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600800.9775Photopolymerized 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
904510.9772Development 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
879620.9770Divergent Roles of Escherichia Coli Encoded Lon Protease in Imparting Resistance to Uncouplers of Oxidative Phosphorylation: Roles of marA, rob, soxS and acrB. Uncouplers of oxidative phosphorylation dissipate the proton gradient, causing lower ATP production. Bacteria encounter several non-classical uncouplers in the environment, leading to stress-induced adaptations. Here, we addressed the molecular mechanisms responsible for the effects of uncouplers in Escherichia coli. The expression and functions of genes involved in phenotypic antibiotic resistance were studied using three compounds: two strong uncouplers, i.e., Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-Dinitrophenol (DNP), and one moderate uncoupler, i.e., Sodium salicylate (NaSal). Quantitative expression studies demonstrated induction of transcripts encoding marA, soxS and acrB with NaSal and DNP, but not CCCP. Since MarA and SoxS are degraded by the Lon protease, we investigated the roles of Lon using a lon-deficient strain (Δlon). Compared to the wild-type strain, Δlon shows compromised growth upon exposure to NaSal or 2, 4-DNP. This sensitivity is dependent on marA but not rob and soxS. On the other hand, the Δlon strain shows enhanced growth in the presence of CCCP, which is dependent on acrB. Interestingly, NaSal and 2,4-DNP, but not CCCP, induce resistance to antibiotics, such as ciprofloxacin and tetracycline. This study addresses the effects of uncouplers and the roles of genes involved during bacterial growth and phenotypic antibiotic resistance. Strong uncouplers are often used to treat wastewater, and these results shed light on the possible mechanisms by which bacteria respond to uncouplers. Also, the rampant usage of some uncouplers to treat wastewater may lead to the development of antibiotic resistance.202438372817
34230.9769Heat-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
847240.9768Genetic 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
517050.9768Synergistic 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
904760.9768Comparison 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
903170.9767EmrR-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
615980.9767Gene expression profiling of Cecropin B-resistant Haemophilus parasuis. Synthetically designed antimicrobial peptides (AMPs) present the potential of replacing antibiotics in the treatment of bacterial infections. However, microbial resistance to AMPs has been reported and little is known regarding the underlying mechanism of such resistance. The naturally occurring AMP cecropin B (CB) disrupts the anionic cell membranes of Gram-negative bacteria. In this study, CB resistance (CBR) was induced in Haemophilusparasuis SH0165 by exposing it to a series of CB concentrations. The CB-resistant H.parasuis strains CBR30 and CBR30-50 were obtained. The growth curves of SH0165 and CBR30 showed that CBR30 displayed lower growth rates than SH0165. The result of transmission electron microscopy showed cell membranes of the CB-resistant CBR30 and CBR30-50 were smoother than SH0165. Microarrays detected 257 upregulated and 254 downregulated genes covering 20 clusters of orthologous groups (COGs) of the CB-resistant CBR30 compared with SH0165 (>1.5-fold change, p < 0.05). Sixty genes were affected in CBR30-50 covering 18 COGs, with 28 upregulated and 32 downregulated genes. Under the COG function classification, the majority of affected genes in the CB-resistant CBR30 and CBR30-50 belong to the category of inorganic ion transport, amino acid transport, and metabolism. The microarray results were validated by real-time quantitative reverse transcription PCR. This study may provide useful guidance for understanding the molecular mechanism underlying H.parasuis resistance to CB.201424862339
882890.9765Phenylalanine 4-Hydroxylase Contributes to Endophytic Bacterium Pseudomonas fluorescens' Melatonin Biosynthesis. Melatonin acts both as an antioxidant and as a growth regulatory substance in plants. Pseudomonas fluorescens endophytic bacterium has been shown to produce melatonin and increase plant resistance to abiotic stressors through increasing endogenous melatonin. However, in bacteria, genes are still not known to be melatonin-related. Here, we reported that the bacterial phenylalanine 4-hydroxylase (PAH) may be involved in the 5-hydroxytryptophan (5-HTP) biosynthesis and further influenced the subsequent production of melatonin in P. fluorescens. The purified PAH protein of P. fluorescens not only hydroxylated phenylalanine but also exhibited l-tryptophan (l-Trp) hydroxylase activity by converting l-Trp to 5-HTP in vitro. However, bacterial PAH displayed lower activity and affinity for l-Trp than l-phenylalanine. Notably, the PAH deletion of P. fluorescens blocked melatonin production by causing a significant decline in 5-HTP levels and thus decreased the resistance to abiotic stress. Overall, this study revealed a possible role for bacterial PAH in controlling 5-HTP and melatonin biosynthesis in bacteria, and expanded the current knowledge of melatonin production in microorganisms.202134868217
