Mechanisms of mepA Overexpression and Membrane Potential Reduction Leading to Ciprofloxacin Heteroresistance in a Staphylococcus aureus Isolate. - Related Documents




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637601.0000Mechanisms of mepA Overexpression and Membrane Potential Reduction Leading to Ciprofloxacin Heteroresistance in a Staphylococcus aureus Isolate. Heteroresistance has seriously affected the evaluation of antibiotic efficacy against pathogenic bacteria, causing misjudgment of antibiotics' sensitivity in clinical therapy, leading to treatment failure, and posing a serious threat to current medical health. However, the mechanism of Staphylococcus aureus heteroresistance to ciprofloxacin remains unclear. In this study, heteroresistance to ciprofloxacin in S. aureus strain 529 was confirmed by antimicrobial susceptibility testing and population analysis profiling (PAP), with the resistance of subclonal 529_HR based on MIC being 8-fold that of the original bacteria. A 7-day serial MIC evaluation and growth curves demonstrate that their phenotype was stable, with 529_HR growing more slowly than 529, but reaching a plateau in a similar proportion. WGS analysis showed that there were 11 nonsynonymous mutations and one deletion gene between the two bacteria, but none of these SNPs were directly associated with ciprofloxacin resistance. Transcriptome data analysis showed that the expression of membrane potential related genes (qoxA, qoxB, qoxC, qoxD, mprF) was downregulated, and the expression of multidrug resistance efflux pump gene mepA was upregulated. The combination of ciprofloxacin and limonene restored the 529_HR MIC from 1 mg/L to 0.125 mg/L. Measurement of the membrane potential found that 529_HR had a lower potential, which may enable it to withstand the ciprofloxacin-induced decrease in membrane potential. In summary, we demonstrated that upregulation of mepA gene expression and a reduction in membrane potential are the main heteroresistance mechanisms of S. aureus to ciprofloxacin. Additionally, limonene may be a potentially effective agent to inhibit ciprofloxacin heteroresistance phenotypes.202540076991
629110.9994Adaptive Resistance of Staphylococcus aureus to Cefquinome Sulfate in an In Vitro Pharmacokinetic Model with Transcriptomic Insights. Cefquinome sulfate has a strong killing effect against Staphylococcus aureus (S. aureus), but bacterial resistance has become increasingly widespread. Experiments were conducted to investigate the pattern of adaptive resistance of S. aureus to cefquinome sulfate under different dosage regimens by using pharmacokinetic-pharmacodynamics (PK-PD) modeling, and the adaptive-resistant bacteria in different states were screened and subjected to transcriptomic sequencing. The results showed that the minimum inhibitory concentration of Staphylococcus aureus under the action of cefquinome sulfate was 0.5 μg/mL, the anti-mutation concentration was 1.6 μg/mL, and the mutation selection window range was 0.5~1.6 μg/mL. In the in vitro pharmacokinetic model to simulate different dosing regimens in the animal body, there are certain rules for the emergence of adaptive drug-resistant bacteria: the intensity of bacterial resistance gradually increased with culture time, and the order of emergence was tolerant bacteria (TO) followed by persistent bacteria (PE) and finally resistant bacteria (RE). The sequence reflected the evolution of adaptive drug resistance. Transcriptome Gene Ontology (GO) analysis revealed that differentially expressed genes were involved in cellular respiration, energy derivation by oxidation of organic compounds, and oxidation-reduction processes. The differentially expressed genes identified functioned in the synthesis of cell membranes, cytoplasm, and intracellular parts. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that 65 genes were differentially expressed after cefquinome sulfate treatment, of which 35 genes were significantly upregulated and 30 genes were significantly downregulated. Five genes, sdhB, sdhA, pdhA, lpdA, and sucC, may be involved in network regulation. This study revealed the cross-regulation of multiple metabolic pathway networks and the targets of network regulation of S. aureus to produce adaptive drug resistance. The results will provide guidance for clinical drug use in animals infected with S. aureus.202540005696
