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The present study investigates the prevalence and etiology of subclinical mastitis in Țurcana sheep flocks located in south-western Romania. Milchtest and California Mastitis Test (CMT), were used for the detection of subclinical mastitis. A number of 360 milk samples across three lactation phases were analyzed. Subclinical mastitis was identified in 146 (40.6%) of sheep milk samples. Only milk samples (n = 146) collected from sheep diagnosed with subclinical mastitis were considered for microbiological analyses. Thus, Staphylococcus aureus (129/146; 88.4%) was identified as the predominant pathogen followed by Streptococcus spp. (14/146; 9.6%) Enterococcus spp. (7/146; 4.8%), Pseudomonas aeruginosa (6/146; 4.1%) and Klebsiella pneumoniae (5/146; 3.4%). Antimicrobial susceptibility of isolated strains of Staphylococcus aureus, Streptococcus spp. and Enterococcus spp. was also tested. The results showed varying resistance patterns depending on the farm and microorganism. In heard A the highest resistance of Staphylococcus aureus was identified to polymyxin B (79.7%) followed by novobiocin (73.4%) and tetracycline (45.3%); Streptococcus spp. to streptomycin (100%), novobiocin (100%), tetracycline (100%) and Enterococcus spp. to tylosin (100%), streptomycin (75.0%) and tetracycline (75.0%). In herd B, Staphylococcus aureus showed high resistance rates to polymyxin B (86.3%), novobiocin (74.5%) and cloxacillin (39.2%); Streptococcus spp. strains to streptomycin (100%), novobiocin (100%), tetracycline (100%) and Enterococcus spp. to gentamicin (100%), streptomycin (66.7%) and erythromycin (66.7%). The obtained preliminary results indicate a potential risk that future treatments of sheep mastitis may not be effective, and demonstrated that raw unpasteurized milk can harbor antimicrobial resistance pathogens posing a threat to public health. However, further investigations involving molecular research on resistance genes are required to draw comprehensive conclusions., Competing Interests: Declarations. Ethical approval: Even though the research entitled “Etiology and Antimicrobial Resistance of Subclinical Mastitis Pathogens Staphylococcus aureus, Streptococcus spp. and Enterococcus spp. in Sheep Milk” included live animals (ovines), however, they were not directly affected or used because the samples processed and analyzed in the study were represented by milk samples collected from sheep during milking stages. The statement was included in the Bioethics Commission Approval No. 5639. The experiments assure the proper accommodation and animal handling in accordance with the Directive 2010/63/EU. Also the experimental protocol includes methods for preventing and reducing animal distress in accordance to the recommendation of European Union and Romanian Legislation. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: A high grain diet causes an ecological imbalance in the gut microbiota and serves as an important endogenous trigger of mastitis in dairy cows, but the underlying mechanisms are unclear. Our previous study revealed that subacute rumen acidosis (SARA)-associated mastitis has distinct metabolic profiles in the rumen, especially a significant increase in succinate, but the role of succinate in the pathogenesis of mastitis remains unclear., Results: Succinate treatment exacerbates low-grade endotoxemia-induced mastitis in mice. Specifically, succinate increased the production of gut microbiota-extracellular vehicles (mEVs) containing lipopolysaccharides, which can diffuse across the damaged intestinal barrier into the mammary glands. Administration of mEVs promotes mammary inflammation via activation of the TLR4/NF-κB pathway., Conclusions: Our findings suggest that succinate promotes mastitis through the proliferation of enteric pathogens and mEVs production, suggesting a potential strategy for mastitis intervention on the basis of intestinal metabolic regulation and pathogen inhibition. The role of mEVs in interspecific communication has also been elucidated., Competing Interests: Declarations Ethics approval and consent to participate All animal husbandry and experimental procedures were approved by the Laboratory Animal Management and Committee of Jilin University and complied with the institution’s Laboratory Animal Use and Welfare Guidelines (20210912). Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The rapid emergence of antibiotic-resistant strains of Staphylococcus aureus presents a substantial challenge to global public health, underscoring the urgent need for novel antibiotics with diverse mechanisms of action. In this study, we conducted mutagenesis on the C -terminal region of the lantibiotic ripcin C to enhance its antimicrobial efficacy against S. aureus . The resulting optimized variant, ripcin C P23A , demonstrated potent and selective antimicrobial activity, with a minimal inhibitory concentration of 2-4 mg/L against S. aureus . Beyond its strong antimicrobial properties, ripcin C P23A exhibited significant antibiofilm activity against methicillin-resistant S. aureus (MRSA). Mechanistic studies revealed that, in addition to targeting lipid II, ripcin C P23A disrupts bacterial membranes, a capability absent in ripcin C, which may contribute to its superior antimicrobial and antibiofilm effects. Moreover, ripcin C P23A displayed favorable biosafety and plasma stability profiles. Notably, in a mouse model of MRSA-induced mastitis, ripcin C P23A effectively reduced bacterial load, alleviated inflammation, and preserved the normal histomorphology of mammary glands. This study introduces ripcin C P23A as a promising antibiotic candidate for the treatment of MRSA-related infections.

Background: Intramammary infections negatively affect milk quality, animal welfare and productivity in the dairy industry. Somatic cell count (SCC) is the most used screening tool to detect subclinical mastitis caused by intramammary infections. In dairy goats, SCC is greatly influenced by non-infectious factors, which complicates the interpretation. The aim of this research paper was to determine the association between SCC, intramammary infections and non-infectious factors including parity, season, lactation stage, and milk yield in dairy goats. In this longitudinal study, 451 goats from four Norwegian dairy goat herds were sampled for bacteriology and SCC up to nine times during two lactations. Factors like parity, milk yield, and stage of lactation were retrieved from the Norwegian goat recording system., Results: The most prevalent udder pathogen findings were Staphylococcus caprae (6.8%), Staphylococcus warneri (6.3%), and Staphylococcus epidermidis (3.8%), all of which had a mild but significant impact on SCC. Staphylococcus aureus was detected in 3.6% of the udder halves and had a major effect on SCC. Parity, stage of lactation, season, and milk yield significantly influenced SCC., Conclusions: This study highlights that intramammary infections caused by Staphylococcus aureus, along with factors such as increasing parity and the seasonal effects of pasturing, significantly influence the SCC. Understanding these key contributors is essential for improving udder health management and improving milk quality in goat milk production., (© 2024. The Author(s).)

The modulation of intestinal flora by various polysaccharides has been shown to mitigate disease progression. Recent research reveals a significant link between intestinal flora and the progression of mastitis. This study demonstrates that the oral administration of Angelica sinensis polysaccharide (ASP) reduces mammary inflammation and blood-milk barrier (BMB) damage induced by Staphylococcus aureus in mice, primarily through the modulation of intestinal flora. The beneficial effects of ASP were negated when antibiotics disrupted the gut microbiota in mice. Furthermore, fecal microbiota transplantation (FMT) from ASP-treated mice to recipients markedly alleviated symptoms of S. aureus -induced mastitis. Oral ASP not only enhances gut microbial diversity but also shifts its composition, increasing the abundance of Lachnospiraceae_NK4A136 while reducing Erysipelatoclostridium . Metabolomic analysis revealed that ASP alters intestinal metabolic pathways, elevating levels of metabolites, such as tabersonine and riboflavin. Notably, tabersonine was found to ameliorate S. aureus -induced mastitis. These results suggest that targeting intestinal flora and metabolism through polysaccharides could serve as a promising strategy for mastitis intervention and potentially for other infectious diseases, as well.

Invasive neonatal infection with Group B Streptococcus (GBS) is a disease of concern that can lead to neurological sequelae. Guidelines for preventing mother-to-child transmission have been introduced to reduce the incidence of early-onset infection, but guidelines for controlling the late-onset form are lacking. Recently, the trans-breastfeeding route of transmission has been highlighted as an example of late-onset infection, but no consensus on how to manage such infections has been reached. In this report, we describe a case of late-onset bacteremia/meningitis in a neonate suspected to have been infected with GBS via breastfeeding. A vaginal culture test of the mother at 35 weeks' gestation was negative for GBS. Since she had symptoms of mastitis, breast milk and nipple cultures were also tested and found to be positive for the strain of GBS identified in the neonate on genetic analysis. Diagnosis of trans-mammary GBS infection is challenging because breastfeeding-related events are difficult to identify. In our case, the diagnosis was based on the mother's history of mastitis, and the patient was treated without escalation to sequelae. When a neonate develops a fever, physicians should consider GBS infection and examine the mother's medical history to facilitate accurate diagnosis, especially if the history includes mastitis. A breast milk culture should be performed if the mother has mastitis, especially in cases of infection in preterm infants and in recurrent cases.

