Polyphosphate Kinase Mediates Antibiotic Tolerance in Extraintestinal Pathogenic Escherichia coli PCN033. - Related Documents




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894401.0000Polyphosphate Kinase Mediates Antibiotic Tolerance in Extraintestinal Pathogenic Escherichia coli PCN033. Extraintestinal pathogenic Escherichia coli (ExPEC) causes a variety of acute infections in its hosts, and multidrug-resistant strains present significant challenges to public health and animal husbandry. Therefore, it is necessary to explore new drug targets to control E. coli epidemics. Previous studies have reported that ppk mutants of Burkholderia pseudomallei and Mycobacterium tuberculosis are more susceptible than the wild types (WTs) to stress. Therefore, we investigated the stress response to antibiotics mediated by polyphosphate kinase (PPK) in ExPEC strain PCN033. We observed that planktonic cells of a ppk knockout strain (Δppk) were more susceptible to antibiotics than was WT. However, biofilm-grown Δppk cells showed similar susceptibility to that of the WT and were more tolerant than the planktonic cells. During the planktonic lifestyle, the expression of genes involved in antibiotic tolerance (including resistance-conferring genes, and antibiotic influx, and efflux genes) did not change in the Δppk mutant without antibiotic treatment. However, the resistance-conferring gene bla and efflux genes were upregulated more in the WT than in the Δppk mutant by treatment with tazobactam. After treatment with gentamycin, the efflux genes and influx genes were upregulated and downregulated, respectively, more in the WT than in the Δppk mutant. The expression of genes involved in biofilm regulation also changed after treatment with tazobactam or gentamycin, and which is consistent with the results of the biofilm formation. Together, these observations indicate that PPK is important for the antibiotic stress response during the planktonic growth of ExPEC and might be a potential drug target in bacteria.201627242742
896410.9996Analysis of the Oxidative Stress Regulon Identifies soxS as a Genetic Target for Resistance Reversal in Multidrug-Resistant Klebsiella pneumoniae. In bacteria, the defense system deployed to counter oxidative stress is orchestrated by three transcriptional factors, SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data are not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH78578 using paraquat and the corresponding oxidative stress regulon recorded using transcriptome sequencing (RNA-seq). The soxS gene was significantly induced during oxidative stress, and a knockout mutant was constructed to explore its functionality. The wild type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress and soxS regulons were identified and denoted as the oxidative SoxS regulon; these included a group of genes specifically regulated by SoxS. Efflux pump-encoding genes and global regulators were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP(+)) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo in a zebrafish embryo model. We conclude that the soxS gene could be considered a genetic target against which an inhibitor could be developed and used in combinatorial therapy to combat infections associated with multidrug-resistant K. pneumoniae. IMPORTANCE Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on K. pneumoniae and uncovers a target that could be useful to harness as a strategy to mitigate resistance.202134098732
895720.9996Transcriptome Profiling Reveals Interplay of Multifaceted Stress Response in Escherichia coli on Exposure to Glutathione and Ciprofloxacin. We have previously reported that supplementation of exogenous glutathione (GSH) promotes ciprofloxacin resistance in Escherichia coli by neutralizing antibiotic-induced oxidative stress and by enhancing the efflux of antibiotic. In the present study, we used a whole-genome microarray as a tool to analyze the system-level transcriptomic changes of E. coli on exposure to GSH and/or ciprofloxacin. The microarray data revealed that GSH supplementation affects redox function, transport, acid shock, and virulence genes of E. coli. The data further highlighted the interplay of multiple underlying stress response pathways (including those associated with the genes mentioned above and DNA damage repair genes) at the core of GSH, offsetting the effect of ciprofloxacin in E. coli. The results of a large-scale validation of the transcriptomic data using reverse transcription-quantitative PCR (RT-qPCR) analysis for 40 different genes were mostly in agreement with the microarray results. The altered growth profiles of 12 different E. coli strains carrying deletions in the specific