628520.9994Triton X-100 counteracts antibiotic resistance of Enterococcus faecalis: An in vitro study. OBJECTIVES: The high prevalence of antibiotic-resistant bacteria poses a threat to the global public health. The appropriate use of adjuvants to restore the antimicrobial activity of antibiotics against resistant bacteria could be an effective strategy for combating antibiotic resistance. In this study, we investigated the counteraction of Triton X-100 (TX-100) and the mechanisms underlying the antibiotic resistance of Enterococcus faecalis (E. faecalis). METHODS: Standard, wild-type (WT), and induced antibiotic-resistant E. faecalis strains were used in this study. In vitro antibacterial experiments were conducted to evaluate the antimicrobial activities of gentamicin sulfate and ciprofloxacin hydrochloride in the presence and absence of 0.02 % TX-100 against both planktonic and biofilm bacteria. Transcriptomic and untargeted metabolomic analyses were performed to explore the molecular mechanisms of TX-100 as an antibiotic adjuvant. Additionally, membrane permeability, membrane potential, glycolysis-related enzyme activity, intracellular adenosine triphosphate (ATP), and expression levels of virulence genes were assessed. The biocompatibility of different drug combinations was also evaluated. RESULTS: A substantially low TX-100 concentration improved the antimicrobial effects of gentamicin sulfate or ciprofloxacin hydrochloride against antibiotic-resistant E. faecalis. Mechanistic studies demonstrated that TX-100 increased cell membrane permeability and dissipated membrane potential. Moreover, antibiotic resistance and pathogenicity of E. faecalis were attenuated by TX-100 via downregulation of the ABC transporter, phosphotransferase system (PTS), and ATP supply. CONCLUSIONS: TX-100 enhanced the antimicrobial activity of gentamicin sulfate and ciprofloxacin hydrochloride at a low concentration by improving antibiotic susceptibility and attenuating antibiotic resistance and pathogenicity of E. faecalis. CLINICAL SIGNIFICANCE: These findings provide a theoretical basis for developing new root canal disinfectants that can reduce antibiotic resistance.202438729285
629830.9993Sublethal Sodium Hypochlorite Exposure: Impact on Resistance-Nodulation-Cell Division Efflux Pump Overexpression and Cross-Resistance to Imipenem. Sodium hypochlorite (NaOCl) is widely used in public healthcare facilities; this exposure can result in the development of bacterial tolerance to disinfectants, which has known links to antibiotic cross-resistance. However, the mechanism through which cross-resistance to antibiotics and disinfectants develops remains ambiguous. Therefore, this study aimed to examine the phenotypic and transcriptomic changes caused by disinfectant exposure in Gram-negative bacteria and determine the cause of cross-resistance to antibiotics. The results demonstrated that the misuse of disinfectants plays an important role in the emergence of disinfectant resistance and in the increase in antibiotic resistance. Antibiotic resistance may occur from the exposure of Gram-negative bacteria to subminimal inhibitory concentrations (MICs) of NaOCl. Ten passages of Gram-negative bacteria in increasingly higher subMICs of the NaOCl disinfectant were sufficient to increase the MIC to >2500 µg/mL NaOCl, particularly in K. pneumoniae and P. aeruginosa. To determine the development of cross-resistance to antibiotics due to NaOCl exposure, the MICs for each antibiotic before and after the exposure of each strain to sublethal concentrations of NaOCl were compared. After overnight incubation with a sublethal concentration of NaOCl, a statistically significant increase in MIC was only observed for imipenem (p < 0.01). An investigation of the mechanism of cross-resistance by means of transcriptome analysis revealed that 1250 µg/mL of NaOCl-adapted K. pneumoniae and P. aeruginosa strains increased resistance to imipenem due to the increased expression of resistance-nodulation-cell division (RND) efflux pumps, such as AcrAB-TolC and MexAB/XY-OprM. Therefore, we suggest that exposure to NaOCl can influence the expression of RND efflux pump genes, contributing to imipenem cross-resistance.202439335002
