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80600.9744A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.200717464069
618910.9741Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus. Resistance nodulation cell division (RND)-type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND-type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND-type transporters were functional as efflux pumps only when co-expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division-type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.201323894076
904620.9739Burkholderia pseudomallei resistance to antibiotics in biofilm-induced conditions is related to efflux pumps. Burkholderia pseudomallei, the causative agent of melioidosis, has been found to increase its resistance to antibiotics when growing as a biofilm. The resistance is related to several mechanisms. One of the possible mechanisms is the efflux pump. Using bioinformatics analysis, it was found that BPSL1661, BPSL1664 and BPSL1665 were orthologous genes of the efflux transporter encoding genes for biofilm-related antibiotic resistance, PA1874-PA1877 genes in Pseudomonas aeruginosa strain PAO1. Expression of selected encoding genes for the efflux transporter system during biofilm formation were investigated. Real-time reverse transcriptase PCR expression of amrB, cytoplasmic membrane protein of AmrAB-OprA efflux transporter encoding gene, was slightly increased, while BPSL1665 was significantly increased during growth of bacteria in biofilm formation. Minimum biofilm inhibition concentration and minimum biofilm eradication concentration (MBEC) of ceftazidime (CTZ), doxycycline (DOX) and imipenem were found to be 2- to 1024-times increased when compared to their MICs for of planktonic cells. Inhibition of the efflux transporter by adding phenylalanine arginine β-napthylamide (PAβN), a universal efflux inhibitor, decreased 2 to 16 times as much as MBEC in B. pseudomallei biofilms with CTZ and DOX. When the intracellular accumulation of antibiotics was tested to reveal the pump inhibition, only the concentrations of CTZ and DOX increased in PAβN treated biofilm. Taken together, these results indicated that BPSL1665, a putative precursor of the efflux pump gene, might be related to the adaptation of B. pseudomallei in biofilm conditions. Inhibition of efflux pumps may lead to a decrease of resistance to CTZ and DOX in biofilm cells.201627702426
873630.9737Effects of intracanal irrigant MTAD Combined with nisin at sub-minimum inhibitory concentration levels on Enterococcus faecalis growth and the expression of pathogenic genes. Exposure to antibiotics is considered to be the major driver in the selection of antibiotic-resistant bacteria and may induce diverse biological responses in bacteria. MTAD is a common intracanal irrigant, but its bactericidal activity remains to be improved. Previous studies have indicated that the antimicrobial peptide nisin can significantly improve the bactericidal activity of MTAD against Enterococcus faecalis. However, the effects of MTAD and its modification at sub-minimum inhibitory concentration (sub-MIC) levels on Enterococcus faecalis growth and the expression of pathogenic genes still need to be explored. In this study, the results of post-antibiotic effects (PAE) and post-antibiotic sub-MIC effects (PASME) showed that MTADN (nisin in combination with MTAD) had the best post-antibiotic effect. E. faecalis after challenge with MTAD was less sensitive to alkaline solutions compared with MTAN (nisin in place of doxycycline in MTAD) and MTADN. E. faecalis induced with sub-MIC of MTAD generated resistance to the higher concentration, but induction of E. faecalis with MTAN did not cause resistance to higher concentrations. Furthermore, real-time polymerase chain reaction (RT-PCR) showed that the stress caused by sub-MIC exposure to MTAD, MTAN, or MTADN resulted in up- or down-regulation of nine stress genes and four virulence-associated genes in E. faecalis and resulted in different stress states. These findings suggested that nisin improved the post-antibacterial effect of MTAD at sub-MIC levels and has considerable potential for use as a modification of MTAD.201424603760
