Brain heart infusion agar

We used corneas from two types of rabbits—wild brown rabbits (Blackface Meat Company, Dumfries, Scotland) and New Zealand rabbits (University of Sheffield, from rabbits sacrificed at the end of a licenced study). There was no difference in the performance of corneas from these two types of rabbits. Cadaveric human corneas unsuitable for transplant were acquired from the Ramayamma International Eye Bank, L V Prasad Eye Institute, Hyderabad, India. All corneas were obtained following procedures approved by the institutional review board for the protection of human subjects.
Dispase II was obtained from Roche Diagnostics (Burgess Hill, UK), and Videne^® antiseptic solution was purchased from Ecolab (St. Paul, MN, USA). Mouse 3T3 fibroblasts (used in India) were from the American Type Culture Collection (ATCC; Manassas, VA, USA), and those used in the UK were an established J2 3T3 cell line originally from Professor Howard Green, USA. Epidermal growth factor was obtained from Invitrogen (Paisley, UK). For the culture of microorganisms, brain-heart infusion (BHI) agar and broth were purchased from Oxoid (Hampshire, UK) or HiMedia (Mumbai, India). All other reagents were obtained from Sigma-Aldrich (Dorset, UK) unless otherwise stated. Calcofluor-white was obtained from Sigma-Aldrich (Dorset, UK) and from HiMedia (Mumbai, India).

Biopsy samples were vortexed vigorously for 5 min and plated on Brain Heart Infusion (BHI) agar (Oxoid Ltd, Hampshire, United Kingdom) supplemented with 7.5% sheep blood, 0.4% Isovitalex, and H. pylori Dent supplement (Oxoid, United Kingdom). Plates were incubated at 37 °C in an atmosphere of 5% O₂, 15% CO₂, and 80% N2 for 3 to 7 days. H. pylori colonies were identified based on their typical morphology, characteristic appearance on Gram staining, a positive urease test, and subsequently confirmed by MALDI_TOF (Bruker, Germany). Isolates were stored at minus 80 °C in 0.5 ml of brain heart infusion (BHI) broth with 20% glycerol.

A readily utilized medium, brain heart infusion agar (Oxoid-UK), was prepared according to the manufacturer’s instructions, and then sterilized at 121 °C for 15 min. Tenfold serial dilutions were carried out in saline by mixing 1 mL of a sample with 9 mL parts of saline so that the new solution was 10 times less concentrated than the original solution. This process was repeated to achieve 10 tubes of saline solution containing the diluted sample. Then, aliquots of 1000 μL of each dilution were plated onto BHI agar plates and incubated at 37 °C for 24 h. The colony-forming units that grew were counted and then transformed into actual counts based on the known dilution factors [30 (link)].

A total of 120 samples were collected from closed abscessed lymph nodes and organs of slaughtered sheep and goats in the slaughterhouse of Matrouh Governorate. Swabs from incised organs and aspirates from an abscess using syringes were immersed into peptone water broth and transferred in an ice box to the laboratory. Samples were subjected to bacteriological examination as soon as possible. The swabs were inoculated onto brain heart infusion agar (Oxoid) supplemented with 200 mg of fosfomycin (Sigma,) and 4 mg/liter of nalidixic acid and incubated at 37 °C for 48 h [4 ].

Two key M. haemolytica reference strains, PH2 and PH202, were used in this study. Isolate PH2 is of serotype A1 and was isolated from the lungs of a confirmed case of bovine pneumonic pasteurellosis, whereas isolate PH202 is of serotype A2 and was recovered from the nasopharynx of a clinically healthy calf on a disease-free farm. The two isolates were characterized in previous comparative studies of M. haemolytica (27 (link), 28 (link), 31 (link), 101 (link)– (link)103 (link)). The bacterial isolates were stored at −80°C in 50% (vol/vol) glycerol–brain heart infusion broth (BHIB; Oxoid) and were subcultured on brain heart infusion agar (Oxoid) containing 5% (vol/vol) defibrinated sheep’s blood (blood agar) overnight at 37°C. Broth cultures were prepared by inoculation of 25-ml volumes of BHIB from overnight growth on blood agar and incubation at 37°C and 120 rpm.

Using the criteria described previously in “Whole-genome sequencing and genomic analyses of enterococcal isolates that harbored oxazolidinone resistance genes,” eight isolates (M4-80, B6, B54, M2-95, M2-77, M2-82, M6-97, and M6-130) carrying different plasmids that were representative of the 175 enterococcal isolates carrying the oxazolidinone resistance gene in this study were selected as donors. The rifampicin-resistant E. faecalis JH2-2 was used as the recipient. Conjugal transfer was performed on a filter membrane as described previously (Brenciani et al., 2016 (link)). The donor and recipient bacteria were mixed in a 1:1 ratio on the filter membrane. Transconjugants were selected on Brain Heart Infusion Agar (Oxoid, Basingstoke, United Kingdom) plates containing 2 mg/l linezolid and 25 mg/l rifampicin. The transfer frequency was expressed as the ratio of the cell number (cfu/ml) of the transconjugant to that of the recipient.
Transconjugants were evaluated for their susceptibility to linezolid, tedizolid, chloramphenicol, florfenicol, tetracycline and doxycycline. Then, the detection of transferable oxazolidinone resistance genes, 16S rDNA sequencing and whole genome sequencing were also used to confirm transconjugants.