476740.9993The impact of probiotic cell-free metabolites in MDR Pseudomonas aeruginosa: antibacterial properties and effect on antibiotic resistance genes expression. There is a significant demand for novel antibacterial agents against multidrug-resistant (MDR) gram-negative bacteria. Recently, probiotics have been noted for their antibacterial properties against various pathogens. This study aimed to investigate the effects of probiotic cell-free supernatants on MDR Pseudomonas aeruginosa. Clinical isolates demonstrating the highest degree of antibiotic resistance were chosen, and the antibacterial effect of probiotic metabolites was evaluated using an agar-well diffusion assay. In addition, the effect of probiotics on the expression of resistance genes was evaluated using real-time PCR. The CFS was assessed using GC-MS to determine the antibacterial compounds. The supernatants inhibited the growth of the isolates (P < 0.0001); however, there was no noticeable difference in the effectiveness of the probiotics. In addition, the supernatants decreased the expression levels of mexD, mexB, mexF, and ampC, and an increase in oprD was observed in some groups. After the assessment of Lactobacillus acidophilus by GC-MS, antibacterial compounds, such as acetamide, nonadecane, 9-methyl, and tetradecane, were determined. Our findings showed that probiotic metabolites can effectively inhibit the growth of MDR P. aeruginosa. Gene expression analysis also revealed that the mechanism of antibacterial action was most likely related to the regulation of efflux pumps.202337742315
622550.9993Genome-Wide Identification of Resveratrol Intrinsic Resistance Determinants in Staphylococcus aureus. Resveratrol has been extensively studied due to its potential health benefits in multiple diseases, for example, cancer, obesity and cardiovascular diseases. Besides these properties, resveratrol displays inhibitory activity against a wide range of bacterial species; however, the cellular effects of resveratrol in bacteria remain incompletely understood, especially in the human pathogen, Staphylococcus aureus. In this study, we aimed to identify intrinsic resistance genes that aid S. aureus in tolerating the activity of resveratrol. We screened the Nebraska Transposon Mutant Library, consisting of 1920 mutants with inactivation of non-essential genes in S. aureus JE2, for increased susceptibly to resveratrol. On agar plates containing 0.5× the minimum inhibitory concentration (MIC), 17 transposon mutants failed to grow. Of these, four mutants showed a two-fold reduction in MIC, being the clpP protease mutant and three mutants with deficiencies in the electron transport chain (menD, hemB, aroC). The remaining 13 mutants did not show a reduction in MIC, but were confirmed by spot-assays to have increased susceptibility to resveratrol. Several genes were associated with DNA damage repair (recJ, xerC and xseA). Treatment of S. aureus JE2 with sub-inhibitory concentrations of resveratrol did not affect the expression of recJ, xerC and xseA, but increased expression of the SOS-stress response genes lexA and recA, suggesting that resveratrol interferes with DNA integrity in S. aureus. Expression of error-prone DNA polymerases are part of the SOS-stress response and we could show that sub-inhibitory concentrations of resveratrol increased overall mutation frequency as measured by formation of rifampicin resistant mutants. Our data show that DNA repair systems are important determinants aiding S. aureus to overcome the inhibitory activity of resveratrol. Activation of the SOS response by resveratrol could potentially facilitate the development of resistance towards conventional antibiotics in S. aureus.202133467002
629760.9993Combined effect of bacteriophage and antibiotic on the inhibition of the development of antibiotic resistance in Salmonella typhimurium. This study was designed to evaluate the combined effects of bacteriophage and antibiotic on the reduction of the development of antibiotic-resistance in Salmonella typhimurium LT2. The susceptibilities of S. typhimurium to ciprofloxacin and erythromycin were increased when treated with bacteriophages, showing more than 10% increase in clear zone sizes and greater than twofold decrease in minimum inhibitory concentration values. The growth of S. typhimurium was effectively inhibited by the combination of bacteriophage P22 and ciprofloxacin. The combination treatment effectively reduced the development of antibiotic resistance in S. typhimurium. The relative expression levels of efflux pump-related genes (acrA, acrB, and tolC) and outer membrane-related genes (ompC, ompD, and ompF) were decreased at all treatments. This study provides useful information for designing new antibiotic therapy to control antibiotic-resistant bacteria.201830263855
