Sheep blood agar

The stored isolates were re-cultured overnight at 35-37 °C in 5% CO₂ on 5% sheep blood agar (Oxoid, Basingstoke, UK; prepared in-house). Single colonies were mixed with matrix (alpha-cyano-4-hydroxycinnamic acid, CHCA) on disposable metallised slides and analysed using the VITEK MS MALDI-TOF system (bioMerieux, Marcy L’ Etoile, France), following manufacturer instructions. Particular care was taken with the preparation of colony-matrix spots, in order to optimise the generation of high-quality mass spectra. To be compatible with routine diagnostic laboratory workflow, a single mass spectrum was generated per isolate. The machine was run in diagnostic (IVD) mode, with automated measurement of proteins in the specific mass range of 2,000 – 20,000 Da. Escherichia coli ATCC 8739 was used for QC and calibration of each slide. Isolates were considered acceptable for analysis if the slide passed QC and the isolate was identified unambiguously as S. pneumoniae by the automated reporting system (bioMerieux Myla, Knowledge Base V3.2.0).

A total of 20 mixed breed dairy cows, ranging in age from 3 to 7 years old, were investigated in private dairy farms at Giza Governorate, Egypt. The farm failed to take the necessary hygienic precautions to prevent mastitis and other infectious illnesses. The milking procedure was carried out in the usual manner. The California mastitis test (CMT) was carried out using the approach outlined by Abdalhamed et al. [12 (link)] and revealed a positive reaction in 34 milk samples. The subclinical mastitis was collected from 20 cows (80 quarters), which had received no medical treatment for 7–10 days. The CMT-positive milk samples were collected aseptically from each teat and kept in sterile plastic bags before being sent to the laboratory under complete aseptic conditions. A loopful of milk sample was inoculated on 5% sheep blood agar and mannitol salt agar (Oxoid, UK). All plates were incubated for 18–24 h at 37°C before being inspected for bacterial growth. Gram staining, hemolysis patron, catalase response, oxidative-fermentative test, and coagulase tests were all performed on suspected colonies [13 (link)].

S. aureus strains isolated from various species and disease presentations (Table 1), were grown on 5% Sheep blood agar (Oxoid) and cultured in brain heart infusion medium (Oxoid) at 37°C. Bacterial genomic DNA was isolated using a Wizard^® Genomic DNA Purification Kit (Promega) following manufacturer’s guidelines. Polymerase chain reaction (PCR) was performed with Easy-A High-Fidelity PCR Mastermix (Agilent) using the primers stated on http://saureus.mlst.net/ (Aanensen and Spratt, 2005 (link)) for the genes arcC, aroE, glpF, gmk, pta, tpi, and tpi. Each primer pair amplified an internal fragment of the housekeeping gene and allowed accurate sequencing of ∼450-bp fragments of each gene on both strands. For each locus, the sequences obtained from all isolates were compared and the different sequences were assigned allele numbers. For each isolate, the alleles at each of the seven loci defined the allelic profile that corresponded to a ST.

Blood culture was performed using Brain Heart Infusion broth (BHI) (Oxoid Ltd) in a ratio of blood to BHI of 1:10 as previously described [3 (link)]. Subsequent sub-culture was done on day 1, 3 and 7 on 5% sheep blood agar, chocolate agar and MacConkey agar (Oxoid, UK). Identification of bacteria was performed using conventional physiological and biochemical methods [13 (link), 14 ]. Repeat blood culture was ordered in all cases where Coagulase negative staphylococcus (CNS) was isolated. Re-isolation of CNS was considered significant blood culture result. Antimicrobial susceptibility of isolates was determined using disk diffusion method according to the Clinical Laboratory standard Institute (CLSI) [15 ].

At the discretion of the healthcare provider, patient blood samples were collected at symptom onset into BacT/Alert bottles (bioMérieux, Saint-Laurent, QC, Canada) according to institutional protocol and incubated using the BacT/Alert blood culture instrument (bioMérieux). Isolates were stored in frozen stocks in skim milk at −70°C and later retrieved by subculture for further analysis.
A total of 92 GCGS isolates were recorded during the study period; 90 were retrieved, 2 were lost in storage, and 1 was identified as S. equi subsp. zooepidemicus by 16S rRNA sequence similarity and excluded from the study. We plated the 89 remaining isolates onto sheep blood agar (Oxoid, Nepean, ON, Canada) and aerobically incubated them for 24 h at 37°C in the presence of 5% CO₂. We confirmed isolate identification by using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry with the MALDI BioTyper system (Bruker, Boston, MA, USA) according to the manufacturer’s protocol. To confirm isolates with ambiguous MALDI-TOF mass spectrometry identifications, we used latex agglutination to Lancefield antigens C and G and the Vitek2 system (bioMérieux) for biochemical identification. All isolates were identified as S. dysgalactiae.

Milk samples were centrifuged (3000 rpm, 15 min) and a loop was taken from the precipitate; the sample was inoculated on tryptone soy broth (Oxoid, Ltd., Basingstoke, Hampshire, UK) and incubated at 37 °C for 18–24 h. All swab samples were also inoculated onto tryptone soy broth and incubated. A loop aliquot from each broth culture was cultivated in 10% sheep blood agar, tryptone soya agar (TSA), mannitol salt agar and Baird–Parker agar (Oxoid, Ltd., Basingstoke, Hampshire, UK) and then incubated at 37 °C for 24–48 h. Staphylococcus species were suspected based on phenotypic characters of the colonies. Bacterial smears were prepared from the suspected colonies and stained with Gram’s stain [56 ].