Novel Class B2 and C β-lactamases harboured by Pseudomonas spp. wastewater isolates

Introduction Antimicrobial resistance has existed in the environment long before its rapid emergence and detection in clinically relevant pathogens. Studying the resistance of environmental bacterial strains may allow novel resistance mechanisms to be identified before they appear in pathogenic strains. Gap Statement Searching for antimicrobial resistance genes in environmental bacteria represents an understudied research area compared to resistance within clinically relevant pathogens. Aim To evaluate resistance genes present within environmental non-aeruginosa Pseudomonas spp. isolates. Methodology We screened a set of bacterial isolates from untreated wastewater from Liverpool, UK, for the presence of extended spectrum β-lactamases and carbapenemases. A sub-set of three resistant Pseudomonas spp. isolates were selected for whole-genome sequencing. We performed minimum inhibitory concentration assays against several β-lactams, and ectopic expression of four novel resistance genes within Escherichia coli . Results Here, we report the discovery of novel class C β-lactamase genes bla PFL7 , bla PFL8 and bla PFL9 , as well as a novel subclass B2 metallo-β-lactamase bla PFM5 present within these strains. The class C genes encoded proteins with between 61-71% amino acid identity to the closest known match, bla PFL-1 . These novel β-lactamases degraded the cephalosporin nitrocefin and confer piperacillin and ceftazidime resistance to susceptible Escherichia coli when ectopically expressed. The β-lactamase inhibitor tazobactam was effective at inhibiting these enzymes. The sub-class B2 metallo-β-lactamase had 88% amino acid identity to its closet match bla PFM-1 and conferred carbapenem resistance to susceptible E. coli . The β-lactamase inhibitors relebactam, vaborbactam, xeruborbactam and captopril had no impact on the carbapenem resistance phenotype. Analogues of all these novel genes (>95% nucleotide sequence identity) were identified within publicly available whole-genome sequencing data, suggesting they are found sporadically. Conclusion Our analysis adds to the growing number of β-lactamase genes found from environmental Pseudomonas spp. and suggests that continued surveillance of this environmental reservoir for novel, clinically relevant, β-lactamase genes is warranted.