637570.9993Role of ppGpp-regulated efflux genes in Acinetobacter baumannii. OBJECTIVES: Treatment of infections caused by Acinetobacter baumannii nosocomial strains has become increasingly problematic owing to their resistance to antibiotics. ppGpp is a secondary messenger involved in growth control and various stress responses in bacteria. The mechanism for inhibition of antibiotic resistance via ppGpp is still unidentified in various pathogenic bacteria including A. baumannii. Here, we investigated the effects of ppGpp on efflux pump (EP)-related genes in A. baumannii. METHODS: ppGpp-deficient and -complementary strains were constructed by conjugation and we confirmed (p)ppGpp measurements by thin-layer chromatography. We observed that the ppGpp-deficient strain (ΔA1S_0579) showed abnormal stretching patterns by transmission electron microscopy analysis. The MICs of antimicrobial agents for the WT A. baumannii (ATCC 17978), ppGpp-deficient and complementary strains were determined by the Etest and broth dilution assay methods. The expression levels of EP-related genes were determined by quantitative RT-PCR. RESULTS: We observed morphological differences between a ppGpp-deficient strain (ΔA1S_0579) and the WT strain. Dramatic reductions of MICs in the ppGpp-deficient strain compared with the WT were observed for gentamicin (2.6-fold), tetracycline (3.9-fold), erythromycin (4-fold) and trimethoprim (>4-fold). Expression of the EP-related genes abeB (2.8-fold), tet(A) (2.3-fold), adeB (10.0-fold), adeI (9.9-fold), adeJ (11.8-fold) and adeK (14.4-fold) was also decreased in the ppGpp-deficient strain. CONCLUSIONS: This study demonstrates that ppGpp regulates EP-related gene expression in A. baumannii, affecting antibiotic susceptibility. To date, treatment for MDR A. baumannii has had no new antimicrobial agents, so the A1S_0579 gene could be a novel therapeutic target for rational drug design by affecting ppGpp production.202032049284
576080.9993Downregulation of Klebsiella pneumoniae RND efflux pump genes following indole signal produced by Escherichia coli. BACKGROUND: More than a century has passed since it was discovered that many bacteria produce indole, but research into the actual biological roles of this molecule is just now beginning. The influence of indole on bacterial virulence was extensively investigated in indole-producing bacteria like Escherichia coli. To gain a deeper comprehension of its functional role, this study investigated how indole at concentrations of 0.5-1.0 mM found in the supernatant of Escherichia coli stationary phase culture was able to alter the virulence of non-indole-producing bacteria, such as Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, which are naturally exposed to indole in mixed infections with Escherichia coli. RESULTS: Biofilm formation, antimicrobial susceptibility, and efflux pump activity were the three phenotypic tests that were assessed. Indole was found to influence antibiotic susceptibly of Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumoniae to ciprofloxacin, imipenem, ceftriaxone, ceftazidime, and amikacin through significant reduction in MIC with fold change ranged from 4 to 16. Biofilm production was partially abrogated in both 32/45 Pseudomonas aeruginosa and all eight Proteus mirabilis, while induced biofilm production was observed in 30/40 Klebsiella pneumoniae. Moreover, acrAB and oqxAB, which encode four genes responsible for resistance-nodulation-division multidrug efflux pumps in five isolates of Klebsiella pneumoniae were investigated genotypically using quantitative real-time (qRT)-PCR. This revealed that all four genes exhibited reduced expression indicated by 2^-ΔΔCT < 1 in indole-treated isolates compared to control group. CONCLUSION: The outcomes of qRT-PCR investigation of efflux pump expression have established a novel clear correlation of the molecular mechanism that lies beneath the influence of indole on bacterial antibiotic tolerance. This research provides novel perspectives on the various mechanisms and diverse biological functions of indole signaling and how it impacts the pathogenicity of non-indole-producing bacteria.202439182027