genes mentioned above with GSH and/or ciprofloxacin supplementation implicate these genes in the GSH-mediated phenotype not only at the molecular level but also at the functional level. We further associated GSH supplementation with increased acid shock survival of E. coli on the basis of our transcriptomic data. Taking the data together, it can be concluded that GSH supplementation influences the expression of genes of multiple stress response pathways apart from its effect(s) at the physiological level to counter the action of ciprofloxacin in E. coli. IMPORTANCE The emergence and spread of multidrug-resistant bacterial strains have serious medical and clinical consequences. In addition, the rate of discovery of new therapeutic antibiotics has been inadequate in last few decades. Fluoroquinolone antibiotics such as ciprofloxacin represent a precious therapeutic resource in the fight against bacterial pathogens. However, these antibiotics have been gradually losing their appeal due to the emergence and buildup of resistance to them. In this report, we shed light on the genome-level expression changes in bacteria with respect to glutathione (GSH) exposure which act as a trigger for fluoroquinolone antibiotic resistance. The knowledge about different bacterial stress response pathways under conditions of exposure to the conditions described above and potential points of cross talk between them could help us in understanding and formulating the conditions under which buildup and spread of antibiotic resistance could be minimized. Our findings are also relevant because GSH-induced genome-level expression changes have not been reported previously for E. coli.201829468195
632830.9995Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis. Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.201828847541
894240.9995Indole-Induced Activities of β-Lactamase and Efflux Pump Confer Ampicillin Resistance in Pseudomonas putida KT2440. Indole, which is widespread in microbial communities, has received attention because of its effects on bacterial physiology. Pseudomonas putida and Pseudomonas aeruginosa can acquire ampicillin (Amp) resistance during growth on indole-Amp agar. Transcriptome, mutant, and inhibitor studies have suggested that Amp resistance induced by indole can be attributed to increased gene expression of ttgAB encoding two genes of RND-type multidrug efflux operons and an ampC encoding β-lactamase. Expression, enzyme activities, and mutational analyses indicated that AmpC β-lactamase is important for acquiring Amp resistance of P. putida in the presence of indole. Here, we show, for the first time, that volatile indole increased Amp-resistant cells. Consistent with results of the volatile indole assay, a low concentration of indole in liquid culture promoted growth initially, but led to mutagenesis after indole was depleted, which could not be observed at high indole concentrations. Interestingly, ttgAB and ampC gene expression levels correlate with the concentration of indole, which might explain the low number of Amp-mutated cells in high indole concentrations. The expression levels of genes involved in mutagenesis, namely rpoS, recA, and mutS, were also modulated by indole. Our data indicates that indole reduces Amp-induced heterogeneity by promoting expression of TtgABC or MexAB-OprM efflux pumps and the indole-induced β-lactamase in P. putida and P. aeruginosa.201728352264
895650.9995Biofilm characteristics and transcriptomic profiling of Acinetobacter johnsonii defines signatures for planktonic and biofilm cells. Most bacteria in the natural environment have a biofilm mode of life, which is intrinsically tolerant to antibiotics. While until now, the knowledge of biofilm formation by Acinetobacter johnsonii is not well understood. In this study, the characteristics and the effect of a sub-inhibitory concentration of antibiotic on A. johnsonii biofilm and planktonic cells were determined. We discovered a positive relationship between biofilm formation and tetracycline resistance, and biofilms rapidly evolve resistance to tetracycline they are treated with. Persister cells commonly exist in both planktonic and biofilm cells, with a higher frequency in the latter. Further transcriptomic analysis speculates that the overexpression of multidrug resistance genes and stress genes were mainly answered to sub lethal concentration of tetracycline in planktonic cells, and the lower metabolic levels after biofilm formation result in high resistance level of biofilm cells to tetracycline. Altogether, these data suggest that A. johnsonii can adjust its phenotype when grown as biofilm and change its metabolism under antibiotic stress, and provide implications for subsequent biofilm control.202235718162
