In vitro efficacy of combinations of antibiotics used in clinical practice on clinical isolates of Helicobacter pylori.

BACKGROUND: The main antibiotics used against Helicobacter pylori have been chosen empirically over time, with few preclinical studies to provide support. The rise in resistance to some of these antibiotics is prompting a reassessment of their use. This work aimed to evaluate the in vitro efficacy of 2 × 2 combinations of the most widely used antibiotics against H. pylori. MATERIALS AND METHODS: J99 reference strains and 19 clinical isolates of H. pylori with various antibiotic resistance phenotypes were used. Minimum inhibitory concentrations were carried out using the microdilution method in 96-well plates. The activity of 15 possible combinations of two antibiotics including amoxicillin, clarithromycin (CLA), levofloxacin, rifampicin, tetracycline, and metronidazole was determined for all strains by the checkerboard method. A mean fractional inhibitory concentration index (FICmean) was calculated for each combination and strain and the type of pharmacodynamic interaction was considered as synergic if FICmean ≤ 0.5, additive if 0.5 < FICmean ≤ 1, indifferent if 1 < FICmean < 4 or antagonistic if FICmean ≥ 4. RESULTS: Most of the 285 pharmacodynamic interactions tested with clinical strains were close to additivity (average FICmean = 0.89 [0.38-1.28]). No interaction was found to be antagonistic. When two antibiotics to which a strain was resistant were combined, the concentrations required to inhibit bacterial growth were higher than their respective breakpoints. CONCLUSION: The present results have shown that in vitro, the different antibiotics used in therapeutics have additive effects. The addition of the effects of two antibiotics to which a strain was resistant was not sufficient to inhibit bacterial growth. In probabilistic treatment, the choice of antibiotics to combine should therefore be based on the local epidemiology of resistance, and on susceptibility testing in the case of CLA therapy, so that at least one antibiotic to which the strain is susceptible is used.

Pan-azole-resistant Meyerozyma guilliermondii clonal isolates harbouring a double F126L and L505F mutation in Erg11.

BACKGROUND: Meyerozyma guilliermondii is a yeast species responsible for invasive fungal infections. It has high minimum inhibitory concentrations (MICs) to echinocandins, the first-line treatment of candidemia. In this context, azole antifungal agents are frequently used. However, in recent years, a number of azole-resistant strains have been described. Their mechanisms of resistance are currently poorly studied. OBJECTIVE: The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii. METHODS: Ten isolates of M. guilliermondii and the ATCC 6260 reference strain were studied. MICs of azoles were determined first. Whole genome sequencing of the isolates was then carried out and the mutations identified in ERG11 were expressed in a CTG clade yeast model (C. lusitaniae). RNA expression of ERG11, MDR1 and CDR1 was evaluated by quantitative PCR. A phylogenic analysis was developed and performed on M. guilliermondii isolates. Lastly, in vitro experiments on fitness cost and virulence were carried out. RESULTS: Of the ten isolates tested, three showed pan-azole resistance. A combination of F126L and L505F mutations in Erg11 was highlighted in these three isolates. Interestingly, a combination of these two mutations was necessary to confer azole resistance. An overexpression of the Cdr1 efflux pump was also evidenced in one strain. Moreover, the three pan-azole-resistant isolates were shown to be genetically related and not associated with a fitness cost or a lower virulence, suggesting a possible clonal transmission. CONCLUSION: In conclusion, this study identified an original combination of ERG11 mutations responsible for pan-azole-resistance in M. guilliermondii. Moreover, we proposed a new MLST analysis for M. guilliermondii that identified possible clonal transmission of pan-azole-resistant strains. Future studies are needed to investigate the distribution of this clone in hospital environment and should lead to the reconsideration of the treatment for this species.