637340.9732Antibiotic resistance and multidrug-resistant efflux pumps expression in lactic acid bacteria isolated from pozol, a nonalcoholic Mayan maize fermented beverage. Pozol is a handcrafted nonalcoholic Mayan beverage produced by the spontaneous fermentation of maize dough by lactic acid bacteria. Lactic acid bacteria (LAB) are carriers of chromosomal encoded multidrug-resistant efflux pumps genes that can be transferred to pathogens and/or confer resistance to compounds released during the fermentation process causing food spoiling. The aim of this study was to evaluate the antibiotic sensibility and the transcriptional expression of ABC-type efflux pumps in LAB isolated from pozol that contributes to multidrug resistance. Analysis of LAB and Staphylococcus (S.) aureus ATCC 29213 and ATCC 6538 control strains to antibiotic susceptibility, minimal inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) to ethidium bromide were based in "standard methods" whereas the ethidium bromide efflux assay was done by fluorometric assay. Transcriptional expression of efflux pumps was analyzed by RT-PCR. LAB showed antibiotic multiresistance profiles, moreover, Lactococcus (L.) lactis and Lactobacillus (L.) plantarum displayed higher ethidium bromide efflux phenotype than S. aureus control strains. Ethidium bromide resistance and ethidium bromide efflux phenotypes were unrelated with the overexpression of lmrD in L. lactics, or the underexpression of lmrA in L. plantarum and norA in S. aureus. These findings suggest that, moreover, the analyzed efflux pumps genes, other unknown redundant mechanisms may underlie the antibiotic resistance and the ethidium bromide efflux phenotype in L. lactis and L. plantarum. Phenotypic and molecular drug multiresistance assessment in LAB may improve a better selection of the fermentation starter cultures used in pozol, and to control the antibiotic resistance widespread and food spoiling for health safety.201627247772
901650.9730Hfq Regulates Efflux Pump Expression and Purine Metabolic Pathway to Increase Trimethoprim Resistance in Aeromonas veronii. Aeromonas veronii (A. veronii) is a zoonotic pathogen. It causes clinically a variety of diseases such as dysentery, bacteremia, and meningitis, and brings huge losses to aquaculture. A. veronii has been documented as a multiple antibiotic resistant bacterium. Hfq (host factor for RNA bacteriophage Qβ replication) participates in the regulations of the virulence, adhesion, and nitrogen fixation, effecting on the growth, metabolism synthesis and stress resistance in bacteria. The deletion of hfq gene in A. veronii showed more sensitivity to trimethoprim, accompanying by the upregulations of purine metabolic genes and downregulations of efflux pump genes by transcriptomic data analysis. Coherently, the complementation of efflux pump-related genes acrA and acrB recovered the trimethoprim resistance in Δhfq. Besides, the accumulations of adenosine and guanosine were increased in Δhfq in metabonomic data. The strain Δhfq conferred more sensitive to trimethoprim after appending 1 mM guanosine to M9 medium, while wild type was not altered. These results demonstrated that Hfq mediated trimethoprim resistance by elevating efflux pump expression and degrading adenosine, and guanosine metabolites. Collectively, Hfq is a potential target to tackle trimethoprim resistance in A. veronii infection.202134899630
901560.9730A D-enantiomer of the antimicrobial peptide GL13K evades antimicrobial resistance in the Gram positive bacteria Enterococcus faecalis and Streptococcus gordonii. Antimicrobial peptides represent an alternative to traditional antibiotics that may be less susceptible to bacterial resistance mechanisms by directly attacking the bacterial cell membrane. However, bacteria have a variety of defense mechanisms that can prevent cationic antimicrobial peptides from reaching the cell membrane. The L- and D-enantiomers of the antimicrobial peptide GL13K were tested against the Gram-positive bacteria Enterococcus faecalis and Streptococcus gordonii to understand the role of bacterial proteases and cell wall modifications in bacterial resistance. GL13K was derived from the human salivary protein BPIFA2. Minimal inhibitory concentrations were determined by broth dilution and a serial assay used to determine bacterial resistance. Peptide degradation was determined in a bioassay utilizing a luminescent strain of Pseudomonas aeruginosa to detect peptide activity. Autolysis and D-alanylation-deficient strains of E. faecalis and S. gordonii were tested in autolysis assays and peptide activity assays. E. faecalis protease inactivated L-GL13K but not D-GL13K, whereas autolysis did not affect peptide activity. Indeed, the D-enantiomer appeared to kill the bacteria prior to initiation of autolysis. D-alanylation mutants were killed by L-GL13K whereas this modification did not affect killing by D-GL13K. The mutants regained resistance to L-GL13K whereas bacteria did not gain resistance to D-GL13K after repeated treatment with the peptides. D-alanylation affected the hydrophobicity of bacterial cells but hydrophobicity alone did not affect GL13K activity. D-GL13K evades two resistance mechanisms in Gram-positive bacteria without giving rise to substantial new resistance. D-GL13K exhibits attractive properties for further antibiotic development.201829566082
