Carbenicillin

The antimicrobial susceptibility testing was done following a previously reported method [26 (link)]. Briefly, the pure colonies obtained from blood agar were resuspended in bacterial suspensions, and were made depositing two or three medium-sized colonies (2 to 3 mm) in BBL Crystal Inoculum Broth (Becton Dickinson; Franklin Lakes, NJ, USA). The obtained inoculum was adjusted while using a Mac Farland 0.5 reading (Expected CFU/mL 1.5 × 108), and cultured in Müeller Hinton 150 × 15 mm² media BD BBL (Becton Dickinson Franklin Lakes, NJ, USA). The antibiotic discs were applied with the Sensi-Disc Designer Dispenser System. The antibiotics panel was conformed by ampicillin (10 µg), ampicillin/sulbactam (10/10 µg), mezlocillin (75 µg), carbenicillin (100 µg), piperacillin/tazobactam (100/10 µg), cefazolin (30 µg), cefaclor (30 µg), cefepime (30 µg), cefoperazone (75 µg), and cefotetan (30 µg) from Becton Dickinson (Franklin Lakes, NJ, USA), Müeller Hinton medium were incubated at 37 °C for 24 h in both aerobic and anaerobic conditions. In anaerobic conditions, the media were placed in an anaerobic jar (BD BBL™ GasPak™), with a C02 gas generators envelope (BD BBL™) and another envelope of BD BBL™ GasPak™ anaerobic indicator placed on them.

Antimicrobial susceptibility testing was carried out by the Kirby–Bauer method under the Clinical and Laboratory Standards Institute protocol (NCCLS standards). The pure colonies obtained from blood agar were resuspended in bacterial suspensions were made depositing two or three medium-sized colonies (2 to 3 mm) in BBL Crystal Inoculum Broth (Becton Dickinson; Franklin Lakes, NJ, USA). The obtained inoculum was adjusted while using a Mac Farland 0.5 reading (Expected CFU/mL 1.5 × 10⁸), and cultured in Müeller Hinton 150 × 15 mm² media BD BBL (Becton Dickinson Franklin Lakes, NJ, USA). The antibiotic discs were applied with the Sensi-Disc Designer Dispenser System. The antibiotics panel was conformed by ampicillin (10 µg), ampicillin/sulbactam (10/10 µg), mezlocillin (75 µg), carbenicillin (100 µg), piperacillin/tazobactam (100/10 µg), cefazolin (30 µg), cefaclor (30 µg), cefepime (30 µg), cefoperazone (75 µg), and cefotetan (30 µg) from Becton Dickinson (Franklin Lakes, NJ, USA)

The production of β-lactamase was tested using Cefinase disks (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) according to the manufacturer’s instructions. Susceptibility to select β-lactam antibiotics was determined as described previously (Letchumanan et al., 2015 (link)). V. antiquarius 939 was grown overnight (18 h) on Mueller-Hinton agar plates (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) (supplemented with 1.5% NaCl) at 37°C. Overnight growth was resuspended in a filter-sterilized 0.85% NaCl solution, normalized to approximate a 0.5 McFarland standard, and fresh Mueller-Hinton agar plates were seeded with a bacterial lawn using a sterile cotton swab. Penicillin (10 units), ampicillin (10 μg), cephalothin (30 μg), and carbenicillin (100 μg) antibiotic disks (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) were dispensed on the lawn and plates were incubated overnight (18 h) at 37°C. The zones of inhibition were measured and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (Clinical and Laboratory Standards Institute [CLSI], 2010 ). The pandemic type strain of V. parahaemolyticus (RIMD2210633) (Nasu et al., 2000 (link)) was tested for comparative purposes.