Search Results (152)

Water buffalo (Bubalus bubalis) are a primary source of milk in Pakistan, where bovine mastitis is a significant health issue among cattle, leading to substantial economic losses. Staphylococcus aureus is a predominant pathogen associated with mastitis; however, a detailed molecular characterization of the strains in the country remains limited. We previously characterized mastitis strains from the Hazara division of Khyber Pakhtunkhwa, Pakistan. In this study, we investigated mastitis cases in the Peshawar division, including samples from both animals and human farm workers for comparison. Higher rates of mastitis (67.27% of animals) and sub-clinical mastitis (91.03% of positive animals) were identified in Peshawar than for those (34.55% and 75.31%, respectively) previously observed in Hazara. Methicillin-susceptible S. aureus (MSSA) belonging to clonal complex 9 (ST2454) were predominant. Methicillin-resistant S. aureus (MRSA) belonging to ST22 and ST8 were also detected in the Nowshera district. While no S. aureus colonization was observed among animal handlers, evidence of hand contamination suggests a potential route for pathogen spread. Low levels of antibiotic resistance were noted amongst isolates, but higher rates were seen in MRSA. This study presents only the second comprehensive molecular investigation of S. aureus isolated from buffalo mastitis in Pakistan and indicates a concerning rise in mastitis within the province. Full article

Staphylococcus aureus frequently causes intramammary infections in dairy cows (bovine mastitis), which impair animal welfare, milk yield, and food safety. This study determined the prevalence and genetic diversity of S. aureus in bulk tank milk (BTM) samples from community-based Alpine dairy pastures in Tyrol, a major milk-producing region in Austria. Throughout the 2023 Alpine season (May–September), 60.3% (94/156) of BTM samples tested positive for S. aureus at least once over the course of up to four samplings. A total of 140 isolates collected from the 94 S. aureus-positive community-based Alpine dairy pastures revealed 33 distinct spa types, with t2953 (n = 33), t529 (n = 12), t267 (n = 11), and t024 (n = 10) being the most common. Selected isolates representing the different spa types were characterised by DNA microarray-based genotyping, multi-locus sequence typing (MLST), and antimicrobial susceptibility testing. Isolates with spa types associated with bovine-adapted CC8 (CC8bov/GTB) were identified as the most common subtype, being detected in BTM samples from 35.3% (55/156) of the pastures. This emphasises the high prevalence of this subtype in dairy herds across European Alpine countries. Other common bovine-associated subtypes were also detected, including CC97, CC151, and CC479. While antimicrobial resistance was rare, enterotoxin-producing genes were detected in all CC8bov-associated spa types. Overall, these findings underscore the importance of rigorous hygiene practices in dairy farming, particularly in community-based Alpine dairy pastures, where the risk of transmission is particularly high. It also emphasises the need for continued surveillance and subtyping to improve animal health, ensure food safety, and promote sustainable milk production. Full article

Lactococcus lactis (L. lactis) is a pathogenic Gram-positive, catalase-negative coccobacillus (GPCN) associated with bovine mastitis. In this study, nine strains of L. lactis were successfully isolated and characterized from 457 milk samples from cows with clinical mastitis in China. All isolates exhibited a high degree of susceptibility to marbofloxacin and vancomycin. A series of molecular and cell biological techniques were used to explore the biological characteristics and pathogenicity of these isolates. The virulence gene profiles of the isolates were analyzed using whole genome resequencing combined with polymerase chain reaction (PCR) to elucidate the differences in virulence gene expression between isolates. To provide a more visual demonstration of the pathogenic effect of L. lactis on bovine mammary epithelial cells, an in vitro infection model was established using MAC-T cells. The results showed that L. lactis rapidly adhered to the surface of bovine mammary epithelial cells and significantly induced the release of lactate dehydrogenase, suggesting that the cell membranes might be damaged. Ultrastructural observations showed that L. lactis not only adhered to MAC-T cells, but also invaded the cells through a perforation mechanism, leading to a cascade of organelle damage, including mitochondrial swelling and ribosome detachment from the endoplasmic reticulum. The objective of this study was to provide strong evidence for the cytotoxic effects of L. lactis on bovine mammary epithelial cells. Based on this research, a prevention and treatment strategy for L. lactis as well as major pathogenic mastitis bacteria should be established, and there is a need for continuous monitoring. Full article

