BACKGROUND: Acinetobacter lwoffii (A. lwoffii) is a Gram-negative bacteria common in the environment, and it is the normal flora in human respiratory and digestive tracts. The bacteria is a zoonotic and opportunistic pathogen that causes various infections, including nosocomial infections. The aim of this study was to identify A. lwoffii strains isolated from bovine milk with subclinical mastitis in China and get a better understanding of its antimicrobial susceptibility and resistance profile. This is the first study to analyze the drug resistance spectrum and corresponding mechanisms of A. lwoffii isolated in raw milk.
RESULTS: Four A. lwoffii strains were isolated by PCR method. Genetic evolution analysis using the neighbor-joining method showed that the four strains had a high homology with Acinetobacter lwoffii. The strains were resistant to several antibiotics and carried 17 drug-resistance genes across them. Specifically, among 23 antibiotics, the strains were completely susceptible to 6 antibiotics, including doxycycline, erythromycin, polymyxin, clindamycin, imipenem, and meropenem. In addition, the strains showed variable resistance patterns. A total of 17 resistance genes, including plasmid-mediated resistance genes, were detected across the four strains. These genes mediated resistance to 5 classes of antimicrobials, including beta-lactam, aminoglycosides, fluoroquinolones, tetracycline, sulfonamides, and chloramphenicol.
CONCLUSIONS: These findings indicated that multi-drug resistant Acinetobacter lwoffii strains exist in raw milk of bovine with subclinical mastitis. Acinetobacter lwoffii are widespread in natural environmental samples, including water, soil, bathtub, soap box, skin, pharynx, conjunctiva, saliva, gastrointestinal tract, and vaginal secretions. The strains carry resistance genes in mobile genetic elements to enhance the spread of these genes. Therefore, more attention should be paid to epidemiological surveillance and drug resistant A. lwoffii.

Non-aureus staphylococci (NAS) are an essential group of bacteria causing antimicrobial resistant intramammary infections in livestock, particularly dairy cows. Therefore, bacteriophages emerge as a potent bactericidal agent for NAS mastitis. This study aimed to obtain NAS-specific bacteriophages using bacterial strains isolated from cows with mastitis, subsequently evaluating their morphological, genomic, and lytic characteristics. Four distinct NAS bacteriophages were recovered from sewage or the environment of Chinese dairy farms; PT1-1, PT94, and PT1-9 were isolated using Staphylococcus chromogenes and PT1-4 using Staphylococcus gallinarum. Both PT1-1 (24/54, 44 %) and PT94 (28/54, 52 %) had broader lysis than PT1-4 (3/54, 6 %) and PT1-9 (10/54, 19 %), but PT1-4 and PT1-9 achieved cross-species lysis. All bacteriophages had a short latency period and good environmental tolerance, including surviving at pH=4-10 and at 30-60℃. Except for PT1-9, all bacteriophages had excellent bactericidal efficacy within 5 h of co-culture with host bacteria in vitro at various multiplicity of infection (MOIs). Based on whole genome sequencing, average nucleotide identity (ANI) analysis of PT1-1 and PT94 can be classified as the same species, consistent with whole-genome synteny analysis. Although motifs shared by the 4 bacteriophages differed little from those of other bacteriophages, a phylogenetic tree based on functional proteins indicated their novelty. Moreover, based on whole genome comparisons, we inferred that cross-species lysis of bacteriophage may be related to the presence of \"phage tail fiber.\" In conclusion 4 novel NAS bacteriophages were isolated; they had good biological properties and unique genomes, with potential for NAS mastitis therapy.

