Bovine Mastitis Pathogens Research Articles

Unravelling the complex mechanisms of multidrug resistance in bovine mastitis pathogens: Insights into antimicrobial resistance genes, biofilm dynamics, and efflux systems

Mastitis remains a paramount economic threat to dairy livestock, with antibiotic resistance severely compromising treatment efficacy. This study provides an in-depth investigation into the multidrug resistance (MDR) mechanisms in bacterial isolates from bovine mastitis, emphasizing the roles of antimicrobial resistance genes (ARGs), biofilm formation, and active efflux systems. A total of 162 Staphylococci, eight Escherichia coli, and seven Klebsiella spp. isolates were obtained from 215 milk samples of clinical and subclinical mastitis cases. Antibiotic susceptibility testing identified Twenty Staphylococci (12.35 %), six E. coli (75 %) and seven Klebsiella (100 %) identified as MDR displaying significant resistance to β-lactams and tetracyclines The Multiple Antibiotic Resistance (MAR) index of these isolates ranged from 0.375 to 1.0, highlighting extensive resistance. Notably, 29 of the 33 MDR isolates produced biofilms on Congo red agar, while all exhibited biofilm formation in the Microtitre Plate assay. Critical ARGs (blaZ, blaTEM, blaCTX-M, tetM, tetA, tetB, tetC, strA/B, aadA) and efflux pump genes (acrB, acrE, acrF, emrB, norB) regulating active efflux were identified. This pioneering study elucidates the synergistic contribution of ARGs, biofilm production, and efflux pump activity to MDR in bovine mastitis pathogens. To our knowledge, this comprehensive study is the first of its kind, offering novel insights into the complex resistance mechanisms. The findings underscore the imperative need for advanced antibiotic stewardship and strategic interventions in dairy farming to curb the rise of antibiotic-resistant infections, thereby protecting both animal and public health.

Distribution of Bovine Mastitis Pathogens in Quarter Milk Samples from Bavaria, Southern Germany, between 2014 and 2023-A Retrospective Study.

The objective of this study was to investigate the distribution of mastitis pathogens in quarter milk samples (QMSs) submitted to the laboratory of the Bavarian Animal Health Service (TGD) between 2014 and 2023 in general, in relation to the clinical status of the quarters, and to analyze seasonal differences in the detection risk. Each QMS sent to the TGD during this period was analyzed and tested using the California Mastitis Test (CMT). Depending on the result, QMSs were classified as CMT-negative, subclinical, or clinical if the milk character showed abnormalities. Mastitis pathogens were detected in 19% of the QMSs. Non-aureus staphylococci (NAS) were the most common species isolated from the culture positive samples (30%), followed by Staphylococcus (S.) aureus (19%), Streptococcus (Sc.) uberis (19%), and Sc. dysgalactiae (9%). In culture-positive QMSs from CMT-negative and subclinically affected quarters, the most frequently isolated pathogens were NAS (44% and 27%, respectively), followed by S. aureus (25% and 17%, respectively) and Sc. uberis (8% and 22%, respectively). In QMSs from clinically affected quarters, the most frequently isolated pathogens were Sc. uberis (32%), S. aureus (13%), Sc. dysgalactiae (11%), and Escherichia (E.) coli (11%). The distribution of NAS and Sc. uberis increased throughout the study period, while that of S. aureus decreased. From June to October, QMSs from subclinically affected quarters increased and environmental pathogens, such as Sc. uberis, were detected more frequently. In conclusion, this study highlights the dynamic nature of the distribution of mastitis pathogens, influenced by mastitis status and seasonal factors. Environmental pathogens still play an important role, especially in clinical mastitis and seasonal dependency, with the number of positive samples continuing to increase. It is therefore essential to continue mastitis control measures and to regularly monitor the spread of mastitis pathogens in order to track trends and adapt targeted prevention measures.

