Staphylococcus aureus is recognized worldwide as one of the major agents of dairy cow intra-mammary infections. This microorganism can express a wide spectrum of pathogenic factors used to attach, colonize, invade and infect the host. The present study evaluated 120 isolates from eight different countries that were genotyped by RS-PCR and investigated for 26 different virulence factors to increase the knowledge on the circulating genetic lineages among the cow population with mastitis. New genotypes were observed for South African strains while for all the other countries new variants of existing genotypes were detected. For each country, a specific genotypic pattern was found. Among the virulence factors, fmtB, cna, clfA and leucocidins genes were the most frequent. The sea and sei genes were present in seven out of eight countries; seh showed high frequency in South American countries (Brazil, Colombia, Argentina), while sel was harboured especially in one Mediterranean country (Tunisia). The etb, seb and see genes were not detected in any of the isolates, while only two isolates were MRSA (Germany and Italy) confirming the low diffusion of methicillin resistance microorganism among bovine mastitis isolates. This work demonstrated the wide variety of S. aureus genotypes found in dairy cattle worldwide. This condition suggests that considering the region of interest might help to formulate strategies for reducing the infection spreading.

Staphylococcus aureus is recognized worldwide as one of the main contagious mastitis agents in cattle and can express a set of antimicrobial resistance genes and virulence-associated genes that explain the wide range of outcomes of intramammary infections. Staphylococcus aureus strains are heterogeneous: their different resistance and virulence patterns, associated with host-level factors and treatment factors, are related to the severity of infection. The aim of this study was to determine phenotypic antibiotic susceptibility, occurrence of selected antimicrobial resistance genes and other virulence genes in 93 S. aureus strains isolated from clinical mastitis in 6 countries: Argentina, Brazil, Germany, Italy, the United States (New York State), and South Africa. These isolates were tested against a total of 16 drugs (amoxicillin-clavulanate, ampicillin, cefazolin, cefoperazone, cefquinome, enrofloxacin, erythromycin, gentamicin, kanamycin, lincomycin, oxacillin, penicillin, rifampin, spiramycin, sulfamethoxazole/trimethoprim, tylosin) by minimum inhibitory concentration (MIC) assay, and examined for the presence of 6 antibiotic-resistance genes (blaZ, mecA, mecC, ermA, ermB, ermC) and 6 virulence-associated genes (scn, chp, sak, hla, hlb, sea) via PCR analysis. The phenotypic results of this study revealed the presence of 19.4% penicillin-resistant strains, whereas 22.6% of the strains were classified as having resistance (5.4%) or intermediate resistance (17.2%) to erythromycin. Most (96.8%) of the isolates were inhibited by cephalosporins, and all were susceptible to amoxicillin-clavulanate. Two strains (1 from Germany, 1 from Italy) were resistant to oxacillin and were positive for mecA. Among the other antimicrobial resistance genes, the most frequently detected was blaZ (46.2%), and 32.3% of the isolates were positive for erm genes: ermC (21.5%) and ermB (10.8%). The most prevalent virulence gene was hla (100%), followed by hlb (84.9%) and sea (65.6%). These results show a low prevalence of antibiotic multidrug resistance in S. aureus isolates, even if the detection of selected antimicrobial resistance genes did not always correspond with the occurrence of phenotypic antibiotic resistance; the immune evasion cluster gene prevalence was quite low in the samples analyzed.

Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of β-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

Staphylococcus aureus is a major pathogen causing intramammary infection and mastitis in dairy cows. S. aureus genotypes (GT) can differ significantly in their ability to diffuse and persist in the herd; while the association of virulence gene carriage with epidemiological behavior remains unclear, a role for secreted proteins has been postulated. We characterized the secretome of six S. aureus strains belonging to two genotypes with opposite within-herd prevalence, GTB (high) and GTS (low), corresponding to sequence types (ST) 8 and 398, by high-resolution tandem mass spectrometry and differential analysis with Proteome Discoverer. Data are available via ProteomeXchange with identifier PXD029571. Out of 720 identified proteins, 98 were unique or more abundant in GTB/ST8 and 68 in GTS/ST398. GTB/ST8 released more immunoglobulin-binding proteins, complement and antimicrobial peptide inhibitors, enterotoxins, and metabolic enzymes, while GTS/ST398 released more leukocidins, hemolysins, lipases, and peptidases. Furthermore, GTB/ST8 released the von Willebrand factor protein, staphylokinase, and clumping factor B, while GTS released the staphylococcal coagulase and clumping factor A. Hence, GTB/ST8 secretomes indicated a higher propensity for immune evasion and chronicity and GTS/ST398 secretomes for cellular damage and inflammation, consistent with their epidemiological characteristics. Accordingly, GTS/ST398 secretions were significantly more cytotoxic against bovine PBMCs in vitro. Our findings confirm the crucial role of extracellular virulence factors in S. aureus pathogenesis and highlight the need to investigate their differential release adding to gene carriage for a better understanding of the relationship of S. aureus genotypes with epidemiological behavior and, possibly, disease severity.

Recently, the use of antimicrobials on dairy farms has been significantly limited from both the legislative and consumer points of view. This study aims to check the efficacy of selective dry cow therapy (SDCT) versus blanket dry cow therapy (BDCT) on bovine udder in healthy animals. SDTC is when an antibiotic is administered only to infected cows, compared with BDCT, where all cows receive an antimicrobial, regardless of their infection status. The milk samples were collected from enrolled Holstein Friesian cows 7 days before dry-off (T0) and 10 days after calving (T1) to assess somatic cell count (SCC), intramammary infections (IMIs), and milk microbiota variation. After pre-drying sampling, cows are randomly assigned to the following treatments: internal teat sealant alone (ITS; 24 cows), which is a treatment in a cow that does not receive antibiotics in SDTC, or in combination with intramammary antibiotic treatment (A+ITS; 22 cows). Non-statistically significant results are found between the two treatment groups at T1 for SCC, milk yield, and alpha diversity in milk microbiota. A statistically (p < 0.033) T1 IMI decrease is reported in the A+ITS group, and a significant beta diversity analysis is shown between the two timepoints (p = 0.009). This study confirms the possibility of selective drying without new IMI risk or increased SCC at calving, considering healthy cows without contagious infections and SCC values >200,000 cells/mL in the previous lactation.

Streptococcus uberis is an important causative agent for clinical and subclinical mastitis in dairy cattle. The aim of this study was to develop 2 multiplex PCR assays (mPCR) for the simultaneous detection of virulence factors and housekeeping genes for use when investigating the genetic variability and distribution of Strep. uberis virulence factors. The tuf, cpn60, pauA, sodA, sua, oppF, and gapC genes were grouped in assay 1 (mPCR1) and the hasA, hasB, and hasC genes were included in assay 2 (mPCR2). The detection limits were 11.8 pg and 5.9 pg of DNA for mPCR1 and mPCR2, respectively. The 2 mPCR assays were validated with 56 Strep. uberis strains isolated from mastitis milk samples collected from different bovine herds in northern Italy. Results revealed that gapC and oppF were detected in 98.2% of the strains, whereas sua and hasC genes were detected in 94.6 and 89.2% of the strains, respectively. The most common pattern was gapC+, oppF+, cpn60+, sua+, sodA+, pauA+, tuf+, hasA+, hasB+, and hasC+, which appeared in 59% of the strains analyzed. The molecular assays developed in the present study represent a powerful tool for the evaluation of virulence pattern distribution in Strep. uberis strains associated with intramammary infections.