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903300.9568Stress Resistance Development and Genome-Wide Transcriptional Response of Escherichia coli O157:H7 Adapted to Sublethal Thymol, Carvacrol, and trans-Cinnamaldehyde. Thymol, carvacrol, and trans-cinnamaldehyde are essential oil (EO) compounds with broad-spectrum antimicrobial activities against foodborne pathogens, including Escherichia coli O157:H7. However, little is known regarding direct resistance and cross-resistance development in E. coli O157:H7 after adaptation to sublethal levels of these compounds, and information is scarce on microbial adaptive responses at a molecular level. The present study demonstrated that E. coli O157:H7 was able to grow in the presence of sublethal thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC), displaying an extended lag phase duration and a lower maximum growth rate. EO-adapted cells developed direct resistance against lethal EO treatments and cross-resistance against heat (58°C) and oxidative (50 mM H(2)O(2)) stresses. However, no induction of acid resistance (simulated gastric fluid, pH 1.5) was observed. RNA sequencing revealed a large number (310 to 338) of differentially expressed (adjusted P value [P(adj) ], <0.05; fold change, ≥5) genes in 1/2T and 1/2C cells, while 1/2TC cells only showed 27 genes with altered expression. In accordance with resistance phenotypes, the genes related to membrane, heat, and oxidative stress responses and genes related to iron uptake and metabolism were upregulated. Conversely, virulence genes associated with motility, biofilm formation, and efflux pumps were repressed. This study demonstrated the development of direct resistance and cross-resistance and characterized whole-genome transcriptional responses in E. coli O157:H7 adapted to sublethal thymol, carvacrol, or trans-cinnamaldehyde. The data suggested that caution should be exercised when using EO compounds as food antimicrobials, due to the potential stress resistance development in E. coli O157:H7.IMPORTANCE The present study was designed to understand transcriptomic changes and the potential development of direct and cross-resistance in essential oil (EO)-adapted Escherichia coli O157:H7. The results demonstrated altered growth behaviors of E. coli O157:H7 during adaptation in sublethal thymol, carvacrol, and trans-cinnamaldehyde. Generally, EO-adapted bacteria showed enhanced resistance against subsequent lethal EO, heat, and oxidative stresses, with no induction of acid resistance in simulated gastric fluid. A transcriptomic analysis revealed the upregulation of related stress resistance genes and a downregulation of various virulence genes in EO-adapted cells. This study provides new insights into microbial EO adaptation behaviors and highlights the risk of resistance development in adapted bacteria.201830217837
904510.9557Development 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
1220.9548A Diketopiperazine, Cyclo-(L-Pro-L-Ile), Derived From Bacillus thuringiensis JCK-1233 Controls Pine Wilt Disease by Elicitation of Moderate Hypersensitive Reaction. Pine wilt disease (PWD) caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus is one of the devastating diseases affecting pine forests worldwide. Although effective control measurements are still missing, induction of resistance could represent a possible eco-friendly alternative. In this study, induced resistance-based in vitro and in vivo screening tests were carried out for selection of bacteria with the ability to suppress PWD. Out of 504 isolated bacteria, Bacillus thuringiensis JCK-1233 was selected for its ability to boost pathogenesis-related 1 (PR1) gene expression, a marker of systemic acquired resistance. Moreover, treatment of pine seedlings with B. thuringiensis JCK-1233 resulted in increased expression of other defense-related genes, and significantly inhibited PWD development under greenhouse conditions. However, B. thuringiensis JCK-1233 showed no direct nematicidal activity against B. xylophilus. To identify the effective compound responsible for the induction of resistance in B. thuringiensis JCK-1233, several diketopiperazines (DPKs) including cyclo-(D-Pro-L-Val), cyclo-(L-Pro-L-Ile), cyclo-(L-Pro-L-Phe), and cyclo-(L-Leu-L-Val) were isolated and tested. Foliar treatment of pine seedlings with Cyclo-(L-Pro-L-Ile) resulted in suppression of PWD severity and increased the expression of defense-related genes similarly to B. thuringiensis JCK-1233 treatment. Interestingly, treatment with B. thuringiensis