9044100.9764Impairment of novel non-coding small RNA00203 inhibits biofilm formation and reduces biofilm-specific antibiotic resistance in Acinetobacter baumannii. Small RNAs (sRNAs) are post-transcriptional regulators of many biological processes in bacteria, including biofilm formation and antibiotic resistance. The mechanisms by which sRNA regulates the biofilm-specific antibiotic resistance in Acinetobacter baumannii have not been reported to date. This study aimed to investigate the influence of sRNA00203 (53 nucleotides) on biofilm formation, antibiotic susceptibility, and expression of genes associated with biofilm formation and antibiotic resistance. The results showed that deletion of the sRNA00203-encoding gene decreased the biomass of biofilm by 85%. Deletion of the sRNA00203-encoding gene also reduced the minimum biofilm inhibitory concentrations for imipenem and ciprofloxacin 1024- and 128-fold, respectively. Knocking out of sRNA00203 significantly downregulated genes involved in biofilm matrix synthesis (pgaB), efflux pump production (novel00738), lipopolysaccharide biosynthesis (novel00626), preprotein translocase subunit (secA) and the CRP transcriptional regulator. Overall, the suppression of sRNA00203 in an A. baumannii ST1894 strain impaired biofilm formation and sensitized the biofilm cells to imipenem and ciprofloxacin. As sRNA00203 was found to be conserved in A. baumannii, a therapeutic strategy targeting sRNA00203 may be a potential solution for the treatment of biofilm-associated infections caused by A. baumannii. To the best of the authors' knowledge, this is the first study to show the impact of sRNA00203 on biofilm formation and biofilm-specific antibiotic resistance in A. baumannii.202337315907
544110.9764Organic Hydroperoxide Induces Prodigiosin Biosynthesis in Serratia sp. ATCC 39006 in an OhrR-Dependent Manner. The biosynthesis of prodigiosin in the model prodigiosin-producing strain, Serratia sp. ATCC 39006, is significantly influenced by environmental and cellular signals. However, a comprehensive regulatory mechanism for this process has not been well established. In the present study, we demonstrate that organic hydroperoxide activates prodigiosin biosynthesis in an OhrR-dependent manner. Specifically, the MarR-family transcriptional repressor OhrR (Ser39006_RS05455) binds to its operator located far upstream of the promoter region of the prodigiosin biosynthesis operon (319 to 286 nucleotides [nt] upstream of the transcription start site) and negatively regulates the expression of prodigiosin biosynthesis genes. Organic hydroperoxide disassociates the binding between OhrR and its operator, thereby promoting the prodigiosin production. Moreover, OhrR modulates the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide by regulating the transcription of its own gene and the downstream cotranscribed ohr gene. These results demonstrate that OhrR is a pleiotropic repressor that modulates the prodigiosin production and the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide stress. IMPORTANCE Bacteria naturally encounter various environmental and cellular stresses. Organic hydroperoxides generated from the oxidation of polyunsaturated fatty acids are widely distributed and usually cause lethal oxidative stress by damaging cellular components. OhrR is known as a regulator that modulates the resistance of bacteria to organic hydroperoxide stress. In the current study, organic hydroperoxide disassociates OhrR from the promoter of prodigiosin biosynthesis gene cluster, thus promoting transcription of pigA to -O genes. In this model, organic hydroperoxide acts as an inducer of prodigiosin synthesis in Serratia sp. ATCC 39006. These results improve our understanding of the regulatory network of prodigiosin synthesis and serve as an example for identifying the cross talk between the stress responses and the regulation of secondary metabolism.202235044847
9046120.9762Burkholderia 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
9035130.9761Involvement 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
661140.9761A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase. The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.201627601569