BACKGROUND: Mammary Pathogenic Escherichia coli (MPEC) is an important pathogen that can escape the attack of the host immune system through biofilm formation and proliferate in the mammary gland continuously, resulting in mastitis in cows and causing enormous economic losses. As an effector of AI-2 quorum sensing, LsrR extensively affects the expression levels of hundreds of genes related to multiple biological processes in model E. coli strain. However, the regulatory role of LsrR in MPEC and whether it is involved in pathogenesis has been seldom reported.
RESULTS: In this study, the function of LsrR in strain MPEC5, obtained from a milk sample in dairy cows with mastitis, was investigated by performing high-throughput sequencing (RNA-seq) assays. The results revealed that LsrR down-regulated the transcript levels of fimAICDFGH (encoding Type 1 pili), which have been reported to be associated with biofilm formation process. Biofilm assays confirmed that deletion of lsrR resulted in a significant increase in biofilm formation in vitro. In addition, electrophoretic mobility shift assay (EMSA) provided evidence that LsrR protein could directly bind to the promoter regions of fimAICDFGH in a dose-dependent manner.
CONCLUSIONS: These results indicate that LsrR protein inhibits the biofilm formation ability of MPEC5 by directly binding to the fimAICDFGH promoter region. This study presents a novel clue for further exploration of the prevention and treatment of MPEC.

Bovine mastitis is a widespread and costly disease that affects dairy farming globally, characterized by mammary gland inflammation. Bovine intramammary gland infection has been associated with more than 135 different pathogens of which Staphylococcus aureus is the main etiology of sub-clinical mastitis (SCM). The current study was designed to investigate the prevalence, antibiotic resistance pattern, and the presence of antibiotic resistance genes (mecA, tetK, aacA-aphD and blaZ) in S. aureus isolated from the raw milk of cows with subclinical mastitis. A total of 543 milk samples were collected from lactating cows such as Holstein Friesian (n = 79), Sahiwal (n = 175), Cholistani (n = 107), and Red Sindhi (n = 182) from different dairy farms in Pakistan. From the milk samples microscopic slides were prepared and the somatic cell count was assessed to find SCM. To isolate and identify S. aureus, milk was streaked on mannitol salt agar (MSA) plates. Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+). All the biochemically confirmed S. aureus isolates were molecularly identified using the thermonuclease (nuc) gene. The antibiotic resistance pattern of all the S. aureus isolates was evaluated through the disc diffusion method. Out of 543 milk samples, 310 (57.09%) were positive for SCM. Among the SCM-positive samples, S. aureus was detected in 30.32% (94/310) samples. Out of 94 isolates, 47 (50%) were determined to be multidrug resistant (MDR). Among these MDR isolates, 11 exhibited resistance to Cefoxitin, and hence were classified as methicillin-resistant Staphylococcus aureus (MRSA). The S. aureus isolates showed the highest resistance to Lincomycin (84.04%) followed by Ampicillin (45.74%), while the least resistance was shown to Sulfamethoxazole/Trimethoprim (3.19%) and Gentamycin (6.38%). Polymerase chain reaction (PCR) analysis revealed that 55.31% of the isolates carried blaZ gene, 46.80% carried tetK gene, 17.02% harbored the mecA gene, whereas, aacA-aphD gene was found in 13.82% samples. Our findings revealed a significant level of contamination of milk with S. aureus and half (50%) of the isolates were MDR. The isolated S. aureus harbored various antibiotic resistance genes responsible for the absorbed phenotypic resistance. The alarmingly high prevalence of MDR S. aureus isolates and MRSA strains in these cases possess a serious risk to public health, emphasizes the urgent need to address this issue to protect both human and animal health in Pakistan.

