# | Rank | Similarity | Title + Abs. | Year | PMID |
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 |
| 6264 | 0 | 1.0000 | Multi-drug resistance pattern and genome-wide SNP detection in levofloxacin-resistant uropathogenic Escherichia coli strains. OBJECTIVES: Antibiotic treatment is extremely stressful for bacteria and has profound effects on their viability. Such administration induces physiological changes in bacterial cells, with considerable impact on their genome structure that induces mutations throughout the entire genome. This study investigated drug resistance profiles and structural changes in the entire genome of uropathogenic Escherichia coli (UPEC) strains isolated from six adapted clones that had evolved under laboratory conditions. METHODS: Eight UPEC strains, including two parental strains and six adapted clones, with different fluoroquinolone resistance levels originally isolated from two patients were used. The minimum inhibitory concentration (MIC) of 28 different antibiotics including levofloxacin was determined for each of the eight strains. In addition, the effects of mutations acquired with increased drug resistance in the levofloxacin-resistant strains on expression of genes implicated to be involved in drug resistance were examined. RESULTS: Of the eight UPEC strains used to test the MIC of 28 different antibiotics, two highly fluoroquinolone-resistant strains showed increased MIC in association with many of the antibiotics. As drug resistance increased, some genes acquired mutations, including the transcriptional regulator acrR and DNA-binding transcriptional repressor marR. Two strain groups with genetically different backgrounds (GUC9 and GFCS1) commonly acquired mutations in acrR and marR. Notably, acquired mutations related to efflux pump upregulation also contributed to increases in MIC for various antibiotics other than fluoroquinolone. CONCLUSIONS: The present results obtained using strains with artificially acquired drug resistance clarify the underlying mechanism of resistance to fluoroquinolones and other types of antibiotics. | 2024 | 38041251 |
| 6263 | 1 | 0.9998 | Gene-Gene Interactions Dictate Ciprofloxacin Resistance in Pseudomonas aeruginosa and Facilitate Prediction of Resistance Phenotype from Genome Sequence Data. Ciprofloxacin is one of the most widely used antibiotics for treating Pseudomonas aeruginosa infections. However, P. aeruginosa acquires mutations that confer ciprofloxacin resistance, making treatment more difficult. Resistance is multifactorial, with mutations in multiple genes influencing the resistance phenotype. However, the contributions of individual mutations and mutation combinations to the amounts of ciprofloxacin that P. aeruginosa can tolerate are not well understood. Engineering P. aeruginosa strain PAO1 to contain mutations in any one of the resistance-associated genes gyrA, nfxB, rnfC, parC, and parE showed that only gyrA mutations increased the MIC for ciprofloxacin. Mutations in parC and parE increased the MIC of a gyrA mutant, making the bacteria ciprofloxacin resistant. Mutations in nfxB and rnfC increased the MIC, conferring resistance, only if both were mutated in a gyrA background. Mutations in all of gyrA, nfxB, rnfC, and parC/E further increased the MIC. These findings reveal an epistatic network of gene-gene interactions in ciprofloxacin resistance. We used this information to predict ciprofloxacin resistance/susceptibility for 274 isolates of P. aeruginosa from their genome sequences. Antibiotic susceptibility profiles were predicted correctly for 84% of the isolates. The majority of isolates for which prediction was unsuccessful were ciprofloxacin resistant, demonstrating the involvement of additional as yet unidentified genes and mutations in resistance. Our data show that gene-gene interactions can play an important role in antibiotic resistance and can be successfully incorporated into models predicting resistance phenotype. | 2021 | 33875431 |