896560.9995Resistance characterization and transcriptomic analysis of imipenem-induced drug resistance in Escherichia coli. BACKGROUND: Bacteria can develop resistance to various antibiotics under selective pressure, leading to multifaceted changes in resistance mechanisms. Transcriptomic sequencing allows for the observation of transcriptional level alterations in cells under antibiotic stress. Understanding the bacterial response to such stress is essential for deciphering their strategy against drug-resistant antibiotics and identifying potential targets for antibiotic development. METHODS: This study using wild-type (WT) Escherichia coli (E. coli) discovered that continuous in vitro induction screening for imipenem-resistant strains resulted in bacteria with enhanced biofilm-forming ability and mutations in antibiotic target sites. Transcriptomic sequencing of the resistant bacteria revealed significant changes in carbon and amino acid metabolism, nutrient assimilation, substance transport, nucleotide metabolism, protein biosynthesis, and cell wall biosynthesis. The up-regulated drug efflux genes were disrupted using gene knockout technology. Drug sensitivity tests indicated that drug efflux has a minimal effect on imipenem resistance. RESULTS: This suggests a strategy for E. coli drug resistance involving the reduction of unnecessary substance synthesis and metabolism, coupled with an increase in activities that aid in resisting foreign threats.202439624129
894370.9995Effects of indole on drug resistance and virulence of Salmonella enterica serovar Typhimurium revealed by genome-wide analyses. BACKGROUND: Many Gram-positive and Gram-negative bacteria produce large quantities of indole as an intercellular signal in microbial communities. Indole demonstrated to affect gene expression in Escherichia coli as an intra-species signaling molecule. In contrast to E. coli, Salmonella does not produce indole because it does not harbor tnaA, which encodes the enzyme responsible for tryptophan metabolism. Our previous study demonstrated that E. coli-conditioned medium and indole induce expression of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium for inter-species communication; however, the global effect of indole on genes in Salmonella remains unknown. RESULTS: To understand the complete picture of genes regulated by indole, we performed DNA microarray analysis of genes in the S. enterica serovar Typhimurium strain ATCC 14028s affected by indole. Predicted Salmonella phenotypes affected by indole based on the microarray data were also examined in this study. Indole induced expression of genes related to efflux-mediated multidrug resistance, including ramA and acrAB, and repressed those related to host cell invasion encoded in the Salmonella pathogenicity island 1, and flagella production. Reduction of invasive activity and motility of Salmonella by indole was also observed phenotypically. CONCLUSION: Our results suggest that indole is an important signaling molecule for inter-species communication to control drug resistance and virulence of S. enterica.201222632036
894680.9995Role of the CpxAR two-component signal transduction system in control of fosfomycin resistance and carbon substrate uptake. Although fosfomycin is an old antibiotic, it has resurfaced with particular interest. The antibiotic is still effective against many pathogens that are resistant to other commonly used antibiotics. We have found that fosfomycin resistance of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by the bacterial two-component signal transduction system CpxAR. A cpxA mutant lacking its phosphatase activity results in constitutive activation of its cognate response regulator, CpxR, and fosfomycin resistance. We have shown that fosfomycin resistance requires CpxR because deletion of the cpxR gene in the cpxA mutant restores fosfomycin sensitivity. We have also shown that CpxR directly represses the expression of two genes, glpT and uhpT, which encode transporters that cotransport fosfomycin with their native substrates glycerol-3-phosphate and glucose-6-phosphate, and repression of these genes leads to a decrease in fosfomycin transport into the cpxA mutant. However, the cpxA mutant had an impaired growth phenotype when cultured with glycerol-3-phosphate or glucose-6-phosphate as a sole carbon substrate and was outcompeted by the parent strain, even in nutrient-rich medium. This suggests a trade-off between fosfomycin resistance and the biological fitness associated with carbon substrate uptake. We propose a role for the CpxAR system in the reversible control of fosfomycin resistance. This may be a beneficial strategy for bacteria to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin.201424163343
