The Contribution of Efflux Pumps in Mycobacterium abscessus Complex Resistance to Clarithromycin. - Related Documents




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625301.0000The Contribution of Efflux Pumps in Mycobacterium abscessus Complex Resistance to Clarithromycin. The basis of drug resistance in Mycobacterium abscessus is still poorly understood. Nevertheless, as seen in other microorganisms, the efflux of antimicrobials may also play a role in M. abscessus drug resistance. Here, we investigated the role of efflux pumps in clarithromycin resistance using nine clinical isolates of M. abscessus complex belonging to the T28 erm(41) sequevar responsible for the inducible resistance to clarithromycin. The strains were characterized by drug susceptibility testing in the presence/absence of the efflux inhibitor verapamil and by genetic analysis of drug-resistance-associated genes. Efflux activity was quantified by real-time fluorometry. Efflux pump gene expression was studied by RT-qPCR upon exposure to clarithromycin. Verapamil increased the susceptibility to clarithromycin from 4- to ≥64-fold. The efflux pump genes MAB_3142 and MAB_1409 were found consistently overexpressed. The results obtained demonstrate that the T28 erm(41) polymorphism is not the sole cause of the inducible clarithromycin resistance in M. abscessus subsp. abscessus or bolletii with efflux activity providing a strong contribution to clarithromycin resistance. These data highlight the need for further studies on M. abscessus efflux response to antimicrobial stress in order to implement more effective therapeutic regimens and guidance in the development of new drugs against these bacteria.201931540480
625910.9997Evidence 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
630020.9996Assessing the role of the RND efflux pump in metronidazole resistance of Helicobacter pylori by RT-PCR assay. INTRODUCTION: Metronidazole is a significant antibiotic used for eradication of Helicobacter pylori infections and it is of notice that metronidazole-resistant clinical isolates have been found in high rates worldwide. While the RND family of efflux pumps plays a central role in drug resistance among Gram-negative bacteria, this is questionable for H. pylori. METHODOLOGY: To understand whether TolC homologues of RND pumps contribute to metronidazole resistance in H. pylori isolates, expression of four TolC homologous genes of five resistant clinical isolates exposed to varying concentrations of metronidazole were evaluated by RT-PCR and transcriptional analysis. RESULTS: The results indicate that excess amounts of metronidazole are able to increase the expression level of these genes at the transcriptional stage. CONCLUSIONS: Therefore, it may be hypothesized that use of metronidazole in H. pyori infection can induce metronidazole resistance. Furthermore, the RND family of efflux pumps may contribute to metronidazole resistance in clinical isolates of H. pylori.201121389587
576330.9996Development 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
625440.9996Role 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
626450.9996Multi-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
618660.9996A triclosan-ciprofloxacin cross-resistant mutant strain of Staphylococcus aureus displays an alteration in the expression of several cell membrane structural and functional genes. Triclosan is an antimicrobial agent found in many consumer products. Triclosan inhibits the bacterial fatty acid biosynthetic enzyme, enoyl-ACP reductase (FabI). Decreased susceptibility to triclosan correlates with ciprofloxacin resistance in several bacteria. In these bacteria, resistance to both drugs maps to genes encoding multi-drug efflux pumps. The focus of this study was to determine whether triclosan resistance contributes to ciprofloxacin resistance in Staphylococcus aureus. In S. aureus, triclosan resistance maps to a fabI homolog and ciprofloxacin resistance maps to genes encoding DNA gyrase, topoisomerase IV and to the multi-drug efflux pump, NorA. Using a norA overexpressing mutant, we demonstrated that upregulation of NorA does not lead to triclosan resistance. To further investigate triclosan/ciprofloxacin resistance in S. aureus, we isolated triclosan/ciprofloxacin-resistant mutants. The mutants were screened for mutations in the genes encoding the targets of triclosan and ciprofloxacin. One mutant, JJ5, was wild-type for all sequences analyzed. We next monitored the efflux of triclosan from JJ5 and determined that triclosan resistance in the mutant was not due to active efflux of the drug. Finally, gene expression profiling demonstrated that an alteration in cell membrane structural and functional gene expression is likely responsible for triclosan and ciprofloxacin resistance in JJ5.200717997080
576170.9996The 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
