Contribution of efflux and mutations in fluoroquinolone susceptibility in MDR enterobacterial isolates: a quantitative and molecular study.

OBJECTIVES: The emergence of MDR strains is a public health problem in the management of associated infections. Several resistance mechanisms are present, and antibiotic efflux is often found at the same time as enzyme resistance and/or target mutations. However, in the laboratory routinely, only the latter two are identified and the prevalence of antibiotic expulsion is underestimated, causing a misinterpretation of the bacterial resistance phenotype. The development of a diagnostic system to quantify the efflux routinely would thus improve the management of patients. METHODS: A quantitative technique based on detection of clinically used fluoroquinolones was investigated in Enterobacteriaceae clinical strains with a high or basal efflux activity. The detail of efflux involvement was studied from MIC determination and antibiotic accumulation inside bacteria. WGS was carried out on selected strains to determine the genetic background associated with efflux expression. RESULTS: Only 1 Klebsiella pneumoniae isolate exhibited a lack of efflux whereas 13 isolates had a basal efflux and 8 presented efflux pump overexpression. The antibiotic accumulation evidenced the efficacy of the efflux mechanism in strains, and the contribution of dynamic expulsion versus target mutations in fluoroquinolone susceptibility. CONCLUSIONS: We confirmed that phenylalanine arginine ß-naphthylamide is not a reliable marker of efflux due to the affinity of the AcrB efflux pump for different substrates. We have developed an accumulation test that can be used efficiently on clinical isolates collected by the biological laboratory. The experimental conditions and protocols ensure a robust assay that with improvements in practice, expertise and equipment could be transferred to the hospital laboratory to diagnose the contribution of efflux in Gram-negative bacteria.

Major discrepancy between factual antibiotic resistance and consumption in South of France: analysis of 539,037 bacterial strains.

The burden of antibiotic resistance is currently estimated by mathematical modeling, without real count of resistance to key antibiotics. Here we report the real rate of resistance to key antibiotics in bacteria isolated from humans during a 5 years period in a large area in southeast in France. We conducted a retrospective study on antibiotic susceptibility of 539,107 clinical strains isolated from hospital and private laboratories in south of France area from January 2014 to January 2019. The resistance rate to key antibiotics as well as the proportion of bacteria classified as Difficult-to-Treat (DTR) were determined and compared with the Mann-Whitney U test, the χ2 test or the Fisher's exact test. Among 539,037 isolates, we did not observe any significant increase or decrease in resistance to key antibiotics for 5 years, (oxacillin resistance in Staphylococcus aureus, carbapenem resistance in enterobacteria and Pseudomonas aeruginosa and 3rd generation cephalosporin resistance in Escherichia coli and Klebsiella pneumoniae). However, we observed a significant decrease in imipenem resistance for Acinetobacter baumannii from 2014 to 2018 (24.19-12.27%; p = 0.005) and a significant increase of ceftriaxone resistance in Klebsiella pneumoniae (9.9-24.03%; p = 0.001) and Enterobacter cloacae (24.05-42.05%; p = 0.004). Of these 539,037 isolates, 1604 (0.3%) had a DTR phenotype. Over a 5-year period, we did not observe a burden of AR in our region despite a high rate of antibiotic consumption in our country. These results highlight the need for implementation of real-time AR surveillance systems which use factual data.