Bacterial strains and antibiotic susceptibility: Thirty-two clinical isolates of Salmonella spp. were isolated from Lebanese and Beninese patients. Salmonella isolates were identified using API 20E system (Biomérieux, France) after isolation on Salmonella Shigella agar (SS) (Biorad- Germany). Antibiotic Susceptibility Testing was performed using the Kirby Bauer technique with different antibiotics disks (Biorad, Germany). The results were interpreted according to EUCAST guidelines (2017). The susceptibility of the isolates to colistin was determined using the Microdilution method (CLSI, 2018)
Phenotypic detection of ESBLand AmpC
Phenotypic detection of extended spectrum beta-lactamase (ESBL) producing bacteria and AmpC beta-lactamases were performed as explained in Dandachi et al. (2018) using the double-disk synergy test or the cefoxitin disk test (Black et al., 2005) respectively. For the detection of carbapenemases, the carba NP test was used as per the manufacturer instructions (Cunningham et al., 2017) to distinguish between KPC, Oxa-48, VIM, IMP, and NDM.
Molecular characterization of beta-Lactamase encoding genes
Isolates that showed cefoxitin resistance or a keyhole effect were further studied using real-time PCR as detailed in Dandachi et al. (2018). Briefly, DNA was extracted from isolates using EZ1 DNA extraction kits (Qiagen, France) and real-time PCR was used for the detection of CTX-M, TEM, SHV, and other resistance genes (Roschanski et al., 2014).
Determination of the Minimum Inhibitory Concentration
The Minimum Inhibitory Concentration (MIC) was performed using broth microdilution method as described in the Clinical and Laboratory Standards Institute (CLSI, 2018). Briefly the highest concentration of the antimicrobial agent was added to the first well of a 96-well plate and a serial dilution was performed. A volume of 100 µl of a 106 CFU/ml bacterial suspension was dispensed into all the wells except for the negative control. The plate was incubated overnight and MIC was recorded as the concentration of the first well that shows no bacterial growth. MIC 90 is defined as the lowest concentration of the extract at which 90% of the bacterial isolates were inhibited.
Mutant Prevention Concentration
The Mutant Prevention Concentration (MPC) was determined using the agar plate dilution method as described by Pasquali & Manfreda (2007). Briefly, the antimicrobial agents were incorporated into Mueller Hinton Agar (MHA) plates to create a concentration gradient from 1000-0.1 μg/ml. The bacterial suspension was then concentrated to 1010 CFU/ml and inoculated on the entire range of antibiotic containing MHA plates. The plates were incubated and the concentration of the first plate that showed no growth after 48 hours was considered as the MPC. All the tests were performed in duplicate.
Detection of virulence genes by Polymerase Chain Reaction.
Salmonella strains was cultured overnight at 37°C in brain heart infusion broth (Oxoid, United Kingdom). An aliquot of 1 ml of each bacterial culture was separated for DNA extraction by heat treatment (Borges et al., 2013). The PCR experiments were conducted in individual reactions using primers for the following genes: sdiA, invA, sopB, invH, sopE, pefA, and sefC (Choudhury et al., 2016). The cycling program was performed in the CFX-96 thermal cycler (BioRad, Germany). The amplified products were separated by electrophoresis in a 1.2% agarose gel and stained with ethidium bromide. Fragments were trans-illuminated with UV light. E. coli ATCC 25922 (stn -ve) and S. enteritidis ATCC 13076 (stn +ve) were used as negative and positive controls respectively for all PCR reactions. A control containing the PCR mix without extracted DNA was also added in all PCR runs. All isolates having colistin MIC > 2mg/ml were subjected to standard PCR for the detection of mcr-1 colistin resistant gene (Rebelo et al. (2018).
Aqueous YM Extraction
Commercial YM dried leaves (brand AMANDA) were purchased from the local market and an aqueous extraction was performed as explained in Noureddine et al. (2018). In brief, the leaves were blended and mixed with distilled water at a ratio of 3.6:1 ml/g. The mixture was heated at 70°C for 2 hours. Extracts were filtered using Whatman (1M) filter papers, dialyzed against distilled water for 48 hours, centrifuged at 3325 G, concentrated using a SpeedVacuum concentrator (Thermo Fisher Scientific, USA), and stored at 4 °C.