629490.9995Comparison of Gene Expression Profiles of Uropathogenic Escherichia Coli CFT073 after Prolonged Exposure to Subinhibitory Concentrations of Different Biocides. Biocides are chemical compounds widely used for sterilization and disinfection. The aim of this study was to examine whether exposure to subinhibitory biocide concentrations influenced transcriptional expression of genes that could improve a pathogen's drug resistance or fitness. We used DNA microarrays to investigate the transcriptome of the uropathogenic Escherichia coli strain CFT073 in response to prolonged exposure to subinhibitory concentrations of four biocides: benzalkonium chloride, chlorhexidine, hydrogen peroxide and triclosan. Transcription of a gene involved in polymyxin resistance, arnT, was increased after treatment with benzalkonium chloride. However, pretreatment of the bacteria with this biocide did not result in cross-resistance to polymyxin in vitro. Genes encoding products related to transport formed the functional group that was most affected by biocides, as 110 out of 884 genes in this category displayed altered transcription. Transcripts of genes involved in cysteine uptake, sulfate assimilation, dipeptide transport, as well as cryptic phage genes were also more abundant in response to several biocides. Additionally, we identified groups of genes with transcription changes unique to single biocides that might include potential targets for the biocides. The biocides did not increase the resistance potential of the pathogen to other antimicrobials.201931569631
8928100.9995Increased survival of antibiotic-resistant Escherichia coli inside macrophages. Mutations causing antibiotic resistance usually incur a fitness cost in the absence of antibiotics. The magnitude of such costs is known to vary with the environment. Little is known about the fitness effects of antibiotic resistance mutations when bacteria confront the host's immune system. Here, we study the fitness effects of mutations in the rpoB, rpsL, and gyrA genes, which confer resistance to rifampin, streptomycin, and nalidixic acid, respectively. These antibiotics are frequently used in the treatment of bacterial infections. We measured two important fitness traits-growth rate and survival ability-of 12 Escherichia coli K-12 strains, each carrying a single resistance mutation, in the presence of macrophages. Strikingly, we found that 67% of the mutants survived better than the susceptible bacteria in the intracellular niche of the phagocytic cells. In particular, all E. coli streptomycin-resistant mutants exhibited an intracellular advantage. On the other hand, 42% of the mutants incurred a high fitness cost when the bacteria were allowed to divide outside of macrophages. This study shows that single nonsynonymous changes affecting fundamental processes in the cell can contribute to prolonged survival of E. coli in the context of an infection.201323089747
8955110.9995Increasing resistance of planktonic and biofilm cultures of Burkholderia cepacia to ciprofloxacin and ceftazidime during exponential growth. The change in resistance of Burkholderia cepacia to ceftazidime and to ciprofloxacin during the exponential phase and up to the onset of stationary phase was assessed along the growth curve in batch culture. B. cepacia was grown in planktonic culture and in a biofilm on a membrane support. Resistance increased progressively during the exponential phase, being increased by ten-fold about every four generations. Bacteria grown in a biofilm were about 15 times more resistant than equivalent planktonic-grown bacteria. The growth rate was not the key factor for the development of resistance. The growth phase and the mode of growth have a fundamental impact on the susceptibility of B. cepacia towards antimicrobial agents. Bacteria growing at the same rate may differ greatly in their resistance to antimicrobial agents.19989738832
