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Antibiotic resistance is one of the greatest threats against human health and the misuse and overuse of antibiotics is a key factor driving resistance development. During prolonged antibiotic treatment of chronic infections, the antimicrobial pressure facilitates selection of antibiotic resistance mutations. It has been suggested that using antibiotics in combinations may reduce the emergence of resistance. Furthermore, antibiotic tolerant persister cells may be a reservoir for resistance development, so targeting persister cells with anti-persister drugs could also reduce the emergence of resistance.In this study, we conducted a 42-day adaptive laboratory evolution experiment using Staphylococcus aureus exposed to common antibiotics and the anti-persister drug mitomycin C, either alone or in combination. We monitored susceptibility daily and assessed phenotypic changes in growth and biofilm formation in evolved strains. Whole-genome sequencing revealed mutations linked to antibiotic resistance and phenotypic shifts.Resistance developed rapidly against rifampicin, while ciprofloxacin and daptomycin showed slower resistance emergence. Treatments with vancomycin or mitomycin C resulted in minimal changes in susceptibility. Combination therapies generally delayed resistance, though resistance was not fully prevented. Notably, mitomycin C combined with rifampicin effectively suppressed rifampicin resistance. Sub-inhibitory antibiotic concentrations were associated with both known and novel mutations, including in the nucleotide excision repair system and azoreductase, following mitomycin C treatment—mutations not previously reported.While combination therapy delayed resistance, mitomycin C’s efficacy and ability to prevent rifampicin resistance highlights its potential in combating antibiotic resistance. Further investigation is needed to evaluate the broader application of anti-persister drugs in resistance prevention.
Antibiotic resistance is one of the greatest threats against human health and the misuse and overuse of antibiotics is a key factor driving resistance development. During prolonged antibiotic treatment of chronic infections, the antimicrobial pressure facilitates selection of antibiotic resistance mutations. It has been suggested that using antibiotics in combinations may reduce the emergence of resistance. Furthermore, antibiotic tolerant persister cells may be a reservoir for resistance development, so targeting persister cells with anti-persister drugs could also reduce the emergence of resistance.In this study, we conducted a 42-day adaptive laboratory evolution experiment using Staphylococcus aureus exposed to common antibiotics and the anti-persister drug mitomycin C, either alone or in combination. We monitored susceptibility daily and assessed phenotypic changes in growth and biofilm formation in evolved strains. Whole-genome sequencing revealed mutations linked to antibiotic resistance and phenotypic shifts.Resistance developed rapidly against rifampicin, while ciprofloxacin and daptomycin showed slower resistance emergence. Treatments with vancomycin or mitomycin C resulted in minimal changes in susceptibility. Combination therapies generally delayed resistance, though resistance was not fully prevented. Notably, mitomycin C combined with rifampicin effectively suppressed rifampicin resistance. Sub-inhibitory antibiotic concentrations were associated with both known and novel mutations, including in the nucleotide excision repair system and azoreductase, following mitomycin C treatment—mutations not previously reported.While combination therapy delayed resistance, mitomycin C’s efficacy and ability to prevent rifampicin resistance highlights its potential in combating antibiotic resistance. Further investigation is needed to evaluate the broader application of anti-persister drugs in resistance prevention.
The pathogenicity of Staphylococcus aureus is largely regulated by the agr quorum sensing (QS) system encoded by agrBDCA, which coordinates virulence factor production through secretion and sensing of auto-inducing peptides (AIPs). agr-like systems are present also in coagulase-negative staphylococci, and several of these encode AIPs that inhibit S. aureus QS. In lactic acid bacteria, a similar locus was previously identified in Lactiplantibacillus plantarum WCSF1 termed lamBDCA. Here, we characterized the lamBDCA locus in L. plantarum LMG 13556 and L. paraplantarum CIRM-BIA 1870, and explored the effects on S. aureus QS. Notably, we found that co-cultivation with L. paraplantarum significantly inhibits S. aureus QS and hemolysin production, while less so for L. plantarum. The inhibition by L. paraplantarum was lost upon disruption of its lamBDCA locus, suggesting that the L. paraplantarum AIP mediates cross-species interference with S. aureus agr activation. Transcriptomic analysis revealed that lamBDCA in L. paraplantarum controls the expression of genes belonging to various functional categories, including stress response and metabolism. The latter includes genes encoding riboflavin (B2 vitamin) biosynthesis, which enabled the growth of the L. paraplantarum lamB mutant in the presence of roseoflavin, a toxic riboflavin analogue. Collectively, our results show that L. paraplantarum CIRM-BIA 1870 interferes with S. aureus virulence gene expression through QS suppression, and they implicate QS in the probiotic properties of L. paraplantarum.
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