The Bifunctional Enzyme SpoT Is Involved in the Clarithromycin Tolerance of Helicobacter pylori by Upregulating the Transporters HP0939, HP1017, HP0497, and HP0471. - Related Documents




#
Rank
Similarity
Title + Abs.
Year
PMID
012345
904301.0000The Bifunctional Enzyme SpoT Is Involved in the Clarithromycin Tolerance of Helicobacter pylori by Upregulating the Transporters HP0939, HP1017, HP0497, and HP0471. Clarithromycin (CLA) is a commonly recommended drug for Helicobacter pylori eradication. However, the prevalence of CLA-resistant H. pylori is increasing. Although point mutations in the 23S rRNA are key factors for CLA resistance, other factors, including efflux pumps and regulation genes, are also involved in the resistance of H. pylori to CLA. Guanosine 3'-diphosphate 5'-triphosphate and guanosine 3',5'-bispyrophosphate [(p)ppGpp)], which are synthesized by the bifunctional enzyme SpoT in H. pylori, play an important role for some bacteria to adapt to antibiotic pressure. Nevertheless, no related research involving H. pylori has been reported. In addition, transporters have been found to be related to bacterial drug resistance. Therefore, this study investigated the function of SpoT in H. pylori resistance to CLA by examining the shifts in the expression of transporters and explored the role of transporters in the CLA resistance of H. pylori A ΔspoT strain was constructed in this study, and it was shown that SpoT is involved in H. pylori tolerance of CLA by upregulating the transporters HP0939, HP1017, HP0497, and HP0471. This was assessed using a series of molecular and biochemical experiments and a cDNA microarray. Additionally, the knockout of genes hp0939, hp0471, and hp0497 in the resistant strains caused a reduction or loss (the latter in the Δhp0497 strain) of resistance to CLA. Furthermore, the average expression levels of these four transporters in clinical CLA-resistant strains were considerably higher than those in clinical CLA-sensitive strains. Taken together, our results revealed a novel molecular mechanism of H. pylori adaption to CLA stress.201728242673
893710.9989Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis. The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.202337065137
629320.9989Gentamicin resistance to Escherichia coli related to fatty acid metabolism based on transcriptome analysis. Antibiotic overuse and misuse have promoted the emergence and spread of antibiotic-resistant bacteria. Increasing bacterial resistance to antibiotics is a major healthcare problem, necessitating elucidation of antibiotic resistance mechanisms. In this study, we explored the mechanism of gentamicin resistance by comparing the transcriptomes of antibiotic-sensitive and -resistant Escherichia coli. A total of 410 differentially expressed genes were identified, of which 233 (56.83%) were up-regulated and 177 (43.17%) were down-regulated in the resistant strain compared with the sensitive strain. Gene Ontology (GO) analysis classifies differential gene expression into three main categories: biological processes, cellular components, and molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the up-regulated genes were enriched in eight metabolic pathways, including fatty acid metabolism, which suggests that fatty acid metabolism may be involved in the development of gentamicin resistance in E. coli. This was demonstrated by measuring the acetyl-CoA carboxylase activity, plays a fundamental role in fatty acid metabolism, was increased in gentamicin-resistant E. coli. Treatment of fatty acid synthesis inhibitor, triclosan, promoted gentamicin-mediated killing efficacy to antibiotic-resistant bacteria. We also found that exogenous addition of oleic acid, which involved in fatty acid metabolism, reduced E. coli sensitivity to gentamicin. Overall, our results provide insight into the molecular mechanism of gentamicin resistance development in E. coli.202337224563
628830.9988Regulation of ofloxacin resistance in Escherichia coli strains causing calf diarrhea by quorum-sensing acyl-homoserine lactone signaling molecules. Escherichia coli is a major pathogen responsible for calf diarrhea. However, it has developed resistance to many antimicrobial drugs for their inappropriate usage. The bacterial quorum sensing system transmits information between bacteria, it's important in regulating bacterial virulence, drug and acid resistance and so on. This system can found in Gram-negative bacteria and operates through acyl-homoserine lactone (AHL) signaling molecules. In this study, a type I quorum sensing AHL, N-Octanoyl-L-Homoserine lactone (C8), was added to E. coli growth medium to investigate its regulatory functions in drug resistance. After screening out the strains of E. coli that showed an obvious regulatory effect to the drug ofloxacin (OFX), transcriptomic sequencing was performed on the E. coli strains from the sub-inhibitory concentration group that concentration plus C8 group, and the control group. It shows that C8 significantly influenced resistance to OFX and the minimum inhibitory concentration of OFX in the tested strain was significantly increased. To Analyze transcriptome sequencing results identified 415 differentially expressed genes between the control and sub-inhibitory concentration groups, of which 201 were up-regulated and 214 were down. There were 125 differentially expressed genes between bacteria treated with a sub-inhibitory concentration of OFX and those treated with C8, of which 102 were up-regulated and 23 were down. Finally, It found that to adding the C8 significantly increased the resistance of tested bacteria to OFX. Data from transcriptome sequencing on differently expressed genes helps to explain how the type I quorum sensing system controls drug resistance in E. coli.202539974163
