Collateral sensitivity in clinical mecillinam resistant isolates of Escherichia coli

Background The rapid increase in antimicrobial resistance (AMR) has become a major threat to the successful management of infectious diseases. To counteract this global threat, development of novel treatment strategies is essential. A promising strategy may be exploiting collateral sensitivity; a phenomenon that occurs when a microorganism that has developed resistance to one antimicrobial agent, exhibits increased susceptibility to another antimicrobial agent. In order to develop novel treatment strategies and prevent further resistance development, we aimed to explore the generality of the concept of collateral sensitivity in clinical urinary tract isolates of E. coli. Furthermore, we wanted to investigate the underlying mechanisms of collateral sensitivity. Methods We evolved resistance to mecillinam in a collection of clinical isolates of E. coli. Ten were selected for further determination of possible collateral sensitivity and cross-resistance networks. The IC90-assay with micro broth dilution was used for this purpose, which we tested for eight different antimicrobial agents. The results were displayed in heat maps and graphs showing the distribution of AMR to various agents. PCR and DNA sequencing were performed for the mrdA gene to detect mutations that may confer mecillinam resistance. Results According to our results both collateral sensitivity and cross-resistance occurred in mecillinam resistant isolates. Chloramphenicol presented the highest tendency of collateral sensitivity, while ciprofloxacin presented the highest tendency of cross-resistance. In general, a substantial tendency for collateral sensitivity frequently appeared compared to cross-resistance. Moreover, 13 synonymous point mutations were observed in the mrdA gene, leading to no alteration in the amino acid sequence. Conclusion Based on our in vitro results, we suggest mecillinam could be a good candidate to be employed as the first drug of choice for UTIs caused by E. coli. Mecillinam resistant isolates exhibited a clear tendency for collateral sensitivity, which we believe would occur on the population level as well. Further investigations of the underlying mechanisms of collateral sensitivity are required

Collateral sensitivity in clinical Escherichia coli isolates

Background At present time, antimicrobial resistance is emerging more rapidly than the development of novel antimicrobials, presenting a serious threat to how we prevent and treat infectious diseases. Several treatment strategies to counteract this development have been proposed, among these is the use of collateral sensitivity in clinical treatment. The ability to predict collateral sensitivity and cross-resistance effects is essential to exploiting this concept. In this study, we aimed to investigate the patterns of collateral sensitivity and cross-resistance in ciprofloxacin resistant isolates carrying gyrA and parC mutations. Method Ciprofloxacin resistant isolates were evolved from three clinical E. coli strain using static and dynamic selection methods. Isolates were selected based on identified mutations and level of ciprofloxacin resistance measured with diffusion gradient strips. DNA sequencing was used to detect mutations in gyrA and parC. Resistant isolates carrying at least one gyrA and parC mutation were characterized by IC90 assays with micro-broth dilutions of six unrelated antimicrobial agents. The observed collateral sensitivity and cross-resistance effects were displayed in a heat map. Results Various non-synonymous point mutations in gyrA and parC were identified in several of the generated ciprofloxacin resistant isolates. These mutants displayed collateral sensitivity and cross-resistance to several unrelated antimicrobials. Collateral sensitivity to gentamicin and trimethoprim was observed in the majority isolates. Cross-resistance effects were found in several mutants, specifically to ceftazidime, chloramphenicol and colistin. Conclusion Our findings suggest that ciprofloxacin resistant mutants with gyrA and parC mutations display a clear tendency of collateral sensitivity to gentamicin, an effect which potentially can be exploited in future treatment. However, we propose further investigation into specific point mutations within these genes, to better understand the observed variations in collateral sensitivity and cross-resistance

Alternative evolutionary paths to bacterial antibiotic resistance cause distinct collateral effects.

