In the current study, we screened a collection of coagulase-negative staphylococci (CoNS) isolates for orthologues of staphylococcal enterotoxins (SEs) involved in S. aureus-related staphylococcal food poisoning (SFP). The amplicons corresponding to SEs were detected in S. chromogenes, S. epidermidis, S. haemolyticus, S. borealis, S. pasteuri, S. saprophyticus, S. vitulinus, S. warneri, and S. xylosus. All amplicons were sequenced and identified as parts of known S. aureus or S. epidermidis SE genes. Quantitative real-time PCR allowed determining the relative copy number of each SE amplicon. A significant portion of the amplicons of the sea, seb, sec, and seh genes occurred at low copy numbers. Only the amplicons of the sec gene identified in three isolates of S. epidermidis displayed relative copy numbers comparable to sec in the reference enterotoxigenic S. aureus and S. epidermidis strains. Consecutive passages in microbiological media of selected CoNS isolates carrying low copy numbers of sea, seb, sec, and seh genes resulted in a decrease of gene copy number. S. epidermidis isolates harbored a high copy number of sec, which remained stable over the passages. We demonstrated that enterotoxin genes may occur at highly variable copy numbers in CoNS. However, we could identify enterotoxin genes only in whole-genome sequences of CoNS carrying them in a stable form at high copy numbers. Only those enterotoxins were expressed at the protein level. Our results indicate that PCR-based detection of enterotoxin genes in CoNS should always require an additional control, like analysis of their presence in the bacterial genome. We also demonstrate S. epidermidis as a CoNS species harboring SE genes in a stable form at a specific chromosome site and expressing them as a protein.

Twelve Staphylococcus borealis strains, isolated in Canada and Poland from milk of cows with intramammary infections, were characterized phenotypically (biochemical reactions on ID 32 STAPH and Biolog Phenotype MicroArrays™ PM1 and PM2A, ability of biofilm production) and genotypically (random amplified polymorphic DNA). In addition, a genomic comparison was done with S. borealis strains of human and porcine origin using the multilocus sequence typing (MLST) technique. The bovine isolates showed a high degree of phenotypic and genotypic diversity, however, they could be differentiated from human strains by the negative test for urease (found in all but one bovine isolate examined with ID 32 STAPH) and positive reaction for D-galactose (on Biolog phenotype microarray PM1) and D-lactose (on both commercial systems). The MLST method, utilizing six concatenated genes of the total length of ∼2930 bp, revealed that bovine strains (irrespective of the country of origin) show a distinctly greater degree of mutual relationship than to the strains of human and porcine origin, suggesting that S. borealis has evolved independently in these hosts. In conclusion, bovine-specific S. borealis can be involved in intramammary infections in cattle.

Conifer-derived essential oils have gained attention as versatile natural additives with potential applications in animal production, including influencing microbial processes and supporting environmental sustainability. This study aimed to characterize the chemical composition of selected conifer essential oils (EOs), evaluate their antimicrobial activity against rumen microorganisms in vitro, and assess the effects of Pinus sylvestris essential oil on rumen fermentation and methane production under in vitro and in vivo conditions. EOs from Thuja occidentalis, Cupressus sempervirens, Juniperus communis, Picea mariana, Pinus sylvestris, and Pinus pinaster were analyzed by GC–MS, and their inhibitory activity against selected rumen bacteria was determined by MIC and IC₅₀ assays. Based on these results, P. sylvestris oil was selected for fermentation experiments. Ninety-two volatile compounds were identified, with monoterpenes as the dominant constituents and α-pinene as the major compound in P. sylvestris oil. In vitro, P. sylvestris oil influenced fermentation in a dose-dependent manner without affecting ruminal pH. In vivo, ruminal pH, ammonia-related parameters, and total VFA concentration were not significantly affected by treatment, whereas several variables showed a significant effect of time. Temporal changes in VFA profiles suggested a transient adaptation of ruminal fermentation. Methane concentration was significantly (p < 0.01) reduced by Pinus sylvestris essential oil supplementation, with a decrease of approximately 28.7% after 14 days. These findings indicate that P. sylvestris EOs may serve as a promising natural modulator of rumen fermentation, although further studies are needed to optimize dosage and confirm long-term effects.