893640.9988Modulation of Iron Import and Metronidazole Resistance in Bacteroides fragilis Harboring a nimA Gene. Bacteroides fragilis is a commensal of the human gut but can also cause severe infections when reaching other body sites, especially after surgery or intestinal trauma. Bacteroides fragilis is an anaerobe innately susceptible to metronidazole, a 5-nitroimidazole drug that is prescribed against the majority of infections caused by anaerobic bacteria. In most of the cases, metronidazole treatment is effective but a fraction of B. fragilis is resistant to even very high doses of metronidazole. Metronidazole resistance is still poorly understood, but the so-called nim genes have been described as resistance determinants. They have been suggested to encode nitroreductases which reduce the nitro group of metronidazole to a non-toxic aminoimidazole. More recent research, however, showed that expression levels of nim genes are widely independent of the degree of resistance observed. In the search for an alternative model for nim-mediated metronidazole resistance, we screened a strain carrying an episomal nimA gene and its parental strain 638R without a nim gene for physiological differences. Indeed, the 638R daughter strain with the nimA gene had a far higher pyruvate-ferredoxin oxidoreductase (PFOR) activity than the parental strain. High PFOR activity was also observed in metronidazole-resistant clinical isolates, either with or without a nim gene. Moreover, the strain carrying a nimA gene fully retained PFOR activity and other enzyme activities such as thioredoxin reductase (TrxR) after resistance had been induced. In the parental strain 638R, these were lost or very strongly downregulated during the development of resistance. Further, after induction of high-level metronidazole resistance, parental strain 638R was highly susceptible to oxygen whereas the daughter strain with a nimA gene was hardly affected. Ensuing RT-qPCR measurements showed that a pathway for iron import via hemin uptake is downregulated in 638R with induced resistance but not in the resistant nimA daughter strain. We propose that nimA primes B. fragilis toward an alternative pathway of metronidazole resistance by enabling the preservation of normal iron levels in the cell.202235756037
894450.9988Polyphosphate Kinase Mediates Antibiotic Tolerance in Extraintestinal Pathogenic Escherichia coli PCN033. Extraintestinal pathogenic Escherichia coli (ExPEC) causes a variety of acute infections in its hosts, and multidrug-resistant strains present significant challenges to public health and animal husbandry. Therefore, it is necessary to explore new drug targets to control E. coli epidemics. Previous studies have reported that ppk mutants of Burkholderia pseudomallei and Mycobacterium tuberculosis are more susceptible than the wild types (WTs) to stress. Therefore, we investigated the stress response to antibiotics mediated by polyphosphate kinase (PPK) in ExPEC strain PCN033. We observed that planktonic cells of a ppk knockout strain (Δppk) were more susceptible to antibiotics than was WT. However, biofilm-grown Δppk cells showed similar susceptibility to that of the WT and were more tolerant than the planktonic cells. During the planktonic lifestyle, the expression of genes involved in antibiotic tolerance (including resistance-conferring genes, and antibiotic influx, and efflux genes) did not change in the Δppk mutant without antibiotic treatment. However, the resistance-conferring gene bla and efflux genes were upregulated more in the WT than in the Δppk mutant by treatment with tazobactam. After treatment with gentamycin, the efflux genes and influx genes were upregulated and downregulated, respectively, more in the WT than in the Δppk mutant. The expression of genes involved in biofilm regulation also changed after treatment with tazobactam or gentamycin, and which is consistent with the results of the biofilm formation. Together, these observations indicate that PPK is important for the antibiotic stress response during the planktonic growth of ExPEC and might be a potential drug target in bacteria.201627242742
516660.9988Illegitimate recombination: an efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis. BACKGROUND: The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. RESULTS: We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). CONCLUSIONS: We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.201222966811
