Biofilm-Producing Ability of Staphylococcus aureus Obtained from Surfaces and Milk of Mastitic Cows.

This study was conducted to evaluate the incidence of mastitis in 153 dairy cows and to evaluate the kinetics of adhesion of isolates obtained from surfaces and milk in comparison with the reference strain (RS), CCM 4223. The surfaces of the floor, teat cup, and cow restraints were aseptically swabbed in three replicates (n = 27). Of the total number of infected cows (n = 43), 11 samples were found to be positive for Staphylococcus aureus, 12 samples tested positive for non-aureus staphylococci, 6 samples tested positive for Streptococcus spp., and 11 samples tested positive for other bacteria (Escherichia coli, Pseudomonas spp.) or a mixed infection. The most represented pathogen in milk (11/43) and on surfaces (14/27) was S. aureus. The kinetics of adhesion of the reference strain and isolates of S. aureus on stainless steel surfaces were determined after 3, 6, 9, 12, 24, and 48 h, and 3, 6, 9, 12, and 15 days of incubation. All strains reached counts higher than 5 Log10 CFU/cm2 needed for biofilm formation, except RS (4.40 Log10 CFU/cm2). The isolates of S. aureus revealed a higher capability to form biofilm in comparison with RS during the first 3 h (p < 0.001). Thus, there is a significant difference between the occurrence of S. aureus on monitored surfaces-floor, teat cup, and cow restraints-and the frequency with which mastitis is caused by S. aureus (p < 0.05). This finding raises the possibility that if various surfaces are contaminated by S. aureus, it can result in the formation of biofilm, which is a significant virulence factor.

Effectiveness of sanitation regime in a milking parlour to control microbial contamination of teats and surfaces teat cups'.

INTRODUCTION AND OBJECTIVE: The major sources of bacterial contamination of raw milk are post-harvest manipulation; therefore the disinfection of teat and teat cups which decrease the bacterial load has a positive impact on minimizing new infection rates. The aim of the study was determination of the incidence of pathogens on investigated surfaces, evaluation of the effectiveness of sanitation regime in the reduction of surface microbial load, and determination of the effectiveness of mechanical cleaning of teats in a milking parlour for dairy cows. MATERIAL AND METHODS: Samples from surfaces were taken by microbiological swabs using a sterile cotton swab from area of 5×2 cm2. Sanitation regime was evaluated based on the effectiveness of active substances - lactic acid and sodium hypochlorite. RESULTS: From a total of 105 swab, 44 samples were found positive for Staphylococcus aureus, 16 samples for E. coli, 15 samples for Micrococcus spp., 8 samples for Staphylococcus xylosus, 9 samples for Staphylococcus cohni urealyticum, 1 sample for Enterococcus faecalis. Among isolates, S. aureus was the predominat species from teats - 19/45, teat cups, 15/45 and from wiping cloths 10/15. Sanitation regime was confirmed by a decrease in the number of coliform bacteria (CB) determined on teat and teat cups from 2.33-0.95 Log10 CFU/cm2 (p<0.001) and 0.90-0.62 Log10 CFU/cm2 (p<0.001), respectively, and in the number of total bacteria count (TBC) determined on teat and teat cups from 4.36-0.99 Log10CFU/cm2 (p<0.001), and 1.85-0.77 Log10 CFU/cm2 (p<0.001), respectively. Incidence of CB (2.53 Log10 CFU/cm2) and TBC (3.83 Log10 CFU/cm2) on wiping cloths after mechanical cleaning of udders stress the importance of this step. CONCLUSIONS: Results show that disinfectant with lactic acid as the main active ingredient is suitable for bacterial reduction. Post-milking disinfection of teat and teat cups reduces bacterial contamination and proves to be most effective against environmental bacteria.

Detection of Microbiota during the Fermentation Process of Wine in Relation to the Biogenic Amine Content.