| 6276 | 2 | 0.9998 | A shared mechanism of multidrug resistance in laboratory-evolved uropathogenic Escherichia coli. The emergence of multidrug-resistant bacteria poses a significant threat to human health, necessitating a comprehensive understanding of their underlying mechanisms. Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, is frequently associated with multidrug resistance and recurrent infections. To elucidate the mechanism of resistance of UPEC to beta-lactam antibiotics, we generated ampicillin-resistant UPEC strains through continuous exposure to low and high levels of ampicillin in the laboratory, referred to as Low Amp(R) and High Amp(R), respectively. Whole-genome sequencing revealed that both Low and High Amp(R) strains contained mutations in the marR, acrR, and envZ genes. The High Amp(R) strain exhibited a single additional mutation in the nlpD gene. Using protein modeling and qRT-PCR analyses, we validated the contributions of each mutation in the identified genes to antibiotic resistance in the Amp(R) strains, including a decrease in membrane permeability, increased expression of multidrug efflux pump, and inhibition of cell lysis. Furthermore, the Amp(R) strain does not decrease the bacterial burden in the mouse bladder even after continuous antibiotic treatment in vivo, implicating the increasing difficulty in treating host infections caused by the Amp(R) strain. Interestingly, ampicillin-induced mutations also result in multidrug resistance in UPEC, suggesting a common mechanism by which bacteria acquire cross-resistance to other classes of antibiotics. | 2024 | 38899601 |
| 6255 | 3 | 0.9998 | Effects of a Mutation in the gyrA Gene on the Virulence of Uropathogenic Escherichia coli. Fluoroquinolones are among the drugs most extensively used for the treatment of bacterial infections in human and veterinary medicine. Resistance to quinolones can be chromosome or plasmid mediated. The chromosomal mechanism of resistance is associated with mutations in the DNA gyrase- and topoisomerase IV-encoding genes and mutations in regulatory genes affecting different efflux systems, among others. We studied the role of the acquisition of a mutation in the gyrA gene in the virulence and protein expression of uropathogenic Escherichia coli (UPEC). The HC14366M strain carrying a mutation in the gyrA gene (S83L) was found to lose the capacity to cause cystitis and pyelonephritis mainly due to a decrease in the expression of the fimA, papA, papB, and ompA genes. The levels of expression of the fimA, papB, and ompA genes were recovered on complementing the strain with a plasmid containing the gyrA wild-type gene. However, only a slight recovery was observed in the colonization of the bladder in the GyrA complement strain compared to the mutant strain in a murine model of ascending urinary tract infection. In conclusion, a mutation in the gyrA gene of uropathogenic E. coli reduced the virulence of the bacteria, likely in association with the effect of DNA supercoiling on the expression of several virulence factors and proteins, thereby decreasing their capacity to cause cystitis and pyelonephritis. | 2015 | 26014933 |
| 6259 | 4 | 0.9998 | Evidence of an efflux pump in Serratia marcescens. Spontaneous mutants resistant to fluoroquinolones were obtained by exposing Serratia marcescens NIMA (wild-type strain) to increasing concentrations of ciprofloxacin both in liquid and on solid media. Frequencies of mutation ranged from 10(-7) to 10(-9). Active expulsion of antibiotic was explored as a possible mechanism of resistance in mutants as well as changes in topoisomerase target genes. The role of extrusion mechanisms in determining the emergence of multidrug-resistant bacteria was also examined. Mutants resistant to high concentrations of fluoroquinolones had a single mutation in their gyrA QRDR sequences, whereas the moderate resistance in the rest of mutants was due to extrusion of the drug. | 2000 | 10990265 |
| 5766 | 5 | 0.9998 | Ceftazidime resistance in Pseudomonas aeruginosa is multigenic and complex. Pseudomonas aeruginosa causes a wide range of severe infections. Ceftazidime, a cephalosporin, is a key antibiotic for treating infections but a significant proportion of isolates are ceftazidime-resistant. The aim of this research was to identify mutations that contribute to resistance, and to quantify the impacts of individual mutations and mutation combinations. Thirty-five mutants with reduced susceptibility to ceftazidime were evolved from two antibiotic-sensitive P. aeruginosa reference strains PAO1 and PA14. Mutations were identified by whole genome sequencing. The evolved mutants tolerated ceftazidime at concentrations between 4 and 1000 times that of the parental bacteria, with most mutants being ceftazidime resistant (minimum inhibitory concentration [MIC] ≥ 32 mg/L). Many mutants were also resistant to meropenem, a carbapenem