Antibacterial activity of natural flavones against bovine mastitis pathogens: in vitro, SAR analysis, and computational study

Bovine mastitis is a worldwide disease affecting dairy cattle and causes major economic losses in the dairy industry. Recently, the emergence of microbial resistance to the current antibiotics complicates the treatment protocol which necessitates antibiotic stewardship and further research to find new active compounds. Recently, phytobiotics have gained interest in being used as an alternative to antibiotics in the poultry industry as an antibiotic stewardship intervention. This study evaluated the in vitro antibacterial activity of 16 flavonoids against bovine mastitis pathogens. Two flavones: 2-(4-methoxyphenyl)chromen-4-one (1) and 2-(3-hydroxyphenyl)chromen-4-one (4) showed inhibition of the growth of Klebsiella oxytoca with MIC values range (25–50 µg mL− 1) followed by a structure-activity relationship (SAR) study indicating that the presence of a hydroxyl group at C-3` or methoxy at C-4` increases the activity against Klebsiella oxytoca while the presence of hydroxyl group at C-7 decreases the activity. Furthermore, a structure-based drug development approach was applied using several in silico tools to understand the interactions of active flavones at the active site of the DNA gyrase protein. Compound (4) showed a higher docking score than quercetin (standard) which is known to have antibacterial activity by inhibiting the DNA gyrase. In addition, the structure-based pharmacophores of compound (4) and quercetin showed similar pharmacophoric features and interactions with DNA gyrase. Based on our findings, compounds (1) and (4) are promising for further study as potential anti-microbial phytochemicals that can have a role in controlling bovine mastitis as well as to investigate their mechanism of action further.

The relationship between resistance evolution and carbon metabolism in Staphylococcus xylosus under ceftiofur sodium stress.

Staphylococcus xylosus has emerged as a bovine mastitis pathogen with increasing drug resistance, resulting in substantial economic impacts. This study utilized iTRAQ analysis to investigate the mechanisms driving resistance evolution in S. xylosus under ceftiofur sodium stress. Findings revealed notable variations in the expression of 143 proteins, particularly glycolysis-related proteins (TpiA, Eno, GlpD, Ldh) and peptidoglycan (PG) hydrolase Atl. Following the induction of ceftiofur sodium resistance in S. xylosus, the emergence of resistant strains displaying characteristics of small colony variants (SCVs) was observed. The transcript levels of TpiA, Eno, GlpD and Ldh were up-regulated, TCA cycle proteins (ICDH, MDH) and Atl were down-regulated, lactate content was increased, and NADH concentration was decreased in SCV compared to the wild strain. That indicates a potential role of carbon metabolism, specifically PG hydrolysis, glycolysis, and the TCA cycle, in the development of resistance to ceftiofur sodium in S. xylosus.

Group B Streptococcus Sequence Type 103 as Human and Bovine Pathogen, Brazil.

Group B Streptococcus sequence type 103 is known primarily as a bovine mastitis pathogen. In Brazil, it has circulated in cattle and humans since the 1990s. It lacks scpB and, in humans, was found only among carriage isolates. Bovine-human interspecies transmission may have contributed to its evolution and spread.

Prevalence, antimicrobial susceptibility profiles and resistant gene identification of bovine subclinical mastitis pathogens in Bangladesh

Subclinical mastitis (SCM), a silent threat in the dairy sector of Bangladesh poses a significant economic impact and serves as a potential source of infection for healthy cows, hindering efforts to achieve milk self-sufficiency. Despite the importance of this issue, limited research has been conducted on mastitis in Sylhet region of Bangladesh. This study aimed to investigate the molecular prevalence, antimicrobial susceptibility profile and resistant genes detection on pathogens (Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae) causing SCM. In a cross-sectional study utilizing convenience sampling, 325 milk samples were collected from apparently healthy dairy cows. Initially, SCM was detected using the modified Whiteside test (MWST) method. Suspected positive samples were then subjected to bacteriological culture and standard biochemical assays, followed by molecular identification through polymerase chain reaction (PCR). Finally, antimicrobial susceptibility testing was conducted on all PCR-positive samples using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar. In the Sylhet district, the prevalence of SCM was 64.92 % (211 out of 325) at the individual animal level and 82.69 % (43 out of 52) at the herd level. Among the SCM-positive animals, S.aureus was found in 31.75 % (67 out of 211) of cases, E.coli in 81.99 % (173 out of 211), and K.pneumoniae in 66.82 % (141 out of 211). K.pneumoniae had the highest prevalence at 60 % in Zakiganj, S.aureus at 45 % in Zakiganj, and E.coli at 72 % in Bishwanath Upazila. Extended spectrum beta lactamase (ESBL) genes blaTEM, blaOXA, blaCTX-M1,blaCTX-M2,MultiCaseDHA were identified. Additionally, antibiotic resistance genes tet(A), AAC(3)-iv, and Sul1 were also detected. The pathogens exhibited resistance to Penicillins (ampicillin, amoxicillin), Cephems (cefuroxime, ceftazidime), and Tetracyclines (tetracycline). However, all three bacteria were highly sensitive to meropenem, amikacin, gentamicin, ciprofloxacin, and sulfamethoxazole-trimethoprim. These findings highlight the critical need for targeted antimicrobial stewardship and effective control measures to mitigate the impact of SCM on dairy production and animal health in the Sylhet region of Bangladesh.