903770.9729Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance. BACKGROUND: Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear. RESULTS: To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed. CONCLUSION: Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.200919761586
903880.9729Molecular mechanisms of chlorhexidine tolerance in Burkholderia cenocepacia biofilms. The high tolerance of biofilm-grown Burkholderia cepacia complex bacteria against antimicrobial agents presents considerable problems for the treatment of infected cystic fibrosis patients and the implementation of infection control guidelines. In the present study, we analyzed the tolerance of planktonic and sessile Burkholderia cenocepacia J2315 cultures and examined the transcriptional response of sessile cells to treatment with chlorhexidine. At low (0.0005%) and high (0.05%) concentrations, chlorhexidine had a similar effect on both populations, but at intermediate concentrations (0.015%) the antimicrobial activity was more pronounced in planktonic cultures. The exposure of sessile cells to chlorhexidine resulted in an upregulation of the transcription of 469 (6.56%) and the downregulation of 257 (3.59%) protein-coding genes. A major group of upregulated genes in the treated biofilms encoded membrane-related and regulatory proteins. In addition, several genes coding for drug resistance determinants also were upregulated. The phenotypic analysis of RND (resistance-nodulation-division) efflux pump mutants suggests the presence of lifestyle-specific chlorhexidine tolerance mechanisms; efflux system RND-4 (BCAL2820-BCAL2822) was more responsible for chlorhexidine tolerance in planktonic cells, while other systems (RND-3 [BCAL1672-BCAL1676] and RND-9 [BCAM1945-BCAM1947]) were linked to resistance in sessile cells. After sessile cell exposure, multiple genes encoding chemotaxis and motility-related proteins were upregulated in concert with the downregulation of an adhesin-encoding gene (BCAM2143), suggesting that sessile cells tried to escape the biofilm. We also observed the differential expression of 19 genes carrying putative small RNA molecules, indicating a novel role for these regulatory elements in chlorhexidine tolerance.201121357299
903590.9728Involvement of a novel efflux system in biofilm-specific resistance to antibiotics. Bacteria growing in biofilms are more resistant to antibiotics than their planktonic counterparts. How this transition occurs is unclear, but it is likely there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. We have identified a novel efflux pump in Pseudomonas aeruginosa that is important for biofilm-specific resistance to a subset of antibiotics. Complete deletion of the genes encoding this pump, PA1874 to PA1877 (PA1874-1877) genes, in an P. aeruginosa PA14 background results in an increase in sensitivity to tobramycin, gentamicin, and ciprofloxacin, specifically when this mutant strain is growing in a biofilm. This efflux pump is more highly expressed in biofilm cells than in planktonic cells, providing an explanation for why these genes are important for biofilm but not planktonic resistance to antibiotics. Furthermore, expression of these genes in planktonic cells increases their resistance to antibiotics. We have previously shown that ndvB is important for biofilm-specific resistance (T. F. Mah, B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A. O'Toole, Nature 426:306-310, 2003). Our discovery that combining the ndvB mutation with the PA1874-1877 gene deletion results in a mutant strain that is more sensitive to antibiotics than either single mutant strain suggests that ndvB and PA1874-1877 contribute to two different mechanisms of biofilm-specific resistance to antibiotics.200818469108