Published onlineJournal ArticleThis is the author accepted manuscript. The final version is available from Oxford University Press via the DOI in this record.When bacteria evolve resistance against a particular antibiotic, they may simultaneously gain increased sensitivity against a second one. Such collateral sensitivity may be exploited to develop novel, sustainable antibiotic treatment strategies aimed at containing the current, dramatic spread of drug resistance. To date, the presence and molecular basis of collateral sensitivity has only been studied in few bacterial species and is unknown for opportunistic human pathogens such as Pseudomonas aeruginosa. In the present study, we assessed patterns of collateral effects by experimentally evolving 160 independent populations of P. aeruginosa to high levels of resistance against eight commonly used antibiotics. The bacteria evolved resistance rapidly and expressed both collateral sensitivity and cross-resistance. The pattern of such collateral effects differed to those previously reported for other bacterial species, suggesting inter-specific differences in the underlying evolutionary trade-offs. Intriguingly, we also identified contrasting patterns of collateral sensitivity and cross-resistance among the replicate populations adapted to the same drug. Whole-genome sequencing of 81 independently evolved populations revealed distinct evolutionary paths of resistance to the selective drug, which determined whether bacteria became cross-resistant or collaterally sensitive towards others. Based on genomic and functional genetic analysis, we demonstrate that collateral sensitivity can result from resistance mutations in regulatory genes such as nalC or mexZ, which mediate aminoglycoside sensitivity in β-lactam-adapted populations, or the two-component regulatory system gene pmrB, which enhances penicillin sensitivity in gentamicin-resistant populations. Our findings highlight substantial variation in the evolved collateral effects among replicates, which in turn determine their potential in antibiotic therapy.We thank Anette Friedrichs, Lutz Becks, and the Schulenburg group for valuable advice and Melanie Vollstedt for technical support during genome sequencing. We are grateful for financial support from the German Science Foundation (DFG grant SCHU 1415/12-1) and the International Max-Planck Research School for Evolutionary Biology at the University of Kiel. We acknowledge infrastructural support by the DFG excellence cluster Inflammation at Interfaces

Collateral sensitivity in clinical Escherichia coli isolates resistant to ciprofloxacin and/or mecillinam

The escalating emergence of AMR is a growing public health concern, and a result of the use and misuse of antibiotics since its introduction. To prevent the development of resistance and preserve the efficacy of antimicrobial agents, new treatment strategies is of utmost importance. One possible approach may be to take advantage of collateral sensitivity, a phenomenon where bacteria acquiring resistance to one antimicrobial drug simultaneously became more sensitive to others. Our aim in this project is to investigate the generality of collateral sensitivity in clinical urinary tract isolates of E. coli. In addition, we wanted to investigate effects of the mutations on biofilm forming ability of the resistant mutants

Polo like kinase 2 tumour suppressor and cancer biomarker: new perspectives on drug sensitivity/resistance in ovarian cancer

The polo-like kinase PLK2 has recently been identified as a potential theranostic marker in the management of chemotherapy sensitive cancers. The methylation status of the PLK2 CpG island varies with sensitivity to paclitaxel and platinum in ovarian cancer cell lines. Importantly, extrapolation of these in vitro data to the clinical setting confirms that the methylation status of the PLK2 CpG island predicts outcomes in patients treated with carboplatin and paclitaxel chemotherapy. A second cell cycle regulator, p57Kip2, is also subject to epigenetic silencing in carboplatin resistance in vitro and in vivo, emphasising that cell cycle regulators are important determinants of sensitivity to chemotherapeutic agents and providing insights into the phenomenon of collateral drug sensitivity in oncology. Understanding the mechanistic basis and identification of robust biomarkers to predict collateral sensitivity may inform optimal use of chemotherapy in patients receiving multiple lines of treatment

Time-programmable drug dosing allows the manipulation, suppression and reversal of antibiotic drug resistance in vitro

Multi-drug strategies have been attempted to prolong the efficacy of existing antibiotics, but with limited success. Here we show that the evolution of multi-drug-resistant Escherichia coli can be manipulated in vitro by administering pairs of antibiotics and switching between them in ON/OFF manner. Using a multiplexed cell culture system, we find that switching between certain combinations of antibiotics completely suppresses the development of resistance to one of the antibiotics. Using this data, we develop a simple deterministic model, which allows us to predict the fate of multi-drug evolution in this system. Furthermore, we are able to reverse established drug resistance based on the model prediction by modulating antibiotic selection stresses. Our results support the idea that the development of antibiotic resistance may be potentially controlled via continuous switching of drugs

Understanding evolution to tackle antibiotic resistance in Pseudomonas aeruginosa