Application of MALDI-TOF MS to Identify and Detect Antimicrobial-Resistant Streptococcus uberis Associated with Bovine Mastitis.

Streptococcus uberis is a common bovine mastitis pathogen in dairy cattle. The rapid identification and characterization of antimicrobial resistance (AMR) in S. uberis plays an important role in its diagnosis, treatment, and prevention. In this study, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify S. uberis and screen for potential AMR biomarkers. Streptococcus uberis strains (n = 220) associated with bovine mastitis in northern Thailand were identified using the conventional microbiological methods and compared with the results obtained from MALDI-TOF MS. Streptococcus uberis isolates were also examined for antimicrobial susceptibility using a microdilution method. Principal component analysis (PCA) and the Mann-Whitney U test were used to analyze the MALDI-TOF mass spectrum of S. uberis and determine the difference between antimicrobial-resistant and -susceptible strains. Using MALDI-TOF MS, 73.18% (161/220) of the sampled isolates were identified as S. uberis, which conformed to the identifications obtained using conventional microbiological methods and PCR. Using PCR, antimicrobial-resistant strains could not be distinguished from antimicrobial-susceptible strains for all three antimicrobial agents, i.e., tetracycline, ceftiofur, and erythromycin. The detection of spectral peaks at 7531.20 m/z and 6804.74 m/z was statistically different between tetracycline- and erythromycin-resistant and susceptible strains, respectively. This study demonstrates a proteomic approach for the diagnosis of bovine mastitis and potentially for the surveillance of AMR among bovine mastitis pathogens.

Light-activated conjugated polymer nanoparticles to defeat pathogens associated with bovine mastitis

Bovine mastitis (BM) represents a significant challenge in the dairy industry. Limitations of conventional treatments have prompted the exploration of alternative approaches, such as photodynamic inactivation (PDI). In this study, we developed a PDI protocol to eliminate BM-associated pathogens using porphyrin-doped conjugated polymer nanoparticles (CPN). The PDI-CPN protocol was evaluated in four mastitis isolates of Staphylococcus and in a hyper-biofilm-forming reference strain. The results in planktonic cultures demonstrated that PDI-CPN exhibited a bactericidal profile upon relatively low light doses (∼9.6 J/cm2). Furthermore, following a seven-hour incubation period, no evidence of cellular reactivation was observed, indicating a highly efficient post-photodynamic inactivation effect. The successful elimination of bacterial suspensions encouraged us to test the PDI-CPN protocol on mature biofilms. Treatment using moderate light dose (∼64.8 J/cm2) reduced biofilm biomass and metabolic activity by up to 74% and 88%, respectively. The impact of PDI-CPN therapy on biofilms was investigated using scanning electron microscopy (SEM), which revealed nearly complete removal of the extracellular matrix and cocci. Moreover, ex vivo studies conducted on bovine udder skin demonstrated the efficacy of the therapy in eliminating bacteria from these scaffolds and its potential as a prophylactic method. Notably, the histological analysis of skin revealed no signs of cellular degeneration, suggesting that the protocol is safe and effective for BM treatment. Overall, this study demonstrates the potential of PDI-CPN in treating and preventing BM pathogens. It also provides insights into the effects of PDI-CPN on bacterial growth, metabolism, and survival over extended periods, aiding the development of effective control strategies and the optimization of future treatments.