622100.9727Small-Molecule Antibiotics Inhibiting tRNA-Regulated Gene Expression Is a Viable Strategy for Targeting Gram-Positive Bacteria. Bacterial infections and the rise of antibiotic resistance, especially multidrug resistance, have generated a clear need for discovery of novel therapeutics. We demonstrated that a small-molecule drug, PKZ18, targets the T-box mechanism and inhibits bacterial growth. The T-box is a structurally conserved riboswitch-like gene regulator in the 5' untranslated region (UTR) of numerous essential genes of Gram-positive bacteria. T-boxes are stabilized by cognate, unacylated tRNA ligands, allowing the formation of an antiterminator hairpin in the mRNA that enables transcription of the gene. In the absence of an unacylated cognate tRNA, transcription is halted due to the formation of a thermodynamically more stable terminator hairpin. PKZ18 targets the site of the codon-anticodon interaction of the conserved stem I and reduces T-box-controlled gene expression. Here, we show that novel analogs of PKZ18 have improved MICs, bactericidal effects against methicillin-resistant Staphylococcus aureus (MRSA), and increased efficacy in nutrient-limiting conditions. The analogs have reduced cytotoxicity against eukaryotic cells compared to PKZ18. The PKZ18 analogs acted synergistically with aminoglycosides to significantly enhance the efficacy of the analogs and aminoglycosides, further increasing their therapeutic windows. RNA sequencing showed that the analog PKZ18-22 affects expression of 8 of 12 T-box controlled genes in a statistically significant manner, but not other 5'-UTR regulated genes in MRSA. Very low levels of resistance further support the existence of multiple T-box targets for PKZ18 analogs in the cell. Together, the multiple targets, low resistance, and synergy make PKZ18 analogs promising drugs for development and future clinical applications.202033077662
9017110.9727Molecular mechanism of Hfq-dependent sRNA1039 and sRNA1600 regulating antibiotic resistance and virulence in Shigella sonnei. Bacillary dysentery caused by Shigella spp. is a significant concern for human health. Small non-coding RNA (sRNA) plays a crucial role in regulating antibiotic resistance and virulence in Shigella spp. However, the specific mechanisms behind this phenomenon are still not fully understood. This study discovered two sRNAs (sRNA1039 and sRNA1600) that may be involved in bacterial resistance and virulence. By constructing deletion mutants (WT/ΔSR1039 and WT/ΔSR1600), this study found that the WT/ΔSR1039 mutants caused a two-fold increase in sensitivity to ampicillin, gentamicin and cefuroxime, and the WT/ΔSR1600 mutants caused a two-fold increase in sensitivity to cefuroxime. Furthermore, the WT/ΔSR1600 mutants caused a decrease in the adhesion and invasion of bacteria to HeLa cells (P<0.01), and changed the oxidative stress level of bacteria to reduce their survival rate (P<0.001). Subsequently, this study explored the molecular mechanisms by which sRNA1039 and sRNA1600 regulate antibiotic resistance and virulence. The deletion of sRNA1039 accelerated the degradation of target gene cfa mRNA and reduced its expression, thereby regulating the expression of pore protein gene ompD indirectly and negatively to increase bacterial sensitivity to ampicillin, gentamicin and cefuroxime. The inactivation of sRNA1600 reduced the formation of persister cells to reduce resistance to cefuroxime, and reduced the expression of type-III-secretion-system-related genes to reduce bacterial virulence by reducing the expression of target gene tomB. These results provide new insights into Hfq-sRNA-mRNA regulation of the resistance and virulence network of Shigella sonnei, which could potentially promote the development of more effective treatment strategies.202438141834
6225120.9727Genome-Wide Identification of Resveratrol Intrinsic Resistance Determinants in Staphylococcus aureus. Resveratrol has been extensively studied due to its potential health benefits in multiple diseases, for example, cancer, obesity and cardiovascular diseases. Besides these properties, resveratrol displays inhibitory activity against a wide range of bacterial species; however, the cellular effects of resveratrol in bacteria remain incompletely understood, especially in the human pathogen, Staphylococcus aureus. In this study, we aimed to identify intrinsic resistance genes that aid S. aureus in tolerating the activity of resveratrol. We screened the Nebraska Transposon Mutant Library, consisting of 1920 mutants with inactivation of non-essential genes in S. aureus JE2, for increased susceptibly to resveratrol. On agar plates containing 0.5× the minimum inhibitory