Antimicrobial-resistant (AMR) Staphylococcus aureus (S. aureus) strains have attained global attention due to their life-threatening zoonotic nature. Being a member of ESKAPE group, S. aureus has an ability to escape the biocidal action of antimicrobial drugs. The current study investigated the prevalence and molecular characterization of methicillin-resistant S. aureus (MRSA), β-lactam-resistant S. aureus (BRSA), aminoglycoside-resistant S. aureus (ARSA), tetracycline-resistant S. aureus (TRSA), and fluoroquinolones-resistant S. aureus (FRSA) associated with goat subclinical mastitis (SCM). Furthermore, the antimicrobial resistance and susceptibility profile of various antibiotics and non-antibiotics (NSAIDs, nisin, N-acetylcysteine, vitamin-C) along with their possible role in modulating the antibiotic resistance of MDR isolates was also investigated. A total of 768 goat milk samples were subjected to California mastitis test for SCM followed by bacteriological and molecular characterization of S. aureus. Moreover, in-vitro susceptibility of resistant antibiotics, non-antibiotics, and their combination against MDR S. aureus were conducted through well diffusion and broth microdilution assays. The results depicted that 55.47 % and 26.82 % of milk samples were positive for SCM and S. aureus, respectively. The molecular assay confirmed 35.92 % of isolates as MRSA, 45.63 % as BRSA, 50.49 % as ARSA, and 32.52 % but no isolate was confirmed as FRSA on molecular basis. The multidrug resistance was observed in 62.13 % and 47.09 % isolates, respectively. Molecular characterized MDR S. aureus revealed high homology of study isolates with the isolates of neighboring countries like India, Korea, Iran, and China. Antimicrobial susceptibility trials on well diffusion assay showed higher efficacy of different non-antibiotics with resistant antibiotics as penicillin with ketoprofen and gentamicin with flunixin meglumine while oxytetracycline with N-acetylcystiene. The synergy testing by checkerboard assay revealed synergistic activity of penicillin with ketoprofen, gentamicin with flunixin meglumine, and oxytetracycline with N-acetylcysteine. The current study highlighted the emergence and spread of AMR S. aureus strains from goat SCM and provided insights into possible drug repurposing of various non-antibiotics to modulate the multidrug resistance of S. aureus which will be helpful in devising the therapeutic options and control measures for this pathogen., Competing Interests: Declaration of competing interest The research article entitled “Molecular insights into antimicrobial resistant Staphylococcus aureus strains: A potential zoonosis of goat origin” is being submitted to Microbial Pathogenesis for possible publication. All authors have read and approved the final version of manuscript and have no conflict of interest. I am submitting the article on behalf of all the co-authors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The wide-spreading of multidrug resistance poses a significant threat to human and animal health. Although antimicrobial peptides (AMPs) show great potential application, their instability has severely limited their clinical application. Here, self-assembled AMPs composed of multiple modules based on the principle of associating natural marine peptide N6 with ß-sheet-forming peptide were designed. It is noteworthy that one of the designed peptides, FFN could self-assemble into nanoparticles at 35.46 µM and achieve a dynamic transformation from nanoparticles to nanofibers in the presence of bacteria, resulting in a significant increase in stability in trypsin and tissues by 1.72-57.5 times compared to that of N6. Additionally, FFN exhibits a broad spectrum of antibacterial activity against multidrug-resistant (MDR) gram-positive (G + ) and gram-negative (G - ) bacteria with Minimum inhibitory concentrations (MICs) as low as 2 µM by membrane destruction and complemented by nanofiber capture. In vivo mouse mastitis infection model further confirmed the therapeutic potential and promising biosafety of the self-assembled peptide FFN, which can effectively alleviate mastitis caused by MDR Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and eliminate pathogenic bacteria. In conclusion, the design of peptide-based nanomaterials presents a novel approach for the delivery and clinical translation of AMPs, promoting their application in medicine and animal husbandry., (© 2024. The Author(s).)

Cell death caused by severe Staphylococcus aureus (S. aureus) infection is a fatal threat to humans and animals. However, whether ferroptosis, an iron-dependent form of cell death, is involved in S. aureus-induced cell death and its role in S. aureus-induced diseases are unclear. Using a mouse mastitis model and mammary epithelial cells (MMECs), we investigated the role of ferroptosis in the pathogenesis of S. aureus infection. The results revealed that S. aureus-induced ferroptosis in vivo and in vitro as demonstrated by dose-dependent increases in cell death; the level of malondialdehyde (MDA), the final product of lipid peroxidation; and dose-dependent decrease the production of the antioxidant glutathione (GSH). Treatment with typical inhibitors of ferroptosis, including ferrostatin-1 (Fer-1) and deferiprone (DFO), significantly inhibited S. aureus-induced death in MMECs. Mechanistically, treatment with S. aureus activated the protein kinase RNA-like ER kinase (PERK)-eukaryotic initiation factor 2, α subunit (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP) pathway, which subsequently upregulated autophagy and promoted S. aureus-induced ferroptosis. The activation of autophagy degraded ferritin, resulting in iron dysregulation and ferroptosis. In addition, we found that excessive reactive oxygen species (ROS) production induced ferroptosis and activated endoplasmic reticulum (ER) stress, manifesting as elevated p-PERK-p-eIF2α-ATF4-CHOP pathway protein levels. Collectively, our findings indicate that ferroptosis is involved in S. aureus-induced mastitis via ER stress-mediated autophagy activation, implying a potential strategy for the prevention of S. aureus-associated diseases by targeting ferroptosis. In conclusion, the ROS-ER stress-autophagy axis is involved in regulating S. aureus-induced ferroptosis in MMECs. These findings not only provide a new potential mechanism for mastitis induced by S. aureus but also provide a basis for the treatment of other ferroptotic-related diseases., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Subclinical mastitis is an asymptomatic inflammatory condition that can be difficult to define and diagnose. In the dairy industry, subclinical mastitis is diagnosed by milk somatic cell counts (SCCs) of ≥250,000 cells mL -1 . In this pilot study, we assessed the efficacy of this index to identify human subclinical mastitis by comparing SCC levels with the inflammatory response [interleukin-8 (IL-8) levels] in 37 samples from asymptomatic and 10 clinical mastitis (CM) lactating women. The milk microbiota was determined by 16S rRNA gene sequencing. The SCC of CM samples ranged from 310,000 to 6,600,000 cells mL -1 . However, 14 of 37 (37.8%) asymptomatic samples had high SCC (250,000-460,000 cells mL -1 ), indicating subclinical mastitis. SCC levels significantly ( P < 0.001) and positively correlated with milk IL-8 levels reflecting the escalating inflammatory response across subclinical and clinical mastitis samples. Samples with an SCC of ≥250,000 cells mL -1 showed significant increases in IL-8 responses when compared with milk samples from healthy women. The milk microbiome of CM samples was dominated by streptococcal and staphylococcal species (89.9% combined median relative abundance). In contrast, the combined median streptococcal/staphylococcal relative levels were 75.4% and 66.3% in milks from asymptomatic (subclinical mastitis) and healthy groups, respectively. The Streptococcus genus was increased in samples with an SCC of ≥250,000, although this should be interpreted with caution. Thus, the index of ≥250,000 somatic cells mL -1 could be a reliable indicator of subclinical mastitis in humans and should aid future studies investigating the impact of subclinical mastitis on maternal health, breastfeeding behaviors, infant health, and development., Importance: This pilot study suggests that SCC at a level of (greater than or equal to) 250,000 cells mL -1 , as used in the dairy industry, is a suitable index to identify asymptomatic subclinical mastitis in lactating women since it reflects a significant increase in the inflammatory response compared to milk samples from healthy women. Using this index should aid studies into the short- and long-term consequences of subclinical mastitis for mother and infant., Competing Interests: The authors declare no conflict of interest.

Mastitis is a common mammalian disease occurring in the mammary tissue and poses a major threat to agriculture and the dairy industry. Hordenine (HOR), a phenylethylamine alkaloid naturally extracted from malt, has various pharmacological effects, but its role in mastitis is unknown. The aim of this study was to investigate the role of HOR and its underlying mechanism in a lipopolysaccharide (LPS)-induced inflammatory response model of mouse mammary epithelial cells (EpH4-Ev) and mouse mastitis model. The experimental results showed that HOR attenuated LPS-induced mammary tissue damage (from 3.75 ± 0.25 to 1.75 ± 0.25) and restored the integrity of the blood-milk barrier. Further mechanistic studies revealed that HOR inhibited LPS-induced overactivation of the TLR4-MAPK/NF-κB signaling pathway and activated the AMPK/Nrf2/HO-1 signaling pathway. Additionally, HOR altered the composition of the intestinal microbiota in mice, ultimately reducing the extent of inflammatory injury (from 3.33 ± 0.33 to 0.67 ± 0.33) and upregulating the expression of tight junction proteins (ZO-1, occludin, and claudin-3). The findings of this study provide a theoretical basis in the rational use of HOR for the prevention and treatment of mastitis and the maintenance of mammalian mammary gland health.

