Per- and polyfluoroalkyl substances (PFAS) as environmental drivers of antimicrobial resistance: insights from genome sequences of Klebsiella grimontii and Citrobacter braakii isolated from contaminated soil

Authors: Matteo Calcagnile, Andrea Giuliano, Maurizio Salvatore Tredici, Davide Gualandris, Davide Rotondo, Antonio Calisi, Chiara Leo, Margherita Martelli, Anna Rocchi, Knud Erik Klint, Francesco Dondero and Pietro Alifano.

Abstract: Per- and poly uoroalkyl substances (PFAS) are man-made chemicals widely used for industrial applications since the 1940s. PFAS are extremely persistent in the environment, to the extent that they have earned the reputation of‘forever chemicals’. There is growing evidence that PFAS have a signi cant impact on the biodiversity, composition, and activity of microbial communities. In this study, we hypothesized that these compounds may increase the abundance of antibiotic-resistant bacteria. To investigate this hypothesis, we employed Winogradsky columns to study the microbial community's response to PFAS- contaminated soil from the Alb¨ ack re drill site (Trelleborg, Sweden). Column amendment with a high amount of per uorooctanoic acid (PFOA) led to selective growth, in the aqueous phase of the columns, of Klebsiella grimontii and Citrobacter braakii, two emerging opportunistic facultative anaerobic pathogens. Whole-genome sequencing of K. grimontii Tre-B and C. braakii Tre-T isolates revealed numerous antibiotic resistance genes (ARGs), with a notable prevalence of resistance to uoroquinolones. Among these genes are those encoding multidrug e ux systems that confer resistance to a wide range of toxic compounds such as antibiotics, surfactants, dyes, detergents, and disinfectants. Both strains contain a large set of features involved in the degradation of aromatic and halogenated compounds, and other recalcitrant chemicals. K. grimontii Tre-B is characterized by the presence of an IncR-group plasmid (named pKGTreB) containing many genes involved in resistance to arsenic, copper, mercury, and silver. This strain also contains a choline utilization (cut) bacterial microcompartment (BMC) locus, which has been implicated in various human diseases as a source of trimethylamine (TMA). Understanding the genomes of these two bacterial strains provides insights into the molecular mechanisms responsible for their pathogenicity, antibiotic resistance, resistance to biocides, and heavy metal tolerance. In this study we also show that when the two bacteria were grown with PFOA, their resistance to certain aminoglycosides, uoroquinolones and macrolides increased, and we found that transcript levels of the kpnF, kpnG, adeF, and oqxA antibiotic-resistance genes of K. grimontii Tre-B increased as a function of PFOA concentration, whereas acrA was upregulated only at low PFOA concentrations. These results indicate that PFOA, in addition to selecting speci c groups of bacteria, may increase antibiotic resistance through upregulation of speci c antibiotic resistance genes and suggest that these genes may also be involved in bacterial resistance to PFAS. Through the exploration of these mechanisms, we can gain valuable insights into how environmental pollutants, such as PFAS and other contaminants, may contribute to the development of antimicrobial resistance.