JCK-1233 or cyclo-(L-Pro-L-Ile) showed moderately enhanced expression of PR-1, PR-2, PR-3, PR-4, PR-5, and PR-9 genes following inoculation with PWN compared to that in the untreated control, indicating that they mitigated the burst of hypersensitive reaction in susceptible pine seedlings. In contrast, they significantly increased the expression levels of PR-6 and PR-10 before PWN inoculation. In conclusion, foliar spraying with either B. thuringiensis JCK-1233 culture suspension or DPKs could induce resistance in pine seedlings, thereby alleviating the serious damage by PWD. Taken together, this study supports aerial spraying with eco-friendly biotic or abiotic agents as a valuable strategy that may mark an epoch for the control of PWD in pine forests.202032849672
12830.9547Identification of a novel system for boron transport: Atr1 is a main boron exporter in yeast. Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Delta mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Delta cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Delta cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.200919414602
80140.9545Redox-sensitive transcriptional regulator SoxR directly controls antibiotic production, development and thiol-oxidative stress response in Streptomyces avermitilis. The redox-sensitive transcriptional regulator SoxR is conserved in bacteria. Its role in mediating protective response to various oxidative stresses in Escherichia coli and related enteric bacteria has been well established. However, functions and regulatory mechanisms of SoxR in filamentous Streptomyces, which produce half of known antibiotics, are unclear. We report here that SoxR pleiotropically regulates antibiotic production, morphological development, primary metabolism and thiol-oxidative stress response in industrially important species Streptomyces avermitilis. SoxR stimulated avermectin production by directly activating ave structural genes. Four genes (sav_3956, sav_4018, sav_5665 and sav_7218) that are homologous to targets of S. coelicolor SoxR are targeted by S. avermitilis SoxR. A consensus 18-nt SoxR-binding site, 5'-VSYCNVVMHNKVKDGMGB-3', was identified in promoter regions of sav_3956, sav_4018, sav_5665, sav_7218 and target ave genes, leading to prediction of the SoxR regulon and confirmation of 11 new targets involved in development (ftsH), oligomycin A biosynthesis (olmRI), primary metabolism (metB, sav_1623, plcA, nirB, thiG, ndh2), transport (smoE) and regulatory function (sig57, sav_7278). SoxR also directly activated three key developmental genes (amfC, whiB and ftsZ) and promoted resistance of S. avermitilis to thiol-oxidative stress through activation of target trx and msh genes. Overexpression of soxR notably enhanced antibiotic production in S. avermitilis and S. coelicolor. Our findings expand our limited knowledge of SoxR and will facilitate improvement of methods for antibiotic overproduction in Streptomyces species.202233951287
80650.9544A 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
34260.9534Heat-shock-increased survival to far-UV radiation in Escherichia coli is wavelength dependent. Heat-shock-induced resistance to far-UV (FUV) radiation was studied in Escherichia coli. The induction of FUV resistance was shown to be dependent on the products of the genes uvrA and polA in bacteria irradiated at 254 nm. Heat shock increased the resistance to 280 nm radiation in a uvrA6 recA13 mutant. Heat shock lowered the mutation frequency (reversion to tryptophan proficiency) in wild-type or uvrA strains irradiated at 254 nm. When these strains were irradiated at 280 nm, heat shock did not interfere with the mutation frequency in the wild-type strain, but greatly enhanced mutations in the uvrA mutant. After heat-shock treatment, the wild-type strain irradiated at 254 nm showed increased DNA degradation, indicating enhanced repair activity. However, heat shock did not stimulate SOS repair triggered by FUV. An increased survival of bacteriophages irradiated with FUV and inoculated into heat-shock-treated bacteria was not detected. The possibility that heat shock enhances excision repair activity in a wavelength-dependent manner is discussed.19948176549
904670.9532Burkholderia 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