Recently studies showed that cow mastitis seriously affected the economic benefit of dairy industry and pathogen infection including S. aureus is the main cause of mastitis. However, there is still a lack of safe and effective treatment for S. aureus-induced mastitis due to its complex pathogenesis. Endogenous retroviruses (ERVs) have long been symbiotic with mammals, and most ERVs still have the ability to produces complementary DNA (cDNA) by reverse transcription, whose induction by commensal or pathogens can regulate host immunity and inflammatory responses through the cGAS-STING pathway. However, whether and how ERVs participate in the pathogenesis of S. aureus-induced mastitis still unclear. In this study, we found that S. aureus treatment increased the levels of ERVs and IFN-β. Inhibition the transcription of ERVs by emtricitabine alleviated S. aureus-induced mammary injury, reduced mammary bacterial burden, and inhibited the production of mammary proinflammatory factors including TNF-α, IL-1β and MPO activity. Moreover, inhibition of ERVs restored the function of blood-milk barrier caused by S. aureus. Next, we showed that S. aureus infection activated mammary cGAS-STING signaling pathway, which was mediated by ERVs, as evidenced by emtricitabine inhibited S. aureus-induced activation of the cGAS-STING pathway. Interestingly, inhibition of cGAS-STING by Ru.521 and H151 respectively, significantly alleviated S. aureus-induced mammary injury and inflammatory responses, which was associated with the inhibition of NF-κB and NLRP3 signaling pathways. In conclusion, our study revealed that ERVs regulate the development of S. aureus-induced mastitis in mice through NF-κB- and NLRP3-mediated inflammatory responses via the activation of cGAS-STING pathway, suggesting that targeting ERVs-cGAS-STING axis may be a potential approach for the treatment of S. aureus-induced mastitis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) act as key gene expression regulators, influencing intracellular biological and pathological processes. They are of significant interest in animal genetics as potential biomarkers for animal selection and health. This study aimed to unravel the complex miRNA signature involved in mastitis in in vitro cell culture. For this purpose, we constructed a control and treatment model in ovarian mammary epithelial cells to analyze miRNA responses upon Staphylococcus aureus (S. aureus) stimulation. The high-throughput Illumina Small RNA protocol was employed, generating an average of 7.75 million single-end reads per sample, totaling 46.54 million reads. Standard bioinformatics analysis, including cleaning, filtering, miRNA quantification, and differential expression was performed using the miRbase database as a reference for ovine miRNAs. The results indicated differential expression of 63 miRNAs, including 33 up-regulated and 30 down-regulated compared to the control group. Notably, miR-10a, miR-10b, miR-21, and miR-99a displayed a significant differential expression (p ≤ 0.05) associated to signal transduction, transcriptional pathways, diseases of signal transduction by growth factor receptors and second messengers, MAPK signaling pathway, NF-κB pathway, TNFα, Toll Like Receptor 4 (TLR4) cascade, and breast cancer. This study contributes expanding miRNA databases, especially for sheep miRNAs, and identifies potential miRNA candidates for further study in biomarker identification for mastitis resistance and diagnosis., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Previous studies have demonstrated that gut microbiota dysbiosis promotes the development of mastitis. The interaction of the vagus nerve and gut microbiota endows host homeostasis and regulates disease development, but whether the vagus nerve participates in the pathogenesis of mastitis is unclear. Here, vagotomized mice exhibit disruption of the blood-milk barrier and mammary gland inflammation. Notably, mastitis and barrier damage caused by vagotomy are dependent on the gut microbiota, as evidenced by antibiotic treatment and fecal microbiota transplantation. Vagotomy significantly alters the gut microbial composition and tryptophan metabolism and reduces the 5-hydroxyindole acetic acid (5-HIAA) level. Supplementation with 5-HIAA alleviates vagotomy-induced mastitis, which is associated with the activation of the aryl hydrocarbon receptor (AhR) and subsequent inhibition of the NF-κB pathway. Collectively, our findings indicate the important role of the vagus-mediated gut-mammary axis in the pathogenesis of mastitis and imply a potential strategy for the treatment of mastitis by targeting the vagus-gut microbiota interaction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Aerococcus viridans ( A. viridans ) is an important opportunistic zoonotic pathogen that poses a potential threat to the animal husbandry industry, such as cow mastitis, due to the widespread development of multidrug-resistant strains. Phage lysins have emerged as a promising alternative antibiotic treatment strategy. However, no lysins have been reported to treat A. viridans infections. In this study, the critical active domain and key active sites of the first A. viridans phage lysin AVPL were revealed. AVPL consists of an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and a C-terminal binding domain comprising two conserved LysM. H40, N44, E52, W68, H147, T157, F60, F64, I77, N92, Q97, H159, V160, D161, and S42 were identified as key sites for maintaining the activity of the catalytic domain. The LysM motif plays a crucial role in binding AVPL to bacterial cell wall peptidoglycan. AVPL maintains stable activity in the temperature range of 4-45°C and pH range of 4-10, and its activity is independent of the presence of metal ions. In vitro, the bactericidal effect of AVPL showed efficient bactericidal activity in milk samples, with 2 µg/mL of AVPL reducing A. viridans by approximately 2 Log 10 in 1 h. Furthermore, a single dose (25 µg) of lysin AVPL significantly reduces bacterial load (approximately 2 Log 10) in the mammary gland of mice, improves mastitis pathology, and reduces the concentration of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in mammary tissue. Overall, this work provides a novel alternative therapeutic drug for mastitis induced by multidrug-resistant A. viridans ., Importance: A. viridans is a zoonotic pathogen known to cause various diseases, including mastitis in dairy cows. In recent years, there has been an increase in antibiotic-resistant or multidrug-resistant strains of this pathogen. Phage lysins are an effective approach to treating infections caused by multidrug-resistant strains. This study revealed the biological properties and key active sites of the first A. viridans phage lysin named AVPL. AVPL can effectively kill multidrug-resistant A. viridans in pasteurized whole milk. Importantly, 25 μg AVPL significantly alleviates the symptoms of mouse mastitis induced by A. viridans . Overall, our results demonstrate the potential of lysin AVPL as an antimicrobial agent for the treatment of mastitis caused by A. viridans ., Competing Interests: The authors declare no conflict of interest.

Objectives: Mastitis is mainly caused by Gram-positive bacteria and usually involves treatment with beta-lactam antibiotics and clindamycin. Oxazolidinones show good results in the treatment of skin and soft tissue infections (SSTIs) due to its pharmacokinetic characteristics. We aimed to describe clinical characteristics and outcomes of patients who received oxazolidinones for the treatment of SSTIs of the mammary tissue., Methods: Retrospective single-centre study of patients with a diagnosis of breast infection who received treatment with oxazolidinones as initial or salvage therapy between September 2016 and November 2022. Patients were identified through the pharmacy database. The primary outcome was clinical cure., Results: Twenty-nine patients received oxazolidinones: 27 received linezolid and 2 tedizolid. Median age was 41 years (IQR 31.0-56.5) and 28 patients were female. Ten patients (35%) had a history of breast cancer, while three (10%) had an immunosuppressive condition. Microbiological isolation was obtained in 24 individuals (83%). Predominant isolations were methicillin-resistant Staphylococcus aureus (8, 28%) and methicillin-susceptible S. aureus (7, 24%). Twenty-four patients (83%) received oxazolidinones as a salvage therapy, with a median duration of 14 days (IQR 10-17). Clinical cure was achieved in 24 patients (83%), while 4 relapsed after a median of 15 days (IQR 4-34). One was lost to follow-up. Three patients (10%) were taking selective serotonin reuptake inhibitors, and one of them concurrently received linezolid for 4 days with no adverse events recorded. Cytopenia during treatment was observed in 2/12 individuals. Oxazolidinones allowed hospital discharge in 11/13 hospitalized patients., Conclusions: Oxazolidinones could be considered as an alternative for treating breast infections., (© 2024. Springer-Verlag GmbH Germany, part of Springer Nature.)