894670.9988Role of the CpxAR two-component signal transduction system in control of fosfomycin resistance and carbon substrate uptake. Although fosfomycin is an old antibiotic, it has resurfaced with particular interest. The antibiotic is still effective against many pathogens that are resistant to other commonly used antibiotics. We have found that fosfomycin resistance of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by the bacterial two-component signal transduction system CpxAR. A cpxA mutant lacking its phosphatase activity results in constitutive activation of its cognate response regulator, CpxR, and fosfomycin resistance. We have shown that fosfomycin resistance requires CpxR because deletion of the cpxR gene in the cpxA mutant restores fosfomycin sensitivity. We have also shown that CpxR directly represses the expression of two genes, glpT and uhpT, which encode transporters that cotransport fosfomycin with their native substrates glycerol-3-phosphate and glucose-6-phosphate, and repression of these genes leads to a decrease in fosfomycin transport into the cpxA mutant. However, the cpxA mutant had an impaired growth phenotype when cultured with glycerol-3-phosphate or glucose-6-phosphate as a sole carbon substrate and was outcompeted by the parent strain, even in nutrient-rich medium. This suggests a trade-off between fosfomycin resistance and the biological fitness associated with carbon substrate uptake. We propose a role for the CpxAR system in the reversible control of fosfomycin resistance. This may be a beneficial strategy for bacteria to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin.201424163343
894580.9988Adaptation of a fluoroquinolone-sensitive Shigella sonnei to norfloxacin exposure. Shigella causes shigellosis that requires antibiotic treatment in severe cases. Sublethal antibiotic concentrations can promote resistance, but their effect on antibiotic-sensitive bacteria before resistance development is unclear. This study investigated the effects of sublethal norfloxacin (NOR) challenges on a NOR-sensitive strain, Shigella sonnei UKMCC1015. Firstly, the whole genome of S. sonnei UKMCC1015 was assembled, and 45 antimicrobial resistance (AMR) genes were identified. Interestingly, transcriptomic analysis showed that low NOR levels do not change either the expression of the AMR genes or NOR targets such as gyrA. Instead, multiple ribosomal protein genes were downregulated, which could be attributed to decreased ribosomal protein promoter activity, modulated by elevated guanosine pentaphosphate and tetraphosphate (ppGpp) levels. This alarmone is involved in the bacterial stringent response during environmental stress, and it is mainly produced from the ppGpp synthetase (relA). Additionally, we observed that a relA overexpression (prolonged period of elevated ppGpp levels) may negatively affect the NOR tolerance of the bacteria. In conclusion, this study revealed that a NOR-sensitive strain responds differently to sublethal NOR than commonly reported in resistant strains.202439100177
903990.9988Resistance of the Burkholderia cepacia complex to fosmidomycin and fosmidomycin derivatives. The Burkholderia cepacia complex (BCC) is a group of 17 closely related opportunistic pathogens that are able to infect the respiratory tract of cystic fibrosis patients. BCC bacteria are intrinsically resistant to many antibiotics and are therefore difficult to eradicate. Fosmidomycin could be a new therapeutic agent to treat BCC infections as it inhibits 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in the non-mevalonate pathway essential in BCC bacteria for isoprenoid synthesis. In this study, the antimicrobial activity of fosmidomycin and eight fosmidomycin derivatives towards 40 BCC strains was investigated. All BCC strains were resistant to fosmidomycin, although addition of glucose-6-phosphate reduced the minimum inhibitory concentration values of FR900098, the fosmidomycin acetyl derivative, from 512 mg/L to 64 mg/L for Burkholderia multivorans and B. cepacia. This enhanced activity was linked to increased expression of the genes involved in glycerol-3-phosphate transport, which appears to be the only route for fosmidomycin import in BCC bacteria. Furthermore, upregulation of a fosmidomycin resistance gene (fsr) encoding an efflux pump was observed during fosmidomycin and FR900098 treatment. These results strongly suggest that the observed resistance in BCC bacteria is due to insufficient uptake accompanied by fosmidomycin and FR900098 efflux.201121724375