8945120.9995Adaptation of a fluoroquinolone-sensitive Shigella sonnei to norfloxacin exposure. Shigella causes shigellosis that requires antibiotic treatment in severe cases. Sublethal antibiotic concentrations can promote resistance, but their effect on antibiotic-sensitive bacteria before resistance development is unclear. This study investigated the effects of sublethal norfloxacin (NOR) challenges on a NOR-sensitive strain, Shigella sonnei UKMCC1015. Firstly, the whole genome of S. sonnei UKMCC1015 was assembled, and 45 antimicrobial resistance (AMR) genes were identified. Interestingly, transcriptomic analysis showed that low NOR levels do not change either the expression of the AMR genes or NOR targets such as gyrA. Instead, multiple ribosomal protein genes were downregulated, which could be attributed to decreased ribosomal protein promoter activity, modulated by elevated guanosine pentaphosphate and tetraphosphate (ppGpp) levels. This alarmone is involved in the bacterial stringent response during environmental stress, and it is mainly produced from the ppGpp synthetase (relA). Additionally, we observed that a relA overexpression (prolonged period of elevated ppGpp levels) may negatively affect the NOR tolerance of the bacteria. In conclusion, this study revealed that a NOR-sensitive strain responds differently to sublethal NOR than commonly reported in resistant strains.202439100177
8968130.9995Antibiotic stress, genetic response and altered permeability of E. coli. BACKGROUND: Membrane permeability is the first step involved in resistance of bacteria to an antibiotic. The number and activity of efflux pumps and outer membrane proteins that constitute porins play major roles in the definition of intrinsic resistance in Gram-negative bacteria that is altered under antibiotic exposure. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the genetic regulation of porins and efflux pumps of Escherichia coli during prolonged exposure to increasing concentrations of tetracycline and demonstrate, with the aid of quantitative real-time reverse transcriptase-polymerase chain reaction methodology and western blot detection, the sequence order of genetic expression of regulatory genes, their relationship to each other, and the ensuing increased activity of genes that code for transporter proteins of efflux pumps and down-regulation of porin expression. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that, in addition to the transcriptional regulation of genes coding for membrane proteins, the post-translational regulation of proteins involved in the permeability of Gram-negative bacteria also plays a major role in the physiological adaptation to antibiotic exposure. A model is presented that summarizes events during the physiological adaptation of E. coli to tetracycline exposure.200717426813
8892140.9995Fur is the master regulator of the extraintestinal pathogenic Escherichia coli response to serum. Drug-resistant extraintestinal pathogenic Escherichia coli (ExPEC) strains are the major cause of colisepticemia (colibacillosis), a condition that has become an increasing public health problem in recent years. ExPEC strains are characterized by high resistance to serum, which is otherwise highly toxic to most bacteria. To understand how these bacteria survive and grow in serum, we performed system-wide analyses of their response to serum, making a clear distinction between the responses to nutritional immunity and innate immunity. Thus, mild heat inactivation of serum destroys the immune complement and abolishes the bactericidal effect of serum (inactive serum), making it possible to examine nutritional immunity. We used a combination of deep RNA sequencing and proteomics in order to characterize ExPEC genes whose expression is affected by the nutritional stress of serum and by the immune complement. The major change in gene expression induced by serum-active and inactive-involved metabolic genes. In particular, the serum metabolic response is coordinated by three transcriptional regulators, Fur, BasR, and CysB. Fur alone was responsible for more than 80% of the serum-induced transcriptional response. Consistent with its role as a major serum response regulator, deletion of Fur renders the bacteria completely serum sensitive. These results highlight the role of metabolic adaptation in colisepticemia and virulence. IMPORTANCE: Drug-resistant extraintestinal pathogenic Escherichia coli (ExPEC) strains have emerged as major pathogens, especially in community- and hospital-acquired infections. These bacteria cause a large spectrum of syndromes, the most serious of which is septicemia, a condition with a high mortality rate. These bacterial strains are characterized by high resistance to serum, otherwise highly toxic to most bacteria. To understand the basis of this resistance, we carried out system-wide analyses of the response of ExPEC strains to serum by using proteomics and deep RNA sequencing. The major changes in gene expression induced by exposure to serum involved metabolic genes, not necessarily implicated in relation to virulence. One metabolic regulator-Fur-involved in iron metabolism was responsible for more than 80% of the serum-induced response, and its deletion renders the bacteria completely serum sensitive. These results highlight the role of metabolic adaptation in virulence.201425118243