Mastitis is a global production disease that needs an intelligent solution to tackle effectively. Infrared Thermography (IRT) is a non-invasive technology that could be incorporated into routine day-to-day farm activities to monitor the health status of the animals. In this study, the udder health status was routinely monitored for 30 days among 41 Murrah buffaloes via IRT and the California Mastitis Test (CMT). Further, somatic cell count (SCC), microbial identification, and milk quality parameters were also estimated for representative samples. The thermal imaging data obtained was tabulated and back propagated from the 0th day to the -10th day and front propagated from the 0th day to +10th day for all the udder quarters. Results revealed that on the 0th day, the mean of udder skin surface temperature (USST) and teat skin surface temperature (TSST) showed a difference (p < 0.05) in the sub-clinical mastitis (SCM) and clinical mastitis (CM) affected quarters to the healthy quarters, and their degree of difference was the highest. The indication of infection was signaled during the -9th to -5th day to th e 0th day in SCM and CM cases. There was a steep increment in the temperature from -2nd and -1st day to the 0th day of infection. Sometimes, some quarters show an increment in temperature due to mastitis during morning hours but recover by evening milking due to the animal's innate immune system. Thus, the initiation period in which the udder gets assaulted is crucial in the early assessment of SCM by monitoring temperature change using IRT., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mastitis is one of the most serious diseases that threatens the health of dairy animals. The somatic cell count (SCC) in milk is widely used to monitor mastitis. This study aimed to reveal the diversity of microorganisms in buffalo milk with high somatic cell count (SCC ≥ 3 × 10 5 cells/mL, n = 30) and low somatic cell count (SCC ≤ 5 × 10 4 cells/mL, n = 10), and identify the dominant bacteria that cause mastitis in a local buffalo farm. We also investigated the potential method to treat bacterial mastitis. The V3-V4 region of 16 S rDNA was sequenced. Results showed that, compared to the milk with low SCC, the high SCC samples showed lower microbial diversity, but a high abundance of bacteria and operational taxonomic units (OTUs). By in vitro isolation and culture, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae were found to be the leading pathogens, which is consistent with the 16 S rDNA sequencing data. We further isolated 3 of the main pathogens and established a pathogen detection method based on ELISA. In addition, the antibacterial effects of 10 antimicrobials and 15 Chinese herbal extracts were also investigated. Results showed that the microbial has developed tolerance to several of the antimicrobials. While the water extracts of Chinese herbal medicine such as Galla Chinensis, Coptis chinensis Franch, Terminalia chebula Retz, and Sanguisorba officinalis L can effectively inhibit the growth of main pathogens. This study provides novel insight into the microbial diversity in buffalo milk and a reference for the prevention, diagnosis, and treatment of mastitis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Mastitis, an inflammation of the mammary gland affecting milk production and quality in dairy herds, is often associated with Staphylococcus spp. in goats. Neutrophils are crucial in combating infections by migrating into milk and deploying various defense strategies, including the release of neutrophil extracellular traps (NETs) composed of DNA, histones, and bactericidal proteins. This study investigated whether NETs are released by goat neutrophils stimulated in vitro by Staphylococcus aureus and Staphylococcus warneri, two common pathogens of goat mastitis. PMNs were isolated from blood from healthy adult goats. We evaluated goat NET formation by stimulating cells with: phorbol 12-myristate 13-acetate (PMA) as a positive control, cytochalasin for inhibition of actin polymerization, S. aureus, and S. warneri. NET formation was observed in response to chemical stimulation and bacterial presence, effectively trapping pathogens. Variations in NET formation between S. aureus and S. warneri suggest pathogen-specific responses. These findings suggest that the formation of NETs may be an important complementary mechanism in the defense against mastitis in goats. In conclusion, this study unveils a novel defense mechanism in goats, indicating the role of NETs against S. aureus and S. warneri in mastitis., Competing Interests: Declaration of Competing Interest none, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Innovative approaches in nanotechnology provide a potentially promising alternative to untreatable cases of mastitis caused by genus Prototheca spp. algae infections. Drying of the teats of the affected animals or culling are typically the outcomes of mastitis in dairy cattle caused by these pathogens. A major issue in both veterinary medicine and animal breeding is the Prototheca species' widespread resistance to the current methods of managing infections and the available drugs, including antibiotics. Commercial antifungal preparations are also ineffective. Nanotechnology, an emerging discipline, has the potential to create an effective alternative treatment for protothecal mastitis . The aim of the paper is to combine the literature data on the use of nanotechnology in the control of mastitis , taking into account data on combating mastitis caused by Prototheca spp. infections. The databases employed were PubMed, Google Scholar, and Scopus, focusing on literature from the last 20 years to ensure relevance and currency. Studies conducted in vitro have demonstrated that nanomaterials have significant biocidal activity against mastitis infections of different etiologies. Analyzed research papers show that (NPs), such as AgNPs, CuNPs, AuNPs, etc., may not negatively impact various cell lines and may be effective agents in reducing the pathogens' viability. However, it is also critical to assess the risks involved in using nanomaterials.

Mastitis typically arises from bacterial invasion, where host cell apoptosis significantly contributes to the inflammatory response. Gram-positive bacteria predominantly utilize the virulence factor lipoteichoic acid (LTA), which frequently leads to chronic breast infections, thereby impacting dairy production and animal husbandry adversely. This study employed LTA to develop models of mastitis in cow mammary gland cells and mice. Transcriptomic analysis identified 120 mRNAs associated with endocytosis and apoptosis pathways that were enriched in the LTA-induced inflammation of the Mammary Alveolar Cells-large T antigen (MAC-T), with numerous differential proteins also concentrated in the endocytosis pathway. Notably, actin-related protein 2/3 complex subunit 3 (ARPC3), actin-related protein 2/3 complex subunit 4 (ARPC4), and the heat shock protein 70 (HSP70) are closely related. STRING analysis revealed interactions among ARPC3, ARPC4, and HSP70 with components of the apoptosis pathway. Histological and molecular biological assessments confirmed that ARPC3, ARPC4, and HSP70 were mainly localized to the cell membrane of mammary epithelial cells. ARPC3 and ARPC4 are implicated in the mechanisms of bacterial invasion and the initiation of inflammation. Compared to the control group, the expression levels of these proteins were markedly increased, alongside the significant upregulation of apoptosis-related factors. While HSP70 appears to inhibit apoptosis and alleviate inflammation, its upregulation presents novel research opportunities. In conclusion, we deduced the development mechanism of ARPC3, ARPC4, and HSP70 in breast inflammation, laying the foundation for further exploring the interaction mechanism between the actin-related protein 2/3 (ARP2/3) complex and HSP70.

Aims: To describe the incidence, aetiology, treatment, and outcomes of farmer-reported clinical mastitis on New Zealand dairy sheep farms., Methods: A prospective cohort study was conducted on 20 spring-lambing New Zealand sheep milking farms over the 2022-2023 season. Clinical mastitis was defined as a change in the appearance of milk and/or signs of inflammation in the gland. Farmers were required to report all cases of clinical mastitis and collect information on affected ewes' demographics, clinical features, treatments (where applicable), and outcomes. Milk samples from mastitic glands were submitted for microbiological culture and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF)., Results: Partial or complete clinical mastitis data were available for 236 cases from 221 ewes on 18/20 study farms. Clinical mastitis was diagnosed in 0-6% of ewes at the farm level, with an overall incidence of 1.8 (95% CI = 1.0-3.2)% using the study data, or 2.3 (95% CI = 1.6-3.3)% using the study data and farmer estimates that included unreported cases. Cases occurred mostly in early lactation, with 59% detected during the lambing period (August-October), at a median of 7 (IQR 3, 40) days in milk. The majority of cases featured clots in the milk (59%), swelling (55%), and unevenness (71%) of the glands. Pyrexia (rectal temperature ≥ 40.0°C) was diagnosed in 25% of cases and depression (lethargy, inappetence, or inability to stand) in 26% of cases. Treatment was given to 46% of cases, with tylosin being the most commonly used treatment (50% of treated cases). The most common outcome was immediate drying off to be culled without treatment (32%), followed by still milking and recovered but with lasting problems (25%). Nearly half of all the milk samples submitted were culture negative. Streptococcus uberis (14%), non-aureus staphylococci (12%), and Staphylococcus aureus (11%) were the most common isolates, found on 12, 8 and 8 of the 16 farms with microbiological data, respectively., Conclusions: Clinical mastitis affected up to 6% of ewes at the farm level. Systemic signs were observed in one quarter of affected ewes, suggesting a role for supportive treatment. Clinical mastitis can be severe and challenging to fully resolve in New Zealand dairy sheep., Clinical Relevance: This is the first systematic study of clinical mastitis in New Zealand dairy ewes. It provides baseline information specific to New Zealand conditions for farmers, veterinarians, and other advisors to guide the management of mastitis for the relatively new dairy sheep industry in New Zealand.