Milk is frequently susceptible to contamination by potential pathogens, posing risks to both food safety and public health. Cheesemaking often relies on raw milk, where microbial communities—including Staphylococcus xylosus—can play a dual role: (i) contributing to fermentation and (ii) acting as opportunistic pathogens that can be often present in subclinical mastitis and be subjected to carry over in dairy products. In this study, Staphylococcus xylosus was isolated from raw bovine milk (preclinical mastitis) and identified via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Biotyper scores: 1.87–2.19). Its susceptibility to erythromycin and to an essential oil blend composed of Myrtus communis, Salvia officinalis, and Cistus ladanifer was evaluated. The essential oil blend produced inhibition zones ranging from 9 mm to 13.3 mm, indicating moderate antibacterial activity. Further Minimum Inhibitory Concentration analysis revealed that Myrtus communis, Salvia officinalis, and the essential oil blend inhibited Staphylococcus xylosus growth at concentrations between 0.5 and 0.25 percent, while Cistus ladanifer required higher levels (1 to 0.5 percent). These findings suggest that selected essential oils—especially in combination—hold promise as complementary antimicrobial agents in food safety and antimicrobial resistance mitigation efforts. Full article

The increasing prevalence of bacterial resistance to conventional disinfectants and antibiotics has intensified the search for effective, natural alternatives in the dairy industry. This study evaluates the bactericidal efficacy of Punica granatum L. (pomegranate) peel ethanolic extract, focusing on its application in pre- and post-dipping procedures for mastitis prevention. The extract exhibited potent activity against Escherichia coli and Staphylococcus aureus, two major mastitis pathogens. At a concentration of 10 mg/mL, the extract induced significant membrane disruption within 30 s of exposure, as evidenced by propidium iodide uptake and elevated extracellular DNA levels (Escherichia coli: 64.25 ng/μL; Staphylococcus aureus: 83.25 ng/μL) compared to controls (11.20 ng/μL and 35.20 ng/μL, respectively; p < 0.05). Complete growth inhibition (100%) was achieved within 30 s at 25 and 50 mg/mL, matching the efficacy of commercial chlorhexidine and high-concentration hypochlorite. Phytochemical analysis identified punicalagin as the predominant bioactive compound. These findings establish Punica granatum peel extract as a fast-acting bactericidal agent, exhibiting an efficacy comparable to or exceeding that of conventional disinfectants. Its rapid action and plant-based origin highlight its potential as a viable alternative for the prevention and control of bovine mastitis in dairy farming. Full article

Staphylococcus aureus is a mastitis pathogen that compromises cow health and causes significant economic losses in the dairy industry. High antimicrobial resistance and biofilm formation by S. aureus limit the efficacy of conventional treatments. This study evaluated the potential of polyhexamethylene biguanide nanoparticles (PHMB NPs) against mastitis-causing S. aureus. PHMB NPs showed low toxicity to bovine mammary epithelial cells (MAC-T cells) at concentrations up to four times higher than the minimum inhibitory concentration (1 µg/mL) against S. aureus. In Experiment 1, PHMB NPs significantly reduced biofilm formation by S. aureus by 50% at concentrations ≥1 µg/mL, though they showed limited efficacy against preformed biofilms. In Experiment 2, using an excised teat model, PHMB NPs reduced S. aureus concentrations by 37.57% compared to conventional disinfectants (chlorhexidine gluconate, povidone–iodine, and sodium dichloroisocyanurate), though limited by short contact time. These findings highlight the potential of PHMB NPs for the control of S. aureus growth and biofilm formation. Full article

Dairy cows are highly susceptible to mastitis caused by Klebsiella pneumoniae, and treating these infections poses a challenge due to the resistance of the bacterium to common antibiotics. This study aimed to evaluate the safety of W. cibaria SDS2.1 and investigate its protective effects against K. pneumoniae-induced mastitis. The safety of W. cibaria SDS2.1 was assessed through comprehensive analyses, including antibiotic resistance profiling, hemolysis assays, cell cytotoxicity tests, and whole-genome sequencing. Furthermore, its ability to protect against cellular and tissue damage caused by K. pneumoniae-induced mastitis was evaluated using both in vitro and in vivo models. Our results revealed that W. cibaria SDS2.1 was non-hemolytic, non-cytotoxic, and significantly inhibited the growth of K. pneumoniae (p < 0.05). Additionally, W. cibaria SDS2.1 effectively reduced the adhesion and invasion of K. pneumoniae. In the K. pneumoniae-induced mouse mastitis model, W. cibaria SDS2.1 significantly reduced myeloperoxidase (MPO) activity, mammary tissue damage, and the expression of inflammatory cytokines (IL-6, IL-1β, and TNF-α) (p < 0.05). In K. pneumoniae-infected bovine mammary epithelial cells (bMECs), W. cibaria SDS2.1 significantly decreased lactate dehydrogenase (LDH) release, indicating reduced cellular damage. These findings demonstrate that W. cibaria SDS2.1 exhibits anti-inflammatory properties in experimental models, suggesting its potential role in mitigating K. pneumoniae-induced mastitis. Full article

