Evolution of Aliarcobacter butzleri under Low Ciprofloxacin Concentrations Associated with the Selection of Multidrug-Resistant Mutants

The threat that repeated exposure to sub-minimum inhibitory concentrations of antibiotics poses to
public health is often overlooked but significant, and the risk to select multidrug-resistant mutants
has recently begun to gain attention. The gastrointestinal pathogen Aliarcobacter butzleri is a
ubiquitous bacterium for which high rates of multidrug resistance have been reported, and the role
of these low concentrations in this phenotype remains unknown. In fact, considering the
environmental distribution of this enteropathogen, resistance selection under low antibiotic
concentrations is highly likely, with the risk of spreading MDR pathogens to human. The main goal
of this work was to unveil the effect of low ciprofloxacin concentrations on the resistance potential
of A. butzleri, with a focus on a One Health perspective. Ciprofloxacin is indicated for the treatment
of infections caused by this microorganism and classified by the WHO as having the highest priority
among the critically important antimicrobials and a member of the Watch category. To achieve the
aim, three strains isolated from humans, food products, and the environment were phenotypically
characterized regarding resistance to ciprofloxacin. Subsequently, short-term evolution
experiments allowed to define the concentrations that select antibiotic resistance in A.butzleri.
Following experimental evolution, the susceptibility of the evolved populations to ciprofloxacin was
determined, as was their cross-resistance profile to several antibiotics, biocides, heavy metals, and
ethidium bromide. The findings of this work showed that the A. butzleri adaptation in the presence
of ciprofloxacin resulted in changes in the susceptibility of the evolved strains to other antibiotics
classes, as well as to acriflavine and ethidium bromide. This suggests that resistance mechanisms
likely involve the activity of efflux pumps. In sum, this work constitutes a further contribution to
understand the harmful effect that antibiotic pollution represents in clinical and non-clinical
ecosystems, particularly for pathogens such as A. butzleri with environmental niches.