Objectives: Staphylococcus aureus (S. aureus) spreads worldwide and occurrence of mastitis caused by it holds significant implications for public health. We aim to reveal the molecular typing, antibiotic resistance and virulence gene profile of S. aureus causing mastitis through investigation., Methods: A total of 200 isolates of S. aureus were collected from outpatients infected with mastitis in a hospital in Beijing from 2020.7 to 2021.7. The molecular characteristics were analyzed by MLST and spa typing, virulence genes were screened by PCR, antibiotic susceptible test was performed by VITEK® 2 Compact system and phylogenetic analysis was performed by MEGA11 and iTOL., Results: Nineteen sequence types (STs) belonging to 9 clone complexes (CCs) were identified. ST22 was the most dominant clone (77.0%, 154/200). MRSA accounted for 19.0% (38/200) and 89.5% (34/38) of MRSA isolates belonged to CC22 and CC59. The isolates had relatively low levels of antibiotic resistance, with the exception of β-lactams and macrolides with resistance rates above 50.0%. The carrying rate of pvl in the ST22-MRSA strains were 84.2% and the detection rates of seb and pvl in the MRSA isolates were significantly higher than those in the MSSA isolates, while the hlg, fnbA and sdrD showed opposite results. Whole genome sequenced specimens of MRSA strains X4 and B5 show the same evolutionary origin as ST22 EMRSA-15 (HE681097), which is popular in Europe., Conclusions: The method based on molecular epidemiology is an important tool for tracking the spread of S. aureus infections. We need to be alert to the major MRSA clones CC22 and CC59 in the region and be vigilant to the possible pandemic and spread of ST22 EMRSA-15., Competing Interests: Declaration of Competing Interest The authors declare no competing conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Mastitis is an inflammatory disease of the mammary gland with a high incidence in lactating animals, significantly impacting their health and breastfeeding. Moreover, mastitis adversely affects milk quality and yield, resulting in substantial economic losses for the dairy farming industry. Forsythiaside A (FTA), a phenylethanol glycoside analog extracted from Forsythia, exhibits notable anti-inflammatory and antioxidant properties. However, its protective effects and specific mechanisms against mastitis remain unclear. In this study, a lipopolysaccharide (LPS)-induced mouse mastitis model was used to investigate the protective effect of FTA on LPS-induced mastitis and its potential mechanism using histological assays, Western blot, qRT-PCR, FITC-albumin permeability test, 16s rRNA gene sequencing analysis and non-targeted metabolomics assays to investigate the protective effect of FTA on LPS-induced mastitis model and its potential mechanism. The results demonstrated that FTA significantly mitigated LPS-induced mouse mastitis by reducing inflammation and apoptosis levels, modulating the PI3K/AKT/mTOR signaling pathways, inducing autophagy, and enhancing antioxidant capacity and the expression of tight junction proteins. Furthermore, FTA increased the abundance of beneficial microbiota while decreasing the levels of harmful microbiota in mice, thus counteracting the gut microbiota disruption induced by LPS stimulation. Intestinal metabolomics analysis revealed that FTA primarily regulated LPS-induced metabolite alterations through key metabolic pathways, such as tryptophan metabolism. This study confirms the anti-inflammatory and antioxidant effects of FTA on mouse mastitis, which are associated with key metabolic pathways, including the restoration of gut microbiota balance and the regulation of tryptophan metabolism. These findings provide a novel foundation for the treatment and prevention of mammalian mastitis using FTA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In herds of dairy goats, mastitis represents a major health and economic problem due to the multiresistance of some microorganisms. In this context, the study aimed to determine the potential of antimicrobial action and antibiofilm of the crude ethanolic extract (CEE) of Hymenaea martiana (jatobá) leaves, as well its fractions, on Staphylococcus sp isolated from bacterial cultures of goat milk. In vitro assays were performed to determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), as well as tests of the effect of CEE on biofilm formation and quantification and the consolidated biofilm. The experimental infection was performed in two groups, each consisting of five goat. Experimental Group 1 (G1) consisted of five females treated with an intramammary ointment based on the CEE, at a concentration of 5%. Experimental Group 2 (G2) consisted of five females treated with a commercial intramammary ointment based on gentamicin, once a day, for six consecutive days. The diagnosis of mastitis was performed using a bacterial culture. The dichloromethane fraction of CEE was the one with the lowest concentrations of MBC, ranging from 195.3 to 781 µg / ml. Concerning to the biofilm, interference of the tested extract was observed for two isolates. In the present study, the ointment prepared from H. martiana extract (jatobá) was able to reduce bacterial infection in mammary glands experimentally infected with S. aureus. Antibacterial activity may be related to the classes of secondary metabolites found., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Beta-glucan (β-glucan) is a polysaccharide containing β-glycosidic bonds that is an important structure part of different yeast cells., Aim: The purpose of the study is to characterize β-glucan obtained from Candida albicans (C. albicans) isolated from caprine mastitis ., Methods: The β-glucan was extracted by using utilizing an Alkaline-acidic extraction technique. The dry weight of extracted β-glucan was 7.47/150 g with 4.98%., Results: The findings demonstrated that the extracted β-glucan had similarity in the primary peak 5.78 of liquid samples using the method of high-performance liquid chromatography when compared to the standard form of β-glucan. However, scanning electron microscopy studies revealed that the standard of β-glucan was distinct in morphology but similar to β-glucan isolated from C. albicans in terms of particle sizes in the range of 1.60-2.65 m and the lack of cell wall traces. The findings of an investigation using energy-dispersive X-ray fluorescence spectroscopy (EDS/EDX) of extracted and standard β-glucan, showed the principal elements discovered were carbon (C), oxygen (O), and nitrogen (N). Aluminum (Al), silicon (Si), nickel (Ni), and gold (Au) were also present, but in less amounts., Conclusion: The extracted β-glucan displayed a high degree of similarity and purity to the standard β-glucan, according to the findings of Fourier transform infrared spectroscopy (FT-IR) research., Competing Interests: All authors have no conflict of interest to disclose.

Mastitis is a disease characterized by congestion, swelling, and inflammation of the mammary gland and usually caused by infection with pathogenic microorganisms. Furthermore, the development of mastitis is closely linked to the exogenous pathway of the gastrointestinal tract. However, the regulatory mechanisms governing the gut-metabolism-mammary axis remain incompletely understood. The present study revealed alterations in the gut microbiota of mastitis rats characterized by an increased abundance of the Proteobacteria phylum. Plasma analysis revealed significantly higher levels of L-isoleucine and cholic acid along with 7-ketodeoxycholic acid. Mammary tissue showed elevated levels of arachidonic acid metabolites and norlithocholic acid. Proteomic analysis showed increased levels of IFIH1, Tnfaip8l2, IRGM, and IRF5 in mastitis rats, which suggests that mastitis triggers an inflammatory response and immune stress. Follistatin (Fst) and progesterone receptor (Pgr) were significantly downregulated, raising the risk of breast cancer. Extracellular matrix (ECM) receptors and focal adhesion signaling pathways were downregulated, while blood-milk barrier integrity was disrupted. Analysis of protein-metabolic network regulation revealed that necroptosis, protein digestion and absorption, and arachidonic acid metabolism were the principal regulatory pathways involved in the development of mastitis. In short, the onset of mastitis leads to changes in the microbiota and alterations in the metabolic profiles of various biological samples, including colonic contents, plasma, and mammary tissue. Key manifestations include disturbances in bile acid metabolism, amino acid metabolism, and arachidonic acid metabolism. At the same time, the integrity of the blood-milk barrier is compromised while inflammation is promoted, thereby reducing cell adhesion in the mammary glands. These findings contribute to a more comprehensive understanding of the metabolic status of mastitis and provide new insights into its impact on the immune system., (© 2024 Federation of American Societies for Experimental Biology.)

A young woman experienced pain and swelling in a non-lactating breast. The culture test result showed an unusual microbe, which is increasingly prevalent in Norway and internationally.

