Comparative Analysis of Bacillus cereus Group Isolates' Resistance Using Disk Diffusion and Broth Microdilution and the Correlation between Antimicrobial Resistance Phenotypes and Genotypes

Abstract

Bacillus cereus group isolates (n = 85) were screened for phenotypic resistance to 18 antibiotics using broth microdilution and CLSI M45 Bacillus spp. breakpoints. The susceptibility to 9 out of 18 antibiotics was tested also using disk diffusion method and M100 Staphylococcus spp. breakpoints. Overall, a high prevalence of susceptibility to clinically relevant antibiotics was identified using broth microdilution. For most tested antibiotics, a poor correlation was found between zones of inhibition and minimum inhibitory concentrations. Using the broth microdilution results as a reference for comparison, we identified high error rates and low categorical agreement between results produced using disk diffusion and broth microdilution for the seven tested antibiotics with defined breakpoints. This suggests that disk diffusion should be avoided for AST of B. cereus group isolates. Further, we detected antimicrobial resistance genes with ARIBA and ABRIcate to calculate the sensitivity and specificity for predicting phenotypic resistance determined using broth microdilution based on the presence of detected antimicrobial resistance genes (ARGs). ARGs with poor sensitivity and high specificity included rph (rifampicin, 0%, 93%), mph (erythromycin, 0%, 99%), bla1 (penicillin, 29%, 100), and blaZ (penicillin, 56%, 100%). Compared to penicillin, bla1 and blaZ had lower specificity for the prediction of ampicillin resistance. Overall, none of the ARGs had both high sensitivity and specificity, suggesting the need for further study of the mechanisms underlying phenotypic antimicrobial resistance in the B. cereus group. IMPORTANCE Bacillus 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, the comparison of antimicrobial susceptibility testing results using the disk diffusion method is complicated by the fact that many previous studies have used Staphylococcus spp. breakpoints to interpret their results. In this study, we compared the results of disk diffusion interpreted using the Staphylococcus spp. breakpoints against the results of broth microdilution interpreted using Bacillus spp. breakpoints. We demonstrated that the disk diffusion method does not produce reliable results for B. cereus group isolates and should therefore be avoided. This study also provides new insight into poor associations between the presence of antimicrobial resistance genes and resistance phenotypes for the B. cereus group.

Elucidating the genomic history of commercially used Bacillus thuringiensis subsp. tenebrionis strain NB176

“…Four beta-lactamase genes were detected, in agreement with previous studies that found beta-lactamase genes as well as phenotypic resistance to be very common in the B. cereus group, including B. thuringiensis ( Luna et al., 2007 ; Park et al., 2009 ; Kim et al., 2015 ; Yim et al., 2015 ; Fiedler et al., 2019 ; Bianco et al., 2021 ; Mills et al., 2022 ). Even though a single vancomycin resistance gene was found, a vancomycin resistance seems unlikely, as individual van genes were previously reported in the B. cereus group but were insufficient to confer phenotypic resistance ( Arthur et al., 1992 ; Patel et al., 1997 ; Bianco et al., 2021 ; Mills et al., 2022 ). Genes associated with resistance to macrolide ( mhpL , abc-f ), lincosamide-streptogramin ( lsa ), streptogramin ( vat ), streptothricin ( sat A ), phenicol ( catA ) and fosfomycin ( fosB ) drug families were also detected in the three analyzed strains.…”

“…Thirteen chromosomally encoded AMR genes were predicted. Four beta-lactamase genes were detected, in agreement with previous studies that found beta-lactamase genes as well as phenotypic resistance to be very common in the B. cereus group, including B. thuringiensis (Luna et al, 2007;Park et al, 2009;Kim et al, 2015;Yim et al, 2015;Fiedler et al, 2019;Bianco et al, 2021;Mills et al, 2022). Even though a single vancomycin resistance gene was found, a vancomycin resistance seems unlikely, as individual van genes were previously reported in the B. cereus group but were insufficient to confer phenotypic resistance (Arthur et al, 1992;Patel et al, 1997;Bianco et al, 2021;Mills et al, 2022).…”

“…Some isolates of the B. cereus group are known for aminoglycoside resistance ( Parulekar and Sonawane, 2018 ; Fiedler et al., 2019 ) whereas others carried nucleotidyltransferase genes but were not phenotypically resistant ( Fiedler et al., 2019 ). Since only few studies associate the presence of AMR ( Mills et al., 2022 ) or virulence genes to phenotypic traits, no direct conclusion can be drawn from in silico studies about the actual functionality of any of these genes or the conditions under which these genes might be expressed.…”

Resistance determinants of emerging pathogens isolated from an intensive care unit as a parameter of population health conditions of the Legal Amazon microregion

“…The broad diversity of Bacillus allows gene regulation that increases its pathogenicity (Bottone, 2010). Previous studies identified MDR phenotypes in this group (Torkar and Bedenic, 2018;Liu et al, 2018, Mills et al, 2022. Some MDR strains of Bacillus sp obtained in the present study cause food poisoning.…”

Prevalence, distribution, enterotoxin profiles, antimicrobial resistance, and genetic diversity of Bacillus cereus group isolates from lettuce farms in Korea

“…Since B. cereus group strains are clinically significant, determining their resistance/susceptibility toward antimicrobial agents is essential for treatment during disease outbreaks. Even though most infections caused by B. cereus group strains may get better without antimicrobial treatment, it is important to be vigilant as new B. cereus group infection strains may emerge in the future ( Mills et al, 2022 ). Based on the antibiotic susceptibility profiles, the B. cereus group isolates were further classified into four different groups (I, II, III, and IV) according to the MDR patterns ( Table 7 ).…”