antibiotic. Twenty-eight genes were mutated in multiple mutants, with dacB and mpl being the most frequently mutated. Mutations in six key genes were engineered into the genome of strain PAO1 individually and in combinations. A dacB mutation by itself increased the ceftazidime MIC by 16-fold although the mutant bacteria remained ceftazidime sensitive (MIC < 32 mg/L). Mutations in ampC, mexR, nalC or nalD increased the MIC by 2- to 4-fold. The MIC of a dacB mutant was increased when combined with a mutation in ampC, rendering the bacteria resistant, whereas other mutation combinations did not increase the MIC above those of single mutants. To determine the clinical relevance of mutations identified through experimental evolution, 173 ceftazidime-resistant and 166 sensitive clinical isolates were analysed for the presence of sequence variants that likely alter function of resistance-associated genes. dacB and ampC sequence variants occur most frequently in both resistant and sensitive clinical isolates. Our findings quantify the individual and combinatorial effects of mutations in different genes on ceftazidime susceptibility and demonstrate that the genetic basis of ceftazidime resistance is complex and multifactorial. | 2023 | 37192202 |
| 5838 | 6 | 0.9998 | Alteration in the Morphological and Transcriptomic Profiles of Acinetobacter baumannii after Exposure to Colistin. Acinetobacter baumannii is often highly resistant to multiple antimicrobials, posing a risk of treatment failure, and colistin is a "last resort" for treatment of the bacterial infection. However, colistin resistance is easily developed when the bacteria are exposed to the drug, and a comprehensive analysis of colistin-mediated changes in colistin-susceptible and -resistant A. baumannii is needed. In this study, using an isogenic pair of colistin-susceptible and -resistant A. baumannii isolates, alterations in morphologic and transcriptomic characteristics associated with colistin resistance were revealed. Whole-genome sequencing showed that the resistant isolate harbored a PmrB(L208F) mutation conferring colistin resistance, and all other single-nucleotide alterations were located in intergenic regions. Using scanning electron microscopy, it was determined that the colistin-resistant mutant had a shorter cell length than the parental isolate, and filamented cells were found when both isolates were exposed to the inhibitory concentration of colistin. When the isolates were treated with inhibitory concentrations of colistin, more than 80% of the genes were upregulated, including genes associated with antioxidative stress response pathways. The results elucidate the morphological difference between the colistin-susceptible and -resistant isolates and different colistin-mediated responses in A. baumannii isolates depending on their susceptibility to this drug. | 2024 | 39203486 |
| 5836 | 7 | 0.9998 | Identification of Pseudomonas aeruginosa genes associated with antibiotic susceptibility. Pseudomonas aeruginosa causes acute and chronic infections in humans and these infections are difficult to treat due to the bacteria's high-level of intrinsic and acquired resistance to antibiotics. To address this problem, it is crucial to investigate the molecular mechanisms of antibiotic resistance in this organism. In this study, a P. aeruginosa transposon insertion library of 17000 clones was constructed and screened for altered susceptibility to seven antibiotics. Colonies grown on agar plates containing antibiotics at minimum inhibitory concentrations (MICs) and those unable to grow at 1/2 MIC were collected. The transposon-disrupted genes in 43 confirmed mutants that showed at least a three-fold increase or a two-fold decrease in susceptibility to at least one antibiotic were determined by semi-random PCR and subsequent sequencing analysis. In addition to nine genes known to be associated with antibiotic resistance, including mexI, mexB and mexR, 24 new antibiotic resistance-associated genes were identified, including a fimbrial biogenesis gene pilY1 whose disruption resulted in a 128-fold increase in the MIC of carbenicillin. Twelve of the 43 genes identified were of unknown function. These genes could serve as targets to control or reverse antibiotic resistance in this important human pathogen. | 2010 | 20953948 |