449080.9995Mutation analysis of mycobacterial rpoB genes and rifampin resistance using recombinant Mycobacterium smegmatis. Rifampin is a major drug used to treat leprosy and tuberculosis. The rifampin resistance of Mycobacterium leprae and Mycobacterium tuberculosis results from a mutation in the rpoB gene, encoding the β subunit of RNA polymerase. A method for the molecular determination of rifampin resistance in these two mycobacteria would be clinically valuable, but the relationship between the mutations and susceptibility to rifampin must be clarified before its use. Analyses of mutations responsible for rifampin resistance using clinical isolates present some limitations. Each clinical isolate has its own genetic variations in some loci other than rpoB, which might affect rifampin susceptibility. For this study, we constructed recombinant strains of Mycobacterium smegmatis carrying the M. leprae or M. tuberculosis rpoB gene with or without mutation and disrupted their own rpoB genes on the chromosome. The rifampin and rifabutin susceptibilities of the recombinant bacteria were measured to examine the influence of the mutations. The results confirmed that several mutations detected in clinical isolates of these two pathogenic mycobacteria can confer rifampin resistance, but they also suggested that some mutations detected in M. leprae isolates or rifampin-resistant M. tuberculosis isolates are not involved in rifampin resistance.201222252831
625290.9995Burkholderia ubonensis High-Level Tetracycline Resistance Is Due to Efflux Pump Synergy Involving a Novel TetA(64) Resistance Determinant. Burkholderia ubonensis, a nonpathogenic soil bacterium belonging to the Burkholderia cepacia complex (Bcc), is highly resistant to some clinically significant antibiotics. The concern is that B. ubonensis may serve as a resistance reservoir for Bcc or B. pseudomallei complex (Bpc) organisms that are opportunistic human pathogens. Using a B. ubonensis strain highly resistant to tetracycline (MIC, ≥256 µg/ml), we identified and characterized tetA(64) that encodes a novel tetracycline-specific efflux pump of the major facilitator superfamily. TetA(64) and associated TetR(64) regulator expression are induced by tetracyclines. Although TetA(64) is the primary tetracycline and doxycycline resistance determinant, maximum tetracycline and doxycycline resistance requires synergy between TetA(64) and the nonspecific AmrAB-OprA resistance nodulation cell division efflux pump. TetA(64) does not efflux minocycline, tigecycline, and eravacycline. Comprehensive screening of genome sequences showed that TetA(64) is unequally distributed in the Bcc and absent from the Bpc. It is present in some major cystic fibrosis pathogens, like Burkholderia cenocepacia, but absent from others like Burkholderia multivorans The tetR(64)-tetA(64) genes are located in a region of chromosome 1 that is highly conserved in Burkholderia sp. Because there is no evidence for transposition, the tetR(64)-tetA(64) genes may have been acquired by homologous recombination after horizontal gene transfer. Although Burkholderia species contain a resident multicomponent efflux pump that allows them to respond to tetracyclines up to a certain concentration, the acquisition of the single-component TetA(64) by some species likely provides the synergy that these bacteria need to defend against high tetracycline concentrations in niche environments.202133318011
9036100.9995Resistance-nodulation-division efflux pump acrAB is modulated by florfenicol and contributes to drug resistance in the fish pathogen Piscirickettsia salmonis. Piscirickettsia salmonis is a fastidious intracellular pathogen responsible for high mortality rates in farmed salmonids, with serious economic consequences for the Chilean aquaculture industry. Oxytetracycline and florfenicol are the most frequently used antibiotics against P. salmonis, but routine use could contribute to drug resistance. This study identified differentiated florfenicol susceptibilities in two P. salmonis strains, LF-89 and AUSTRAL-005. The less susceptible isolate, AUSTRAL-005, also showed a high ethidium bromide efflux rate, indicating a higher activity of general efflux pump genes than LF-89. The P. salmonis genome presented resistance nodulation division (RND) family members, a family containing typical multidrug resistance-related efflux pumps in Gram-negative bacteria. Additionally, efflux pump acrAB genes were overexpressed in AUSTRAL-005 following exposure to the tolerated maximal concentration of florfenicol, in contrast to LF-89. These results indicate that tolerated maximum concentrations of florfenicol can modulate RND gene expression and increase efflux pump activity. We propose that the acrAB efflux pump is essential for P. salmonis survival at critical florfenicol concentrations and for the generation of antibiotic-resistant bacterial strains.201627190287
6263110.9995Gene-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
