Development of in vitro resistance to fluoroquinolones in Pseudomonas aeruginosa. - Related Documents




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576301.0000Development 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.202032758289
625910.9998Evidence 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.200010990265
576520.9998Expression 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.201830201819
576630.9998Ceftazidime 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.202337192202
583640.9998Identification 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.201020953948
576150.9998The 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.202134987489
576460.9998Aminoglycoside-Modifying Enzymes Are Sufficient to Make Pseudomonas aeruginosa Clinically Resistant to Key Antibiotics. Aminoglycosides are widely used to treat infections of Pseudomonas aeruginosa. Genes encoding aminoglycoside-modifying enzymes (AMEs), acquired by horizontal gene transfer, are commonly associated with aminoglycoside resistance, but their effects have not been quantified. The aim of this research was to determine the extent to which AMEs increase the antibiotic tolerance of P. aeruginosa. Bioinformatics analysis identified AME-encoding genes in 48 out of 619 clinical isolates of P. aeruginosa, with ant(2')-Ia and aac(6')-Ib3, which are associated with tobramcyin and gentamicin resistance, being the most common. These genes and aph(3')-VIa (amikacin resistance) were deleted from antibiotic-resistant strains. Antibiotic minimum inhibitory concentrations (MICs) were reduced by up to 64-fold, making the mutated bacteria antibiotic-sensitive in several cases. Introduction of the same genes into four antibiotic-susceptible P. aeruginosa strains increased the MIC by up to 128-fold, making the bacteria antibiotic-resistant in all cases. The cloned genes also increased the MIC in mutants lacking the MexXY-OprM efflux pump, which is an important contributor to aminoglycoside resistance, demonstrating that AMEs and this efflux pump act independently in determining levels of aminoglycoside tolerance. Quantification of the effects of AMEs on antibiotic susceptibility demonstrates the large effect that these enzymes have on antibiotic resistance.202235884138
626470.9998Multi-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.202438041251
626380.9998Gene-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.202133875431
584090.9997Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa. Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. To reduce the selection pressure for resistance, it is important to determine the antibiotic susceptibility pattern of bacteria so that hospital patients can be treated with more narrow-spectrum and target-specific antibiotics. This study describes the development of a technique for detecting point muations in the fluoroquinolone resistance-determining region of the gyrA and parC genes as well as the efflux regulatory genes mexR, mexZ and mexOZ that are associated with fluoroquinolone and aminoglycoside resistance. The assay is based on a short DNA sequencing method using multiplex-fast polymerase chain reaction (PCR) and Pyrosequencing for amplification and sequencing of the selected genes. Fifty-nine clinical isolates of P. aeruginosa were examined for mutations in the abovementioned genes. Mutations related to antibiotic resistance were detected in codons 83 and 87 of gyrA and codon 126 of the mexR regulatory gene. Results of this study suggest Pyrosequencing as a substitute for traditional methods as it provides a rapid and reliable technique for determining the antibiotic resistance pattern of a given bacterial strain in <1 h.200919656662
5757100.9997The expression regulation of recA gene and bacterial class 2 integron-associated genes induced by antibiotics. OBJECTIVE: To investigate the effects and mechanisms of common antibiotics induction on the expression of class 2 integron integrase and variable region resistance genes in bacteria, as well as potential structural mutations. METHODS: Clinical isolates containing non-functional class 2 integrons and functional class 2 integrons were selected. Strains containing non-functional class 2 integrons or functional class 2 integrons were constructed using isolated DNA templates. These strains were subjected to continuous induction with drug concentrations of 1/2 MIC and 1/4 MIC (ciprofloxacin, ampicillin, and kanamycin) and a concentration of 0.2 μg/ml (mitomycin C) over 8 days. The relative expression levels of relevant genes were measured on days 1, 3, and 8. Drug resistance in the experimental