| 6319 | 8 | 0.9998 | Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica. Heteroresistance, a phenomenon where subpopulations of a bacterial isolate exhibit different susceptibilities to an antibiotic, is a growing clinical problem where the underlying genetic mechanisms in most cases remain unknown. We isolated colistin resistant mutants in Escherichia coli and Salmonella enterica serovar Typhimurium at different concentrations of colistin. Genetic analysis showed that genetically stable pmrAB point mutations were responsible for colistin resistance during selection at high drug concentrations for both species and at low concentrations for E. coli. In contrast, for S. Typhimurium mutants selected at low colistin concentrations, amplification of different large chromosomal regions conferred a heteroresistant phenotype. All amplifications included the pmrD gene, which encodes a positive regulator that up-regulates proteins that modify lipid A, and as a result increase colistin resistance. Inactivation and over-expression of the pmrD gene prevented and conferred resistance, respectively, demonstrating that the PmrD protein is required and sufficient to confer resistance. The heteroresistance phenotype is explained by the variable gene dosage of pmrD in a population, where sub-populations with different copy number of the pmrD gene show different levels of colistin resistance. We propose that variability in gene copy number of resistance genes can explain the heteroresistance observed in clinically isolated pathogenic bacteria. | 2016 | 27381382 |
| 6266 | 9 | 0.9998 | Bacterial gene loss as a mechanism for gain of antimicrobial resistance. Acquisition of exogenous DNA by pathogenic bacteria represents the basis for much of the acquired antimicrobial resistance in pathogenic bacteria. A more extreme mechanism to avoid the effect of an antibiotic is to delete the drug target, although this would be predicted to be rare since drug targets are often essential genes. Here, we review and discuss the description of a novel mechanism of resistance to the cephalosporin drug ceftazidime caused by loss of a penicillin-binding protein (PBP) in a Gram-negative bacillus (Burkholderia pseudomallei). This organism causes melioidosis across south-east Asia and northern Australia, and is usually treated with two or more weeks of ceftazidime followed by oral antibiotics for three to six months. Comparison of clinical isolates from six patients with melioidosis found initial ceftazidime-susceptible isolates and subsequent ceftazidime-resistant variants. The latter failed to grow on commonly used culture media, rendering these isolates difficult to detect in the diagnostic laboratory. Genomic analysis using pulsed-field gel electrophoresis and array based genomic hybridisation revealed a large-scale genomic deletion comprising 49 genes in the ceftazidime-resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a PBP 3 present within the region of genomic loss. This provides one explanation for ceftazidime treatment failure, and may be a frequent but undetected event in patients with melioidosis. | 2012 | 23022568 |
| 6273 | 10 | 0.9998 | Burkholderia multivorans Exhibits Antibiotic Collateral Sensitivity. Burkholderia multivorans is a member of the Burkholderia cepacia complex whose members are inherently resistant to many antibiotics and can cause chronic lung infections in patients with cystic fibrosis. A possible treatment for chronic infections arises from the existence of collateral sensitivity (CS)-acquired resistance to a treatment antibiotic results in a decreased resistance to a nontreatment antibiotic. Determining CS patterns for bacteria involved in chronic infections may lead to sustainable treatment regimens that reduce development of multidrug-resistant bacterial strains. CS has been found to occur in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Here, we report that B. multivorans exhibits antibiotic CS, as well as cross-resistance (CR), describe CS and CR networks for six antibiotics (ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, and trimethoprim-sulfamethoxazole), and identify candidate genes involved in CS. Characterization of CS and CR patterns allows antibiotics to be separated into two clusters based on the treatment drug to which the evolved strain developed primary resistance, suggesting an antibiotic therapy strategy of switching between members of these two clusters. | 2020 | 31393205 |