6299120.9995The effects of antidepressants fluoxetine, sertraline, and amitriptyline on the development of antibiotic resistance in Acinetobacter baumannii. This study investigates the effects of antidepressants fluoxetine, sertraline, and amitriptyline on the development of antibiotic resistance in clinical Acinetobacter baumannii isolates. The isolates were exposed to fluoxetine, sertraline, and amitriptyline for 30 days, respectively. The bacteria that developed resistance to gentamicin, imipenem, colistin, and ciprofloxacin were isolated and expression levels of some antibiotic-resistance genes were determined by quantitative reverse-transcriptase PCR. Before and after the exposure, minimum inhibitory concentration (MIC) values of the bacteria were determined by the microdilution method. The statistical analysis was performed using Student's t test. A time-dependent increase was observed in the number of bacteria that developed resistance and increased the MIC value. After exposure to fluoxetine and sertraline, decreases were observed for efflux and outer membrane porin genes in isolates that developed colistin resistance, and increases were observed in isolates that developed ciprofloxacin resistance. These observations suggest that these antidepressants have similar effects on the development of resistance. While the exposure to fluoxetine did not result in the development of resistance to imipenem, it was observed after exposure to sertraline and amitriptyline, and a common decrease in ompA gene expression was determined in these isolates. To our knowledge, the comparative effects of selected antidepressants on the development of antibiotic resistance in A. baumannii are reported and presented in the literature here for the first time.202235355118
6268130.9995Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them is a concern during therapy. A recent study revealed that mutation of the tlyA gene, encoding a putative rRNA methyltransferase, confers capreomycin and viomycin resistance in Mycobacterium tuberculosis bacteria. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs; however, reports of cross-resistance to the drugs have been variable. We investigated the role of rrs mutations in capreomycin resistance and examined the molecular basis of cross-resistance to the four drugs in M. tuberculosis laboratory-generated mutants and clinical isolates. Spontaneous mutants were generated to the drugs singularly and in combination by plating on medium containing one or two drugs. The frequencies of recovery of the mutants on single- and dual-drug plates were consistent with single-step mutations. The rrs genes of all mutants were sequenced, and the tlyA genes were sequenced for mutants selected on capreomycin, viomycin, or both; MICs of all four drugs were determined. Three rrs mutations (A1401G, C1402T, and G1484T) were found, and each was associated with a particular cross-resistance pattern. Similar mutations and cross-resistance patterns were found in drug-resistant clinical isolates. Overall, the data implicate rrs mutations as a molecular basis for resistance to each of the four drugs. Furthermore, the genotypic and phenotypic differences seen in the development of cross-resistance when M. tuberculosis bacteria were exposed to one or two drugs have implications for selection of treatment regimens.200516048924
6187140.9995Mechanisms of fluoroquinolone resistance: an update 1994-1998. Fluoroquinolone resistance is mediated by target changes (DNA gyrase and/or topoisomerase IV) and/or decreased intracellular accumulation. The genes (gyrA/gyrB/parC/parE) and proteins of DNA topoisomerase IV show great similarity, both at the nucleotide and amino acid sequence level to those of DNA gyrase. It has been shown that there are hotspots, called the quinolone resistance determining region (QRDR), for mutations within gyrA and parC. Based on the Escherichia coli co-ordinates, the hotspots most favoured for giving rise to decreased susceptibility and/or full resistance to quinolones are at serine 83 and aspartate 87 of gyrA, and at serine 79 and aspartate 83 for parC. Few mutations in gyrB or parE/grlB of any bacteria have been described. Efflux of fluoroquinolones is the major cause of decreased accumulation of these agents; for Staphylococcus aureus, the efflux pump involved in norfloxacin resistance is NorA, and for Streptococcus pneumoniae, PmrA. By analysis of minimum inhibitory concentration (MIC) data derived in the presence and absence of the efflux inhibitor reserpine, it has been shown that up to 50% of ciprofloxacin-resistant clinical isolates of S. pneumoniae may possess enhanced efflux. This suggests that efflux may be an important mechanism of clinical resistance in this species. In Pseudomonas aeruginosa, several efflux operons have been demonstrated genetically and biochemically. These operons are encoded by mex (Multiple EffluX) genes: mexAmexB-oprM, mexCD-OprJ system and mexEF-oprN system. The E. coli efflux pump is the acrAB-tolC system. Both the mar operon and the sox operon can give rise to multiple antibiotic resistance. It has been shown that mutations giving rise to increased expression of the transcriptional activators marA and soxS affect the expression of a variety of different genes, including ompF and acrAB. The net result is that expression of OmpF is reduced and much less drug is able to enter the cell; expression of acrAB is increased, enhancing efflux from the cell.199910553699
