16 isolates harboured methicillin-resistant (mecA) and panton-valentine leucocidin (pvl) genes, respectively, and 46 plasmid-bearing isolates were MDR. Nine Staphylococcal enterotoxins (SEs/SEls) including sea (11.9%), sec (7.4%), sed (4.6%), seg (3.7%), and seh (3.7%) were detected with 72.48% toxinotypes comprising a single gene., Conclusion: This study therefore suggests S. aureus as the single-most aetiology (∼37.0%) of SCM in riverine buffaloes, and emergence of MDR, enterotoxin producing, and virulent S. aureus strains could impose potential threats to animal welfare and public health., (© 2022 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The milk of lactating cows presents a complex ecosystem of interconnected microbial communities which can influence the pathophysiology of mastitis. We hypothesized possible dynamic shifts of microbiome composition and genomic features with different pathological conditions of mastitis (Clinical Mastitis; CM, Recurrent CM; RCM, Subclinical Mastitis; SCM). To evaluate this hypothesis, we employed whole metagenome sequencing (WMS) in 20 milk samples (CM, 5; RCM, 6; SCM, 4; H, 5) to unravel the microbiome dynamics, interrelation, and relevant metabolic functions. The WMS data mapped to 442 bacterial, 58 archaeal and 48 viral genomes with distinct variation in microbiome composition (CM > H > RCM > SCM). Furthermore, we identified a number of microbial genomic features, including 333, 304, 183 and 50 virulence factors-associated genes (VFGs) and 48, 31, 11 and 6 antibiotic resistance genes (ARGs) in CM, RCM, SCM, and H-microbiomes, respectively. We also detected different metabolic pathway and functional genes associated with mastitis pathogenesis. Therefore, profiling microbiome dynamics in different conditions of mastitis and associated microbial genomic features contributes to developing microbiome-based diagnostics and therapeutics for bovine mastitis., (Copyright © 2020. Published by Elsevier Inc.)

Milk microbiomes significantly influence the pathophysiology of bovine mastitis. To assess the association between microbiome diversity and bovine mastitis, we compared the microbiome of clinical mastitis (CM, n = 14) and healthy (H, n = 7) milk samples through deep whole metagenome sequencing (WMS). A total of 483.38 million reads generated from both metagenomes were analyzed through PathoScope (PS) and MG-RAST (MR), and mapped to 380 bacterial, 56 archaeal, and 39 viral genomes. We observed distinct shifts and differences in abundance between the microbiome of CM and H milk in phyla Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria with an inclusion of 68.04% previously unreported and/or opportunistic strains in CM milk. PS identified 363 and 146 bacterial strains in CM and H milk samples respectively, and MR detected 356 and 251 bacterial genera respectively. Of the identified taxa, 29.51% of strains and 63.80% of genera were shared between both metagenomes. Additionally, 14 archaeal and 14 viral genera were found to be solely associated with CM. Functional annotation of metagenomic sequences identified several metabolic pathways related to bacterial colonization, proliferation, chemotaxis and invasion, immune-diseases, oxidative stress, regulation and cell signaling, phage and prophases, antibiotic and heavy metal resistance that might be associated with CM. Our WMS study provides conclusive data on milk microbiome diversity associated with bovine CM and its role in udder health.

Identification of cows with subclinical mastitis (SCM) is an important tool for sustainable dairying and implementing effective mastitis control strategies. A total of 892 quarters milk samples from 228 lactating cows were screened by California mastitis test (CMT), White side test (WST), Surf field mastitis test (SFMT), and somatic cell count (SCC) to study the prevalence of bovine SCM in some selected areas of Bangladesh. Out of 228 cows, 148 (64.9%), 138 (60.5%), 132 (57.9%), and 164 (71.9%) were found positive for SCM by CMT, WST, SFMT, and SCC, respectively. The prevalence of bovine SCM was diagnosed 45.7, 40.2, 36.6, and 29.6% in Chittagong, Sirajgonj, Mymensingh, and Gazipur districts, respectively, based on a combination of all tests. The overall quarter-wise prevalence of SCM was 45.7, 43.5, 41.2, and 55.0% for CMT, WST, SFMT, and SCC. Single quarters and left front quarters were more prone to SCM (P < 0.05). Friesian crossbred cows (56.4%), BCS 2.0-2.5 (55.4%), and parity 4-6 (52.4%), the late lactation stage (5-8 months; 64.7%) and high yielding cows (16-20 L/day; 65.3%) were more susceptible to SCM (P < 0.05). The sensitivity of the CMT, WST, SFMT, and SCC was 65.8, 57.9, 51.0, and 82.5%; specificity 76.2, 72.4, 69.5, and 89.4%; percentage accuracy 70.0, 64.8, 59.9, and 85.2%; positive predictive value 75.2, 69.8, 64.9, and 92.7%, respectively. The categories of CMT reactions were strongly correlated with SCC (P < 0.05). Kappa value of SCC was higher than that of other tests (SCC>CMT>WST>SFMT). Thus, CMT was concluded to be the most accurate (r = 0.782) field diagnostic test after laboratory test like SCC (r = 0.924). However, the use of any single test may not be reliable in diagnosing SCM, while the result of CMT supported by SCC might be used effectively to pinpoint diagnosis of SCM in dairy animals than alone.