Comparative analysis of Bacillus cereus group isolates' resistance using disk diffusion and broth microdilution and the correlation between resistance phenotypes and genotypes

A collection of 85 Bacillus cereus group isolates were screened for phenotypic resistance to nine antibiotics using disk diffusion and broth microdilution. The broth microdilution antimicrobial results were interpreted using the CLSI M45 breakpoints for Bacillus spp. Due to the lack of Bacillus spp. disk diffusion breakpoints, the results obtained with the disk diffusion assay were interpreted using the CLSI M100 breakpoints for Staphylococcus spp. We identified significant (p < 0.05) discrepancies in resistance interpretation between the two methods for ampicillin, gentamicin, rifampicin, tetracycline, and trimethoprim/sulfamethoxazole. Antimicrobial resistance genes were detected using unassembled and assembled whole-genome sequences with Ariba and Abricate, respectively, to assess the sensitivity and specificity for predicting phenotypic resistance based on the presence of antimicrobial resistance genes. We found antimicrobial resistance gene presence to be a poor indicator for phenotypic resistance, calling for further investigation of mechanisms underlying antimicrobial resistance in the B. cereus group. Genes with poor sensitivity and/or specificity, as determined based on broth microdilution results included rph (rifampicin, 0%, 95%), mph genes (erythromycin, 0%, 96%), and all van genes (vancomycin, 100%, 35%). However, Bc (ampicillin, 64%, 100%) and tet genes (tetracycline, 67%, 100%) were highly specific, albeit moderately sensitive indicators of phenotypic resistance based on broth microdilution results. Only beta-lactam resistance genes (Bc, BcII, and blaTEM) were highly sensitive (94%) and specific (100%) markers of resistance to ceftriaxone based on the disk diffusion results, providing further evidence of these beta-lactams role in nonsusceptibility of Bacillus cereus group isolates to ceftriaxone. IMPORTANCEBacillus cereus group includes human pathogens that can cause severe infections requiring antibiotic treatment. Screening of environmental and food isolates for antimicrobial resistance can provide insight into what antibiotics may be more effective therapeutic options based on the lower prevalence of resistance. Currently, interpretation of antimicrobial susceptibility testing results using the disk diffusion method is complicated by the fact that there are no standard disk diffusion resistance breakpoints defined for Bacillus spp. Hence, the breakpoints for Staphylococcus are often used in research studies. By comparing the results of disk diffusion interpreted using the Staphylococcus spp. breakpoints against broth microdilution interpreted using Bacillus spp. breakpoints, this study demonstrated that disk diffusion results interpretation with Staphylococcus spp. breakpoints are inconsistent. This study also provides new insight into the poor associations between antimicrobial resistance genotypes and phenotypes for the B. cereus group.