strains was assessed before and after induction to identify any differences. Finally, the sequence of the non-functional class 2 integron integrase gene was analyzed for structural changes that occurred as a result of induction. RESULTS: All drugs selected in this study increased the relative expression levels of recA, intI2, dfrA1, sat2, and aadA1. Significant differences in inductive abilities were observed among the drugs. The 1/2 MIC concentrations were more effective than 1/4 MIC concentrations in increasing the relative expression levels of target genes and enhancing the resistance of the experimental strains. The relative expression levels of recA, intI2, and dfrA1 rose on day 1, peaked on day 3, and slightly declined by day 8. Induced strains exhibited increased resistance to the drugs, with the most significant changes observed in the clinical isolates, particularly concerning CIP resistance. Notably, clinical isolate 7b induced with 1/2 MIC KAN exhibited the loss of one base at position 12bp in the integrase sequence. However, none of the four drugs induced mutations at the 444 bp position of class 2 integrons. CONCLUSION: Sub-MIC concentrations of drugs have been shown to induce an increase in the relative expression level of the SOS response-related gene recA, as well as the integrase and resistance genes of class 2 integrons. Continuous induction leads to sustained upregulation of these genes, which stabilizes or slightly decreases upon reaching a plateau. However, the capacity of different drugs to induce expression varies significantly. Short-term antibiotic exposure did not result in critical mutations that convert class 2 integrons into functional forms.202540950603
5762110.9997Evolution of antimicrobial resistance in E. coli biofilm treated with high doses of ciprofloxacin. The evolution of antimicrobial resistance (AMR) has mainly been studied in planktonic bacteria exposed to sub-inhibitory antimicrobial (AM) concentrations. However, in a number of infections that are treated with AMs the bacteria are located in biofilms where they tolerate high doses of AM. In the present study, we continuously exposed biofilm residing E. coli at body temperature to high ciprofloxacin (CIP) concentrations increasing from 4 to 130 times the minimal inhibitory concentration (MIC), i.e., from 0.06 to 2.0 mg/L. After 1 week, the biofilms were full of CIP resistant bacteria. The evolutionary trajectory observed was the same as described in the literature for planktonic bacteria, i.e., starting with a single mutation in the target gene gyrA followed by mutations in parC, gyrB, and parE, as well as in genes for regulation of multidrug efflux pump systems and outer membrane porins. Strains with higher numbers of these mutations also displayed higher MIC values. Furthermore, the evolution of CIP resistance was more rapid, and resulted in strains with higher MIC values, when the bacteria were biofilm residing than when they were in a planktonic suspension. These results may indicate that extensive clinical AM treatment of biofilm-residing bacteria may not only fail to eradicate the infection but also pose an increased risk of AMR development.202337731931
5759120.9997The Relationship between Antibiotic Susceptibility and pH in the Case of Uropathogenic Bacteria. Urinary tract infections (UTIs) are common bacterial infections caused mainly by enteric bacteria. Numerous virulence factors assist bacteria in the colonization of the bladder. Bacterial efflux pumps also contribute to bacterial communication and to biofilm formation. In this study, the phenotypic and genetic antibiotic resistance of clinical UTI pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis were determined by disk diffusion method and polymerase chain reaction (PCR). Following this, different classes of antibiotics were evaluated for their antibacterial activity at pH 5, 6, 7 and 8 by a microdilution method. Gentamicin (GEN) was the most potent antibacterial agent against E. coli strains. The effect of GEN on the relative expression of marR and sdiA genes was evaluated by quantitative PCR. The slightly acidic pH (pH 6) and GEN treatment induced the upregulation of marR antibiotic resistance and sdiA QS activator genes in both E. coli strains. Consequently, bacteria had become more susceptible to GEN. It can be concluded that antibiotic activity is pH dependent and so the artificial manipulation of urinary pH can contribute to a more effective therapy of multidrug resistant bacterial infections.202134943643
6258130.9997Alterations 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.199910103206
