Pseudomonas aeruginosa, a major cause of nosocomial and chronic infections, is considered a paradigm of antimicrobial resistance development. However, the evolutionary trajectories of antimicrobial ...resistance and the impact of mutator phenotypes remain mostly unexplored. Therefore, whole-genome sequencing (WGS) was performed in lineages of wild-type and mutator (ΔmutS) strains exposed to increasing concentrations of relevant antipseudomonal agents. WGS provided a privileged perspective of the dramatic effect of mutator phenotypes on the accumulation of random mutations, most of which were transitions, as expected. Moreover, a frameshift mutagenic signature, consistent with error-prone DNA polymerase activity as a consequence of SOS system induction, was also seen. This effect was evidenced for all antibiotics tested, but it was higher for fluoroquinolones than for cephalosporins or carbapenems. Analysis of genotype versus phenotype confirmed expected resistance evolution trajectories but also revealed new pathways. Classical mechanisms included multiple mutations leading to AmpC overexpression (ceftazidime), quinolone resistance-determining region (QRDR) mutations (ciprofloxacin), oprD inactivation (meropenem), and efflux pump overexpression (ciprofloxacin and meropenem). Groundbreaking findings included gain-of-function mutations leading to the structural modification of AmpC (ceftazidime), novel DNA gyrase (GyrA) modification (ciprofloxacin), and the alteration of the β-lactam binding site of penicillin-binding protein 3 (PBP3) (meropenem). A further striking finding was seen in the evolution of meropenem resistance, selecting for specific extremely large (>250 kb) genomic deletions providing a growth advantage in the presence of the antibiotic. Finally, fitness and virulence varied within and across evolved antibiotic-resistant populations, but mutator lineages showed a lower biological cost for some antibiotics.
Abstract One of the most terrible consequences of Darwinian evolution is arguably the emergence and spread of antibiotic resistance, which is becoming a serious menace to modern societies. While ...spontaneous mutation, recombination and horizontal gene transfer are recognized as the main causes of this notorious phenomenon; recent research has raised awareness that sub-lethal concentrations of antibiotics can also foster resistance as an undesirable side-effect. They can produce genetic changes by different ways, including a raise of free radicals within the cell, induction of error-prone DNA-polymerases mediated by SOS response, imbalanced nucleotide metabolism or affect directly DNA. In addition to certain environmental conditions, subinhibitory concentrations of antimicrobials may increase, even more, the mutagenic effect of antibiotics. Here, we review the state of knowledge on antibiotics as promoters of antibiotic resistance.
In recent years, there has been accumulating evidence that antibiotics, besides their antimicrobial action, potentially have a number of undesired side effects that can, at least in some cases, ...promote genetic variability of bacteria. In addition to resistant variants, antibiotics have also been shown to select mutator clones, thus stimulating evolution towards further resistance. Furthermore, mutations, recombination and horizontal gene transfer have been reported to be somehow affected when bacteria are exposed to subinhibitory concentrations of certain antibiotics. These findings may have implications for the use of antibiotics, because they may have undesired side effects, such as enhancing antibiotic resistance evolution. Here we present data supporting (or not) this fearsome possibility and discuss whether this potential threat should be taken into consideration.
Bacterial infections caused by antibiotic-resistant isolates have become a major health problem in recent years, since they are very difficult to treat, leading to an increase in morbidity and ...mortality. Fosfomycin is a broad-spectrum bactericidal antibiotic that inhibits cell wall biosynthesis in both Gram-negative and Gram-positive bacteria. This antibiotic has a unique mechanism of action and inhibits the initial step in peptidoglycan biosynthesis by blocking the enzyme, MurA. Fosfomycin has been used successfully for the treatment of urinary tract infections for a long time, but the increased emergence of antibiotic resistance has made fosfomycin a suitable candidate for the treatment of infections caused by multidrug-resistant pathogens, especially in combination with other therapeutic partners. The acquisition of fosfomycin resistance could threaten the reintroduction of this antibiotic for the treatment of bacterial infection. Here, we analyse the mechanism of action and molecular mechanisms for the development of fosfomycin resistance, including the modification of the antibiotic target, reduced antibiotic uptake and antibiotic inactivation. In addition, we describe the role of each pathway in clinical isolates.
It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for beta-lactams is an important mechanism (target modification) by which ...Gram-positive cocci acquire antibiotic resistance. Among Gram-negative rods (GNR), however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in beta-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical) beta-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for beta-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex beta-lactam resistance response, triggering overproduction of the chromosomal beta-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of beta-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.
Mycobacterium abscessus is an opportunistic, extensively drug-resistant non-tuberculous mycobacterium. Few genomic studies consider its diversity in persistent infections. Our aim was to characterize ...microevolution/reinfection events in persistent infections. Fifty-three sequential isolates from 14 patients were sequenced to determine SNV-based distances, assign resistance mutations and characterize plasmids. Genomic analysis revealed 12 persistent cases (0-13 differential SNVs), one reinfection (15,956 SNVs) and one very complex case (23 sequential isolates over 192 months), in which a first period of persistence (58 months) involving the same genotype 1 was followed by identification of a genotype 2 (76 SNVs) in 6 additional alternating isolates; additionally, ten transient genotypes (88-243 SNVs) were found. A macrolide resistance mutation was identified from the second isolate. Despite high diversity, the genotypes shared a common phylogenetic ancestor and some coexisted in the same specimens. Genomic analysis is required to access the true intra-patient complexity behind persistent infections involving M. abscessus.
Antimicrobial resistance is a continuously increasing threat that severely compromises our antibiotic arsenal and causes thousands of deaths due to hospital-acquired infections by pathogens such as ...Pseudomonas aeruginosa, situation further aggravated by the limited development of new antibiotics. Thus, alternative strategies such as those targeting bacterial resistance mechanisms, virulence or potentiating the activity of our immune system resources are urgently needed. We have recently shown that mutations simultaneously causing the peptidoglycan recycling blockage and the β-lactamase AmpC overexpression impair the virulence of P.aeruginosa. These findings suggested that peptidoglycan metabolism might be a good target not only for fighting antibiotic resistance, but also for the attenuation of virulence and/or potentiation of our innate immune weapons. Here we analyzed the activity of the innate immune elements peptidoglycan recognition proteins (PGRPs) and lysozyme against P. aeruginosa. We show that while lysozyme and PGRPs have a very modest basal effect over P. aeruginosa, their bactericidal activity is dramatically increased in the presence of subinhibitory concentrations of the permeabilizing agent colistin. We also show that the P. aeruginosa lysozyme inhibitors seem to play a very residual protective role even in permeabilizing conditions. In contrast, we demonstrate that, once the permeability barrier is overpassed, the activity of lysozyme and PGRPs is dramatically enhanced when inhibiting key peptidoglycan recycling components (such as the 3 AmpDs, AmpG or NagZ), indicating a decisive protective role for cell-wall recycling and that direct peptidoglycan-binding supports, at least partially, the activity of these enzymes. Finally, we show that recycling blockade when occurring simultaneously with AmpC overexpression determines a further decrease in the resistance against PGRP2 and lysozyme, linked to quantitative changes in the cell-wall. Thus, our results help to delineate new strategies against P. aeruginosa infections, simultaneously targeting β-lactam resistance, cell-wall metabolism and virulence, ultimately enhancing the activity of our innate immune weapons.
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