Point-of-need mastitis pathogen biosensing in bovine milk: From academic sample preparation novelty to industry prototype field testing

Bovine mastitis is an inflammation of the mammary gland, and it is the most common infectious disease in dairy cattle. Mastitis reduces milk yield and quality, costing dairy farmers millions of dollars each year. The aim of this study was to develop a point-of-need test for identifying mastitis pathogens that is field portable, cost-effective and can be used with minimal training. Using a proprietary polymer-based milk sample preparation method to rapidly extract pathogen DNA in milk samples, we demonstrated quantitative Polymerase Chain Reaction (qPCR) assays for six common bovine bacterial mastitis pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Mycoplasma bovis and Escherichia coli. We also implemented this sample preparation method on a prototype point-of-need system in a proof-of-concept field trial to evaluate user experience. Importantly, the protype system enabled a sample-to-result turnaround time of within 70 min to quantitatively detect all six target pathogens. The key advantage of our point-of-need prototype system is being culture-independent yet providing automated milk sample preparation for molecular identification of key mastitis pathogens by non-expert users. Our point-of-need prototype system showed a good correlation to laboratory-based qPCR for target pathogen detection outcomes, thus potentially removing the need for milk samples to be transported off-site for laboratory testing. Above all, we successfully achieved our objective of developing a point-of-need biosensor technology for mastitis and increased its readiness level with industry partners towards technology commercialization.

Draft genome sequences of 148 Klebsiella pneumoniae species complex members from bovine and human hosts.

Klebsiella pneumoniae species complex members, particularly K. pnemoniae sensu stricto, are common bovine clinical mastitis pathogens and often the cause of hospital- and community-acquired infections in humans. Here, we present 148 draft genome assemblies and annotations of K. pneumoniae species complex members from bovine and human hosts in Canada.

Discriminating bovine mastitis pathogens by combining loop-mediated isothermal amplification and amplicon-binding split trehalase assay.

Bovine mastitis is predominantly caused by intramammary infections with various Gram-positive and Gram-negative bacteria, requiring accurate pathogen identification for effective treatment and antimicrobial resistance prevention. Here, a novel diagnostic method was developed to detect mastitis pathogens in milk samples by combining loop-mediated isothermal amplification with a split enzyme biosensor whereby trehalase fragments were fused with a DNA-binding protein, SpoIIID. Three primer sets, LAMPstaph, LAMPstrep, and LAMPneg, harboring SpoIIID recognition sequences targeted Staphylococcus, Streptococcus, and Gram-negative pathogens, respectively. Limits of detection were determined for DNA extracted from bacterial culture and bacteria-spiked milk. The combined method detected as low as 2, 24, and 10 copies of genomic DNA of staphylococci, streptococci and Escherichia coli and 11 CFU/ml for milk spiked with Escherichia coli. Higher detection limits were observed for Gram-positive bacteria in spiked milk. When testing genomic DNA of 10 mastitis isolates at concentrations of 106 and 103 copies per reaction, no cross-reactivity was detected for LAMPstaph nor LAMPstrep, whereas the LAMPneg assay cross-reacted only with Corynebacterium sp. at the highest concentration. This combined method demonstrated the potential to distinguish mastitis pathogenic Gram types for a rapid decision of antimicrobial treatment without culturing.