5760140.9997Downregulation of Klebsiella pneumoniae RND efflux pump genes following indole signal produced by Escherichia coli. BACKGROUND: More than a century has passed since it was discovered that many bacteria produce indole, but research into the actual biological roles of this molecule is just now beginning. The influence of indole on bacterial virulence was extensively investigated in indole-producing bacteria like Escherichia coli. To gain a deeper comprehension of its functional role, this study investigated how indole at concentrations of 0.5-1.0 mM found in the supernatant of Escherichia coli stationary phase culture was able to alter the virulence of non-indole-producing bacteria, such as Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, which are naturally exposed to indole in mixed infections with Escherichia coli. RESULTS: Biofilm formation, antimicrobial susceptibility, and efflux pump activity were the three phenotypic tests that were assessed. Indole was found to influence antibiotic susceptibly of Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumoniae to ciprofloxacin, imipenem, ceftriaxone, ceftazidime, and amikacin through significant reduction in MIC with fold change ranged from 4 to 16. Biofilm production was partially abrogated in both 32/45 Pseudomonas aeruginosa and all eight Proteus mirabilis, while induced biofilm production was observed in 30/40 Klebsiella pneumoniae. Moreover, acrAB and oqxAB, which encode four genes responsible for resistance-nodulation-division multidrug efflux pumps in five isolates of Klebsiella pneumoniae were investigated genotypically using quantitative real-time (qRT)-PCR. This revealed that all four genes exhibited reduced expression indicated by 2^-ΔΔCT < 1 in indole-treated isolates compared to control group. CONCLUSION: The outcomes of qRT-PCR investigation of efflux pump expression have established a novel clear correlation of the molecular mechanism that lies beneath the influence of indole on bacterial antibiotic tolerance. This research provides novel perspectives on the various mechanisms and diverse biological functions of indole signaling and how it impacts the pathogenicity of non-indole-producing bacteria.202439182027
6254150.9997Role of the multidrug efflux system MexXY in the emergence of moderate resistance to aminoglycosides among Pseudomonas aeruginosa isolates from patients with cystic fibrosis. This study investigates the role of active efflux system MexXY in the emergence of aminoglycoside (AG) resistance among cystic fibrosis (CF) isolates of Pseudomonas aeruginosa. Three genotypically related susceptible and resistant (S/R) bacterial pairs and three other AG-resistant CF strains were compared to four non-CF strains moderately resistant to AGs. As demonstrated by immunoblot experiments, pump MexY was strongly overproduced in all of the resistant bacteria. This MexXY upregulation was associated with a 2- to 16-fold increase in the MICs of AGs in the S/R pairs and lower intracellular accumulation of dihydrostreptomycin. Alterations in mexZ, the repressor gene of operon mexXY, were found in all of the AG-resistant CF isolates and in one non-CF strain. Complementation of these bacteria with a plasmid-borne mexZ gene dramatically reduced the MICs of AGs, thus highlighting the role played by MexXY in the development of moderate resistance in CF patients. In contrast, complementation of the three non-CF strains showing wild-type mexZ genes left residual levels of resistance to AGs. These data indicate that a locus different from mexZ may be involved in overproduction of MexXY and that other nonenzymatic mechanisms contribute to AG resistance in P. aeruginosa.200415105120
6262160.9997Potential of Tetracycline Resistance Proteins To Evolve Tigecycline Resistance. Tigecycline is a glycylcycline antibiotic active against multidrug-resistant bacterial pathogens. The objectives of our study were to examine the potential of the Tet(A), Tet(K), Tet(M), and Tet(X) tetracycline resistance proteins to acquire mutations causing tigecycline resistance and to determine how this affects resistance to earlier classes of tetracyclines. Mutations in all four tet genes caused a significant increase in the tigecycline MIC in Escherichia coli, and strains expressing mutant Tet(A) and Tet(X) variants reached clinically relevant MICs (2 mg/liter and 3 mg/liter, respectively). Mutations predominantly accumulated in transmembrane domains of the efflux pumps, most likely increasing the accommodation of tigecycline as a substrate. All selected Tet(M) mutants contained at least one mutation in the functionally most important loop III