Cephalothin

All third-generation cephalosporin-resistant E. coli isolates were investigated for their antimicrobial resistance using the disc diffusion test with the following 19 discs (BD Biosciences): amikacin (30 μg), amoxicillin/clavulanate (20/10 μg), ampicillin (10 μg), cefazolin (30 μg), cefepime (30 μg), cefoxitin (30 μg), cephalothin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), colistin (10 μg), doxycycline (30 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), neomycin (30 μg), norfloxacin (10 μg), streptomycin (10 μg), tetracycline (30 μg), and trimethoprim/sulfamethoxazole (1.25/23.75 μg). Results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [15 , 16 ]. The minimum inhibitory concentrations (MICs) for cefazolin, cephalothin, cefoxitin, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, and cefepime were determined by standard broth microdilution methods with Mueller–Hinton broth (BD Biosciences) according to the recommendations of the CLSI [15 , 16 ]. Escherichia coli ATCC 25,922 strain was used the control organisms in the antimicrobial susceptibility tests. Multi-drug resistance (MDR) was defined as acquired non-susceptibility to at least 1 agent in 3 or more antimicrobial categories [17 (link)].

Samples were first plated on Chromocult agar, and then presumptive E. coli colonies were transferred onto MacConkey lactose (MKL) agar for confirmation before replating on Chromocult was performed to ensure pure isolates. We selected up to four E. coli colonies from soil and fecal samples and two from water and surface samples to test for AR. More isolates from soil samples than from water or surfaces were used because we expected higher microbial diversity in soil. Antibiotic sensitivity was assessed using the Kirby-Bauer disc diffusion method (43 (link)) and 12 antibiotics: ampicillin, amoxicillin/clavulanate, cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, enrofloxacin, gentamicin, streptomycin, sulfisoxazole, trimethoprim-sulfamethoxazole, and tetracycline (Becton, Dickinson, Franklin Lakes, NJ). Zones of inhibition were measured after a 24-h incubation period using digital calipers.

To assess antibiotic resistance profiles of E. coli isolates, about every fifth of the enrichments prepared for the detection of Shiga toxin genes were selected (EE broth; 18–24 h, 37 °C). The enriched samples (one loopful: approx. 10 µl) were subcultured for 24 h at 37 °C on chromogenic RAPID’ E. coli 2 agar (Bio-Rad Laboratories, Reinach, Switzerland). One E. coli colony (violet to pink on RAPID’ E. coli 2 agar) from each of the 95 samples was selected and subjected to susceptibility testing against 12 antimicrobial agents by the disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) protocols and criteria [8 ]. The panel included ampicillin (AM, 10 µg), amoxicillin-clavulanic acid (AMC, 30 µg), chloramphenicol (C, 30 µg), cephalothin (CF, 30 µg), ciprofloxacin (CIP, 5 µg), cefotaxime (CTX, 30 µg), gentamicin (GM, 10 µg), kanamycin (K, 30 µg), nalidixic acid (NA, 30 µg), streptomycin (S, 10 µg), tetracycline (TE, 30 µg), and trimethoprim (TMP, 5 µg) (Becton–Dickinson).

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.

Water was collected from 12 sink faucets in a hospital administrative and outpatient building, twice a week over 6.5 weeks for 13 sampling dates, for a total of 156 samples. The hot water outlets were opened, and 500 mL of the immediate water flow was collected into sterile bottles containing sodium thiosulfate to neutralize any residual chlorine in the water. The samples were cultured using a modified ISO method [21 ] immediately on site (T0), then again at 1 h (T1), 24 h (T24), and 48 h (T48) from collection for a total of 624 cultures. Water samples were held at room temperature throughout the testing period. On site at T0, 0.1 mL aliquots of each sample were plated onto two selective agar media: DGVP and BCYE with colistin, cephalothin, vancomycin, and cycloheximide (CCVC) (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA). Upon returning to the laboratory, the T0 agar plates were placed into a humidified incubator at 36.5 ± 1 °C. After holding times of 1 h, 24 h, and 48 h (T1, T24, and T48), each water sample was plated directly and after filter concentration (100 mL was concentrated as above) onto BCYE and to DGVP agar plates. If the initial culture plates were overgrown, the filter concentrates were re-plated after pre-treatment with acid (0.2 M KCl, pH 2.2 ± 1). All sets of cultures were incubated for 7 days and examined for Legionella as above.