6292100.9988Genome-Wide Screening and Characterization of Genes Involved in Response to High Dose of Ciprofloxacin in Escherichia coli. The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Inevitably, considering its extensive use and misuse, resistance toward ciprofloxacin has increased in almost all clinically relevant bacteria. This study aimed to investigate the transcriptome changes at a high concentration of ciprofloxacin in Escherichia coli. In brief, 1,418 differentially expressed genes (DEGs) were identified, from which 773 genes were upregulated by ciprofloxacin, whereas 651 genes were downregulated. Enriched biological pathways reflected the upregulation of biological processes such as DNA damage and repair system, toxin/antitoxin systems, formaldehyde detoxification system. With kyoto encyclopedia of genes and genomes pathway analysis, higher expressed DEGs were associated with "LPS biosynthesis," "streptomycin biosynthesis," and "polyketide sugar unit biosynthesis." Lower expressed DEGs were associated with "biosynthesis of amino acids" and "flagellar assembly" pathways. After treatment of ciprofloxacin, lipopolysaccharide (LPS) release was increased by two times, and the gene expression level of LPS synthesis was elevated (p < 0.05) in both reference and clinical strains. Our results demonstrated that transient exposure to high-dose ciprofloxacin is a double-edged sword. Cautions should be taken when administering high-dose antibiotic treatment for infectious diseases.202235512736
6300110.9988Assessing 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
9041120.9987Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis. BACKGROUND: Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes. RESULTS: Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance. CONCLUSIONS: Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.201121781329
8867130.9987Hfq plays important roles in virulence and stress adaptation in Cronobacter sakazakii ATCC 29544. Cronobacter spp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated with Cronobacter infection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq in C. sakazakii virulence. In the absence of hfq, C. sakazakii was highly attenuated in dissemination in vivo, showed defects in invasion (3-fold) into animal cells and survival (10(3)-fold) within host cells, and exhibited low resistance to hydrogen peroxide (10(2)-fold). Remarkably, the loss of hfq led to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lacking hfq. Together, these data strongly suggest that hfq plays important roles in the virulence of C. sakazakii by participating in the regulation of multiple genes.201525754196
9040140.9987Gene expression changes linked to antimicrobial resistance, oxidative stress, iron depletion and retained motility are observed when Burkholderia cenocepacia grows in cystic fibrosis sputum. BACKGROUND: Bacteria from the Burkholderia cepacia complex (Bcc) are the only group of cystic fibrosis (CF) respiratory pathogens that may cause death by an invasive infection known as cepacia syndrome. Their large genome (> 7000 genes) and multiple pathways encoding the same putative functions make virulence factor identification difficult in these bacteria. METHODS: A novel microarray was designed to the genome of Burkholderia cenocepacia J2315 and transcriptomics used to identify genes that were differentially regulated when the pathogen was grown in a CF sputum-based infection model. Sputum samples from CF individuals infected with the same B. cenocepacia strain as genome isolate were used, hence, other than a dilution into a minimal growth medium (used as the control condition), no further treatment of the sputum was carried out. RESULTS: A total of 723 coding sequences were significantly altered, with 287 upregulated and 436 downregulated; the microarray-observed expression was validated by quantitative PCR on five selected genes. B. cenocepacia genes with putative functions in antimicrobial resistance, iron uptake, protection against reactive oxygen and nitrogen species, secretion and motility were among the most altered in sputum. Novel upregulated genes included: a transmembrane ferric reductase (BCAL0270) implicated in iron metabolism, a novel protease (BCAL0849) that may play a role in host tissue destruction, an organic hydroperoxide resistance gene (BCAM2753), an oxidoreductase (BCAL1107) and a nitrite/sulfite reductase (BCAM1676) that may play roles in resistance to the host defenses. The assumptions of growth under iron-depletion and oxidative stress formulated from the microarray data were tested and confirmed by independent growth of B. cenocepacia under each respective environmental condition. CONCLUSION: Overall, our first full transcriptomic analysis of B. cenocepacia demonstrated the pathogen alters expression of over 10% of the 7176 genes within its genome when it grows in CF sputum. Novel genetic pathways involved in responses to antimicrobial resistance, oxidative stress, and iron metabolism were revealed by the microarray analysis. Virulence factors such as the cable pilus and Cenocepacia Pathogenicity Island were unaltered in expression. However, B. cenocepacia sustained or increased expression of motility-associated genes in sputum, maintaining a potentially invasive phenotype associated with cepacia syndrome.200818801206