879080.9528Bacillus circulans GN03 Alters the Microbiota, Promotes Cotton Seedling Growth and Disease Resistance, and Increases the Expression of Phytohormone Synthesis and Disease Resistance-Related Genes. Plant growth-promoting bacteria (PGPB) are components of the plant rhizosphere that promote plant growth and/or inhibit pathogen activity. To explore the cotton seedlings response to Bacillus circulans GN03 with high efficiency of plant growth promotion and disease resistance, a pot experiment was carried out, in which inoculations levels of GN03 were set at 10(4) and 10(8) cfu(⋅)mL(-1). The results showed that GN03 inoculation remarkably enhanced growth promotion as well as disease resistance of cotton seedlings. GN03 inoculation altered the microbiota in and around the plant roots, led to a significant accumulation of growth-related hormones (indole acetic acid, gibberellic acid, and brassinosteroid) and disease resistance-related hormones (salicylic acid and jasmonic acid) in cotton seedlings, as determined with ELISA, up-regulated the expression of phytohormone synthesis-related genes (EDS1, AOC1, BES1, and GA20ox), auxin transporter gene (Aux1), and disease-resistance genes (NPR1 and PR1). Comparative genomic analyses was performed between GN03 and four similar species, with regards to phenotype, biochemical characteristics, and gene function. This study provides valuable information for applying the PGPB alternative, GN03, as a plant growth and disease-resistance promoting fertilizer.202133936131
847290.9527Genetic architecture of resistance to plant secondary metabolites in Photorhabdus entomopathogenic bacteria. BACKGROUND: Entomopathogenic nematodes of the genus Heterorhabditis establish a symbiotic association with Photorhabdus bacteria. Together, they colonize and rapidly kill insects, making them important biological control agents against agricultural pests. Improving their biocontrol traits by engineering resistance to plant secondary metabolites (benzoxazinoids) in Photorhabdus symbiotic bacteria through experimental evolution has been shown to increase their lethality towards benzoxazinoid-defended larvae of the western corn rootworm, a serious crop pest of maize, and it is therefore a promising approach to develop more efficient biocontrol agents to manage this pest. To enhance our understanding of the genetic bases of benzoxazinoid resistance in Photorhabdus bacteria, we conducted an experimental evolution experiment with a phylogenetically diverse collection of Photorhabdus strains from different geographic origins. We cultured 27 different strains in medium containing 6-methoxy-2-benzoxazolinone (MBOA), a highly active benzoxazinoid breakdown product, for 35 24 h-cycles to select for benzoxazinoid-resistant strains. Then, we carried out genome-wide sequence comparisons to uncover the genetic alterations associated with benzoxazinoid resistance. Lastly, we evaluated the resistance of the newly isolated resistant Photorhabdus strains to eight additional bioactive compounds, including 2-benzoxazolinone (BOA), nicotine, caffeine, 6-chloroacetyl-2-benzoxazolinone (CABOA), digitoxin, fenitrothion, ampicillin, and kanamycin. RESULTS: We found that benzoxazinoid resistance evolves rapidly in Photorhabdus in a strain-specific manner. Across the different Photorhabdus strains, a total of nineteen nonsynonymous point mutations, two stop codon gains, and one frameshift were associated with higher benzoxazinoid resistance. The different genetic alterations were polygenic and occurred in genes coding for the EnvZ/OmpR two-component regulatory system, the different subunits of the DNA-directed RNA polymerase, and the AcrABZ-TolC multidrug efflux pump. Apart from increasing MBOA resistance, the different mutations were also associated with cross-resistance to 2-benzoxazolinone (BOA), nicotine, caffeine, and 6-chloroacetyl-2-benzoxazolinone (CABOA) and with collateral sensitivity to fenitrothion, ampicillin, and kanamycin. Targeted mutagenesis will provide a deeper mechanistic understanding, including the relative contribution of the different mutation types. CONCLUSIONS: Our study reveals several genomic features that are associated with resistance to xenobiotics in this important group of biological control agents and enhances the availability of molecular tools to develop better biological control agents, which is essential for more sustainable and ecologically friendly agricultural practices.202541168779