Background: The emergence of multidrug-resistant (MDR) Staphylococcus aureus presents a critical global health challenge due to treatment failures and high mortality rates. Faced with this growing threat, new antimicrobials with original modes of action are urgently needed, and antimicrobial peptides proved to be promising alternatives. Objectives: The aim of this study is to explore the structure–function relationship of the lipopeptide humimycin A, compare the spectrum of activity of the synthetic analogs against a panel of S. aureus isolates, and investigate their binding to the humimycin target, the lipid II flippase MurJ. Methods: Humimycin A and 15 analogs were produced by solid-phase peptide synthesis, and their antimicrobial activity was evaluated by agar diffusion and microtitration assays against 19 S. aureus isolates from bovine mastitis and other pathogens. Results: Among the synthesized peptides, four humimycin analogs exhibited activity against methicillin-sensitive and methicillin-resistant S. aureus, as well as several isolates in the panel, including MDR S. aureus, with minimal inhibitory concentration values ranging from 0.5 to 256 µg/mL. Results from the structure–activity relationship study showed that the β-hydroxymyristoyl lipid chain and C-terminal carboxylic acid are essential for antimicrobial efficacy. In presence of human erythrocytes, the active humimycin analogs exhibited moderate hemolytic activity, suggesting selectivity indexes ranging from 3 to 27 against the more sensitive S. aureus strains. Critical micelle concentration measurements elucidated micelle formation and proved to not be essential for the antibacterial activity. Molecular docking and 100 ns simulations with the lipid II flippase MurJ (PDB: 5T77) provided favorable binding energy. Conclusions: The findings underscore the potential of humimycin analogs as antimicrobials for preventing and treating MDR S. aureus infections in veterinary, animal husbandry, and human medicine. Full article

K. pneumoniae is a major cause of bovine mastitis worldwide, making it difficult to control due to its resistance to multiple drugs. L. paraplantarum has been explored as a promising new approach to fighting bovine mastitis. In this study, the probiotic potential and safety of L. paraplantarum SDN1.2, as well as its ex vivo and in vivo anti-inflammatory effects against K. pneumoniae-induced mastitis, were comprehensively investigated using bioinformatics analyses and experimental validation methods. The results revealed that L. paraplantarum SDN1.2 exhibits non-hemolytic activity, is not cytotoxic, lacks virulence genes (e.g., adhesion factors, toxins, and invasion factors) and antibiotic resistance genes (e.g., beta-lactamases and tetracycline resistance genes), as supported by whole-genome sequencing, and significantly inhibits the growth of K. pneumoniae, as evaluated by antimicrobial tests. Following further validation in vitro, L. paraplantarum SDN1.2 demonstrated the capability to inhibit the adhesion and invasion of K. pneumoniae to bMECs. In a mouse model of K. pneumoniae-induced mastitis, L. paraplantarum SDN1.2 reduced the extent of neutrophil infiltration and inflammatory lesions. Furthermore, L. paraplantarum SDN1.2 pretreatment significantly reduced myeloperoxidase (MPO) activity and the expression of inflammatory cytokines (IL-6, IL-1β, and TNF-a) in mouse mammary gland tissue. In K. pneumoniae-infected bMECs, L. paraplantarum SDN1.2 significantly lowered lactate dehydrogenase (LDH) levels and expression of inflammatory cytokines such as IL-6, IL-1β, and TNF-α. The results demonstrated that the newly isolated L. paraplantarum SDN1.2 from bovine sources exhibits promising characteristics as a safe probiotic for the alleviation of bovine mastitis due to its safety profile and anti-inflammatory and antibacterial properties. Full article