Viticulture is one of the traditional industries in Slovakia, where there are six wine-growing regions: Malokarpatska, Southern Slovakia, Central Slovakia, Nitra, Eastern Slovakia, and Tokaj. This study focuses on the detection of microbiota in soil samples, grape leaves and berries, and samples taken from fermenting must and young wine (the variety Tramín cervený) in relation to the detected concentrations of biogenic amines during the fermentation process. In the examined samples, the number of yeasts and molds (from 3.8 to 6.8 log cfu/g or mL) and TVC (from 3.7 to 6.5 log cfu/g or mL) were determined via culture examination. At the same time, the number of LAB (from ˂3.0 to 4.4 log cfu/g or mL) was determined, which was the highest on day 4 of the must fermentation process and was related to the detected of the highest concentration of biogenic amines (histamine and tyramine) on day 6 in the investigated must samples using the UHPLC system. Mycobiota species were identified by MALDI-TOF MS, PCR, ITS-PCR-RFLP, and PCR sequencing of the amplified products. The study confirmed the presence of the yeasts Saccharomyces cerevisiae, Metschnikowia pulcherrima, Hanseniospora uvarum, Pichia kudriavzevii, Pichia kluyveri, Pichia fermentas, Torulaspora delbrueckii, and Candida tenuis. At the same time, the presence of molds (Cladosporium herbarum, Cladosporium cladosporioides, Penicillium granulatum, Penicillium mononematosum, Botritis cinerea, and Penicillium glabrum) was also confirmed in soil samples, leaves, grape berries, and fresh grape must. The study confirmed the reduction in the species diversity of the microbiota during the must fermentation process, which resulted in decreases in the concentrations of the monitored biogenic amines in the early stages of the must fermentation process and young wine of the variety Tramín cervený.

Detection of Gluten in Gluten-Free Foods of Plant Origin.

The work deals with the issue of standardization and more accurate methodology for the isolation of gluten DNA in gluten-free products of plant origin, which is more demanding due to the more complex structure of plant cells. Three isolation methods were compared, of which the combination of glass and zirconium beads, Proteinase K and a commercially produced isolation kit was confirmed to be the most effective procedure. The given isolation procedure was more effective in one-component gluten-free foods, where the concentration of the obtained DNA ranged from 80.4 ± 0.7 to 99.0 ± 0.0 ng/µL. The subsequent PCR reaction revealed the presence of gluten not only in guaranteed gluten-free products (40%), but also in naturally gluten-free foods (50%). These were mainly gluten-free sponge cakes, gluten-free biscuits "Cranberries", cocoa powder, coffee "3in1", and instant coffee.

Detection of Resistant and Enterotoxigenic Strains of Staphylococcus warneri Isolated from Food of Animal Origin.

The topic of this work is the detection of antimicrobial resistance to Staphylococcus warneri strains and the genes encoding staphylococcal enterotoxins. It is considered a potential pathogen that can cause various-mostly inflammatory-diseases in immunosuppressed patients. The experimental part of the paper deals with the isolation of individual isolates from meat samples of Oryctolagus cuniculus, Oncorhynchus mykiss, Scomber scombrus, chicken thigh, beef thigh muscle, pork thigh muscle, and bryndza cheese. In total, 45 isolates were obtained and subjected to phenotypic (plasma coagulase activity, nuclease, pigment, hemolysis, lecithinase, and lipase production) and genotypic analyses to confirm the presence of the S. warneri species. The presence of genes encoding staphylococcal enterotoxins A (three isolates) and D (six isolates) was determined by PCR. Using the Miditech system, the minimum inhibitory concentration for various antibiotics or antibiotics combinations was determined, namely for ampicillin; ampicillin + sulbactam; oxacillin; cefoxitin; piperacillin + tazobactam; erythromycin; clindamycin; linezolid; rifampicin; gentamicin; teicoplanin; vancomycin; trimethoprim; chloramphenicol; tigecycline; moxifloxacin; ciprofloxacin; tetracycline; trimethoprim + sulfonamide; and nitrofurantoin. Resistance to ciprofloxacin and tetracycline was most common (73%). At the same time, out of a total of 45 isolates, 22% of the isolates were confirmed as multi-resistant. Isolates that showed phenotypic resistance to ß-lactam antibiotics were subjected to mecA gene detection by PCR.