Lactococcus garvieae (L. garvieae) is a pathogenic bacterium that is Gram-positive and catalase-negative (GPCN), and it is capable of growing in a wide range of environmental conditions. This bacterium is associated with significant mortality and losses in fisheries, and there are concerns regarding its potential as a zoonotic pathogen, given its presence in cattle and dairy products. While we have identified and characterized virulent strains of L. garvieae through phenotyping and molecular typing studies, their impact on mammary tissue remains unknown. This study aims to investigate the pathogenicity of strong and weak virulent strains of L. garvieae using in vivo mouse models. We aim to establish MAC-T cell model to examine potential injury caused by the strong virulent strain LG41 through the TLR2/NLRP3/NF-kB pathway. Furthermore, we assess the involvement of NLRP3 inflammasome-mediated pyroptosis in dairy mastitis by silencing NLRP3. The outcomes of this study will yield crucial theoretical insights into the potential mechanisms involved in mastitis in cows caused by the L. garvieae-induced inflammatory response in MAC-T cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Mastitis in goats is unquestionably a grave concern, with far-reaching implications for both animal well-being and productivity, while also presenting a potential threat to public health., Aim: The study aimed to compare culture methods and multiplex PCR (m-PCR) in the detection of the most three common mastitis-causing pathogens ( Staphylococcus aureus , Escherichia coli , and Streptococcus spp.) and investigate the gene expression, single nucleotide polymorphisms (SNPs), serum concentrations of immunological and antioxidant indicators linked to mastitis in Shami goats., Methods: One hundred Shami do (50 Shami goats with clinical mastitis and 50 normal goats taken as control group). The culture methods and m-PCR analysis were used to find the bacteria in the milk samples. Blood samples were obtained to assess some hemato-biochemical parameters, detect SNPs, and determine the expression of certain immunological and antioxidant indicators in the genes., Results: The culture method detected the pathogens causing mastitis in 90% of the milk samples, but m-PCR detected them in 100% of the milk samples. SNPs linked to mastitis resistance/susceptibility in examined does were detected through DNA sequencing of immunological and antioxidant indicators. The magnitude of gene expression varied significantly between the resistant and mastitis-affected groups. Significant ( P ˂ 0.05) elevations were noticed in WBCs count, mainly neutsrophils count, serum levels of BHB, NEFA, triglycerides, LDL-C, AST, ALT, ALP, creatinine, total protein, globulin, Ca, K, GPx, MDA, acute phase proteins, and cytokines in mastitis affected does as compared to control. While RBCs count, PCV%, lymphocytes count, serum concentration of glucose, cholesterol, HDL-C, albumin, Na, Cl, P, GSH, SOD, and catalase significantly ( P ˂ 0.05) diminished in mastitis affected does compared to healthy ones. APPs and pro-inflammatory cytokines scored high sensitivities and NPVs but TNF-α and serum amyloid A (SAA) had the highest percentages of increase., Conclusion: The study confirmed that m-PCR is the most sensitive method for bacteria identification ( S. aureus , E. coli, and Strept. spp.) while SNPs in antioxidant and immunological genes may be important genetic indicators for mastitis risk or resistance in Shami does. To establish an effective management plan and forecast the most sensitive risk time for illness onset, gene expression profiles of the tested genes may also be employed as proxy biomarkers. TNF-α and SAA may be precious indicators for the detection of caprine mastitis., Competing Interests: The authors declare that they have no conflict of interest.

Mastitis is one of the highly devastating issues responsible for production and economic losses in all dairy animals including sheep. This study was designed to investigate subclinical mastitis (SCM) associated with S. aureus in lactating nomadic ewes, along with the associated risk factors analysis. Furthermore, molecular characterization and antibiogram profiling of local methicillin-resistant S. aureus (MRSA) isolates of ovine origin were also performed. A total of 384 milk samples (n = 384) were collected from 13 nomadic sheep flocks using a convenient sampling technique. SCM was evaluated using a Surf Field Mastitis test and the S. aureus was isolated using standard microbiological techniques. Kirby-Bauer disc diffusion assay was used for phenotypic identification of MRSA while the mecA gene was tested through PCR. Study results revealed that SCM was prevalent at 34.37% while S. aureus association was recorded at 39.39%. MRSA prevalence was 36.53% and 21.15% using phenotypic and genotypic tests, respectively. The mecA gene sequences of study isolates showed maximum resemblance with already reported sequences from Pakistan, China, and Myanmar. MRSA isolates showed maximum resistance towards penicillin, ceftriaxone sodium, and trimethoprim + sulphamethoxazole while gentamicin, ciprofloxacin, and tylosin showed maximum efficacy. Risk factors analysis revealed that various flock management, housing, and host-related factors positively influenced the incidence of S. aureus-associated SCM. This study is the first report on the prevalence of S. aureus and MRSA associated with SCM in lactating ewes in Pakistan. This study will help to devise effective treatment and control strategies for S. aureus-associated SCM., Competing Interests: Declaration of Competing Interest Authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mastitis is a common and serious bacterial infection of the mammary gland. Saikosaponin A (SSA) is a triterpenoid saponin isolated from Bupleurum falcatum that has the ability to treat various diseases. However, little is known about the role of SSA in achieving mastitis remission. Here, we found that SSA alleviated Staphylococcus aureus (S. aureus)-induced mastitis by attenuating inflammation and maintaining blood-milk barrier integrity. Furthermore, S. aureus activated nuclear factor kappa B (NF-κB) pathway by upregulated p-p65 and p-IκB. S. aureus also induced ferroptosis in mammary gland in mice, mainly characterized by excessive iron accumulation, mitochondrial morphological changes and impaired antioxidant production. However, S. aureus-induced NF-κB activation and ferroptosis were prevented by SSA. Moreover, SAA could upregulate the expression of SIRT1, Nrf2, HO-1 and GPX4. And the inhibitory effects of SAA on inflammation and ferroptosis were reversed by SIRT1 inhibitor EX-527. In conclusion, SAA protected S. aureus-induced mastitis through suppressing inflammation and ferroptosis by activating SIRT1/Nrf2 pathway., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Dairy products play a crucial role in human nutrition as they provide essential nutrients. However, the presence of diverse microorganisms in these products can pose challenges to food safety and quality. Here, we provide a comprehensive molecular characterization of a diverse collection of lactic acid bacteria (LAB) and staphylococci isolated from raw sheep's milk. Whole-genome sequencing, phenotypic characterization, and bioinformatics were employed to gain insight into the genetic composition and functional attributes of these bacteria. Bioinformatics analysis revealed the presence of various genetic elements. Important toxin-related genes in staphylococci that contribute to their pathogenic potential were identified and confirmed using phenotypic assays, while adherence-related genes, which are essential for attachment to host tissues, surfaces in the dairy environment, and the creation of biofilms, were also present. Interestingly, the Staphylococcus aureus isolates belonged to sequence type 5, which largely consists of methicillin-susceptible isolates that have been involved in severe nosocomial infections. Although genes encoding methicillin resistance were not identified, multiple resistance genes (RGs) conferring resistance to aminoglycosides, macrolides, and fluroquinolones were found. In contrast, LAB had few inherently present RGs and no virulence genes, suggesting their likely safe status as food additives in dairy products. LAB were also richer in bacteriocins and carbohydrate-active enzymes, indicating their potential to suppress pathogens and effectively utilize carbohydrate substrates, respectively. Additionally, mobile genetic elements, present in both LAB and staphylococci, may facilitate the acquisition and dissemination of genetic traits, including RGs, virulence genes, and metabolic factors, with implications for food quality and public health. The molecular and phenotypic characterization presented herein contributes to the effort to mitigate risks and infections (e.g., mastitis) and enhance the safety and quality of milk and products thereof.

We evaluated the presence of bacterial pathogens in the milk of goats and their relationship with somatic cell count (SCC) and milk composition. The study was performed on a dairy farm in northern Slovakia. Half udder milk samples were collected from goats in June and July. The samples were divided on the basis of SCC into 4 bands (SCC1 lowest to SCC4 highest). Bacterial pathogens were only detected in 13% of samples. SCC3 and SCC4 had 15 and 25% positive samples respectively compared with SCC1 (2%) and SCC2 (14%). Coagulase-negative staphylococci (CNS) were the most common isolates (73%), of which Staphylococcus caprae was the most frequently isolated (65%). In samples with ≥ 1000 × 10 3 cells ml - 1 (SCC3, SCC4) there was higher somatic cell score (SCS) in the presence of a pathogen (7.48 ± 0.11) than without a pathogen (7.16 ± 0.05, P < 0.01). Statistically significant but weak negative correlations were observed between SCS and lactose, dry matter and non-fat dry matter. In conclusion, a higher percentage of bacteriologically positive milk samples was observed in both SCC3 and SCC4 groups but this does not explain the aetiology of high SCC in the milk of goats that are apparently free of bacteria. As a diagnostic tool, SCC is probably less useful in goats than in cows.

Actinomycosis is an infection characterized by abscess formation, draining sinuses, and tissue fibrosis. The causative bacterium is a Gram-positive facultative anaerobe from the genus Actinomyces . Infections classically affect the cervicofacial, thoracic, or pelvic region and often require prolonged antibiotic therapy. Actinomycosis of the breast is a rare condition that may present as a recurrent breast abscess. We present a 33-year-old female with a recurrent breast abscess which grew A. radingae and A. israeli on aspirated fluid cultures. Treatment with surgical aspiration and a 6-week course of oral amoxicillin/clavulanic acid 875 mg twice daily resulted in clinical improvement. Our case demonstrates how recurrent breast abscesses caused by Actinomyces can be difficult to manage. Long-term antibiotic therapy with surgical aspiration and regular follow-up offer the best chance of clinical resolution., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Context: Breast abscess is the most common complication of acute bacterial mastitis usually referred to as pyogenic mastitis. It is usually encountered during lactation due to an infection with Staphylococcus aureus and streptococcal bacteria. These bacteria produce a severe inflammatory reaction leading to pus formation which is mainly treated by ultrasound-guided drainage or fine needle aspirations. We find that in this condition homoeopathic treatment can play an important role as it avoids such surgical procedures and helps in healing in a most gentle and rapid way. We report a successful single case study that opens up opportunities to take up more such cases to strengthen the results of this report., Case Summary: A 23-year-old lactating mother came with complaints of pain and swelling with a collection of pus in the breast region and decreased breast milk secretion. Individualised homoeopathic medicine Silicea 200C was given to hasten suppuration and Belladonna 200C to treat inflammation.Thus, this case shows us how only few doses of medicine may be quickly helpful in managing a case of breast abscess without the need of any surgical procedures.