13100.9525Streptomyces sp. JCK-6131 Protects Plants Against Bacterial and Fungal Diseases via Two Mechanisms. Plant bacterial and fungal diseases cause significant agricultural losses and need to be controlled. Beneficial bacteria are promising candidates for controlling these diseases. In this study, Streptomyces sp. JCK-6131 exhibited broad-spectrum antagonistic activity against various phytopathogenic bacteria and fungi. In vitro assays showed that the fermentation filtrate of JCK-6131 inhibited the growth of bacteria and fungi with minimum concentration inhibitory (MIC) values of 0.31-10% and 0.31-1.25%, respectively. In the in vivo experiments, treatment with JCK-6131 effectively suppressed the development of apple fire blight, tomato bacterial wilt, and cucumber Fusarium wilt in a dose-dependent manner. RP-HPLC and ESI-MS/MS analyses indicated that JCK-6131 can produce several antimicrobial compounds, three of which were identified as streptothricin E acid, streptothricin D, and 12-carbamoyl streptothricin D. In addition, the disease control efficacy of the foliar application of JCK-6131 against tomato bacterial wilt was similar to that of the soil drench application, indicating that JCK-6131 could enhance defense resistance in plants. Molecular studies on tomato plants showed that JCK-6131 treatment induced the expression of the pathogenesis-related (PR) genes PR1, PR3, PR5, and PR12, suggesting the simultaneous activation of the salicylate (SA) and jasmonate (JA) signaling pathways. The transcription levels of PR genes increased earlier and were higher in treated plants than in untreated plants following Ralstonia solanacearum infection. These results indicate that Streptomyces sp. JCK-6131 can effectively control various plant bacterial and fungal diseases via two distinct mechanisms of antibiosis and induced resistance.202134603354
7828110.9525Simultaneous elimination of antibiotic-resistant bacteria and antibiotic resistance genes by different Fe-N co-doped biochars activating peroxymonosulfate: The key role of pyridine-N and Fe-N sites. The coexistence of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in the environment poses a potential threat to public health. In our study, we have developed a novel advanced oxidation process for simultaneously removing ARGs and ARB by two types of iron and nitrogen-doped biochar derived from rice straw (FeN-RBC) and sludge (FeN-SBC). All viable ARB (approximately 10(8) CFU mL(-1)) was inactivated in the FeN-RBC/ peroxymonosulfate (PMS) system within 40 min and did not regrow after 48 h even in real water samples. Flow cytometry identified 96.7 % of dead cells in the FeN-RBC/PMS system, which verified the complete inactivation of ARB. Thorough disinfection of ARB was associated with the disruption of cell membranes and intracellular enzymes related to the antioxidant system. Whereas live bacteria (approximately 200 CFU mL(-1)) remained after FeN-SBC/PMS treatment. Intracellular and extracellular ARGs (tetA and tetB) were efficiently degraded in the FeN-RBC/PMS system. The production of active species, primarily •OH, SO(4)(•-) and Fe (IV), as well as electron transfer, were essential to the effective disinfection of FeN-RBC/PMS. In comparison with FeN-SBC, the better catalytic performance of FeN-RBC was mainly ascribed to its higher amount of pyridine-N and Fe(0), and more reactive active sites (such as CO group and Fe-N sites). Density functional theory calculations indicated the greater adsorption energy and Bader charge, more stable Fe-O bond, more easily broken OO bond in FeN-RBC/PMS, which demonstrated the stronger electron transfer capacity between FeN-RBC and PMS. To encapsulate, our study provided an efficient and dependable method for the simultaneous elimination of ARGs and ARB in water.202438669989
7842120.9525Removal of Antibiotic Resistant Bacteria and Genes by UV-Assisted Electrochemical Oxidation on Degenerative TiO(2) Nanotube Arrays. Antibiotic resistance has become a global crisis in recent years, while wastewater treatment plants (WWTPs) have been identified as a significant source of both antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). However, commonly used disinfectants have been shown to be ineffective for the elimination of ARGs. With the goal of upgrading the conventional UV disinfection unit with stronger capability to combat ARB and ARGs, we developed a UV-assisted electrochemical oxidation (UV-EO) process that employs blue TiO(2) nanotube arrays (BNTAs) as photoanodes. Inactivation of tetracycline- and sulfamethoxazole-resistant E. coli along with degradation of the corresponding plasmid coded genes (tetA and sul1) is measured by plate counting on selective agar and qPCR, respectively. In comparison with UV(254) irradiation alone, enhanced ARB inactivation and ARG degradation is achieved by UV-EO. Chloride significantly promotes the inactivation efficiency due to the electrochemical production of free chlorine and the subsequent UV/chlorine photoreactions. The fluence-based first-order kinetic rate coefficients of UV-EO in Cl(-) are larger than those of UV(254) irradiation alone by a factor of 2.1-2.3 and 1.3-1.8 for the long and short target genes, respectively. The mechanism of plasmid DNA damage by different radical species is further explored using gel electrophoresis and computational kinetic modeling. The process can effectively eliminate ARB and ARGs in latrine wastewater, though the kinetics were retarded.202139605952