Dairy Cows' Udder Pathogens and Occurrence of Virulence Factors in Staphylococci.

This study investigated 960 Slovak and Czech spotted cattle from four different conventional (non-organic) dairy herds located in Eastern Slovakia and Czechia during early lactation (14-100 days after calving). Dairy cows were examined clinically; milk from fore-stripping of each udder quarter was subjected to sensory examination and assessed by the California mastitis test (CMT), and laboratory analyses of bacterial pathogens in milk, including virulence factors, were conducted. Positive CMT scores (1-3) for one or more quarters were detected in 271 (28.2%) of the examined animals. Out of 230 infected milk samples, representing 24.0% of all dairy cows, staphylococci (59.1% of positive findings) were the most commonly isolated organisms, followed by E. coli (11.3%), streptococci Str. uberis (9.1%) and Str. agalactiae (3.4%), and enterococci (6.1%). From 136 isolates of S. aureus (38 isolates) and non-aureus staphylococci (NAS; 98 isolates), virulence factors and their resistance to 14 antimicrobials were detected using the disk diffusion method, with PCR detection of the methicillin resistance gene, mecA. An increased incidence of clinical and chronic forms of mastitis has been reported in mastitic cows in which staphylococci, especially S. aureus and NAS (S. chromogenes, S. warneri, and S. xylosus), have been detected and compared to other isolated udder pathogens. From those species, S. aureus and isolates of NAS mentioned above showed multiple virulence factors that are more likely to hydrolyze DNA, hemolysis, produce gelatinase and biofilm, and have multi-drug resistance as compared to other less virulent staphylococci. Generally, the isolated staphylococci showed 77.2% resistance to one or more antimicrobials, in particular to aminoglycosides, ß-lactams, macrolides, or cephalosporins. Isolates that showed the ability to form a biofilm were more resistant to more than one antimicrobial than isolates without biofilm production. Multi-drug resistance to three or more antimicrobial classes was recorded in 16 isolates (11.7%), and the presence of the mecA gene was also confirmed in two isolates of S. aureus and two species of NAS.

Antimicrobial Resistance of Enterococcus sp. Isolated from Sheep and Goat Cheeses.

This study aimed to calculate the proportion of antibiotic resistance profiles of Enterococcus faecium, E. faecalis, and E. durans isolated from traditional sheep and goat cheeses obtained from a selected border area of Slovakia with Hungary (region Slanské vrchy). A total of 110 Enterococcus sp. were isolated from cheese samples, of which 52 strains (E. faecium (12), E. faecalis (28), E. durans (12)) were represented. After isolation and identification by polymerase chain reaction and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, the enterococci (E. faecium, E. faecalis, and E. durans) were submitted to susceptibility tests against nine antimicrobial agents. In general, strains of E. faecalis were more resistant than E. durans and E. faecium. A high percentage of resistance was noted in E. faecalis to rifampicin (100%), vancomycin (85.7%), teicoplanin (71.4%), erythromycin (71.4%), minocycline (57.1%), nitrofurantoin (57.1%), ciprofloxacin (14.3%), and levofloxacin (14.3%). E. durans showed resistance to rifampicin (100%), teicoplanin (100%), vancomycin (66.7%), erythromycin (66.7%), nitrofurantoin (66.7%), and minocycline (33.3%), and E. faecium showed resistance to vancomycin, teicoplanin, and erythromycin (100%). Multidrug-resistant strains were confirmed in 80% of the 52 strains in this study. Continuous identification of Enterococcus sp. and monitoring of their incidence and emerging antibiotic resistance is important in order to prevent a potential risk to public health caused by the contamination of milk and other dairy products, such as cheeses, made on farm level.