656150.9761HflXr, a homolog of a ribosome-splitting factor, mediates antibiotic resistance. To overcome the action of antibiotics, bacteria have evolved a variety of different strategies, such as drug modification, target mutation, and efflux pumps. Recently, we performed a genome-wide analysis of Listeria monocytogenes gene expression after growth in the presence of antibiotics, identifying genes that are up-regulated upon antibiotic treatment. One of them, lmo0762, is a homolog of hflX, which encodes a heat shock protein that rescues stalled ribosomes by separating their two subunits. To our knowledge, ribosome splitting has never been described as an antibiotic resistance mechanism. We thus investigated the role of lmo0762 in antibiotic resistance. First, we demonstrated that lmo0762 is an antibiotic resistance gene that confers protection against lincomycin and erythromycin, and that we renamed hflXr (hflX resistance). We show that hflXr expression is regulated by a transcription attenuation mechanism relying on the presence of alternative RNA structures and a small ORF encoding a 14 amino acid peptide containing the RLR motif, characteristic of macrolide resistance genes. We also provide evidence that HflXr is involved in ribosome recycling in presence of antibiotics. Interestingly, L. monocytogenes possesses another copy of hflX, lmo1296, that is not involved in antibiotic resistance. Phylogenetic analysis shows several events of hflXr duplication in prokaryotes and widespread presence of hflXr in Firmicutes. Overall, this study reveals the Listeria hflXr as the founding member of a family of antibiotic resistance genes. The resistance conferred by this gene is probably of importance in the environment and within microbial communities.201830545912
6180160.9760Mab2780c, 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
9020170.9760Transcriptome Analysis Reveals the Resistance Mechanism of Pseudomonas aeruginosa to Tachyplesin I. BACKGROUND: Tachyplesin I is a cationic antimicrobial peptide with a typical cyclic antiparallel β-sheet structure. We previously demonstrated that long-term continuous exposure to increased concentration of tachyplesin I can induce resistant Gram-negative bacteria. However, no significant information is available about the resistance mechanism of Pseudomonas aeruginosa (P. aeruginosa) to tachyplesin I. MATERIALS AND METHODS: In this study, the global gene expression profiling of P. aeruginosa strain PA-99 and P. aeruginosa CGMCC1.2620 (PA1.2620) was conducted using transcriptome sequencing. For this purpose, outer membrane permeability and outer membrane proteins (OMPs) were further analyzed. RESULTS: Transcriptome sequencing detected 672 upregulated and 787 downregulated genes, covering Clusters of Orthologous Groups (COGs) of P. aeruginosa strain PA-99 compared with PA1.2620. Totally, 749 differentially expressed genes (DEGs) were assigned to 98 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and among them, a two-component regulatory system, a beta-lactam resistance system, etc. were involved in some known genes resistant to drugs. Additionally, we further attempted to indicate whether the resistance mechanism of P. aeruginosa to tachyplesin I was associated with the changes of outer membrane permeability and OMPs. CONCLUSION: Our results indicated that P. aeruginosa resistant to tachyplesin I was mainly related to reduced entry of tachyplesin I into the bacterial cell due to overexpression of efflux pump, in addition to a decrease of outer membrane permeability. Our findings were also validated by pathway enrichment analysis and quantitative reverse transcription polymerase chain reaction (RT-qPCR). This study may provide a promising guidance for understanding the resistance mechanism of P. aeruginosa to tachyplesin I.202032021330
9043180.9760The 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
9062190.9760Biological cost of pyocin production during the SOS response in Pseudomonas aeruginosa. LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance to clinically important antibiotics and impairs the survival of bacteria exposed to one of several genotoxic agents. Two distinct mechanisms mediated these effects. Cell lysis genes that are induced following PrtR autocleavage reduced resistance to bactericidal levels of ciprofloxacin, and production of extracellular R2 pyocin was lethal to cells that initially survived UV light treatment. Although typically resistant to R2 pyocin, P. aeruginosa becomes transiently sensitive to R2 pyocin following UV light treatment, likely because of the strong downregulation of lipopolysaccharide synthesis genes that are required for resistance to R2 pyocin. Our results demonstrate that pyocin production during the P. aeruginosa SOS response carries both expected and unexpected costs.201425022851