Long non-coding RNAs (LncRNAs) are dysregulated in a variety of human diseases and are highly involved in the development and progression of tumors. Studies on lncRNAs associated with cow mastitis have been lagging behind compared to humans or model animals, therefore, the aim of this study was to explore the mechanism of LncRNAs (CMR) involved in autoprotection against S. aureus mastitis in Bovine Mammary Epithelial Cells (BMECs). First, qRT-PCR was used to examine the relative expression of CMR in a S. aureus mastitis model of BMECs. Then, cell proliferation and apoptosis were detected by EdU and apoptosis assay. Finally, the targeting relationship between miRNAs and mRNA/LncRNAs was determined by dual luciferase reporter gene, qRT-PCR and western blotting techniques. The results showed that CMR was upregulated in the S. aureus mastitis model of BMECs and promoted the expression of inflammatory factors, and SiRNA-mediated CMR inhibited the proliferation of mammary epithelial cells and induced apoptosis. Mechanistically, CMR acts as a competitive endogenous RNA (ceRNA) sponge miR-877, leading to upregulation of FOXM1, a target of miR-877. Importantly, either miR-877 overexpression or FOXM1 inhibition abrogated CMR knockdown-induced apoptosis promoting cell proliferation and reducing inflammatory factor expression levels. In summary, CMR is involved in the regulation of autoprotection against S. aureus mastitis through the miR-877/FOXM1 axis in BMECs and induces immune responses in mammary tissues and cells of dairy cows, providing an important reference for subsequent prevention and control of cow mastitis and the development of targeted drugs.

Escherichia coli (E. coli) can induce severe clinical bovine mastitis, which is to blame for large losses experienced by dairy farms. Macrophage polarization into various states is in response to pathogen infections. Lycopene, a naturally occurring hydrocarbon carotenoid, relieved inflammation by controlling M1/M2 status of macrophages. Thus, we wanted to explore the effect of lycopene on polarization states of macrophages in E. coli-induced mastitis. Macrophages were cultivated with lycopene for 24, before E. coli inoculation for 6 h. Lycopene (0.5 μmol/L) significantly enhanced cell viabilities and significantly reduced lactic dehydrogenase (LDH) levels in macrophages, whereas 2 and 3 μmol/L lycopene significantly enhanced LDH activities. Lycopene treatment significantly reduced the increase in LDH release, iNOS, CD86, TNF-α, IL-1β and phosphatase and tensin homolog (PTEN) expressions in E. coli group. 0.5 μmol/L lycopene significantly increased E. coli-induced downregulation of CD206, arginase I (ARG1), indoleamine 2,3-dioxygenase (IDO), chitinase 3-like 3 (YM1), PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, jumonji domain-containing protein-3 (JMJD3) and interferon regulatory factor 4 (IRF4) levels. Moreover, Ginkgolic acid C17:1 (a specific PTEN inhibitor), 740YPDGFR (a specific PI3K activator), SC79 (a specific AKT activator) or CHPG sodium salt (a specific NF-κB activator) significantly decreased CD206, AGR1, IDO and YM1 expressions in lycopene and E. coli-treated macrophages. Therefore, lycopene increased M2 macrophages via inhibiting NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to E. coli infection in macrophages. These results contribute to revealing the pathogenesis of E. coli-caused bovine mastitis, providing the new angle of the prevention and management of mastitis.

Bovine mastitis, caused by Streptococcus agalactiae (Group B Streptococcus; GBS), poses significant economic challenges to the global dairy industry. Mouse models serves as valuable tools for assessing GBS-induced infections as an alternative to large animals. This study aimed to investigate the LD50 dose, organ bacterial load, and quantification of peritoneal leukocyte populations for GBS serotypes Ia and II isolates from China and Pakistan. Additionally, we measured indicators such as lactoferrin, albumin, and myeloperoxidase (MPO) activity. Pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-2) and anti-inflammatory cytokines (IL-10 and TGF-β) in serum and tissue samples were evaluated using ELISA and qPCR, respectively. BALB/c mice (4 mice per group) received individual intraperitoneal injections of 100 μl containing specific bacterial inoculum concentrations (ranging from 105 to 109 CFU per mouse) of Chinese and Pakistani GBS isolates (serotypes Ia and II). Control groups received 100 μL of sterile PBS. Results revealed that the LD50 bacterial dose causing 50 % mortality in mice was 107 CFU. The highest bacterial load in all experimental groups was quantified in the peritoneum, followed by blood, mammary gland, liver, spleen, lungs, and brain. The most significant bacterial dissemination was observed in mice inoculated with Pakistani serotype Ia at 24 h, with a subsequent notable decline in bacterial counts at day 3. Notably, infection with Pakistani serotype Ia showed a trend of increased total leukocyte counts, significantly higher than Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II. A substantial influx of neutrophils and lymphocytes was observed in response to all tested serotypes, with Pakistani serotype Ia inducing a significantly higher influx compared to other groups (Pakistani serotype II, Chinese serotype Ia, and Chinese serotype II). Furthermore, TNF-α, IL-1β, IL-2, and IL-6 expressions were significantly increased in mice one day after infection with the Pakistani serotype Ia. Compared to mice infected with the Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II, those infected with the Pakistani serotype Ia isolate exhibited the highest production of IL-10 and TGF-β, along with significantly increased concentrations of lactoferrin, albumin, and MPO. These findings suggest that the persistence and severity of infection caused by the Pakistani serotype Ia may be linked to its ability to spread to deeper tissues. This study enhances our understanding of the clinical characteristics of bovine mastitis caused by S. agalactiae in China and Pakistan.

