Nalidixic acid na

The organism used in all experiments was a poultry isolate of Salmonella enterica serovar Enteritidis (S. Enteritidis), bacteriophage type 13A, obtained from the USDA National Veterinary Services Laboratory (Ames, IA, United State). This strain was resistant to 25 μg/mL of novobiocin (NO, catalog no.N-1628, Sigma) and was selected for resistance to 20 μg/mL of nalidixic acid (NA, catalog no.N-4382, Sigma) in our laboratory. For the present studies, 100 μL of S. Enteritidis from a frozen aliquot was added to 10 mL of tryptic soy broth (Catalog no. 22092, Sigma), incubated at 37°C for 8 h, and passed three times every 8 h to ensure that all bacteria were in log phase as previously described (28 (link)). Post-incubation, bacterial cells were washed three times with sterile 0.9% saline by centrifugation at 1,864 × g for 10 min, reconstituted in saline, quantified by densitometry with a spectrophotometer (Spectronic 20D+, Spectronic Instruments Thermo Scientific, Rochester, NY, United States), and finally diluted to an approximate concentration of 1 × 10⁸, 4 × 10⁴, and 4 × 10⁷ cfu/mL. Concentrations of S. Enteritidis were further verified by serial dilution and plating on brilliant green agar (BGA, Catalog no. 70134, Sigma) with NO and NA for enumeration of actual cfu used to in the experiments.

One isolate each from six different serovars of S. enterica (S. Montevideo, S. Poona, S. Newport, S. Baildon, S. Braenderup, and S. Saintpaul – tomato outbreak isolates) were used in the study. These isolates were kindly provided by Dr. Venkitanarayanan (Department of Animal Science, University of Connecticut, Storrs, CT, United States). Since S. Newport and S. Branderup have been previously associated with Salmonella outbreaks associated with mangoes, although isolated from tomatoes, similar serovars were employed in the study. Further, this study was done as a follow up to a recent study investigating the efficacy of commercially employed wash water disinfectants in controlling Salmonella in wash water and on mangoes (Mathew et al., 2018 (link)). Therefore, in order to understand the behavior of these isolates on mangoes during the post-harvest handling and storage of mangoes, the same bacterial cultures were utilized in the present study. All the six isolates were induced for resistance to nalidixic acid (NA; Sigma–Aldrich, St. Louis, MO, United States; 50 μg/ml) to facilitate selective enumeration of the inoculated pathogens (Harris et al., 2001 (link)).

E. coli O157:H7 strains used in the study were as follows: E. coli O157:H7 (Odwalla strain), a clinical isolated from an outbreak associated with apple juice, E. coli O157:H7 T-50 (apple isolate); E. coli O157:H7 7927, a clinical isolate from an outbreak associated with apple cider, and E. coli O157:H7 EDL933, a clinical isolate from an outbreak associated with ground beef. In order to selectively enumerate pathogen populations on the nuts, a stepwise procedure was used to isolate mutants of all strains that were able to grow in media supplemented with nalidixic acid (NA; Sigma-Aldrich, St. Louis, MO, USA; 50 μg/mL; [35 (link)]). All bacteriological media used in the study were procured from Difco (Becton, Dickson and Company, Franklin Lakes, NJ, USA).