8929150.9995Interplay in the selection of fluoroquinolone resistance and bacterial fitness. Fluoroquinolones are antibacterial drugs that inhibit DNA Gyrase and Topoisomerase IV. These essential enzymes facilitate chromosome replication and RNA transcription by regulating chromosome supercoiling. High-level resistance to fluoroquinolones in E. coli requires the accumulation of multiple mutations, including those that alter target genes and genes regulating drug efflux. Previous studies have shown some drug-resistance mutations reduce bacterial fitness, leading to the selection of fitness-compensatory mutations. The impact of fluoroquinolone-resistance on bacterial fitness was analyzed in constructed isogenic strains carrying up to 5 resistance mutations. Some mutations significantly decreased bacterial fitness both in vitro and in vivo. We identified low-fitness triple-mutants where the acquisition of a fourth resistance mutation significantly increased fitness in vitro and in vivo while at the same time dramatically decreasing drug susceptibility. The largest effect occurred with the addition of a parC mutation (Topoisomerase IV) to a low-fitness strain carrying resistance mutations in gyrA (DNA Gyrase) and marR (drug efflux regulation). Increased fitness was accompanied by a significant change in the level of gyrA promoter activity as measured in an assay of DNA supercoiling. In selection and competition experiments made in the absence of drug, parC mutants that improved fitness and reduced susceptibility were selected. These data suggest that natural selection for improved growth in bacteria with low-level resistance to fluoroquinolones could in some cases select for further reductions in drug susceptibility. Thus, increased resistance to fluoroquinolones could be selected even in the absence of further exposure to the drug.200919662169
8939160.9995YmdB-mediated down-regulation of sucA inhibits biofilm formation and induces apramycin susceptibility in Escherichia coli. Antibiotic resistance associated with biofilm formation is a major concern when treating bacterial infections with drugs. The genes and pathways involved in biofilm formation have been extensively studied and are also involved in antibiotic resistance. Recent studies show that overexpression of Escherichia coli (E. coli) YmdB protein alters gene expression profiles and inhibits biofilm formation. Therefore, it is expected that YmdB and its regulated genes play a key role in development of biofilm and antibiotic resistance phenotypes. The present study screened antibiotics to identify those whose susceptibility profiles were regulated by YmdB levels. This protocol identified apramycin. Additional screening for genes negatively regulated by inactivation of RNase III activity via YmdB overexpression revealed that a gene associated with the tricarboxylic acid cycle gene, sucA, was necessary for the YmdB-like phenotype. Taken together, these data suggest that regulation of RNase III activity by trans-acting factors may be the key to identifying genes or pathways connecting biofilm and antibiotic resistance phenotypes. This information could be used to reduce the emergence of biofilm-associated multidrug-resistant bacteria.201728034758
6292170.9995Genome-Wide Screening and Characterization of Genes Involved in Response to High Dose of Ciprofloxacin in Escherichia coli. The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Inevitably, considering its extensive use and misuse, resistance toward ciprofloxacin has increased in almost all clinically relevant bacteria. This study aimed to investigate the transcriptome changes at a high concentration of ciprofloxacin in Escherichia coli. In brief, 1,418 differentially expressed genes (DEGs) were identified, from which 773 genes were upregulated by ciprofloxacin, whereas 651 genes were downregulated. Enriched biological pathways reflected the upregulation of biological processes such as DNA damage and repair system, toxin/antitoxin systems, formaldehyde detoxification system. With kyoto encyclopedia of genes and genomes pathway analysis, higher expressed DEGs were associated with "LPS biosynthesis," "streptomycin biosynthesis," and "polyketide sugar unit biosynthesis." Lower expressed DEGs were associated with "biosynthesis of amino acids" and "flagellar assembly" pathways. After treatment of ciprofloxacin, lipopolysaccharide (LPS) release was increased by two times, and the gene expression level of LPS synthesis was elevated (p < 0.05) in both reference and clinical strains. Our results demonstrated that transient exposure to high-dose ciprofloxacin is a double-edged sword. Cautions should be taken when administering high-dose antibiotic treatment for infectious diseases.202235512736