Mastitis is a major infectious disease that causes significant economic losses in the dairy industry. Current control programs primarily rely on antimicrobials, contributing to the growing concerns of the resistance situation and drug residues in milk. This study aimed to identify the bacterial pathogens responsible for subclinical/clinical bovine mastitis, assess their antimicrobial resistance profiles, and evaluate the antimicrobial effects of Caesalpinia sappan in treating mastitis through both in vitro and in vivo studies. A total of 138 bacterial isolates, representing 40 species, were identified from 100 milk samples collected from dairy cows under the Maejo Cooperative Group in Chiang Mai, Thailand, between May 2021 and February 2022. The most prevalent species was Escherichia coli (10.87%), followed by Bacillus cereus (9.42%) and Staphylococcus sciuri (7.97%). The highest resistance rates were observed for penicillin (65.21%), followed by streptomycin and lincomycin (61.59%). Nine isolates resisted all 18 antimicrobials tested. The average minimum inhibitory concentration (MIC) of C. sappan against the identified pathogens ranged from 0.63 to 17.68 mg/mL, with the highest MIC observed against Pseudomonas luteola. In the animal experiment, treatment with a prototype of an intramammary infusion compound primarily formulated from C. sappan significantly reduced the total bacterial count and California Mastitis Test (CMT) scores (p < 0.01). These results suggest that C. sappan exhibits antimicrobial efficacy against various mastitis bacteria and could serve as a potential alternative treatment to control bovine mastitis in dairy cattle within the study region. Full article

Staphylococcus aureus-induced mastitis is a significant cause of economic losses in the dairy industry, yet its molecular mechanisms remain poorly defined. Although ferroptosis, a regulated cell death process, is associated with inflammatory diseases, its role in bovine mastitis is unknown. In this study, 11 S. aureus strains were isolated from milk samples obtained from cows with clinical or subclinical mastitis. Transcriptome analysis of Mac-T cells challenged with isolated S. aureus identified differentially expressed genes (DEGs). Enrichment analysis revealed significant associations between DEG clusters and traits related to bovine mastitis. KEGG pathway enrichment revealed ferroptosis, Toll-like receptor, and TNF signaling as significantly enriched pathways. Weighted gene co-expression network analysis (WGCNA) further prioritized ferroptosis-related genes (HMOX1, SLC11A2, STEAP3, SAT1, and VDAC2) involved in iron metabolism. Notably, the expression levels of HMOX1 and SAT1 were significantly increased in S. aureus-challenged Mac-T cells, and this upregulation was consistent with trends observed in transcriptome data from mother–daughter pairs of cows with subclinical mastitis caused by S. aureus infection. Furthermore, Ferrostatin-1 treatment significantly reduced the expression of HMOX1 and SAT1 in S. aureus-challenged cells, confirming the involvement of ferroptosis in this process. This study reveals that ferroptosis plays a key role in S. aureus-induced mastitis and highlights its potential as a target for molecular breeding strategies aimed at improving bovine mastitis resistance. Full article

Background/Objectives: Bovine mastitis remains a challenge for the Irish dairy industry. This study aimed to explore the seasonality and antimicrobial resistance of mastitis pathogens obtained by the regional laboratories (RVL) of the Department of Agriculture, Food and the Marine. Methods: Seasonality of isolation of the most common bacterial species and antimicrobial resistance of those species repeatedly obtained in the same herds in different years were explored using the RVL diagnostic data. Additionally, whole genome sequencing (WGS) was employed to establish the persistency of Staphylococcus aureus strains within the same herd. Results: A clear seasonality was observed in the isolation of Staphylococcus aureus, Streptococcus uberis, and Escherichia coli from milk. Seasonal differences were statistically significant within and between bacterium. Persistence of resistance within herds was highest in S. aureus against penicillin (35.5% of herds) and in S. uberis against pirlimycin (14% of herds), while E. coli did not show persistence of resistance to any antimicrobials. Sequencing of S. aureus isolates revealed that the strains causing mastitis in ten out of twenty-one herds were similar genetically in different years. In seven of these herds, S. aureus was persistently resistant to penicillin. Isolates from two different herds were practically identical and carried the human immune evasion cluster genes (IEC, scn, sak, chp and sea) suggesting a recent human-bovine host switch event. Conclusions: These findings underscore the importance of implementing targeted biosecurity measures and monitoring programs to mitigate the spread of mastitis-causing pathogens and enhance antimicrobial stewardship in the Irish dairy industry, while it also highlights the significance of including a One Health perspective in surveillance programs. Full article

