Baird parker agar

The extraction solvents used, as well as specific chemical reagents, included PBS, Na₂CO₃ ≥ 99.5% (Cas No.497-19-8), AlCl₃, Folin–Ciocalteu (Cat. No 109001), ferric chloride (Cas No.7705-08-0), potassium acetate, quercetin (Cas No.117-39-5), gallic acid (Cas No. 149-91-7), vanillin (Cas No. 121-33-5), sulfuric acid, hydrochloric acid (HCl), sodium phosphate, ammonium molybdate, and DPPH (2,2-diphenyl-1-picrylhydrazyl, Cas No. 1898-66-4), (Sigma-Aldrich, St. Louis, MO, USA). HPLC-grade solvents were procured from Merck (Darmstadt, Germany). Water purification was carried out using the Direct-Q UV system from Millipore, (St. Louis, MO, USA). High-purity standard compounds were used for HPLC, specifically, gallic acid, chlorogenic acid (with 99% HPLC purity), and rutin (with 99% HPLC purity) (Sigma, St. Louis, MO, USA). Various culture mediums were used, including nutrient broth, Baird-Parker agar, TBX agar, XLD agar, Palcam agar, and Muller Hinton agar (Oxoid Ltd., Basingstoke, Hampshire, UK). All chemicals and reagents used in this study met analytical grade standards.

Before conducting the challenge test, OFSP puree was assessed microbiologically for E. coli and S. aureus and then microwaved and assessed again. The initial enumeration of E. coli and S. aureus in the puree was carried out as described in previous studies [22 ]. Puree sample (25 g) was homogenized with 0.85 % NaCl and serial dilutions were prepared up to 10⁻⁶. A volume of 0.1 mL from each dilution was spread in triplicate onto Brilliance E. coli/Coliform agar (Oxoid, Hampshire, England) and incubated at 37°C for 24 hours for enumeration of E. coli. Similarly, 0.1 mL of each dilution was spread in triplicate onto Baird parker agar (Oxoid, Hampshire, England) and incubated at 37°C for 48 hours for the enumeration of S. aureus. Enumeration was done for plates with 30-300 colonies. All microbial counts were expressed as mean base-10 logarithms of colony forming units per gram (log cfu/g). Data points were expressed as means from the triplicate analysis. The results indicated high levels of E. coli and S. aureus in puree before treatment with preservatives. E. coli and S. aureus were not detected after microwaving OFSP puree as shown in Table 1. This formed the basis for the level of inoculation of E. coli and S. aureus into the puree.

From the same herds and period, the nares/muzzles of dairy cows were sampled by swabbing the muzzle and the inner nares with a single moistened sterile cotton swab, as previously described [8 (link)]. Then, the swabs were spread onto the surface of Baird-Parker agar (Oxoid) plates with 5% egg yolk tellurite emulsion and incubated at 37 °C for 24–48 h under aerobic conditions. From each plate, if present, three gray-to-black colonies surrounded by clear zones, as a result of proteolysis and lipolysis, and three gray-to-black colonies without clear zones were selected and transferred separately to microtubes containing 900 μL of brain heart infusion (BHI), and incubated overnight at 37 °C, then glycerol (10% final concentration) was added, and the samples were stored at 80 °C until identification. Afterwards, for bacterial identification, the bacterial isolates (n = 159) were spread onto BHI agar plates for 24–48 h at 37 °C. The bacterial isolates were first subjected to Gram staining and catalase and coagulase tests and further identified by MALDI-TOF MS [6 (link)]. Furthermore, S. aureus identification was confirmed by PCR analysis targeting a portion of the S. aureus nuc gene [7 (link)]. Among the 159 bacterial isolates, all S. aureus isolates identified by both MALDI-TOF MS and PCR as S. aureus were subjected to spa typing and antimicrobial susceptibility tests.

Firstly, S. aureus isolates were isolated onto mannitol salt agar and Baird Parker agar supplemented with an egg yolk–tellurite emulsion (Oxoid, UK). Standard bacteriological procedures were applied to make a preliminary phenotypic identification of S. aureus based on their growth on selective media, β-hemolysis on blood agar and production of golden yellow pigments. Furthermore, microscopical examination of Gram stained films from colonies grown onto mannitol salt agar were observed for the formation of Gram-positive grape-like clusters (Becker et al., 2015 (link); Abd El-Hamid et al., 2019 (link)). The recovered isolates were then confirmed to be S. aureus based on their positive reactions for catalase and coagulase tests. The isolates were finally identified using PCR assay to detect nuc gene (Brakstad et al., 1992 (link)). All the isolates were preserved frozen in brain heart infusion broth (Oxoid, UK) containing 30% glycerol at - 80°C prior to subsequent detailed analysis.

Two bacterial species, XDR P. aeruginosa and VRSA (n = 10 each), were gratefully acquired from the Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University. The bacterial isolates were obtained from infrequent cases of bovine clinical mastitis at Zagazig City, Egypt. The presumptive bacterial isolates were confirmed by implementing the standard microbiological techniques [17 ]. P. aeruginosa isolates were cultivated on pseudomonas cetrimide agar (Oxoid, Cambridge, UK). Meanwhile, Baird–Parker agar supplemented with tellurite–egg yolk emulsion (Oxoid, UK) was used to isolate S. aureus. Genomic DNA was isolated from fresh bacterial isolates using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s guidelines. Species-specific primer pairs (Metabion, Planegg, Germany) (Supplementary Table S1) [18 (link),19 (link)] were used for the identification of the oprL gene of P. aeruginosa, whereas the nuc gene was amplified for the confirmation of S. aureus.

TMC was quantified using GTK-M agar plates (MILCOM a.s., Tábor, Czech Republic) by culturing at 30 °C for 72 h under the aerobic conditions, according to ČSN EN ISO 4833-1 [14 ], which is the Czech equivalent of ISO 4833-1.
Identification of bacteria and their count: pathogenic bacteria in milk were determined by the Veterinary Laboratory Vedia s.r.o. (Strakonice, Czech Republic). The presence of coliform bacteria, staphylococci, streptococci, and corynebacteria was monitored. Basic cultivation of bacteria occurred on the Columbia blood agar (Oxoid, UK). ENDO agar (Oxoid, UK) was used to cultivate coliform bacteria. Enterococcus Selective Agar-BAA (Oxoid, UK) was used to distinguish streptococci and enterococci, and Edwards agar (Oxoid, UK) was used for streptococcal culture, and for staphylococci, Baird-Parker agar (Oxoid, UK) and Staphylococcus agar (Sigma-Aldrich, USA) was applied. A coagulase test was used to distinguish between coagulase-negative and -positive staphylococci (Staphylase Test, Oxoid, UK).
When necessary, a microflex LT MALDI TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with a microSCOUT ion source and a TOF flight time analyzer (Bruker Daltonics, Bremen, Germany) in conjunction with the MALDI Biotyper software system (Bruker Daltonics, Bremen, Germany) was used to identify bacterial species.

Serial two-fold dilutions of up to 10⁻⁶ were made from the initial dilution (1:10) and 0.1 mL aliquots of these dilutions were inoculated onto Baird Parker agar (Oxoid). The bacteria suspected to be S. aureus count were found by examining the plates at typical black colonies, convex shape, with a shiny halo zone, and these were checked for positive Gram coagulase reaction and catalase test (Bactident Coagulase Biolife, Milan, Italy). The identification of the bacterial isolates was then carried out by API-Kits (Biomereux, Montaliea, France) as given by the manufacturer’s instruction.