Bacterial strains resistant to different antimicrobials were isolated from water samples in the second sampling campaign (S2, low rainfall sampling). The triplicates were mixed (750 mL of water from each replicate), and 1000 mL of water from each pond was filtered through 0.45 μm pore size Millipore® (Barueri, SP, Brazil) membranes using a Kitassato connected to a vacuum pump. The four membranes were washed individually in 20 mL of saline (NaCl 0.85%).
Aliquots of 100 µL of each sample and their respective dilutions (10−1 and 10−2) were plated in Petri dishes containing CHROMagar Orientation culture medium (BD Diagnostics) supplemented with 50 μg/mL ciprofloxacin (Sigma®, Saint Louis, MI, USA), 60 μg/mL sulfamethoxazole (Sigma®, Buchs, Switzerland) or 8 μg/mL ceftriaxone (Sigma® Saint Louis, MI, USA) [43 (link),44 (link)]. The plates were incubated at 37 °C for 24 h. After determination of colony forming units per milliliter (CFU/mL), three colonies of each morphology were reinoculated on CHROMagar and further identified at the genus level by MALDI-TOF (Bruker Daltonics Bremen, Germany). All isolated strains were maintained at −80 °C in tryptic soy broth medium (TSB) supplemented with the antibiotic used for their isolation, and 20% glycerol.
Aliquots of 100 µL of each sample and their respective dilutions (10−1 and 10−2) were plated in Petri dishes containing CHROMagar Orientation culture medium (BD Diagnostics) supplemented with 50 μg/mL ciprofloxacin (Sigma®, Saint Louis, MI, USA), 60 μg/mL sulfamethoxazole (Sigma®, Buchs, Switzerland) or 8 μg/mL ceftriaxone (Sigma® Saint Louis, MI, USA) [43 (link),44 (link)]. The plates were incubated at 37 °C for 24 h. After determination of colony forming units per milliliter (CFU/mL), three colonies of each morphology were reinoculated on CHROMagar and further identified at the genus level by MALDI-TOF (Bruker Daltonics Bremen, Germany). All isolated strains were maintained at −80 °C in tryptic soy broth medium (TSB) supplemented with the antibiotic used for their isolation, and 20% glycerol.
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