Nanoparticles in Managing Bovine Mastitis Pathogens—Isolation, Identification, and Therapeutic Strategies

Nanoparticles in Managing Bovine Mastitis Pathogens—Isolation, Identification, and Therapeutic Strategies

Antimicrobial resistance of bovine mastitis pathogens

Antimicrobial resistance of bovine mastitis pathogens

Comparative anti-microbial and anti-biofilm activities of postbiotics derived from kefir and normal raw milk lactic acid bacteria against bovine mastitis pathogens

Bovine mastitis, caused by pathogens including Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli, poses a significant challenge to the dairy industry. This study aimed to investigate the underlying mechanisms of the anti-microbial and anti-biofilm activities of postbiotics derived from lactic acid bacteria against bovine mastitis pathogens. Kefir-derived Lentilactobacillus kefiri LK1 produced significantly more lactic acid than normal raw milk-derived Enterococcus faecium EFM2, which was attributed to differences in biochemical activities. At an optimum concentration of 25%, both L. kefiri LK1 (POS_LK1) and E. faecium EFM2 postbiotics (POS_EFM2) exhibited anti-microbial and anti-biofilm activities by modulating hydrophobicity, auto-aggregation, and exopolysaccharide (EPS) production phenotypes and genotypes of bovine mastitis pathogens. POS_LK1 treatment reduced auto-aggregation and EPS production by E. faecalis and downregulated wspA and pelA expression in P. aeruginosa compared to those in POS_EFM2. Therefore, our results suggest that POS_LK1 could be used in the dairy industry as an anti-biofilm agent to effectively prevent pathogenic biofilm-induced bovine mastitis.

Antimicrobial resistance profiles of common mastitis pathogens on large Chinese dairy farms

The primary objective of this study was to determine the antimicrobial resistance (AMR) profile of common mastitis pathogens on large Chinese dairy farms. A total of 673 isolates, including Staphylococcus aureus (14.41%, 97/673), coagulase-negative staphylococci (CNS, 52.30%, 352/673), Streptococcus agalactiae (5.64%, 38/673), non-agalactiae streptococci (7.42%, 50/673), Acinetobacter spp. (7.72%, 52/673), Escherichia spp. (6.39%, 43/673), and Klebsiella spp. (6.09%, 41/673), were collected from 15 large Chinese dairy farms in 12 provinces. The AMR profiles were measured using a microdilution method. Our results showed that more than 75% of Staph. aureus (87/97) and CNS (291/352) were resistant to penicillin (PEN). More than 30% of Escherichia spp. (15/43) showed resistance to ampicillin (AMP). However, less than 10% CNS and non-agalactiae streptococci showed resistance to amoxicillin/clavulanate (AMC; 1/352; 0/50), cephalexin (LEX; 1/352; 0/50), ceftiofur (EFT; 10/352; 0/50), and rifaximin (RIX; 21/352; 2/50); less than 10% Staph. aureus showed resistance to AMC (1/97), oxacillin (OX; 3/97), LEX (1/97), EFT (2/97), and RIX (2/97); less than 10% Strep. agalactiae showed resistance to PEN (3/38), AMC (0/38), LEX (0/38), EFT (0/38), and RIX (0/38); and less than 10% Escherichia spp. showed resistance to AMC (1/43) and EFT (4/43). These results suggested that most mastitis pathogens were susceptible to most antimicrobials with exceptions of Staph. aureus tested against penicillin or ampicillin and CNS against penicillin or oxacillin. To control the AMR threat in Chinese dairy farms, a nationwide surveillance program for AMR of bovine mastitis pathogens is needed.

Development of engineered endolysins with in vitro intracellular activity against streptococcal bovine mastitis-causing pathogens.

Bacteriophage-derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic-resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae, also targeting intracellular survival and biofilm formation. For this purpose, high-throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram-positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time-kill assays in phosphate-buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis, also showing similar activity against S. agalactiae and S. dysgalactiae. This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta-lactam penicillin used to intramammarily treat bovine Gram-positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add-on in the treatment of bovine streptococcal mastitis.