8729130.9525Protein S-Acyl Transferase GhPAT27 Was Associated with Verticillium wilt Resistance in Cotton. Protein palmitoylation is an ability of the frame of the cell marker protein is one of the most notable reversible changes after translation. However, studies on protein palmitoylation in cotton have not yet been performed. In our current research, the PAT gene family was systematically identified and bioinformatically analyzed in G. arboreum, G. raimondii, G. barbadense and G. hirsutum, and 211 PAT genes were authenticated and classified into six subfamilies. Sixty-nine PAT genes were identified in upland cotton, mainly at the ends of its the 26 chromosomes of upland cotton. The majority of these genes are located in the nucleus of the plant. Gene structure analysis revealed that each member encodes a protein that which contains at least one DHHC structural domain. Cis-acting element analysis indicated that GhPATs genes are mainly involved in hormone production, light response and stress response. Gene expression pattern analysis indicated that most GhPATs genes were differentially expressed upon induction by pathogenic bacteria, drought, salt, hot and cold stresses, and some GhPATs could be induced by multiple abiotic stresses simultaneously. GhPATs genes showed different expression patterns in tissue-specific assays and were found to be preferentially expressed in roots, followed by expression in stems and leaves. Virus-induced gene silencing (VIGS) experiments showed that cotton was significantly less resistant to Verticillium dahliae when GhPAT27 was silenced. We conclude that the GhPAT27 gene, which mediates S-palmitoylation acetylation, may be involved in the regulation of upland cotton resistance to Verticillium wilt (VW). Overall, this work has provided a fundamental framework for understanding the latent capabilities of GhPATs and a solid foundation for molecular breeding and plant pathogen resistance in cotton.202236297782
6008140.9524Photopolymerized keratin-PGLa hydrogels for antibiotic resistance reversal and enhancement of infectious wound healing. Infectious wounds have become serious challenges for both treatment and management in clinical practice, so development of new antibiotics has been considered an increasingly difficult task. Here, we report the design and synthesis of keratin 31 (K31)-peptide glycine-leucine-amide (PGLa) photopolymerized hydrogels to rescue the antibiotic activity of antibiotics for infectious wound healing promotion. K31-PGLa displayed an outstanding synergistic effect with commercial antibiotics against drug-resistant bacteria by down-regulating the synthesis genes of efflux pump. Furthermore, the photopolymerized K31-PGLa/PEGDA hydrogels effectively suppressed drug-resistant bacteria growth and enhanced skin wound closure in murine. This study provided a promising alternative strategy for infectious wound treatment.202337810750
8717150.9522Protective Effect of Pediococcus pentosaceus Li05 on Constipation via TGR5/TPH1/5-HT Activation. Pediococcus pentosaceus Li05, a strain of lactic acid bacteria isolated from the faeces of healthy volunteers, exhibited potential protective effects against various diseases. This study performed third-generation sequencing and detailed characterisation of its genome. The Li05 chromosome harboured conserved genes associated with acid resistance (atp), bile salt resistance (bsh), oxidative stress resistance (hsl, dltA, and et al.), and adhesion (nrd, gap, and et al.), whereas the plasmid did not contain antibiotic resistance or virulence genes. Following intervention with Li05 in loperamide-induced constipated mice, constipation symptoms improved. Meanwhile, alterations in gut microbiota, increased BSH activity in faeces, and modifications to the faecal bile acid profile were observed. Additionally, expression levels of TGR5 and TPH1 in the colon of the mice increased, leading to elevated 5-HT levels. When the TGR5 gene was knocked out or the TPH1 inhibitor LX1606 was administered to suppress 5-HT synthesis in constipated mice, the beneficial effects of Li05 on gastrointestinal motility and mucus secretion were reversed. Culturing intestinal organoids demonstrated that increased bile acids such as DCA, Iso-LCA, and EALCA could enhance 5-HT levels through the TGR5/TPH1 axis. Therefore, we concluded that Li05 regulated bile acid metabolism, subsequently increasing 5-HT levels through the TGR5/TPH1 axis, thus alleviating constipation.202541159760
