Plasmids of the multidrug-resistant <i>Citrobacter portucalensis</i> KOS1-1 strain isolated from a wastewater treatment plant harbor antibiotic resistance genes and gene clusters involved in carbon metabolism

The growing interest in multidrug-resistant (MDR) <i>Citrobacter</i> species stems from their epidemiological significance and their potential to harbor antibiotic resistance genes (ARGs), with mobile genetic elements playing a central role in their dissemination. Wastewater treatment plants play an important role in the formation of such MDR strains due to the high rate of horizontal gene transfer in these environments. In this study, we isolated the MDR strain KOS1-1 of <i>Citrobacter portucalensis</i> from wastewater, sequenced its genome, and characterized its ARG content and plasmid profile. This strain was resistant to ampicillin, cefazolin, cefaclor, cefatrizine, ciprofloxacin, kanamycin, streptomycin, spectinomycin, erythromycin, chloramphenicol, tetracycline, sulfamethoxazole, and trimethoprim. The KOS1-1 strain harbored five low copy number plasmids ranging in size from 77,569 to 289,033 bp. Genome analysis revealed the presence of multiple ARGs both on the chromosome and on plasmids, conferring resistance to β-lactams, quinolones, aminoglycosides, macrolides, sulfonamides, trimethoprim, phenicols, and tetracyclines. Many of these genes were associated with pseudo-composite transposon-like structures, emphasizing the role of mobile elements in ARGs dissemination. Plasmids harbored a bacterial cellulose biosynthesis operon and genes involved in mannose/fucose metabolism that could facilitate biofilm formation and a glycerol dissimilation gene cluster. Bacterial cellulose production was confirmed using electron and atomic force microscopy. Homologus gene clusters were identified on various plasmids of <i>Enterobacteriales</i>, suggesting their distribution via horizontal gene transfer. The presence of plasmids carrying ARGs and adaptive accessory genes increases the competitive fitness of <i>C. portucalensis</i> KOS1-1.IMPORTANCEAntimicrobial resistance represents a silent epidemic that has emerged as a critical global concern in recent years, underscoring the need for further research in this field. This study aimed to isolate and characterize multidrug-resistant bacteria from municipal wastewater, a huge reservoir of antibiotic resistance genes and resistant strains, from which they became disseminated into the environment. The isolated <i>Citrobacter portucalensis</i> strain KOS1-1 exhibits resistance to multiple antibiotics, arsenate, and mercury. It harbors five megaplasmids containing most of the resistance genes, along with laterally acquired bacterial cellulose biosynthesis operon and genes associated with mannose/fucose metabolism, which may facilitate biofilm formation. These plasmids may not only confer a selective advantage to host strains but also promote transfer of resistance determinants in high-density microbial communities of activated sludge at wastewater treatment plants. This work contributes to the understanding of the mechanisms of dissemination of bacterial resistance and virulence factors in municipal wastewater environments.