Escherichia coli (E. coli) has the ability to induce clinical and subclinical mastitis in dairy cows, causing a huge loss for the dairy industry. In this study, 51 subclinical mastitis isolates and 36 clinical mastitis isolates from eight provinces of China between 2019 and 2021 were used to investigate the differences in their biological characteristics. The results showed that B1 (52.9%) and A (39.1%) were the predominant phylogroups; R1 (50.6%) was the predominant lipopolysaccharide (LPS) core type; and 44 STs (ST10 and ST58 were the most sequence-prevalent STs) and 2 new STs (ST14828 and ST14829) were identified; however, no significant difference was observed between the clinical and subclinical group strains. To compare the virulence gene differences between the clinical and subclinical mastitis-related isolates, 18 common virulence genes (including afaE, eaeA, papC, saa, sfa, ompA, aer, irp2, iucD, escV, sepD, east1, estB, stx2e, CNF1, cba, hlyA and traT) were determined using the PCR method. The results showed that the detection rates of traT, irp2 and iucD in clinical mastitis isolates were significantly higher than those in subclinical mastitis isolates (p ˂ 0.05). Meanwhile, subclinical-group E. coli had stronger biofilm formation abilities than the clinical group (p < 0.05) in 78 (89.7%) mastitis-related E. coli that could form biofilms. Furthermore, 87 mastitis-related E. coli showed severe resistance against tetracycline (37.9%), ampicillin (36.8%), streptomycin (34.5%) and cotrimoxazole (28.7%); their most prevalent resistance genes were blaCTX-M (33.3%), tetA (27.6%), sul2 (18.4%) and strB (28.7%). It was noteworthy that the clinical-group strains had a higher resistance against ampicillin and possessed higher amounts of the resistance gene blaCTX-M (p < 0.05) compared to the subclinical group. This study aims to provide references for preventing the E. coli isolates from inducing different types of mastitis. Full article

Background/Objectives: Pasteurella multocida commonly colonizes the bovine respiratory tract and can occasionally cause intramammary infections. Here, eight P. multocida isolates from clinical cases of bovine mastitis were investigated for their molecular characteristics as well as phenotypic and genotypic antimicrobial resistance (AMR) properties. Methods: The isolates originated from quarter milk samples obtained in Germany for diagnostic purposes. Antimicrobial susceptibility testing (AST) by broth microdilution was performed according to the Clinical and Laboratory Standards Institute. Closed whole-genome sequences were generated by hybrid assembly of Illumina MiSeq short-reads and Oxford Nanopore MinION long-reads, followed by consecutive sequence analysis. Results: The P. multocida isolates belonged either to capsular:lipopolysaccharide type A:3 (n = 7) or A:6 (n = 1), and multi-locus sequence types 1 (n = 7) or 7 (n = 1). Seven isolates carried AMR genes, such as mef(C), mph(G), strA, strB, aphA1, aadA31, tet(H), tet(Y), floR, catA3, and sul2, as part of an integrative and conjugative element (ICE). These mobile genetic elements, 58,382–78,401 bp in size, were highly similar to the ICEs Tn7406 or Tn7407 that have been previously described in bovine Mannheimia haemolytica and P. multocida, respectively. Moreover, the isolates showed elevated minimal inhibitory concentrations corresponding to the identified AMR determinants. Conclusions: Molecular typing and ICE organization suggest the bovine respiratory tract as reservoir of the investigated mastitis-associated P. multocida. Horizontal cross-genus transfer of multidrug-resistance-mediating ICEs seems to occur under in vivo conditions among different pathogens from cattle in Germany, which underlines the importance of pathogen identification followed by AST for successful bovine mastitis therapy. Full article

Background/Objectives: Bovine mastitis (BM), a prevalent and economically burdensome bacterial infection affecting dairy cattle, poses a significant challenge to the dairy industry. The traditional approach to combating BM, relying heavily on antibiotics, faces growing concerns due to the increasing antibiotic resistance exhibited by pathogens. The objective of this study was to evaluate and determine the antimicrobial and anti-biofilm potential of chitosan nanoparticles (NQo) on S. aureus strains isolated from milk samples obtained from dairy areas in southern Chile from cows diagnosed with BM. Methods: NQo were synthesized using the ionotropic gelation method and thoroughly characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Results: The NQo exhibit a robust positive charge (Z-potential of +55.4 ± 2.5 mV) and an exceptionally small size (20.3 ± 3.2 nm). This unique combination of properties makes NQo particularly well-suited for targeting and interacting with bacterial pathogens. To assess the effectiveness of NQo against BM, we conducted a series of experiments using a Staphylococcus aureus strain isolated from milk samples of cows diagnosed with BM in southern Chile. NQo demonstrated a remarkable ability to inhibit bacterial proliferation and effectively modulate biofilm formation in the S. aureus strains. Furthermore, the performance of NQo in comparison to established antibiotics like ampicillin and gentamicin strongly suggests that these nanoparticles hold immense potential as an attractive alternative for the control, prevention, and/or treatment of BM. Conclusions: NQo exhibit both antimicrobial and antibiofilm activity against a clinically relevant BM pathogen. Further investigations are necessary to develop a hydrogel formulation optimized for effective delivery to the target diseased tissue. Full article