9031160.9521EmrR-Dependent Upregulation of the Efflux Pump EmrCAB Contributes to Antibiotic Resistance in Chromobacterium violaceum. Chromobacterium violaceum is an environmental Gram-negative bacterium that causes infections in humans. Treatment of C. violaceum infections is difficult and little is known about the mechanisms of antibiotic resistance in this bacterium. In this work, we identified mutations in the MarR family transcription factor EmrR and in the protein GyrA as key determinants of quinolone resistance in C. violaceum, and we defined EmrR as a repressor of the MFS-type efflux pump EmrCAB. Null deletion of emrR caused increased resistance to nalidixic acid, but not to other quinolones or antibiotics of different classes. Moreover, the ΔemrR mutant showed decreased production of the purple pigment violacein. Importantly, we isolated C. violaceum spontaneous nalidixic acid-resistant mutants with a point mutation in the DNA-binding domain of EmrR (R92H), with antibiotic resistance profile similar to that of the ΔemrR mutant. Other spontaneous mutants with high MIC values for nalidixic acid and increased resistance to fluoroquinolones presented point mutations in the gene gyrA. Using DNA microarray, Northern blot and EMSA assays, we demonstrated that EmrR represses directly a few dozen genes, including the emrCAB operon and other genes related to transport, oxidative stress and virulence. This EmrR repression on emrCAB was relieved by salicylate. Although mutation of the C. violaceum emrCAB operon had no effect in antibiotic susceptibility or violacein production, deletion of emrCAB in an emrR mutant background restored antibiotic susceptibility and violacein production in the ΔemrR mutant. Using a biosensor reporter strain, we demonstrated that the lack of pigment production in ΔemrR correlates with the accumulation of quorum-sensing molecules in the cell supernatant of this mutant strain. Therefore, our data revealed that overexpression of the efflux pump EmrCAB via mutation and/or derepression of EmrR confers quinolone resistance and alters quorum-sensing signaling in C. violaceum, and that point mutation in emrR can contribute to emergence of antibiotic resistance in bacteria.201830498484
9041170.9519Spontaneous 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
5170180.9519Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori. BACKGROUND: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism. RESULTS: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs. CONCLUSION: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.200919594901
9047190.9518Comparison of transcriptomes of wild-type and isothiazolone-resistant Pseudomonas aeruginosa by using RNA-seq. Isothiazolone biocides (such as Kathon) are widely used in a variety of industrial and domestic applications. However, the mechanisms through which bacteria develop resistance to these biocides are not completely clear. A better understanding of these mechanisms can contribute to optimal use of these biocides. In this study, transcription profiles of a Kathon-resistant strain of Pseudomonas aeruginosa (Pa-R) and the wild-type strain were determined using RNA sequencing (RNA-Seq) with the Illumina HiSeq 2000 platform. RNA-Seq generated 18,657,896 sequence reads aligned to 7093 genes. In all, 1550 differently expressed genes (DEGs, log2 ratio ≥1, false discovery rate (FDR) ≤0.001) were identified, of which 482 were up-regulated and 1068 were down-regulated. Most Kathon-induced genes were involved in metabolic and cellular processes. DEGs significantly enriched nitrogen metabolism and oxidative phosphorylation pathways. In addition, Pa-R showed cross-resistance to triclosan and ciprofloxacin and showed repressed pyocyanin production. These results may improve our understanding of the resistance mechanisms of P. aeruginosa against isothiazolones, and provide insight into the development of more efficient isothiazolones.201627072374