Comparison between broth microdilution and agar disk diffusion methods for antimicrobial susceptibility testing of bovine mastitis pathogens

In order to counter the antibiotic resistance phenomenon, a prudent and rational use of antimicrobials should be driven by an accurate clinical diagnosis and, when possible, by the isolation of the etiological agent followed by susceptibility testing, with the aim to select the most suitable molecule for therapy. Cow mastitis is considered the main cause of antibiotic use in the cattle breeding sector. The purpose of this study was to compare the broth microdilution (BMD) method performed with Sensititre Custom Plates and the agar disk diffusion (ADD) method in determining antimicrobial susceptibility of 215 isolates from bovine mastitis, including contagious pathogens (Staphylococcus aureus, Streptococcus agalactiae) and environmental (Streptococcus uberis, Streptococcus dysgalactiae, Enterococcus spp., Escherichia coli, Serratia marcescens, Klebsiella pneumoniae). We compared results of the following antimicrobials: amoxicillin/clavulanic acid, ampicillin, cefazolin, ceftiofur, enrofloxacin, erythromycin, kanamycin, oxacillin, penicillin, pirlimycin, rifampicin and trimethoprim/sulphonamides. We applied MIC breakpoints and zone diameter breakpoints as recommended by CLSI and EUCAST. MIC and disk diffusion diameters were compared for 1839 microorganism/antimicrobial combination and discrepancies between the two methods were classified as very major discrepancy (VMD), major discrepancy (MD) and minor discrepancy (MiD). The overall agreement between the two methods was found to be 80.7% with a Cohen's kappa coefficient of 0.397, thus indicating a fair concordance. BMD method and ADD method demonstrated a satisfactory agreement (89 to 100%) for S. aureus and S. marcescens and all antimicrobial agents tested. Low agreement was observed for S. uberis and rifampicin (20%), enrofloxacin (49%), penicillin (51%) and pirlimycin (52%), E. coli and ampicillin (20%), S. dysgalactiae and enrofloxacin (44%), S. agalactiae and rifampicin (25%). A possible explanation for the discrepancies detected could be found in the breakpoints used which, sometimes, are not specific for the tissue-matrix of isolation/animal species/pathogen agent. The majority of the discrepancies found were MiD and MD, revealing a higher restrictiveness of the BMD method, while VMD represented only 0.2% of the total observations, a comforting fact since this type of error may result in treatment failure.

Therapeutic potential of natural products and antibiotics against bovine mastitis pathogen of cows and buffaloes.

The present study aims toevaluate the prevalence and antimicrobial sensitivity ofStaphylococcus aureus associated with bovine mastitis toselected antibiotics and plant extracts. Inthe current study, 140milk samples were collected from cows and buffaloes. Among the 140samples, 93samples were positive for sub-clinical mastitis based onthe California Mastitis Test (CMT). Out ofthe total positive samples, 45 were confirmed for S.aureus onaMannitol salt agar media. The antimicrobial susceptibility test revealed that 44.82% ofthe isolates were resistant tocefoxitin (oxacillin) confirming methicillin-resistant S.aureus (MRSA) with ahigher percentage (51.61%) inthe buffalo than inthe cow samples. Furthermore, the PCR assay confirmed the presence ofthemecA gene inall the MRSA isolates. Among the seven tested antibiotics, sulfamethoxazole + trimethoprim showed high efficacy (71.1%) against methicillin-susceptible S.aureus isolates (MSSA). Oxytetracycline and sulfamethoxazole+ trimethoprim showed 20% efficacy against MRSA followed byenrofloxacin (10%). Onthe other hand, the tested samples from Pistacia chinensis revealed that the ethyl acetate extract ofbark showed amaximum zone ofinhibition of21.3mm against MSSA and MRSA isolates at3000μg/disc. Moreover, the methanol extract ofCotoneaster microphyllus formed a12.3mm and 9.1mm zone ofinhibition against the MSSA and MRSA isolates, respectively.

Gene co-expression in response to Staphylococcus aureus infection reveals networks of genes with specific functions during bovine subclinical mastitis