| 3805 | 11 | 0.9998 | De Novo Characterization of Genes That Contribute to High-Level Ciprofloxacin Resistance in Escherichia coli. Sensitization of resistant bacteria to existing antibiotics depends on the identification of candidate targets whose activities contribute to resistance. Using a transposon insertion library in an Escherichia coli mutant that was 2,000 times less susceptible to ciprofloxacin than its parent and the relative fitness scores, we identified 19 genes that contributed to the acquired ciprofloxacin resistance and mapped the shortest genetic path that increased the antibiotic susceptibility of the resistant bacteria back to a near wild-type level. | 2016 | 27431218 |
| 6265 | 12 | 0.9998 | Fitness costs of fluoroquinolone resistance in Streptococcus pneumoniae. The fitness cost of the genes responsible for resistance to fluoroquinolones in clinical isolates of Streptococcus pneumoniae were estimated in vitro in a common genetic background. Naturally occurring parC, parE, and gyrA loci containing mutations in the quinolone-resistance-determining regions were introduced by transformation into S. pneumoniae strain R6 individually and in combinations. The fitness of these transformants was estimated by pairwise competition experiments with a common R6 strain. On average, single par and gyr mutants responsible for low-level MIC resistance (first-step resistance) impose a fitness burden of approximately 8%. Some of these mutants engender no measurable cost, while one, a parE mutant, reduces the fitness of these bacteria by more than 40%. Most interestingly, the addition of the second par or gyr mutations required for clinically significant, high-MIC fluoroquinolone resistance does not increase the fitness burden imposed by these single genes and can even reduce it. We discuss the implications of these results for the epidemiology of fluoroquinolone resistance and the evolution of acquired resistance in treated patients. | 2007 | 17116668 |
| 5763 | 13 | 0.9998 | Development of in vitro resistance to fluoroquinolones in Pseudomonas aeruginosa. Fluoroquinolone resistance in Pseudomonas aeruginosa typically arises through site-specific mutations and overexpression of efflux pumps. In this study, we investigated the dynamics of different resistance mechanisms in P. aeruginosa populations that have evolved under fluoroquinolone pressure, as well as the interactions between these mechanisms in evolutionary trajectories. Bacteria of strain ATCC27853 were selected under different concentrations of ciprofloxacin and levofloxacin for six parallel lineages, followed by amplification of four target genes in the quinolone-resistance determining region (QRDR) and Sanger sequencing to identify the mutations. The expression of four efflux pump proteins was evaluated by real-time polymerase chain reaction using the relative quantitation method, with the ATCC27853 strain used as a control. We found that ciprofloxacin killed P. aeruginosa sooner than did levofloxacin. Further, we identified five different mutations in three subunits of QRDRs, with gyrA as the main mutated gene associated with conferring fluoroquinolone resistance. Additionally, we found a larger number of mutations appearing at 2 mg/L and 4 mg/L of ciprofloxacin and levofloxacin, respectively. Moreover, we identified the main efflux pump being expressed as MexCD-OprJ, with initial overexpression observed at 0.25 mg/L and 0.5 mg/L of ciprofloxacin and levofloxacin, respectively. These results demonstrated gyrA(83) mutation and MexCD-OprJ overexpression as the primary mechanism conferring ciprofloxacin and levofloxacin resistance in P. aeruginosa. In addition, we also show that ciprofloxacin exhibited a stronger ability to kill the bacteria while potentially rendering it more susceptible to resistance. | 2020 | 32758289 |
| 6258 | 14 | 0.9997 | Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium. High-level quinolone resistance in Enterococcus faecium was associated with mutations in both gyrA and parC genes in 10 of 11 resistant strains. On low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as Enterococcus faecalis. | 1999 | 10103206 |
