Cm0001b

Four pathogenic bacterial species (E. faecalis, E. coli, P. aeruginosa and S. aureus) were procured from MB Laboratory, Institute of Zoology, PU, Lahore, Pakistan and stored at low temperature (4 °C) in a refrigerator. The bacterial growth was revived in nutrient broth (CM0001B, Oxoid) prior to all further experiments of antibacterial susceptibility. The composition of nutrient broth is shown in Table 1.Table 1

Composition of nutrient broth.

Chemical constituent	Quantity (g/L)
‘Lab-Lemco’ powder	1.0
Peptone	5.0
Sodium chloride	5.0
Yeast extract	2.0
pH: 7.4 ± 0.2 at room temperature

Cultures were prepared from frozen stocks by aseptically placing one bead (TSC, Lancashire, UK) of each isolate in 25 ml Hunts broth containing 0.65 g nutrient broth (CM0001B, Oxoid, Cambridge, UK) and 0.15 g Yeast extract (CM0019B, Oxoid, Cambridge, UK), 5% Lysed Horse Blood (SR048C, Lennox, Dublin), and 0.4% Campylobacter growth supplement (SR0232E, Oxoid, Cambridge, UK). The inoculated broths were incubated under microaerobic conditions at 42°C for 48 h. After incubation, broths were vortexed for 30 s followed by centrifugation at 10,000 rpm for 10 min at 4°C. The supernatant was discarded and the pellet was suspended in 25 ml phosphate-buffered saline (P4417, Sigma-Aldrich Arklow, Wicklow, Ireland) and vortexed. Cell suspensions were diluted to 10⁻³ in 9 ml Maximum Recovery Diluent (MRD, CM0733B Oxoid, Cambridge, UK). A 1 ml volume of the suspensions were then transferred to 99 ml Hunts broth to give a final cell concentration of 3 log₁₀ CFU/ml for spiking of the caeca. Plate counts were carried to confirm spiking concentrations.