concentration (MIC), 17 transposon mutants failed to grow. Of these, four mutants showed a two-fold reduction in MIC, being the clpP protease mutant and three mutants with deficiencies in the electron transport chain (menD, hemB, aroC). The remaining 13 mutants did not show a reduction in MIC, but were confirmed by spot-assays to have increased susceptibility to resveratrol. Several genes were associated with DNA damage repair (recJ, xerC and xseA). Treatment of S. aureus JE2 with sub-inhibitory concentrations of resveratrol did not affect the expression of recJ, xerC and xseA, but increased expression of the SOS-stress response genes lexA and recA, suggesting that resveratrol interferes with DNA integrity in S. aureus. Expression of error-prone DNA polymerases are part of the SOS-stress response and we could show that sub-inhibitory concentrations of resveratrol increased overall mutation frequency as measured by formation of rifampicin resistant mutants. Our data show that DNA repair systems are important determinants aiding S. aureus to overcome the inhibitory activity of resveratrol. Activation of the SOS response by resveratrol could potentially facilitate the development of resistance towards conventional antibiotics in S. aureus.202133467002
9036130.9726Resistance-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
6180140.9726Mab2780c, a TetV-like efflux pump, confers high-level spectinomycin resistance in mycobacterium abscessus. Mycobacterium abscessus is highly resistant to spectinomycin (SPC) thereby making it unavailable for therapeutic use. Sublethal exposure to SPC strongly induces whiB7 and its regulon, and a ΔMab_whiB7 strain is SPC sensitive suggesting that the determinants of SPC resistance are included within its regulon. In the present study we have determined the transcriptomic changes that occur in M. abscessus upon SPC exposure and have evaluated the involvement of 11 genes, that are both strongly SPC induced and whiB7 dependent, in SPC resistance. Of these we show that MAB_2780c can complement SPC sensitivity of ΔMab_whiB7 and that a ΔMab_2780c strain is ∼150 fold more SPC sensitive than wildtype bacteria, but not to tetracycline (TET) or other aminoglycosides. This is in contrast to its homologues, TetV from M. smegmatis and Tap from M. tuberculosis, that confer low-level resistance to TET, SPC and other aminoglycosides. We also show that the addition of the efflux pump inhibitor (EPI), verapamil results in >100-fold decrease in MIC of SPC in bacteria expressing Mab2780c to the levels observed for ΔMab_2780c; moreover a deletion of MAB_2780c results in a decreased efflux of the drug into the cell supernatant. Together our data suggest that Mab2780c is an SPC antiporter. Finally, molecular docking of SPC and TET on models of TetV(Ms) and Mab2780c confirmed our antibacterial susceptibility findings that the Mab2780c pump preferentially effluxes SPC over TET. To our knowledge, this is the first report of an efflux pump that confers high-level drug resistance in M. abscessus. The identification of Mab2780c in SPC resistance opens up prospects for repurposing this relatively well-tolerated antibiotic as a combination therapy with verapamil or its analogs against M. abscessus infections.202336584486
9041150.9726Spontaneous 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
9045160.9725Development of Resistance in Escherichia coli ATCC25922 under Exposure of Sub-Inhibitory Concentration of Olaquindox. Quinoxaline1,4-di-N-oxides (QdNOs) are a class of important antibacterial drugs of veterinary use, of which the drug resistance mechanism has not yet been clearly explained. This study investigated the molecular mechanism of development of resistance in Escherichia coli (E. coli) under the pressure of sub-inhibitory concentration (sub-MIC) of olaquindox (OLA), a representative QdNOs drug. In vitro challenge of E. coli with 1/100× MIC to 1/2× MIC of OLA showed that the bacteria needed a longer time to develop resistance and could only achieve low to moderate levels of resistance as well as form weak biofilms. The transcriptomic and genomic profiles of the resistant E. coli induced by sub-MIC of OLA demonstrated that genes involved in tricarboxylic acid cycle, oxidation-reduction process, biofilm formation, and efflux pumps were up-regulated, while genes involved in DNA repair and outer membrane porin were down-regulated. Mutation rates were significantly increased in the sub-MIC OLA-treated bacteria and the mutated genes were mainly involved in the oxidation-reduction process, DNA repair, and replication. The SNPs were found in degQ, ks71A, vgrG, bigA, cusA, and DR76(-)4702 genes, which were covered in both transcriptomic and genomic profiles. This study provides new insights into the resistance mechanism of QdNOs and increases the current data pertaining to the development of bacterial resistance under the stress of antibacterials at sub-MIC concentrations.202033182563
