Pilot-testing a protocol for antibiotic-resistant Pseudomonas aeruginosa monitoring in aquatic environments

Recent efforts have focused on modifying fecal indicator bacteria (FIB) targets for monitoring antimicrobial resistance (AMR) in water environments, as exemplified by the recent World Health Organization TriCycle Protocol for cefotaxime-resistant Escherichia coli. However, given that certain FIB are not well-adapted to survival in aquatic environments, there is interest in alternative targets that persist once released from a wastewater treatment plant (WWTP) or other source and are thus presented with greater opportunity to engage in the exchange of antibiotic resistance genes (ARGs) with other environmental bacteria. Pseudomonas spp. have been identified as a promising target for this purpose, given that they are well-adapted to aquatic environments and notorious for carrying multiple, mobile ARGs. However, there are no reliable standard methods for culture-based monitoring of Pseudomonas in aquatic environments. Here, we pilot-tested an AMR P. aeruginosa monitoring protocol for aquatic environments using Pseudomonas CN agar, consisting of Pseudomonas agar base with cetrimide and nalidixic acid supplement. The CN agar was applied directly for total P. aeruginosa counts, with ceftazidime (32 μg/mL) or imipenem (8 μg/mL) amendment for counts of respective resistant strains. We sampled influent and effluent from four WWTPs across Virginia, capturing a range of plant sizes and treatment levels, along with corresponding surface water and sediment samples from locations upstream and downstream of discharge from each plant. P. aeruginosa was recovered across all sample types amongst the various WWTPs, with 24.13% and 0.14% growing on imipenem or ceftazidime-amended plates, respectively. This study helps take a step towards developing comparable protocols for culture methods targeting P. aeruginosa and antibiotic-resistant P. aeruginosa in aquatic and sediment environments, where non-target microorganisms are especially prone to interfere with selective media.