RESUMO
The global spread of antimicrobial resistance has become a prominent issue in both veterinary and public health in the 21st century. The extensive use of amoxicillin, a beta-lactam antibiotic, and consequent resistance development are particularly alarming in food-producing animals, with a focus on the swine and poultry sectors. Another beta-lactam, cefotaxime, is widely utilized in human medicine, where the escalating resistance to third- and fourth-generation cephalosporins is a major concern. The aim of this study was to simulate the development of phenotypic and genotypic resistance to beta-lactam antibiotics, focusing on amoxicillin and cefotaxime. The investigation of the minimal inhibitory concentrations (MIC) of antibiotics was performed at 1×, 10×, 100×, and 1000× concentrations using the modified microbial evolution and growth arena (MEGA-plate) method. Our results indicate that amoxicillin significantly increased the MIC values of several tested antibiotics, except for oxytetracycline and florfenicol. In the case of cefotaxime, this increase was observed in all classes. A total of 44 antimicrobial resistance genes were identified in all samples. Chromosomal point mutations, particularly concerning cefotaxime, revealed numerous complex mutations, deletions, insertions, and single nucleotide polymorphisms (SNPs) that were not experienced in the case of amoxicillin. The findings suggest that, regarding amoxicillin, the point mutation of the acrB gene could explain the observed MIC value increases due to the heightened activity of the acrAB-tolC efflux pump system. However, under the influence of cefotaxime, more intricate processes occurred, including complex amino acid substitutions in the ampC gene promoter region, increased enzyme production induced by amino acid substitutions and SNPs, as well as mutations in the acrR and robA repressor genes that heightened the activity of the acrAB-tolC efflux pump system. These changes may contribute to the significant MIC increases observed for all tested antibiotics. The results underscore the importance of understanding cross-resistance development between individual drugs when choosing clinical alternative drugs. The point mutations in the mdtB and emrR genes may also contribute to the increased activity of the mdtABC-tolC and emrAB-tolC pump systems against all tested antibiotics. The exceptionally high mutation rate induced by cephalosporins justifies further investigations to clarify the exact mechanism behind.
RESUMO
The issue of antimicrobial resistance is becoming an increasingly serious challenge in both human and veterinary medicine. Prudent antimicrobial use in veterinary medicine is warranted and supported by international guidelines, with the Antimicrobial Advice Ad Hoc Expert Group (AMEG) placing particular emphasis on the critically important group B antimicrobials. These antimicrobials are commonly employed, especially in the poultry and swine industry. The impact of florfenicol, a veterinary antibiotic, was studied on the resistance development of Escherichia coli. The aim of the study was to investigate the effect of the use of florfenicol on the development of phenotypic and genomic resistances, not only to the drug itself but also to other drugs. The minimum inhibitory concentrations (MICs) of the antibiotics were investigated at 1×, 10×, 100× and 1000× concentrations using the adapted Microbial Evolution and Growth Arena (MEGA-plate) method. The results demonstrate that florfenicol can select for resistance to fluoroquinolone antibiotics (167× MIC value increase) and cephalosporins (67× MIC value increase). A total of 44 antimicrobial resistance genes were identified, the majority of which were consistent across the samples. Chromosomal point mutations, including alterations in resistance-associated and regulatory genes (acrB, acrR, emrR and robA), are thought to trigger multiple drug efflux pump activations, leading to phenotypically increased resistance. The study underscores the impact of florfenicol and its role in the development of antimicrobial resistance, particularly concerning fluoroquinolone antibiotics and cephalosporins. This study is the first to report florfenicol's dose-dependent enhancement of other antibiotics' MICs, linked to mutations in SOS-box genes (mdtABC-tolC, emrAB-tolC and acrAB-tolC) and increased multidrug efflux pump genes. Mutations in the regulatory genes acrR, emrR and rpbA support the possibility of increased gene expression. The results are crucial for understanding antimicrobial resistance and its development, highlighting the promising potential of in vitro evolutionary and coselection studies for future research.
