Selection of a Gentamicin-Resistant Variant Following Polyhexamethylene Biguanide (PHMB) Exposure in Escherichia coli Biofilms

Abstract

Antibiotic resistance is one of the most important issues facing modern medicine. Some biocides have demonstrated the potential of selecting resistance to antibiotics in bacteria, but data are still very scarce and it is important to better identify the molecules concerned and the underlying mechanisms. This study aimed to assess the potential of polyhexamethylene biguanide (PHMB), a widely used biocide in a variety of sectors, to select antibiotic resistance in Escherichia coli grown in biofilms. Biofilms were grown on inox coupons and then exposed daily to sublethal concentrations of PHMB over 10 days. Antibiotic-resistant variants were then isolated and characterized phenotypically and genotypically to identify the mechanisms of resistance. Repeated exposure to PHMB led to the selection of an E. coli variant (Ec04m1) with stable resistance to gentamycin (8-fold increase in minimum inhibitory concentration (MIC) compared to the parental strain. This was also associated with a significant decrease in the growth rate in the variant. Sequencing and comparison of the parental strain and Ec04m1 whole genomes revealed a nonsense mutation in the aceE gene in the variant. This gene encodes the pyruvate dehydrogenase E1 component of the pyruvate dehydrogenase (PDH) complex, which catalyzes the conversion of pyruvate to acetyl-CoA and CO₂. A growth experiment in the presence of acetate confirmed the role of this mutation in a decreased susceptibility to both PHMB and gentamicin (GEN) in the variant. This work highlights the potential of PHMB to select resistance to antibiotics in bacteria, and that enzymes of central metabolic pathways should be considered as a potential target in adaptation strategies, leading to cross-resistance toward biocides and antibiotics in bacteria.

Keywords: adaptation; aminoglycoside; antibiotic resistance; biocide; biofilm; cross-resistance; pyruvate cycle.

Figure 1

(A) Colony morphotypes obtained after plating of Ec04 biofilm exposed to PHMB over 9 days. Ec04m1 corresponds to a small colony variant. (B) ERIC-PCR profiles of Ec04 and Ec04m1.

Figure 2

Maximum growth rate (µmax) and lag time (lag) for parental strain Ec04 and Ec04m1 variant in the presence of (A) PHMB concentrations from 0 to 3.125 mg L⁻¹, or (B) GEN from 0 to 8 mg L⁻¹. Different letters upon the SD bars indicate significant differences between µmax or lag mean values (T-test. p < 0.05).

Figure 3

Detection of a nonsense mutation in the Ec04m1 variant in aceE gene encoding the pyruvate dehydrogenase E1 component of the pyruvate dehydrogenase (PDH) complex that catalyzes the conversion of pyruvate to acetyl-CoA and CO₂. The mutation (C > T at position 421) results in a premature stop codon at the location of glutamine, leading to a truncated aceE protein (140aa). (A) Schematic representation of the aceE gene and its genetic environment (pdhR-aceEF-lpdA operon) in Ec04 and Ec04m1 strains. (B) Protein sequence of aceE in Ec04 (887aa) and its alignment with the truncated protein in Ec04m1 (140aa); stars symbolize stop codons. (C) Schematic representation of the central metabolic pathway altered by the mutation in the Ec04m1 variant.

Figure 4

Effect of acetate medium supplementation (30 mM) on maximal growth rate (µmax) for parental strain Ec04 and Ec04m1 variant in the presence of PHMB or GEN. Different letters upon the SD bars indicate significant differences between µmax or lag mean values (T-test. p < 0.05).