Background: Enterococcus faecalis is one of the most frequent causes of catheter-associated urinary tract infections, largely due to its ability to form biofilms on indwelling urinary catheter surfaces, which enhance bacterial persistence and antimicrobial tolerance. Sub-minimum inhibitory concentrations (sub-MICs) of antimicrobials frequently occur in clinical settings, and growing evidence suggests that such suboptimal exposures can induce bacterial biofilm formation. We hypothesized that exposure to sub-MICs of amoxicillin, ciprofloxacin, and nitrofurantoin, antimicrobials commonly employed in the treatment of urinary tract infections, would enhance the biofilm-forming capacity of E. faecalis strains. Objective: To investigate the effects of sub-MICs of amoxicillin, ciprofloxacin, and nitrofurantoin on biofilm formation and biofilm-associated gene expression. The study focused on key biofilm-related genes, including those encoding aggregation substance protein (asa1), collagen adhesin (ace), E. faecalis surface protein (esp), gelatinase (gelE), cytolysin activator A (cylA), endocarditis antigen A (efaA), and the endocarditis- and biofilm-associated pili subunit A (ebpA) in E. faecalis. Methods: Two strains, E. faecalis ATCC 29212 and strain 54, were exposed to 1/8× and 1/4× MIC of amoxicillin, ciprofloxacin, and nitrofurantoin in either artificial urine medium (AUM) or tryptone soya broth (TSB). Bacterial growth kinetics were monitored by optical density measurements, while biofilm formation was quantified using a microtiter plate biofilm assay. The expression of biofilm-associated genes was analyzed using quantitative reverse transcription PCR (RT-qPCR) at 24 and 48 h following exposure to sub-MICs of amoxicillin under flow conditions mimicking the urinary tract milieu. Results: Exposure to sub-MICs of the three antimicrobials did not significantly affect bacterial growth in either strain or culture medium. Sub-MICs of amoxicillin significantly enhanced biofilm formation, with the most pronounced effect observed at 1/4× MIC in both AUM and TSB. In contrast, ciprofloxacin and nitrofurantoin exerted inhibitory effects on biofilm formation across both media. Gene expression analysis demonstrated time- and strain-dependent responses to amoxicillin exposure. E. faecalis ATCC 29212 exhibited a moderate, coordinated upregulation of adhesion- and biofilm-associated genes, particularly at 48 h. By comparison, E. faecalis strain 54 showed a stronger and more dynamic transcriptional response, characterized by early and sustained induction of key biofilm-related genes, including esp and gelE, as well as a pronounced late upregulation of ebpA. Conclusions: These findings emphasize the importance of maintaining therapeutically effective antimicrobial concentrations, as sub-inhibitory amoxicillin exposure may promote biofilm-associated persistence and potentially compromise treatment efficacy.

In this study, we characterized the chemical composition of lime essential oil (Citrus aurantifolia) by GC–MS and evaluated the effects of lime essential oil and its major constituent, (R)-(+)-limonene, on rumen fermentation, methane production, microbial community structure and metabolic responses in dairy cows. In vitro assays were used to establish effective doses for in vivo application. In vivo supplementation with lime essential oil on day 14 significantly increased ruminal pH (6.96 vs. 6.66; p < 0.05), total VFA concentration (4.84 vs. 4.11 mg mL⁻¹; p < 0.05), serum glucose (3.92 vs. 3.63 mmol L⁻¹; p < 0.05) and bicarbonate concentration (26.75 vs. 22.35 mmol L⁻¹; p < 0.05), indicating improved fermentation efficiency and buffering capacity. Metabolic profiling revealed elevated glucose and reduced non-esterified fatty acids, suggesting enhanced energy utilization and decreased lipid mobilization, without adverse effects on liver enzymes (AST, ALT, GGT), lipid profile or acid–base balance. Rumen microbiota remained stable and was dominated by Bacteroidota and Bacillota; no significant changes occurred in alpha or beta diversity. (R)-(+)-limonene selectively reduced the abundance of Methanobacteriota (p = 0.04). Methane concentrations were not significantly affected. Both additives exerted beneficial, dose-dependent effects on rumen function and host metabolism and show promise as natural feed additives in dairy cows.

Red blood cell (RBC) concentrates remain at risk of bacterial contamination during cold storage. Although infrequent, Yersinia enterocolitica poses a significant blood safety risk. This study aimed to assess Y. enterocolitica bioserotype growth in RBC concentrates, serum sensitivity, and genetic diversity including iron metabolism genes. Ten Y. enterocolitica isolates from bioserotypes 1A, 1B/O:8, 4/O:3, and 2/O:9 were incubated in RBC concentrates and counted on days 3, 7, 14, 21, and 28. After incubation, the isolates were tested in human serum (NHS). Eight genomes were sequenced, analyzed using cgMLST, and screened for iron metabolism genes. The isolates formed two clusters, with 186dz (1A) and Ye8 (1B/O:8) as singletons. After 28 days in the RBC concentrates, the bacterial counts ranged from 1.98 × 10⁵ to 1.2 × 10⁹ CFU/mL, with Ye8 (1B/O:8) achieving the highest growth and one 4/O:3 isolate showing the lowest. All isolates survived 15–30 min in NHS, but the 28s isolate did not survive at 60 min. Serum sensitivity increased in two isolates, decreased in three, and remained unchanged in five. Isolates contained 27–42 iron metabolism genes with multiple allelic variants. The iron metabolism gene content or variants may influence the growth of Y. enterocolitica in RBC.