| 5761 | 15 | 0.9997 | The Effects of Sub-inhibitory Antibiotic Concentrations on Pseudomonas aeruginosa: Reduced Susceptibility Due to Mutations. Pseudomonas aeruginosa chronically infects in the lungs of people with cystic fibrosis and other forms of lung disease. Infections are treated with antibiotics, but over time, the bacteria acquire mutations that reduce their antibiotic susceptibility. The effects of inhibitory amounts of antibiotics in selecting for antibiotic-resistant mutants have been well studied. However, the concentrations of antibiotics that reach infecting bacteria can be sub-inhibitory and but may nonetheless promote emergence of antibiotic-resistant bacteria. Therefore, the aim of this research was to investigate the effects of sub-inhibitory concentrations of antibiotics on the antibiotic susceptibility of P. aeruginosa. Two P. aeruginosa reference strains, PAO1 and PA14, and six isolates from individuals with cystic fibrosis were studied. The bacteria were passaged in the presence of antibiotics (ceftazidime, ciprofloxacin, meropenem or tobramycin) at sub-inhibitory amounts. Fifteen populations of bacteria (up to five per strain) were exposed to each of the four antibiotics. Antibiotic susceptibility was determined following 10 passages on agar supplemented with antibiotic and compared with susceptibility prior to antibiotic exposure. Antibiotic exposure resulted in susceptibility being significantly (>2-fold) reduced for 13 of the 60 populations. Seven samples had reduced susceptibility to ciprofloxacin, three to tobramycin, two to ceftazidime and one to meropenem. Whole-genome sequencing revealed the mutations arising following antibiotic exposure. Mutants with reduced antibiotic susceptibility had mutations in genes known to affect antibiotic resistance, including regulators of efflux pumps (mexR, mexS, mexZ and nalC) and the fusA1 gene that is associated with aminoglycoside resistance. Genes not previously associated with resistance, including gacS, sigX and crfX and two genes with no known function, were also mutated in some isolates with reduced antibiotic susceptibility. Our results show that exposure to sub-inhibitory amounts of antibiotics can select for mutations that reduce the susceptibility of P. aeruginosa to antibiotics and that the profile of mutations is different from that arising during selection with inhibitory antibiotic concentrations. It is likely that exposure to sub-inhibitory amounts of antibiotics during infection contributes to P. aeruginosa becoming antibiotic-resistant. | 2021 | 34987489 |
| 5765 | 16 | 0.9997 | Expression of Pseudomonas aeruginosa Antibiotic Resistance Genes Varies Greatly during Infections in Cystic Fibrosis Patients. The lungs of individuals with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa that is difficult to eradicate by antibiotic treatment. Two key P. aeruginosa antibiotic resistance mechanisms are the AmpC β-lactamase that degrades β-lactam antibiotics and MexXYOprM, a three-protein efflux pump that expels aminoglycoside antibiotics from the bacterial cells. Levels of antibiotic resistance gene expression are likely to be a key factor in antibiotic resistance but have not been determined during infection. The aims of this research were to investigate the expression of the ampC and mexX genes during infection in patients with CF and in bacteria isolated from the same patients and grown under laboratory conditions. P. aeruginosa isolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription-quantitative PCR (RT-qPCR). The expression of ampC varied over 20,000-fold and that of mexX over 2,000-fold between isolates. The median expression levels of both genes were increased by the presence of subinhibitory concentrations of antibiotics. To measure P. aeruginosa gene expression during infection, we carried out RT-qPCR using RNA extracted from fresh sputum samples obtained from 31 patients. The expression of ampC varied over 4,000-fold, while mexX expression varied over 100-fold, between patients. Despite these wide variations, median levels of expression of ampC in bacteria in sputum were similar to those in laboratory-grown bacteria. The expression of mexX was higher in sputum than in laboratory-grown bacteria. Overall, our data demonstrate that genes that contribute to antibiotic resistance can be highly expressed in patients, but there is extensive isolate-to-isolate and patient-to-patient variation. | 2018 | 30201819 |