6251150.9995Overexpression of Resistance-Nodulation-Division Efflux Pump Genes Contributes to Multidrug Resistance in Aeromonas hydrophila Clinical Isolates. Aeromonas hydrophila is a Gram-negative bacterium that is a critical causative agent of infections in fish and is occasionally responsible for human infections following contact with contaminated water or food. Currently, the extensive use of antibiotics in clinical practice has led to increased number of isolates of multidrug-resistant (MDR) Aeromonas and has posed a serious public health challenge. The efflux pump system is a critical mechanism of antibiotic resistance in most Gram-negative bacteria. However, the role of resistance-nodulation-division (RND)-type efflux pumps in MDR A. hydrophila is not fully understood. We aimed to evaluate the contribution of the RND efflux pump system to MDR A. hydrophila clinical isolates. PCR results indicated a considerable variation in the presence of RND efflux pump genes in clinical isolates compared to that of the environmental reference strain ATCC7966(T). Compared to non-MDR clinical isolates, the expression levels of three putative RND efflux pump genes, AHA0021, AHA1320, and AheB, were significantly elevated in MDR strains. The minimal inhibitory concentrations of piperacillin/tazobactam, imipenem, erythromycin, and polymyxin B were significantly reduced by phenylalanine-arginine β-naphthylamide (PAβN), further supporting the contribution of the RND efflux system in MDR A. hydrophila. We provided evidence supporting the contribution of the RND efflux system to multidrug resistance in A. hydrophila clinical isolates. Further studies are warranted to elucidate the detailed mechanisms that confer intrinsic resistance to antimicrobials in A. hydrophila.202234609911
5766160.9995Ceftazidime 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
6260170.9995Mechanisms of resistance to fluoroquinolones: state-of-the-art 1992-1994. This paper gives an update on the mechanisms of bacterial resistance to fluoroquinolones. The laboratory techniques currently used to determine the mechanism(s) of resistance are outlined, including the use of restriction fragment length polymorphism and single-stranded conformational polymorphism analysis of mutations in gyrA. Alterations in gyrA have continued to be the most reported cause of resistance, with high level resistance due to 2 or more mutations in this gene. Recently, mutations in gyrA of Mycobacterium tuberculosis and Campylobacter jejuni have been described. Complementation studies with plasmid encoded cloned gyrB from Escherichia coli suggest that high fluoroquinolone resistance (minimum inhibitory concentration = 32 mg/L) in Salmonella typhimurium can be due to mutation in both gyrA and gyrB. Decreased fluoroquinolone accumulation into E. coli has been shown to be due to mutations in a number of genes at different loci. Current interest has focused upon the marRAB and soxRS loci, with mutations in genes of either loci giving rise to decreased susceptibility to several unrelated drugs, including fluoroquinolones, tetracycline, chloramphenicol and some beta-lactams, and decreased expression of OmpF. The genetic characterisation of fluoroquinolone efflux from Staphylococcus aureus has shown that efflux occurs in both fluoroquinolone-susceptible and -resistant bacteria. The most likely cause of resistance is overexpression of NorA, giving rise to increased efflux. Recently, 2 efflux systems in Pseudomonas aeruginosa have been proposed, MexA-MexB-OprK and MexC-MexD-OprM, conferring decreased susceptibility to fluoroquinolones, tetracycline, chloramphenicol and some beta-lactams.(ABSTRACT TRUNCATED AT 250 WORDS)19958549336
4705180.9995Upregulation of outer membrane porin gene ompC contributed to enhancement of azithromycin susceptibility in multidrug-resistant Escherichia coli. The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.202438441474
4703190.9995Positive adaptive state: microarray evaluation of gene expression in Salmonella enterica Typhimurium exposed to nalidixic acid. The emergence of antimicrobial resistance among foodborne bacteria associated with food animal production is an important global issue. We hypothesised that antibiotics generate a positive adaptive state in Salmonella that actively contributes to the development of antimicrobial resistance. This is opposed to common views that antimicrobials only act as a passive selective pressure. Microarray analysis was used to evaluate changes in gene expression that occur upon exposure of Salmonella enterica Typhimurium ATCC 14028 to 1.6 microg/mL of nalidixic acid. The results showed a significant (P < 0.02) difference (fold expression differences >2.0) in the expression of 226 genes. Comparatively repressed transcripts included Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Induced genes included efflux pumps representing all five families of multidrug-resistance efflux pumps, outer membrane lipoproteins, and genes involved in regulating lipopolysaccharide chain length. This profile suggests both enhanced antimicrobial export from the cell and membrane permeability adaptations to limit diffusion of nalidixic acid into the cell. Finally, increased expression of the error-prone DNA repair mechanisms were also observed. From these data we show a highly integrated genetic response to nalidixic acid that places Salmonella into a positive adaptive state that elicits mutations. Evaluation of gene expression profile changes that occur during exposure to antibiotics will continue to improve our understanding of the development of antibiotic resistance.200717600486