of domain IV. Deletion of leucine 505 of this loop led to the highest increase of the tigecycline MIC (0.5 mg/liter) among Tet(M) mutants. It also caused collateral sensitivity to earlier classes of tetracyclines. A majority of the Tet(X) mutants showed increased activity against all three classes of tetracylines. All tested Tet proteins have the potential to acquire mutations leading to increased MICs of tigecycline. As tet genes are widely found in pathogenic bacteria and spread easily by horizontal gene transfer, resistance development by alteration of existing Tet proteins might compromise the future medical use of tigecycline. We predict that Tet(X) might become the most problematic future Tet determinant, since its weak intrinsic tigecycline activity can be mutationally improved to reach clinically relevant levels without collateral loss in activity to other tetracyclines.201626596936
3805170.9997De 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.201627431218
5768180.9997The resistance mechanism of Escherichia coli induced by ampicillin in laboratory. BACKGROUND: Multi-drug-resistant Escherichia coli poses a great threat to human health, especially resistant to ampicillin (AMP), but the mechanism of drug resistance is not very clear. PURPOSE: To understand the mechanism of resistance of E. coli to beta-lactam antibiotics by inducing drug resistance of sensitive bacteria in laboratory. METHODS: Clinical sensitive E. coli strain was induced into resistance strain by 1/2 minimum inhibitive concentration (MIC) induced trails of AMP. The drug resistance spectrum was measured by modified K-B susceptibility test. Whole-genome sequencing analysis was used to analyze primary sensitive strain, and resequencing was used to analyze induced strains. Protein tertiary structure encoded by the gene containing single nucleotide polymorphism (SNP) was analyzed by bioinformatics. RESULTS: After 315 hrs induced, the MIC value of E. coli 15743 reached to 256 µg/mL, 64 times higher than that of the sensitive bacteria. During the induction process, the bacterial resistance process is divided into two stages. The rate of drug resistance occurs rapidly before reaching the critical concentration of 32 µg/mL, and then the resistance rate slows down. Sequencing of the genome of resistant strain showed that E. coli 15743 drug-resistant strain with the MIC values of 32 and 256 µg/mL contained four and eight non-synonymous SNPs, respectively. These non-synonymous SNPs were distributed in the genes of frdD, ftsI, acrB, OmpD, marR, VgrG, and envZ. CONCLUSION: These studies will improve our understanding of the molecular mechanism of AMP resistance of E. coli, and may provide the basis for prevention and control of multi-drug-resistant bacteria and generation of new antibiotics to treat E. coli infection.201931571941
5758190.9997RND pump inhibition: in-silico and in-vitro study by Eugenol on clinical strain of E. coli and P. aeruginosa. Multidrug-resistant (MDR) gram-negative bacteria pose significant challenges to the public health. Various factors are involved in the development and spread of MDR strains, including the overuse and misuse of antibiotics, the lack of new antibiotics being developed, and etc. Efflux pump is one of the most important factors in the emergence of antibiotic resistance in bacteria. Aiming at the introduction of novel plant antibiotic, we investigated the effect of eugenol on the MexA and AcrA efflux pumps in Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). Molecular docking was performed using PachDock Server 1.3. The effect of eugenol on bacteria was determined by disk diffusion, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). A cartwheel test was also performed to evaluate efflux pump inhibition. Finally, the expression of the MexA and AcrA genes was examined by real-time PCR. The results of molecular docking showed that eugenol interacted with MexA and AcrA pumps at - 29.28 and - 28.59 Kcal.mol(-1), respectively. The results of the antibiogram test indicated that the antibiotic resistance of the treated bacteria decreased significantly (p < 0.05). The results of the cartwheel test suggested the inhibition of efflux pump activity in P. aeruginosa and E. coli. Analysis of the genes by real-time PCR demonstrated that the expression of MexA and AcrA genes was significantly reduced, compared to untreated bacteria (p < 0.001). The findings suggest, among other things, that eugenol may make P. aeruginosa and E. coli more sensitive to antibiotics and that it could be used as an inhibitor to prevent bacteria from becoming resistant to antibiotics.202337587975