Enterococci are a group of microorganisms that have a controversial position from some scientific points of view. The species of the greatest clinical importance are E. faecalis and E. faecium, which are common agents of nosocomial infections. However, enterococci also have important applications in the dairy industry, as they are used as non-starter lactic acid bacteria (NSLAB) in a variety of cheeses, especially artisanal cheeses. The aim of this study was to determine the presence of representatives from the Enterococcus genus using PCR and MALDI-TOF MS methods on samples of raw milk, processing environment swabs, and cheese from four different artisanal dairy plants in Slovakia. Among the 136 isolates of enterococci, 9 species of genus Enterococci (E. faecalis, E. faecium, E. durans, E. devriesi, E. hirae, E. italicus, E. casseliflavus, E. malodoratus, and E. gallinarum) were identified and were tested for their antimicrobial resistance (AMR) to 8 antibiotics (amoxicillin, penicillin, ampicillin, erythromycin, levofloxacin, vancomycin, rifampicin, and tetracycline); most of them were resistant to rifampicin (35.3%), ampicillin (22.8%), and tetracycline (19.9%). A PCR analysis of vanA (4.41%) and tetM (14.71%) revealed that antimicrobial resistance genes were present in not only phenotypic resistant isolates of enterococci but also susceptible isolates. The investigation of antimicrobial resistance in enterococci during the cheesemaking process can be a source of valuable information for public health in the concept of “One Health”.

Staphylococci are common pathogens in dogs, causing a variety of dermatological problems. This study aimed to characterize the prevalence, antibiotic resistance, and biofilm-forming potential of Staphylococcus species isolated from the skin of shelter dogs. Overall, 108 samples were collected from the hairless skin areas of dogs in a shelter over one year. Isolates were cultured using standard microbiological methods and identified through biochemical testing, MALDI-TOF MS, and multiplex PCR. A total of 67 Staphylococcus isolates were identified, with S. pseudintermedius being the most prevalent. Antibiotic susceptibility was assessed using disk diffusion and MIC methods, revealing high resistance to ampicillin, erythromycin, and tetracycline. Notably, 12 multidrug-resistant SIG (S. intermedius group; S. pseudintermedius) and 4 CoNS strains (coagulase-negative staphylococci; S. equorum) were identified. Biofilm production was evaluated using a crystal violet assay, showing variable biofilm-forming capabilities among isolates and PCR, to confirm genes associated with biofilm formation. These findings highlight the presence of multidrug-resistant Staphylococcus species in shelter dogs, emphasizing the need for careful monitoring and antibiotic stewardship to manage potential risks to both animal and human health.

The work studies the survival of added selected probiotic bacteria Lactobacillus acidophilus (S1), Lactobacillus casei (S2), and Lactobacillus plantarum96 (S3) in semi-hard cheese with low-cooking curd during the maturation process. Cheeses were made according to the standard procedure (Polyfood SI 050 device). Probiotic lactobacilli strains Lactobacillus acidophilus (S1), Lactobacillus casei (S2), and Lactobacillus plantarum96 (S3) used in this study were added into the milk before the renneting process. The manufactured cheeses were matured for 6 months at the temperature of 10 °C. Cheese samples were taken for pH and titratable acidity measurements, lactobacilli enumeration, and chemical analysis at 30, 60, 90, 120, 150 and 180 days of maturation. At the end of the experiment (180 days) the cheese samples were analyzed also for the amount of lactic acid and protein contents. Initial numbers of lactobacilli inoculated into the milk (10⁸ CFU mL⁻¹) decreased during the first 2 weeks of maturation and reached from 2.15 10⁷ CFU g⁻¹ in S1 cheese to 4.32 10⁷ CFU g⁻¹ in S3 cheese. The number of Lactobacillus acidophilus strain bacteria at the beginning of the maturation period was 2.47.10⁷ CFU g⁻¹ and declined until day 120 of maturation to the number of 0.45 10⁶ CFU g⁻¹. In the last month of the experiment day 180 the viable cell numbers started to rise up to the final number of 0.41 10⁷ CFU g⁻¹. The numbers of Lactobacillus plantarum96 varied around 10⁸ CFU g⁻¹ during the whole period of the experiment. According to our results it was detected that in all experimental cheeses, the used probiotic lactobacilli reached the values above 10⁶ CFU g⁻¹. Thus the legislated and therapeutic minimum limits set for the products containing probiotic bacteria for human diet were fulfilled.

Dysbiosis of the gut microbiota, caused by antibiotics, plays a key role in the establishment of Clostridioides difficile CD). Toxin-producing strains are involved in the pathogenesis of Clostridioides difficile infection (CDI), one of the most common hospital-acquired infections. We cultured a total of 84 C. difficile isolates from stool samples of patients hospitalized at Louis Pasteur University Hospital in Kosice, Slovakia, that were suspected of CDI and further characterized by molecular methods. The presence of genes encoding toxin A, toxin B, and binary toxin was assessed by toxin-specific PCR. CD ribotypes were detected using capillary-based electrophoresis ribotyping. A total of 96.4% of CD isolates carried genes encoding toxins A and B, and 54.8% of them were positive for the binary toxin. PCR ribotyping showed the presence of three major ribotypes: RT 176 (n = 40, 47.6%); RT 001 (n = 23, 27.4%); and RT 014 (n = 7, 8.3%). Ribotype 176 predominated among clinical CD isolates in our hospital. The proportion of RT 176 and RT 001 in four hospital departments with the highest incidence of CDI cases was very specific, pointing to local CDI outbreaks. Based on our data, previous use of antibiotics represents a significant risk factor for the development of CDI in patients over 65 years of age.

Methicillin-resistant coagulase-negative staphylococci (MRCoNS) are a major cause of infectious diseases, owing to their ability to form biofilms and colonize community and hospital environments. MRCoNS strains were identified using biochemical tests, an MALDI-TOF MS analyzer, and PCR-based 16S rRNA gene confirmation. This study was designed to assess antibiotic resistance and biofilm-forming capacity and to determine the presence of the mecA, mecC, agrA, srtA, icaABCD, bap, fnbAB, and clfAB genes in MRCoNS isolates. From patients undergoing random screening during hospitalization in the Orthopedics Clinic in Slovakia, 28 strains of MRCoNS were identified: S. epidermidis (n = 10), S. hominis (n = 8), S. haemolyticus (n = 4), S. lugdunensis (n = 3), while S. simulans, S. pasteuri, and S. warneri were detected only once. The highest rates of resistance were observed for ampicillin, oxacillin, rifampicin, trimethoprim (100%), and erythromycin (62%). The mecA gene was detected in 12 analyzed isolates. In 12 isolates, MDR, strong efflux pump activity, and strong or moderate biofilm formation were simultaneously detected. Our findings highlight the problems posed by biofilm-forming, resistant CoNS in hospitalized patients and the importance of diagnostics, separation, rapid treatment, and proper hospital hygiene.