804170.9725Cloning, mutagenesis, and characterization of the microalga Parietochloris incisa acetohydroxyacid synthase, and its possible use as an endogenous selection marker. Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic-resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched-chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the wild-type and mutant proteins were characterized in the AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wild-type and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild-type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type.201222488216
807180.9725Transcriptomic analysis of Saccharomyces cerevisiae upon honokiol treatment. Honokiol (HNK), one of the main medicinal components in Magnolia officinalis, possesses antimicrobial activity against a variety of pathogenic bacteria and fungi. However, little is known of the molecular mechanisms underpinning the antimicrobial activity. To explore the molecular mechanism of its antifungal activity, we determined the effects of HNK on the mRNA expression profile of Saccharomyces cerevisiae using a DNA microarray approach. HNK markedly induced the expression of genes related to iron uptake and homeostasis. Conversely, genes associated with respiratory electron transport were downregulated, mirroring the effects of iron starvation. Meanwhile, HNK-induced growth deficiency was partly rescued by iron supplementation and HNK reacted with iron, producing iron complexes that depleted iron. These results suggest that HNK treatment induced iron starvation. Additionally, HNK treatment resulted in the upregulation of genes involved in protein synthesis and drug resistance networks. Furthermore, the deletion of PDR5, a gene encoding the plasma membrane ATP binding cassette (ABC) transporter, conferred sensitivity to HNK. Overexpression of PDR5 enhanced resistance of WT and pdr5Δ strains to HNK. Taken together, these findings suggest that HNK, which can be excluded by overexpression of Pdr5, functions in multiple cellular processes in S. cerevisiae, particularly in inducing iron starvation to inhibit cell growth.201728499955
6376190.9725Mechanisms of mepA Overexpression and Membrane Potential Reduction Leading to Ciprofloxacin Heteroresistance in a Staphylococcus aureus Isolate. Heteroresistance has seriously affected the evaluation of antibiotic efficacy against pathogenic bacteria, causing misjudgment of antibiotics' sensitivity in clinical therapy, leading to treatment failure, and posing a serious threat to current medical health. However, the mechanism of Staphylococcus aureus heteroresistance to ciprofloxacin remains unclear. In this study, heteroresistance to ciprofloxacin in S. aureus strain 529 was confirmed by antimicrobial susceptibility testing and population analysis profiling (PAP), with the resistance of subclonal 529_HR based on MIC being 8-fold that of the original bacteria. A 7-day serial MIC evaluation and growth curves demonstrate that their phenotype was stable, with 529_HR growing more slowly than 529, but reaching a plateau in a similar proportion. WGS analysis showed that there were 11 nonsynonymous mutations and one deletion gene between the two bacteria, but none of these SNPs were directly associated with ciprofloxacin resistance. Transcriptome data analysis showed that the expression of membrane potential related genes (qoxA, qoxB, qoxC, qoxD, mprF) was downregulated, and the expression of multidrug resistance efflux pump gene mepA was upregulated. The combination of ciprofloxacin and limonene restored the 529_HR MIC from 1 mg/L to 0.125 mg/L. Measurement of the membrane potential found that 529_HR had a lower potential, which may enable it to withstand the ciprofloxacin-induced decrease in membrane potential. In summary, we demonstrated that upregulation of mepA gene expression and a reduction in membrane potential are the main heteroresistance mechanisms of S. aureus to ciprofloxacin. Additionally, limonene may be a potentially effective agent to inhibit ciprofloxacin heteroresistance phenotypes.202540076991