β-lactamase-mediated hydrolysis of cloxacillin increases tolerance in Escherichia coli

Antimicrobial resistance is a global health threat, partly driven by the spread of extended-spectrum β-lactamase (ESBL)-producing bacteria. The use of β-lactam antibiotics in healthcare and agriculture contributes to this emergence. The β-lactam antibiotic cloxacillin, a narrow-spectrum antibiotic, is widely applied in dairy farms during dry cow treatment (DCT). Paralleling this use is an increasing incidence of ESBL producing gram-negative bacteria detected in the Dutch dairy sector. To investigate whether cloxacillin inadvertently affects the growth of E. coli and ESBL-producing E. coli, we measured the hydrolytic activity of purified β-lactamase enzymes, assessed their impact on the antibiotic susceptibility and growth kinetics of wild-type and recombinant E. coli and determined cloxacillin concentrations in colostrum. Our findings show that β-lactamases bind and hydrolyze cloxacillin, resulting in an increase in MIC when recombinantly expressed in hypersensitive E. coli. These results suggest that cloxacillin, although not clinically active against gram-negative bacteria, may influence the growth of β-lactamase-producing strains, warranting further investigations, specifically of the possible selective effect on ESBL-producing bacteria.