Assessment of a plasmid conjugation procedure to monitor horizontal transfer of an extended-spectrum β-lactamase resistance gene under food chain scenarios

Abstract

Plasmids are relevant reservoirs of antimicrobial resistance genes (ARGs) which confer adaptive advantages to their host and can be horizontally transferred. The aims of this study were to develop a conjugation procedure to monitor the horizontal transfer of a 193 kb plasmid containing the extended-spectrum β-lactamase production gene bla _CTX-M-14 between two Escherichia coli strains under a range of food chain-related scenarios, including temperature (20-37 °C), pH (5.0-9.0) or the presence of some biocidal agents (benzalkonium chloride, sodium hypochlorite or peracetic acid). The average conjugation rate in LB broth after 18 h at 37 °C was 2.09e-04 and similar rates were observed in a food matrix (cow's milk). The conjugation was reduced at temperatures below 37 °C, at alkaline pH (especially at pH 9.0) or in the presence of benzalkonium chloride. Peracetic acid and sodium hypochlorite slightly increased conjugation rates, which reached 5.59e-04 and 6.77e-03, respectively. The conjugation procedure described can be used to identify risk scenarios leading to an enhanced ARGs transmission via plasmid conjugation, as well as to identify novel intervention strategies impairing plasmid conjugation and tackling antimicrobial resistance.

Keywords: Conjugation; E. coli; ESBL; Horizontal gene transfer; Plasmid.

Graphical abstract

Fig. 1

Schematic representation of the conjugation protocol: Arrows show steps carried out with the donor (red arrows) and recipient (green arrows) strains, respectively. Bacteria were initially grown on 10 mL of LB broth. To obtain cultures in the exponential phase of growth, 400 μl of a grown broth culture were inoculated in tubes filled in with 40 mL of fresh LB supplemented with antibiotics, and optical density was monitored at 610 nm.

Fig. 2

Conjugation rates under different conditions: Bacterial counts (upper row) and average conjugation rates with their 95% confidence interval (bottom row) obtained when the conjugation protocol was applied under different conditions of temperature (first column), pH (second column), conjugation matrix (third column), or presence of biocides (fourth column). The dashed line represents the lower limit of detection. BAC = Benzalkonium chloride, NaClO = sodium hypochlorite, PAA = peracetic acid, cfu = colony forming units. In those cases were no transconjugants were detected, conjugation rates were estimated using the limit of enumeration of the transconjugants detection method (10 cfu/mL).