Methodological approaches to fluoroquinolone susceptibility testing of Salmonella

Abstract

Objective. To evaluate effectiveness of the various approaches to fluoroquinolone susceptibility testing of Salmonella, including S. typhi, taking into account molecular resistance mechanisms. Materials and Methods. The MIC values of ciprofloxacin were compared with the inhibition zone for pefloxacin disc, 5 mg (310 isolates) and nalidixic acid, 30 mg (420 isolates). MIC of ciprofloxacin was determined by gradient diffusion test and broth microdilution method. Muller – Hinton agar and Muller – Hinton broth, antibiotic discs and MICE-tests (Oxoid, UK) were used. Chromosomal mutations in QRDR of gyrA, gyrB, parC, and parE genes and plasmid-mediated quinolone resistance genes (qnr, aac-(6’)-1b etc.) were detected in 19 Salmonella isolates by analysis using ResFinder service (Center of Genomic Epidemiology). Genomic DNA libraries were prepared using the MiSeq Nextera XT Library Preparation Kit (Illumina, USA). WGS was performed on MiSeq (Illumina, USA) with MiSeq Reagent Kit v3 600 cycles (Illumina, USA). Genome assembly and analysis were performed using CLC Genomics Workbench 8.0 (Qiagen, USA). Results. Despite the high concordance of ciprofloxacin MIC values and the results of disc diffusion screening with pefloxacin (96.5% of isolates) and nalidixic acid (98.1% of isolates), the results obtained for some resistant isolates were inconsistent. When those isolates were tested by a single method, there was a possibility of incorrect susceptibility categorization. Discordant results were obtained for 19 isolates and had the objective reason (paradoxical resistance phenotype due to the plasmid-mediated resistance, qnrS) in 3 cases. Other discrepancies were noted when the values were equal to the clinical breakpoints: ciprofloxacin MIC – 0.06 mg/l, inhibition zone for pefloxacin – 24 mm. Repeated testing revealed the variations around the clinical breakpoints: the MIC values of 0.06–0.12 mg/l, and inhibition zone of 23 to 25 mm. Conclusions. When performing fluoroquinolone susceptibility testing of Salmonella, it is reasonable to add the category “Area of Technical Uncertainty”: ciprofloxacin MIC value of 0.06 mg/l, and inhibition zone for pefloxacin of 23 to 25 mm. Two discs (pefloxacin and nalidixic acid) should be used for fluoroquinolone resistance screening by disk diffusion method.