Water samples (400 mL) were serially diluted in sterile distilled water and filtered through 0.45 µm cellulose nitrate filters (Sartorius) and GN-6 Metricel MCE Membrane Disc Filters (Pall), which were placed on Chromocult® Coliform Agar (Merck Millipore) for 24 h at 37 °C for detection of E. coli (blue colonies), total coliforms (salmon-colored colonies) and other Gram-negative bacteria (transparent colonies). Resistant bacteria were recovered by plating on chromogenic media selective for β-lactamase producing Enterobacteriaceae (CARBA Agar, ChromID, BioMerieux) and extended-spectrum β-lactamases (ESBL Agar, ChromID, BioMerieux), methicillin-resistant S. aureus (MRSA Agar, ChromID, BioMerieux), and vancomycin-resistant enterococci (VRE Agar, ChromID, BioMerieux). Recovered colonies were subjected to previously reported PCR assays for species identification and resistance detection53 –60 (link).
Carba agar
Manufactured by bioMérieux
Sourced in France
CARBA Agar is a chromogenic culture medium used for the detection and differentiation of carbapenem-resistant Gram-negative bacteria. It is designed to facilitate the identification of various carbapenemase-producing organisms from clinical specimens.
Automatically generated - may contain errors
3 protocols using carba agar
1
Comprehensive Microbial Analysis of Water Samples
2
Carbapenemase-Producing Enterobacteriaceae Isolation
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Stool samples (200 mg) were plated, transferred into 1 mL phosphate-buffered saline buffer, and agitated to release the microorganisms. An inoculum volume of 100 μL was directly plated onto chromID Carba agar (bioMérieux, France), which consists of a nutrient base combining different peptones, three chromogenic substrates enabling the detection of activities of specific metabolic enzymes for Escherichia coli , Klebsiella /Enterobacter /Serratia/Citrobacter, and Proteeae, and a proprietary mixture of antibiotics favoring the selective growth of carbapenemase-producing Enterobacteriaceae. Species identification was further performed using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) (Table S1 ). Carbapenemase genes (blaKPC, blaVIM, blaIMP, blaNDM, and blaOXA-48-like) were detected by multiplex PCR (46 (link)– (link)48 (link)). Antibiotic susceptibility test results for CRE isolates are shown in Table S2 . The distribution of the genetic determinants of resistance detected by whole-genome sequencing (WGS) in each CRE isolate is shown in Fig. S5 .
3
Carbapenem-Resistant Bacteria Isolation from Lake Biwa
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In total, 72 water samples were collected from 12 sites in Lake Biwa and 13 water samples were collected from 8 sites in its surrounding rivers between February and November 2020 (Figure 1). All samples were collected in sterile 1-L sampling bottles. Samples (100 mL to 600 mL) were processed using the membrane filter method with chromID CARBA agar (bioMérieux, Marcy-l'Étoile, France). After 20 h of incubation at 37 °C, for each sample, up to three colonies showing Escherichia coli profiles (pink to burgundy) or KESC (Klebsiella, Enterobacter, Serratia, Citrobacter) profiles (bluish green to bluish grey) with different colony morphologies were isolated. The oxidase test was performed using a cytochrome oxidase test strip (Nissui, Tokyo, Japan), and isolates with negative test results were stored at -85°C in 35% glycerol. Colonies suspected to be non-Enterobacterales species were not saved because some non-Enterobacterales species prevalent in surface waters are intrinsically resistant to carbapenems. 15, 16
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