The increase in bacterial resistance to antibiotics is a global problem for public health. In our previous works, it was shown that the application of AgNPs in cow mastitis treatment increased S. aureus and S . dysgalactiae susceptibility to 31 antibiotics due to a decrease in the bacterial efflux effect. The aim of the present work was to shed light on whether the change in adhesive and anti-lysozyme activities caused by AgNPs also contribute to the restoration of bacterial susceptibility to antibiotics. In vivo sampling was performed before and after cow mastitis treatments with antibiotics or AgNPs. The isolates were identified, and the adhesive and anti-lysozyme activities were assessed. These data were compared with the results obtained for in vitro pre-treatment of reference bacteria with AgNPs or antibiotics. The present study revealed that bacterial treatments in vitro and in vivo with AgNPs: (1) decrease the bacterial ability to adhere to cells to start an infection and (2) decrease bacterial anti-lysozyme activity, thereby enhancing the activity of lysozyme, a natural "antibiotic" present in living organisms. The obtained data contribute to the perspective of the future application of AgNPs for recovering the activity of antibiotics rapidly disappearing from the market.

Although emerging evidence shows that gut microbiota-mediated metabolic changes regulate intestinal pathogen invasions, little is known about whether and how gut microbiota-mediated metabolites affect pathogen infection in the distal organs. In this study, untargeted metabolomics was performed to identify the metabolic changes in a subacute ruminal acidosis (SARA)-associated mastitis model, a mastitis model with increased susceptibility to Staphylococcus aureus (S. aureus). The results showed that cows with SARA had reduced cholic acid (CA) and deoxycholic acid (DCA) levels compared to healthy cows. Treatment of mice with DCA, but not CA, alleviated S. aureus-induced mastitis by improving inflammation and the blood-milk barrier integrity in mice. DCA inhibited the activation of NF-κB and NLRP3 signatures caused by S. aureus in the mouse mammary epithelial cells, which was involved in the activation of TGR5. DCA-mediated TGR5 activation inhibited the NF-κB and NLRP3 pathways and mastitis caused by S. aureus via activating cAMP and PKA. Moreover, gut-dysbiotic mice had impaired TGR5 activation and aggravated S. aureus-induced mastitis, while restoring TGR5 activation by spore-forming bacteria reversed these changes. Furthermore, supplementation of mice with secondary bile acids producer Clostridium scindens also activated TGR5 and alleviated S. aureus-induced mastitis in mice. These results suggest that impaired secondary bile acid production by gut dysbiosis facilitates the development of S. aureus-induced mastitis and highlight a potential strategy for the intervention of distal infection by regulating gut microbial metabolism., (© 2023. The Author(s).)

Cefquinome is a new generation cephalosporin that is effective in the treatment of mastitis in animals. In this study, we evaluated the associations between the specific pharmacokinetics and pharmacodynamics (PK/PD) of cefquinome and its antibacterial activity against Streptococcus agalactiae in a mouse model of mastitis. After a single intramammary dose of cefquinome (30, 60, 120, and 240 μg/mammary gland), the concentration of cefquinome in plasma was analysed by liquid chromatography with tandem mass spectrometry (HPLC/MS-MS). The PK parameters were calculated using a one-compartment first-order absorption model. Antibacterial activity was defined as the maximum change in the S. agalactiae population after each dose. An inhibitory sigmoid Emax model was used to evaluate the relationships between the PK/PD index values and antibacterial effects. The duration for which the concentration of the antibiotic (%T) remained above the minimum inhibitory concentration (MIC) was defined as the optimal PK/PD index for assessing antibacterial activity. The values of %T > MIC to reach 0.5-log10CFU/MG, 1-log10 CFU/MG and 2-log10 CFU/MG reductions were 31, 47, and 81%, respectively. When the PK/PD index %T > MIC of cefquinome was >81% in vivo, the density of the Streptococcus agalactiae was reduced by 2-log10. These findings provide a valuable understanding to optimise the dose regimens of cefquinome in the treatment of S. agalactiae infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Long-term breastfeeding is beneficial for both mothers and infants and mastitis is associated with the premature interruption of breastfeeding. Mastitis can be infectious or noninfectious. However, the effect of noninfectious mastitis on milk microbiota is not well-understood. In this study, we aimed to clarify the relationship between noninfectious mastitis and the microbiota by conducting breast milk culture tests., Methods: We compared the milk microbiota between women with noninfectious mastitis and without mastitis. Bacterial cultures were compared in 143 milk samples from January to November 2022, and bacterial diversity was evaluated based on the total number of bacterial species and bacterial species found per specimen., Results: Women with noninfectious mastitis provided samples at a significantly later stage postpartum (p < 0.01). The total bacterial count was significantly lower in samples from participants with noninfectious mastitis (p < 0.01). The bacterial diversity of milk from participants with noninfectious mastitis was lower than that without mastitis: nine bacterial species identified in the former and 21 in the latter. The number of Rothia spp. was significantly higher, whereas the number of Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas fluorescens was significantly lower in samples from women with mastitis. There was no correlation between postpartum week and the number of bacterial species or presence of Rothia spp., Conclusions: Noninfectious mastitis is associated with a decrease in the diversity of human milk microbiota, indicating impaired immune, metabolic, and neuroendocrine development functions in infants. Rothia spp. may also be associated with noninfectious mastitis, suggesting a possible target for future research., (© 2023 Japan Pediatric Society.)

Escherichia coli ( E. coli ) is a major environmental pathogen causing coliform mastitis, characterized by cell death and mammary tissue damage. Our previous study has shown the antimicrobial effect of Zophobas morio ( Z. morio ) hemolymph against mastitis pathogens. In this study, we established E. coli -induced cellular and animal models for mastitis, aiming to evaluate the protective effect of Z. morio hemolymph against E. coli -induced mastitis in vivo and in vitro. In mice with E. coli , Z. morio hemolymph attenuated bacterial burden and histopathological impairment, reduced the production of interleukin (IL)-1β, IL-18, tumor necrosis factor-α (TNF-α) and the ratio of CD4 + T/CD8 + T, and increased the production of IL-2 triggered by E. coli . Z. morio hemolymph also enhanced the integrity of the blood-milk barrier in E. coli -induced mastitis. In E. coli -stimulated porcine mammary epithelial cells, Z. morio hemolymph inhibited E. coli -induced inflammatory responses and upregulated tight junction proteins (ZO-1, Claudin-3 and Occludin). Moreover, we found that the anti-inflammatory effect of Z. morio hemolymph was mediated by inhibiting E. coli -induced NLRP3 inflammasome assembly, Caspase-1 activation, and reversing the inhibitory effect of E. coli on autophagy. Besides, Z. morio hemolymph augmented ATG5/ATG16L1-mediated autophagy activation, negatively regulated NLRP3 inflammasome activation. Our results reveal that Z. morio hemolymph alleviates E. coli -induced mastitis via lessening the inflammatory response by regulating the NLRP3 and ATG5/ATG16L1 signaling pathway, as well as repairing the blood-milk barrier.

Background: Breast abscess is developed on the basis of acute mastitis, which will cause damage to the physical and mental health of lactating women and is an important factor affecting the rate of breastfeeding. This study examined the risk factors for mastitis to develop into breast abscess, and analyzed the distribution of pathogenic bacteria, bacterial resistance, and treatment outcome., Methods: The medical records of 316 cases of mastitis and 219 cases of breast abscess were retrospectively collected. We analyzed the bacterial distribution of mastitis and breast abscess, and compared the differences of bacterial drug resistance. Univariate analysis and binary logistic regression were used to analyze the following aspects: age, primiparity or not, history of breast surgery, body temperature, puerperium or not, onset time, located in the nipple/areolar complexe area or not, history of massage by non-professionals, staphylococcus aureus/methicillin-resistant staphylococcus aureus (MRSA) infection or not, diabetes and white blood cell count., Results: Of the 535 patients, 203 (37.9%) were positive for staphylococcus aureus. There were 133 (65.5%) cases of methicillin-sensitive staphylococcus aureus (MSSA) and 70 (34.5%) cases of MRSA. Concerning bacterial drug resistance, a statistical analysis showed that MSSA had high resistance rate to penicillin (96.2%), ampicillin (91%), clindamycin (42.9%) and erythromycin (45.9%). MRSA had a high resistance rate to penicillin (100%), ampicillin (98.6%), oxacillin (95.7%), erythromycin (81.4%), clindamycin (80%), and amoxicillin (31.7%). Risk factors for progression of mastitis to breast abscess include a body temperature<38.5°C, a postpartum time ≥ 42 days, an onset time ≥ 2 days, lesions in the nipple/areolar complex area, a history of massage by non-medical staff and bacterial cultures for milk or pus that test positive for staphylococcus aureus or MRSA (P < 0.001)., Conclusions: The most common pathogenic bacteria of mastitis and breast abscess is staphylococcus aureus. There are many risk factors for mastitis to develop into breast abscess. We should take effective measures for its risk factors and select sensitive antibiotics according to the results of bacterial culture to reduce the formation of breast abscess., Competing Interests: The authors have declared that no competing interests exist.