Xinjiang brown cattle are highly resistant to disease and tolerant of roughage feeding. The identification of genes regulating mastitis resistance in Xinjiang brown cattle is a novel means of genetic improvement. In this study, the blood levels of IL-1β, IL-6, IL-10, TNF-α, and TGF-β in Xinjiang brown cattle with high and low somatic cell counts (SCCs) were investigated, showing that cytokine levels were higher in cattle with high SCCs. The peripheral blood transcriptomic profiles of healthy and mastitis-affected cattle were constructed by RNA-seq. Differential expression analysis identified 1632 differentially expressed mRNAs (DE-mRNAs), 1757 differentially expressed lncRNAs (DE-lncRNAs), and 23 differentially expressed circRNAs (DE-circRNAs), which were found to be enriched in key pathways such as PI3K/Akt, focal adhesion, and ECM-receptor interactions. Finally, ceRNA interaction networks were constructed using the differentially expressed genes and ceRNAs. It was found that keynote genes or mRNAs were also enriched in pathways such as PI3K-Akt, cholinergic synapses, cell adhesion molecules, ion binding, cytokine receptor activity, and peptide receptor activity, suggesting that the key genes and ncRNAs in the network may play an important role in the regulation of bovine mastitis.

Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.

BACKGROUND: Staphylococcus aureus is one of the most prevalent pathogens in bovine mastitis, which leads to substantial financial losses for the dairy industry.
RESULTS: In this study, S. aureus (n = 72) was isolated from 18 dairy farms in 15 provinces across China in 2021. The identification of these isolates at the species level was achieved by employing 16S rRNA sequencing. An isothermal amplification method for auxiliary detection of S. aureus was established, which can be employed not only for laboratory detection but also for point-of-care testing (POCT). Molecular characteristics of S. aureus mastitis in Chinese dairy cows were determined through MLST and spa typing. Finally, methicillin-resistant Staphylococcus aureus (MRSA) and MRSA resistance genes were detected using MIC and PCR amplification techniques. 72 isolates were identified as 12 sequence types (STs) and 7 clonal complexes (CC). ST1/CC1 was the dominant prevalent accounting for 33.3 % of the total, and exhibiting a wide distribution range. In terms of spa types, t114 was the dominant type, accounting for 31.9 % of the total, followed by t529 as the second major type. Four S. aureus strains were classified as MRSA according to their levels of oxacillin resistance (MIC ≥4 μg/mL). Among these four MRSA strains, one of them was found to be mecA positive. However, the presence of drug-resistance genes mecA and mecC was not detected in the remaining three MRSA strains, indicating the possible existence of new resistance genes.
CONCLUSIONS: Our study investigated the prevalence of S. aureus mastitis in dairy cows in China, while also examined the molecular characteristics and MRSA strains. This information will help with the clinical monitoring, prevention, and control of S. aureus mastitis in dairy cattle.