4708150.9987Proteomic analysis of nalidixic acid resistance in Escherichia coli: identification and functional characterization of OM proteins. The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. To understand the mechanisms of the resistance is extremely important to the control of these bacteria. In the current study, proteomic methodologies were utilized to characterize OM proteome of Escherichia coli with nalidixic acid (NA) resistance. The OM proteins TolC, OmpT, OmpC and OmpW were found to be up-regulated, and FadL was down-regulated in the NA-resistant E. coli strains. The changes at the level of protein expression were validated using Western blotting. Furthermore, the possible roles these altered proteins played in regulation of NA resistance were investigated using genetically modified strains with the deletion of these genes. The results obtained from functional characterization of these genetically modified strains suggest that TolC and OmpC may play more important roles in the control of NA resistance than other OM proteins identified. To gain better understanding of the mechanisms of NA resistance, we also characterized the role of the two-component system EnvZ/OmpR which is responsible for the regulation of OmpC and OmpF expression in response to NA resistance using their genetically modified strains. Our results suggest that OmpF and the EnvZ/OmpR are also important participants of the pathways regulating the NA resistance of E. coli.200818438992
6295160.9987Anaerobiosis and Mutations Can Reduce Susceptibility of Pseudomonas aeruginosa to Tobramycin Without Reducing the Cellular Concentration of the Antibiotic. Chronic infections of Pseudomonas aeruginosa are commonly treated with tobramycin. During infections, the bacteria can exist under conditions of oxygen deprivation that render them less susceptible to this antibiotic. The aims of this research were to investigate the genetic basis of tobramycin resistance under anaerobic conditions, and to investigate the effects of anaerobiosis and mutations on the cellular concentration of tobramycin. Ten mutants with lowered susceptibility to tobramycin than wild-type bacteria were evolved from a laboratory reference strain under anaerobic conditions. Mutations were identified by genome sequencing. Mutations had arisen most frequently in the fusA1 gene that encodes elongation factor EF-G1A and in genes involved in twitching motility. Cellular concentrations of tobramycin were then measured. Mutations in fusA1 or absence of the MexXY efflux pump that is associated with tobramycin resistance did not alter the cellular tobramycin concentration under either anaerobic or aerobic conditions. Anaerobic growth reduced the cellular concentration of tobramycin, relative to aerobically grown bacteria, in some but not all of five tested P. aeruginosa isolates. Overall, our findings indicate that anaerobiosis and mutations that reduce aminoglycoside effectiveness do not lower the cellular concentration of antibiotic but instead reduce susceptibility through other mechanisms.202540005562
4705170.9987Upregulation 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
6328180.9987Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis. Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.201828847541
4707190.9987Comparative transcriptome analyses of magainin I-susceptible and -resistant Escherichia coli strains. Antimicrobial peptides (AMPs) have attracted considerable attention because of their multiple and complex mechanisms of action toward resistant bacteria. However, reports have increasingly highlighted how bacteria can escape AMP administration. Here, the molecular mechanisms involved in Escherichia coli resistance to magainin I were investigated through comparative transcriptomics. Sub-inhibitory concentrations of magainin I were used to generate four experimental groups, including magainin I-susceptible E. coli, in the absence (C) and presence of magainin I (CM); and magainin I-resistant E. coli in the absence (R) and presence of magainin I (RM). The total RNA from each sample was extracted; cDNA libraries were constructed and further submitted for Illumina MiSeq sequencing. After RNA-seq data pre-processing and functional annotation, a total of 103 differentially expressed genes (DEGs) were identified, mainly related to bacterial metabolism. Moreover, down-regulation of cell motility and chaperone-related genes was observed in CM and RM, whereas cell communication, acid tolerance and multidrug efflux pump genes (ABC transporter, major facilitator and resistance-nodulation cell division superfamilies) were up-regulated in these same groups. DEGs from the C and R groups are related to basal levels of expression of homeostasis-related genes compared to CM and RM, suggesting that the presence of magainin I is required to change the transcriptomics panel in both C and R E. coli strains. These findings show the complexity of E. coli resistance to magainin I through the rearrangement of several metabolic pathways involved in bacterial physiology and drug response, also providing information on the development of novel antimicrobial strategies targeting resistance-related transcripts and proteins herein described.201830277857