The lactation capacity of dairy cows is critical to the productivity of the animals. Mastitis is a disease that directly affects the lactation capacity of cows. Staphylococcus aureus (S. aureus) is one of the most important pathogens that causes mastitis in dairy cows. The anti-inflammatory effect of Salvia miltiorrhiza polysaccharides (SMPs) has been demonstrated in mice and chickens. However, the effectiveness of SMPs in preventing and treating mastitis is unclear. Therefore, the purpose of this study was to explore the protective effect and mechanism of SMPs on mastitis caused by S. aureus. S. aureus was used to induce mastitis in rats, and three doses of SMPs (87.5, 175, 350 mg/kg, BW/d) were administered as treatments. The bacterial load, histopathology, and myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAGase) activities of mammary glands were observed and measured. Cytokines, including interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis factor α (TNF-α), were examined by qRT-PCR and ELISA. Key proteins in the NF-κB and MAPK signaling pathways were analyzed by Western blotting. The results showed that SMP supplementation could significantly reduce the colonization of S. aureus and the recruitment of inflammatory cells in mammary glands. S. aureus-induced gene transcription and protein expression of IL-1β, IL-6, and TNF-α were significantly suppressed in mammary glands. In addition, the increase in NF-κB and MAPK protein phosphorylation was inhibited by SMPs. These results revealed that supplementation with SMPs protected the mammary gland of rats against damage caused by S. aureus and alleviated the inflammatory response. This study provides a certain experimental basis for the treatment of S. aureus-induced mastitis with SMPs in the future., (© 2022. The Author(s).)

The recognition of lesions of the mammary gland in small ruminants is a useful diagnostic procedure that can aid in the identification of several udder diseases. This article reviews the main pathological lesions in this organ in sheep and goats. Mastitis is, by far, the most commonly diagnosed change. Acute clinical mastitis is associated with bacterial infections, mainly Staphylococcus aureus or Mannheimia haemolytica. Lesions related to subclinical and chronic mastitis are also described, either as localized cases or as a part of systemic diseases such as contagious agalactia, maedi-visna or tuberculosis. Neoplasia is rare in the mammary gland of sheep and goats with sporadic mammary adenocarcinomas most commonly reported. Teat lesions, including those due to trauma, orf virus infection or papillomas, are predisposing factors for the subsequent development of mastitis., Competing Interests: Conflict of Interest Statement The authors declared no potential conflicts of interest with respect to the research, authorship or publication of this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Streptococcus uberis (S. uberis) is an environmentally important pathogenic bacterium and is the main pathogenic microorganism responsible for mastitis, which causes significant economic losses worldwide. Currently, there is no particularly effective treatment other than antibiotic therapy. Angiotensin-converting enzyme 2 (ACE2) plays an anti-inflammatory as well as an anti-injury role in numerous inflammatory diseases. Therefore, this study aimed to assess the hypothesis that S. uberis-induced mammary epithelial cells injury associated with ACE2, angiotensin II (Ang II) as well as angiotensin 1-7 (Ang-(1-7)) imbalance and that overexpression of ACE2 can repair S. uberis-induced mammary epithelial cells injury. We observed that the expression level of ACE2 was significantly downregulated after treatment of EpH4-Ev cells with S. uberis. Next, this assay verified the role of ACE2 in S. uberis-induced inflammatory injury in EpH4-Ev cells by overexpressing the ACE2 gene as well as its silencing. The results showed that overexpression of the ACE2 gene significantly activated the interleukin-10/signal transducer and activator of transcription 3/suppressors-of-cytokine-signaling 3 (IL-10/STAT3/SOCS3) pathway, thereby inhibiting the nuclear factor-κB (NF-κB) as well as pyroptosis pathways. Furthermore, overexpression of the ACE2 gene reversed the downregulation of zonula occludens 1 (ZO-1), Occludin, Claudin-1, and Claudin-2 caused by S. uberis, suggesting that ACE2 could promote to repair the blood-milk barrier. However, siRNA silencing of the ACE2 gene produced the opposite effect. These results suggest that ACE2 ameliorates S. uberis-induced mammary epithelial cells injury. AVAILABILITY OF DATA: All data generated or analyzed during this study are included within the article and its additional information file., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mastitis is part of the aggressive diseases that affecting the development of dairy farming. Lactic acid bacteria (LAB), an important microbiological agent of gastrointestinal flora, can effectively promote the development of the immune system. Herein, the objectives of this study is to explore the protective role of LAB on Staphylococcus aureus(S. aureus)-induced mastitis in mice. 88 strains of suspected LAB were isolated from the milk of healthy dairy cows. Antibacterial activity was screened, and the 16S rRNA sequence analysis showed that the bacteria were Enterococcus mundtii H81 (E. mundtii H81). Furthermore, the model of mastitis has been established by nipple duct injection of S. aureus in mice, while E. mundtii H81 was treated 2 h before S. aureus injection. Twenty-four hours later of S. aureus infection, the mammary gland tissues were collected. The pathological changes of the mammary gland were observed by H&E staining. The levels of TNF-α and IL-1β were measured by ELISA and the myeloperoxidase (MPO) activity was measured by the MPO assay kit. We also observed the changes of nuclear transcription factor kappa B (NF-κB) by using western blotting. The results showed that E. mundtii H81 pretreatment reduced neutrophil infiltration, and significantly reduce the secretion of TNF-α and IL-1β, down-regulate the phosphorylation of p65 NF-κB and IκB, and the expression of tight junction protein Claudin 3 and ZO-1 was up-regulated. Collectively, our findings showed that E. mundtii H81 protects mammary gland from S. aureus-induced mastitis, which may be a candidate of treatment for mastitis infected by S. aureus., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The symptoms of mastitis caused by Staphylococcus aureus ( S. aureus ) in dairy cows are not obvious and difficult to identify, resulting in major economic losses. N 6 -Methyladenosine (m 6 A) modification has been reported to be closely associated with the occurrence of many diseases. However, only a few reports have described the role of m 6 A modification in S. aureus -induced mastitis. In this study, after 24 h of treatment with inactivated S. aureus , MAC-T cells (an immortalized bovine mammary epithelial cell line) showed increased expression levels of the inflammatory factors IL-1 β , IL-6, TNF- α , and reactive oxygen species. We found that the mRNA levels of METLL3, METLL14, WTAP, and ALKBH5 were also upregulated. Methylated RNA immunoprecipitation sequencing analysis revealed that 133 genes were m 6 A hypermethylated, and 711 genes were m 6 A hypomethylated. Biological functional analysis revealed that the differential m 6 A methylated genes were mainly related to oxidative stress, lipid metabolism, inflammatory response, and so on. In the present study, we also identified 62 genes with significant changes in m 6 A modification and mRNA expression levels. These findings elucidated the m 6 A modification spectrum induced by S. aureus in MAC-T cells and provide the basis for subsequent m 6 A research on mastitis., Competing Interests: There is no conflict of interest., (Copyright © 2022 Ting Li et al.)

Lactational mastitis is an excellent target to study possible interactions between HMOs, immune factors and milk microbiota due to the infectious and inflammatory nature of this condition. In this work, microbiological, immunological and HMO profiles of milk samples from women with (MW) or without (HW) mastitis were compared. Secretor status in women (based on HMO profile) was not associated to mastitis. DFLNH, LNFP II and LSTb concentrations in milk were higher in samples from HW than from MW among Secretor women. Milk from HW was characterized by a low bacterial load (dominated by Staphylococcus epidermidis and streptococci), high prevalence of IL10 and IL13, and low sialylated HMO concentration. In contrast, high levels of staphylococci, streptococci, IFNγ and IL12 characterized milk from MW. A comparison between subacute (SAM) and acute (AM) mastitis cases revealed differences related to the etiological agent (S. epidermidis in SAM; Staphylococcus aureus in AM), milk immunological profile (high content of IL10 and IL13 in SAM and IL2 in AM) and milk HMOs profile (high content of 3FL in SAM and of LNT, LNnT, and LSTc in AM). These results suggest that microbiological, immunological and HMOs profiles of milk are related to mammary health of women., (© 2022. The Author(s).)