Staphylococcus aureus is one of the most prevalent contagious bacterial pathogen of bovine mastitis. The subclinical mastitis it causes has long-term economic implications and it is difficult to control. To further understanding of the genetic basis of mammary gland defense against S. aureus infection, the transcriptomes of milk somatic cells from 15 cows with persistent natural S. aureus infection (S. aureus-positive, SAP) and 10 healthy control cows (HC) were studied by deep RNA-sequencing technology. Comparing the transcriptomes of SAP to HC group revealed 4,077 differentially expressed genes (DEG; 1,616 up- and 2,461 downregulated). Functional annotation indicated enrichment of DEG in 94 Gene Ontology (GO) and 47 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Terms related to the immune response and disease processes were mostly enriched for by upregulated DEG, whereas biological process terms related to cell adhesion, cell movement and localization, and tissue development were mostly enriched for by downregulated DEG. Weighted gene co-expression network analysis grouped DEG into 7 modules, the most important module (colored turquoise by software and here referred to as Turquoise module) was positively significantly correlated with S. aureus subclinical mastitis. The 1,546 genes in the Turquoise module were significantly enriched in 48 GO terms and 72 KEGG pathways, with 80% of them being disease- and immune-related terms [e.g., immune system process (GO:0002376), cytokine-cytokine receptor interaction (bta04060) and S. aureus infection (bta05150)]. Some DEG such as IFNG, IL18, IL1B, NFKB1, CXCL8, and IL12B were enriched in immune and disease pathways suggesting their possible involvement in the regulation of the host response to S. aureus infection. Four modules (Yellow, Brown, Blue, and Red) were negatively correlated (significantly) with S. aureus subclinical mastitis, and were enriched in functional annotations involved in the regulation of cell migration, cell communication, metabolic process, and blood circulatory system development, respectively. Application of sparse partial least squares discriminant analysis to genes of the Turquoise module identified 5 genes (NR2F6, PDLIM5, RAB11FIP5, ACOT4, and TMEM53) capable of explaining the majority of the differences in the expression patterns between SAP and HC cows. In conclusion, this study has furthered understanding of the genetic changes in the mammary gland and the molecular mechanisms underlying S. aureus mastitis, as well as revealed a list of candidate discriminant genes with potential regulatory roles in response to S. aureus infection.

Comparative Genomic Analyses of Lactococcus garvieae Isolated from Bovine Mastitis in China.

Lactococcus garvieae is an emerging zoonotic pathogen, but there are few reports regarding bovine mastitis. The prevalence of L. garvieae represents an increasing disease threat and global public health risk. Thirty-nine L. garvieae isolates were obtained from 2,899 bovine clinical mastitis milk samples in 6 provinces of China from 2017 to 2021. Five clonal complexes were determined from 32 multilocus sequence types (MLSTs) of L. garvieae: sequence type 46 (ST46) was the predominant sequence type, and 13 novel MLSTs were identified. All isolates were resistant to chloramphenicol and clindamycin, but susceptible to penicillin, ampicillin, amoxicillin-clavulanic acid, imipenem, ceftiofur, enrofloxacin, and marbofloxacin. Based on genomic analyses, L. garvieae had 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase. Most isolates had lsaD and mdtA antimicrobial resistance (AMR) genes. Based on COG (Clusters of Orthologous Genes database) results, the functions of defense, transcription and replication, and recombination and repair were enhanced in unique genes, whereas functions of translation, ribosomal structure, and biogenesis were enhanced in core genes. The KEGG functional categories enriched in unique genes included human disease and membrane transport, whereas COG functional categories enriched in core genes included energy metabolism, nucleotide metabolism, and translation. No gene was significantly associated with host specificity. In addition, analysis of core genome single nucleotide polymorphisms (SNPs) implied potential host adaptation of some isolates in several sequence types. In conclusion, this study characterized L. garvieae isolated from mastitis and detected potential adaptations of L. garvieae to various hosts. IMPORTANCE This study provides important genomic insights into a bovine mastitis pathogen, Lactococcus garvieae. Comprehensive genomic analyses of L. garvieae from dairy farms have not been reported. This study is a detailed and comprehensive report of novel features of isolates of L. garvieae, an important but poorly characterized bacterium, recovered in the past 5 years in 6 Chinese provinces. We documented diverse genetic features, including predominant sequence type ST46 and 13 novel MLSTs. Lactococcus garvieae had 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase and resistance to chloramphenicol and clindamycin. Most isolates had lsaD and mdtA antimicrobial resistance genes. However, no gene was significantly associated with host specificity. This is the first report that characterized L. garvieae isolates from bovine mastitis and revealed potential host adaptations of L. garvieae to various hosts.