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 04-11-2022Antibiotic resistance (AR) constitutes a major public health concern, which has been aggravated in recent decades due to the emergence and spread of multidrug-resistant microorganisms, especially Gram-negative bacteria. Among them, Pseudomonas aeruginosa stands out; it is an opportunistic pathogen, widely distributed in nature, that frequently infects hospitalized patients and presents low susceptibility to many antimicrobials, as well as an overwhelming capacity to develop AR via mutation, mainly during chronic infections. Hence, novel treatment strategies are needed to deal with the infections caused by this bacterium. Collateral sensitivity, whereby acquiring resistance to one drug increases susceptibility to a second drug, is an evolutionary trade-off that may be exploited for treating bacterial infections by the combination or sequential use of drugs' pairs. This application is only possible if those collateral sensitivity phenotypes are conserved within different genetic contexts, environments and situations; robust collateral sensitivity events were searched for during this thesis. We determined that tobramycin, tigecycline and ceftazidime resistance acquisition in P. aeruginosa is associated with a robust fosfomycin collateral sensitivity and ascertained the mechanism responsible for this event. Further, we observed that ciprofloxacin exposure selects distinct mutations in different genetic backgrounds of P. aeruginosa, all of them leading to a robust tobramycin and aztreonam collateral sensitivity, and we proposed tobramycin-ciprofloxacin and ciprofloxacin-aztreonam combinations as promising therapies against infections caused by this bacterium. We also determined that media composition and nutrients’ availability constrain the pathways towards tobramycin, ceftazidime and ceftazidime-avibactam resistance in P. aeruginosa, but fosfomycin collateral sensitivity associated with ceftazidime resistance robustly emerges when P. aeruginosa evolves in different media mimicking those that can be encountered during infection. The compensation of fitness costs associated with the acquisition of AR in the absence of selective pressure could cause a decline of AR, which may also be used for designing therapeutic strategies considering those specific antibiotics whose resistance is robustly unstable in absence of selection. In this thesis, we observed that compensatory evolution of fitness costs associated with ceftazidime resistance in P. aeruginosa leads to a ceftazidime resistance decline in distinct genetic backgrounds, both in antibiotic-free and in sublethal tobramycin environments. The alternation of ceftazidime with drug restriction periods or the switch back to ceftazidime after a ceftazidime-tobramycin alternation may be feasible therapeutic approaches against P. aeruginosa infections. For its part, AR may be transiently induced by some conditions encountered by bacteria during infection, compromising the antibiotic treatments. In this thesis we identified dequalinium chloride, procaine and atropine, which can be present in P. aeruginosa site infections, as inducers of the expression of MexCD-OprJ efflux pump encoding genes, hence transiently increasing ciprofloxacin resistance of this bacterium. Finally, by further studying efflux pumps regulation and considering their ancestral function, we determined that the identification of compounds which are both substrates and inducers of efflux pumps of P. aeruginosa constitutes an effective strategy for finding molecules that reduce the virulence potential of this pathogen. Overall, the results of this thesis allow us to propose novel treatment strategies against P. aeruginosa infections, based on the identification of novel drugs and on the rational use of the antibiotics that we already have, as well as to better understand AR evolutio

Bacterial evolution of antibiotic hypersensitivity.

The evolution of resistance to a single antibiotic is frequently accompanied by increased resistance to multiple other antimicrobial agents. In sharp contrast, very little is known about the frequency and mechanisms underlying collateral sensitivity. In this case, genetic adaptation under antibiotic stress yields enhanced sensitivity to other antibiotics. Using large-scale laboratory evolutionary experiments with Escherichia coli, we demonstrate that collateral sensitivity occurs frequently during the evolution of antibiotic resistance. Specifically, populations adapted to aminoglycosides have an especially low fitness in the presence of several other antibiotics. Whole-genome sequencing of laboratory-evolved strains revealed multiple mechanisms underlying aminoglycoside resistance, including a reduction in the proton-motive force (PMF) across the inner membrane. We propose that as a side effect, these mutations diminish the activity of PMF-dependent major efflux pumps (including the AcrAB transporter), leading to hypersensitivity to several other antibiotics. More generally, our work offers an insight into the mechanisms that drive the evolution of negative trade-offs under antibiotic selection