8949180.9995Potential Risk of Spreading Resistance Genes within Extracellular-DNA-Dependent Biofilms of Streptococcus mutans in Response to Cell Envelope Stress Induced by Sub-MICs of Bacitracin. Antibiotics are used to treat or prevent some types of bacterial infection. The inappropriate use of antibiotics unnecessarily promotes antibiotic resistance and increases resistant bacteria, and controlling these bacteria is difficult. While the emergence of drug-resistant bacteria is a serious problem, the behavior of drug-resistant bacteria is not fully understood. In this study, we investigated the behavior of Streptococcus mutans, a major etiological agent of dental caries that is resistant to bacitracin, which is a cell wall-targeting antibiotic, and focused on biofilm formation in the presence of bacitracin. S. mutans UA159 most strongly induced extracellular DNA (eDNA)-dependent biofilm formation in the presence of bacitracin at 1/8× MIC. The ΔmbrC and ΔmbrD mutant strains, which lack bacitracin resistance, also formed biofilms in the presence of bacitracin at 1/2× MIC. This difference between the wild type and the mutants was caused by the induction of atlA expression in the mid-log phase. We also revealed that certain rgp genes involved in the synthesis of rhamnose-glucose polysaccharide related to cell wall synthesis were downregulated by bacitracin. In addition, glucosyltransferase-I was also involved in eDNA-dependent biofilm formation. The biofilm led to increased transformation efficiencies and promoted horizontal gene transfer. Biofilms were also induced by ampicillin and vancomycin, antibiotics targeting cell wall synthesis, suggesting that cell envelope stress triggers biofilm formation. Therefore, the expression of the atlA and rgp genes is regulated by S. mutans, which forms eDNA-dependent biofilms, promoting horizontal gene transfer in response to cell envelope stress induced by sub-MICs of antibiotics.IMPORTANCE Antibiotics have been reported to induce biofilm formation in many bacteria at subinhibitory concentrations. Accordingly, it is conceivable that the MIC against drug-sensitive bacteria may promote biofilm formation of resistant bacteria. Since drug-resistant bacteria have spread, it is important to understand the behavior of resistant bacteria. Streptococcus mutans is bacitracin resistant, and the 1/8× MIC of bacitracin, which is a cell wall-targeted antibiotic, induced eDNA-dependent biofilm formation. The ΔmbrC and ΔmbrD strains, which are not resistant to bacitracin, also formed biofilms in the presence of bacitracin at 1/2× MIC, and biofilms of both the wild type and mutants promoted horizontal gene transfer. Another cell wall-targeted antibiotic, vancomycin, showed effects on biofilms and gene transfer similar to those of bacitracin. Thus, treatment with cell wall-targeted antibiotics may promote the spread of drug-resistant genes in biofilms. Therefore, the behavior of resistant bacteria in the presence of antibiotics at sub-MICs should be investigated when using antibiotics.202032532873
8894190.9995Genome Recombination-Mediated tRNA Up-Regulation Conducts General Antibiotic Resistance of Bacteria at Early Stage. Bacterial antibiotic resistance sets a great challenge to human health. It seems that the bacteria can spontaneously evolve resistance against any antibiotic within a short time without the horizontal transfer of heterologous genes and before accumulating drug-resistant mutations. We have shown that the tRNA-mediated translational regulation counteracts the reactive oxygen species (ROS) in bacteria. In this study, we demonstrated that isolated and subcultured Escherichia coli elevated its tRNAs under antibiotic stress to rapidly provide antibiotic resistance, especially at the early stage, before upregulating the efflux pump and evolving resistance mutations. The DNA recombination system repaired the antibiotic-induced DNA breakage in the genome, causing numerous structural variations. These structural variations are overrepresented near the tRNA genes, which indicated the cause of tRNA up-regulation. Knocking out the recombination system abolished the up-regulation of tRNAs, and coincidently, they could hardly evolve antibiotic resistance in multiple antibiotics, respectively. With these results, we proposed a multi-stage model of bacterial antibiotic resistance in an isolated scenario: the early stage (recombination-tRNA up-regulation-translational regulation); the medium stage (up-regulation of efflux pump); the late stage (resistant mutations). These results also indicated that the bacterial DNA recombination system and tRNA could be targeted to retard the bacterial spontaneous drug resistance.202135126332