Meropenem

Solanum lycopersicum L. cv. Micro-Tom was used in this study. Tomato plants were grown in a growth chamber under conditions of 21–25°C with 16 h light at 4000–6000 l×/8 h dark. CRISPR/Cas9 vectors were transformed into Agrobacterium tumefaciens strain GV2260 and introduced into tomato cotyledons by the leaf disk method according to a previous study with slight modification (Sun et al., 2006 (link)). Sterilized tomato seeds were germinated on MS medium and cotyledons (5–7 days after germination) were cut into small pieces of approximately 0.5–0.7 cm and then transformed with Agrobacterium (OD₆₀₀ = 0.01) in 40 ml infection medium [1X MS liquid medium (pH5.7), 1.2 μl 2-mercaptoethanol (Sigma-Aldrich), 100 μM acetosyringone (TCI chemicals)]. The explants were transferred to MS medium containing 40 μM acetosyringone and cultured in the dark for 3 days in a growth chamber, then transferred onto MS-agar medium containing 100 mg/L kanamycin, 1.0 mg/L trans-zeatin (Wako), and 25 mg/L meropenem (Wako). Four weeks after transformation, transgenic calli were selected using kanamycin and GFP selection as a Cas9 expression marker (Ueta et al., 2017 (link)). GFP positive calli were cut using a scalpel under a fluorescence stereoscopic microscope M165FC (Leica) for use in further mutation analysis.

Transfer of the bla_KHM-1 carrying plasmid was confirmed by a filter-mating method described by Kudo [9 (link)] with slight modifications. Briefly, the recipient was a rifampicin-resistant Escherichia coli K-12 DH5α strain [10 (link)]. The donor, recipient, and transconjugant were selected on MacConkey agar (OXOID) supplemented with 0.5 μg/mL meropenem (Wako) and 50 μg/mL rifampicin (Wako) [11 (link)]. Colonies were counted after overnight incubation at 37°C. The transfer frequency was reported as the ratio of the numbers of transconjugant to recipient colonies (transconjugant/recipient). The procedure was repeated in triplicate. The transconjugant was tested for antimicrobial sensitivity, and the presence of carbapenemase was confirmed by the CarbaNP test II and PCR of bla_KHM-1 as described above.

Antimicrobial susceptibility testing was performed for the S. algae clade isolates and E. coli DH5α isolates carrying bla_OXA-55-like cloned from S. algae isolates by the broth microdilution method using BBL Mueller-Hinton II broth, which was cation adjusted (Becton Dickinson and Co., USA) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (19 ). The following antimicrobial agents were used for antibiotic susceptibility testing: ampicillin, piperacillin, cefazolin, cefotaxime, ceftazidime (Sigma-Aldrich, St. Louis, MO, USA), aztreonam (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), imipenem (Banyu Pharmaceutical, Tokyo, Japan), clavulanic acid, and meropenem (Wako Pure Chemical Industry, Ltd., Tokyo, Japan). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. The results were interpreted according to CLSI guidelines (20 ).

Cefiderocol, ceftolozane, and avibactam were synthesized at the research laboratories of Shionogi & Co., Ltd. (Osaka, Japan). Commercial-grade antibiotics were obtained as follows: ceftazidime, tazobactam, amikacin, and aztreonam from Chem-Impex International, Inc. (Wood Dale, IL); cefepime and metronidazole from U.S. Pharmacopeia (Rockville, MD); meropenem, colistin, and gentamicin from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); and ciprofloxacin and piperacillin from LKT Laboratories, Inc. (St. Paul, MN).

MICs of antibiotics and disinfectants were determined by microdilution in LB broth for antibiotics or Mueller-Hinton broth (BD Biosciences) for disinfectants, as described previously (35 (link)). Antibiotic agents, such as colistin, meropenem, amikacin, and ciprofloxacin, and disinfectants, such as ethanol, H₂O₂, SDS, and benzalkonium, were purchased from Fujifilm Wako Pure Chemical Industries. Tigecycline was purchased from the Tokyo Chemical Industry (Tokyo, Japan). The bacterial culture was adjusted to an OD₆₀₀ of 0.001 from the overnight cultures. MICs were monitored using a 2-fold dilution series for each antibiotic or disinfectant.

Meropenem was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and dissolved in physiologic saline (0.9% NaCl). Micafungin was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), dissolved in distilled water, and stored at −80°C until use.

Agrobacterium-mediated transformation of rice (Oryza sativa L. cv. Nipponbare) using scutellum-derived calli was performed as described previously (Toki 1997 , Toki, et al. 2006 (link)). One-month-cultured rice calli were infected by Agrobacterium carrying the pZH_p-gRNA_MMCas9-Nuclease or pZH_p-gRNA_MMCas9-Nickase vectors. After 3 days of co-cultivation, infected calli were transferred to fresh callus induction medium (CIM) (Toki 1997 ) containing 50 mg l^–1 hygromycin B (Wako Pure Chemicals) and 25 mg l^–1 meropenem (Wako) to remove Agrobacterium. Transgenic calli were selected on hygromycin-containing medium for 3 weeks. Proliferating calli were then transferred to fresh CIM without meropenem and cultured for 1 week. After a total of 4 weeks of selection, transgenic calli of pZH_p-gRNA_MMCas9-Nuclease or pZH_p-gRNA_MMCas9-Nickase were used for analysis of mutation frequency.