| 5977 | 17 | 0.9997 | Methods to determine antibiotic resistance gene silencing. The occurrence of antibiotic-resistant bacteria is an increasingly serious problem world-wide. In addition, to phenotypically resistant bacteria, a threat may also be posed by isolates with silent, but intact, antibiotic resistance genes. Such isolates, which have recently been described, possess wild-type genes that are not expressed, but may convert to resistance by activating expression of the silent genes. They may therefore compromise the efficacy of antimicrobial treatment, particularly if their presence has not been diagnosed. This chapter describes the detection of silent resistance genes by PCR and DNA sequencing. A method to detect five potentially silent acquired resistance genes; aadA, bla (OXA-2), strAB, sul1, and tet(A) is described. First, the susceptibility of the isolates to the relevant antibiotics is determined by an appropriate susceptibility testing method, such as E-test. Then the presence of the genes is investigated by PCR followed by agarose gel electrophoresis of the amplification products. If a resistance gene is detected in a susceptible isolate, the entire open-reading frame and promoter sequence of the gene is amplified by PCR and their DNA sequences obtained. The DNA sequences are then compared to those of known resistant isolates, to detect mutations that may account for susceptibility. If no mutations are detected the expression of the gene is investigated by RT-PCR following RNA extraction. The methods described here can be applied to all acquired resistance genes for which sequence and normal expression data are available. | 2010 | 20401584 |
| 6267 | 18 | 0.9997 | Beta-lactamase dependent and independent evolutionary paths to high-level ampicillin resistance. The incidence of beta-lactam resistance among clinical isolates is a major health concern. A key method to study the emergence of antibiotic resistance is adaptive laboratory evolution. However, in the case of the beta-lactam ampicillin, bacteria evolved in laboratory settings do not recapitulate clinical-like resistance levels, hindering efforts to identify major evolutionary paths and their dependency on genetic background. Here, we used the Microbial Evolution and Growth Arena (MEGA) plate to select ampicillin-resistant Escherichia coli mutants with varying degrees of resistance. Whole-genome sequencing of resistant isolates revealed that ampicillin resistance was acquired via a combination of single-point mutations and amplification of the gene encoding beta-lactamase AmpC. However, blocking AmpC-mediated resistance revealed latent adaptive pathways: strains deleted for ampC were able to adapt through combinations of changes in genes involved in multidrug resistance encoding efflux pumps, transcriptional regulators, and porins. Our results reveal that combinations of distinct genetic mutations, accessible at large population sizes, can drive high-level resistance to ampicillin even independently of beta-lactamases. | 2024 | 38918379 |
| 6277 | 19 | 0.9997 | A large-scale whole-genome comparison shows that experimental evolution in response to antibiotics predicts changes in naturally evolved clinical Pseudomonas aeruginosa. Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have mutations that make them antibiotic resistant, making treatment difficult. To identify resistance-associated mutations we experimentally evolved the antibiotic sensitive strain P. aeruginosa PAO1 to become resistant to three widely used anti-pseudomonal antibiotics, ciprofloxacin, meropenem and tobramycin. Mutants could tolerate up to 2048-fold higher concentrations of antibiotic than strain PAO1. Genome sequences were determined for thirteen mutants for each antibiotic. Each mutant had between 2 and 8 mutations. For each antibiotic at least 8 genes were mutated in multiple mutants, demonstrating the genetic complexity of resistance. For all three antibiotics mutations arose in genes known to be associated with resistance, but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in this study we analysed the corresponding genes in 558 isolates of P. aeruginosa from patients with chronic lung disease and in 172 isolates from the general environment. Many genes identified through experimental evolution had predicted function-altering changes in clinical isolates but not in environmental isolates, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. Additionally, large deletions of up to 479kb arose in experimentally evolved meropenem resistant mutants and large deletions were present in 87 of the clinical isolates. These findings significantly advance understanding of antibiotic resistance in P. aeruginosa and demonstrate the validity of experimental evolution in identifying clinically-relevant resistance-associated mutations. | 2019 | 31570397 |