Detection of Antimicrobial Resistance of Bacteria Staphylococcus chromogenes Isolated from Sheep's Milk and Cheese.

Antimicrobial and multidrug resistance is detected in nonaureus staphylococci, including Staphylococcus chromogenes, which commonly causes intramammary infections. Recent clinical studies point to the presence of methicillin-resistant S. chromogenes. Therefore, this study aims to determine the prevalence of this species in samples of sheep's milk and cheeses made from them. Isolates were identified by polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). A total of 208 staphylococcal isolates were identified. Of these, 18% were identified as S. chromogenes. The antimicrobial resistance of the identified isolates was determined using the agar dilution method against penicillin, ceftaroline, teicoplanin, gentamicin, erythromycin, tetracycline, and ofloxacin. The highest resistance was found to penicillin (95%), tetracycline (86%), and oxacillin (81%). The highest sensitivity was confirmed for gentamicin (55%). The study also confirmed the presence of methicillin resistant staphylococcal isolates (30%) based on the phenotypic manifestation of antimicrobial resistance and detection of the presence of the mecA gene. The study shows that the tested isolates (62%) were multidrug resistant. Resistance to two antibiotics was most often found (39%).

Maintaining Optimal Mammary Gland Health and Prevention of Mastitis.

In dairy industry, quality of produced milk must be more important than quantity without a high somatic cells count (SCC) or pathogens causing mastitis of dairy cows and consumer diseases. Preserving the good health of dairy cows is a daily challenge for all involved in primary milk production. Despite the increasing level of technological support and veterinary measures, inflammation of the mammary gland-mastitis, is still one of the main health problems and reasons for economic losses faced by cow farmers. The mammary gland of high-yielding dairy cows requires making the right decisions and enforcing the proper measures aimed at minimizing external and internal factors that increase the risk of intramammary infection. Due to the polyfactorial nature of mastitis related to its reduction, the effectiveness of commonly used antimastitis methods tends to be limited and therefore it is necessary to find the areas of risk in udder health programs and monitoring systems. Only by implementing of complete udder health programs should be accompanied by research efforts to further development these complete udder health control. The present review analyses the current knowledge dealing with damping and prevention of mastitis include SCC control, proper nutrition, housing and management, milking and drying as practiced in dairy farming conditions. This information may help to improve the health of the mammary gland and the welfare of the dairy cows as well as the production of safe milk for consumers.

Antimicrobial Resistance of Staphylococcus sp. Isolated from Cheeses.

S. aureus and some species of coagulase-negative staphylococci, including S. chromogenes and S. simulans, commonly cause intramammary infections. However, little attention was paid to the antimicrobial resistance of these species with respect to their occurrence in dairy products, for example, popular sheep and goat cheeses made from unpasteurized milk. The aim of this study was to investigate such sheep and goat cheeses for the occurrence and antimicrobial resistance of the relevant staphylococci species. The staphylococcal isolates were identified by polymerase chain reaction (130 isolates) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The most common species of S. aureus (56 isolates) were identified, as well as S. chromogenes (16 isolates) and S. simulans (10 isolates). Antimicrobial resistance to penicillin, oxacilin, ceftaroline, teicoplanin, gentamicin, erythromycin, tetracycline and ofloxacin was subsequently determined in these species using the agar dilution method. The highest resistance was confirmed in all species, especially to penicillin (91%) and erythromycin (67%). The highest sensitivity was confirmed to ofloxacin (83%). Due to the high incidence of penicillin and oxacilin-resistant staphylococci, the mecA gene was detected by polymerase chain reaction, which was confirmed only in S. aureus isolates (19%). Our study shows that the tested strains (77%) were resistant to more than one antibiotic at a time.