624690.9993The CRISPR System and MepA Multidrug Efflux Pump Linked to Antibiotic Resistance in Staphylococcus aureus. Staphylococcus aureus (S. aureus) is a major zoonotic pathogen. To investigate CRISPR carriage in S. aureus isolates from cows with mastitis and the role of the CRISPR system and efflux pumps in antibiotic resistance. We analyzed antibiotic resistance genes and CRISPR loci, sequenced spacers, and assessed correlations between CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) presence and antibiotic resistance in 234 S. aureus isolates. The changes in CRISPR sequences were examined by continuous passage of 360 generations without antibiotic pressure. Subsequently, variations in CRISPR loci and transcript levels were measured under ciprofloxacin (CIP) exposure. In addition, an S. aureus-25-mepA was constructed to evaluate changes in antimicrobial sensitivity and mepA transcript levels in both planktonic and biofilm states. Our results revealed a CRISPR loci detection rate of 7.69% among the 234 S. aureus isolates, with significantly lower rates of the antibiotic resistance genes gyrA, grlA, norA, and tet(M) in CRISPR-positive isolates compared to those in CRISPR-negative isolates (p < 0.05). CIP-resistant strains exhibited loss of repeat and spacer sequence in CRISPR loci, and the transcript abundance of these loci gradually decreased under CIP pressures, indicating that CRISPR loci deletion or transcript level downregulation under antibiotic stress may be a potential regulatory mechanism of antibiotic resistance. Correlation analysis linked CIP resistance in both planktonic and biofilm S. aureus to mepA transcript levels and biofilm integrity. Our study provides insight into the mechanism by which S. aureus develops antibiotic resistance via the CRISPR system and the MepA efflux pump, offering a theoretical foundation for monitoring the prevalence and resistance of pathogenic bacteria.202539977007
4768100.9993Attenuating the virulence of the resistant superbug Staphylococcus aureus bacteria isolated from neonatal sepsis by ascorbic acid, dexamethasone, and sodium bicarbonate. BACKGROUND: Infections affecting neonates caused by Staphylococcus aureus are widespread in healthcare facilities; hence, novel strategies are needed to fight this pathogen. In this study, we aimed to investigate the effectiveness of the FDA-approved medications ascorbic acid, dexamethasone, and sodium bicarbonate to reduce the virulence of the resistant Staphylococcus aureus bacteria that causes neonatal sepsis and seek out suitable alternatives to the problem of multi-drug resistance. METHODS: Tested drugs were assessed phenotypically and genotypically for their effects on virulence factors and virulence-encoding genes in Staphylococcus aureus. Furthermore, drugs were tested in vivo for their ability to reduce Staphylococcus aureus pathogenesis. RESULTS: Sub-inhibitory concentrations (1/8 MIC) of ascorbic acid, dexamethasone, and sodium bicarbonate reduced the production of Staphylococcus aureus virulence factors, including biofilm formation, staphyloxanthin, proteases, and hemolysin production, as well as resistance to oxidative stress. At the molecular level, qRT-PCR was used to assess the relative expression levels of crtM, sigB, sarA, agrA, hla, fnbA, and icaA genes regulating virulence factors production and showed a significant reduction in the relative expression levels of all the tested genes. CONCLUSIONS: The current findings reveal that ascorbic acid, dexamethasone, and sodium bicarbonate have strong anti-virulence effects against Staphylococcus aureus. Thus, suggesting that they might be used as adjuvants to treat infections caused by Staphylococcus aureus in combination with conventional antimicrobials or as alternative therapies.202236348266
4724110.9993Transcriptomic analysis of sub-MIC Eugenol exposition on antibiotic resistance profile in Multidrug Resistant Enterococcus faecalis E9.8. The spread of multidrug-resistant (MDR) bacteria and their resistance genes along the food chain and the environment has become a global threat aggravated by incorrect disinfection strategies. This study analysed the effect of induction by sub-inhibitory concentrations of eugenol - a major ingredient in clove essential oil commonly used in disinfectant agents - on the phenotypic and genotypic response of MDR Enterococcus faecalis E9.8 strain, selected based on the phenotypic response of other enterococci. Eugenol treatment irreversibly reduced several antibiotics' minimum inhibitory concentration (MIC), confirmed by kinetic studies for kanamycin, erythromycin, and tetracycline. Furthermore, transcriptomic analysis indicated the reversion of antibiotic resistance through direct and indirect measures, such as down-regulation of genes coding for proteins involved in antibiotic resistance, toxin resistance and virulence factors. Regarding antibiotic resistance genes (ARGs), ten differentially expressed genes (five down-regulated and five up-regulated genes) were related to the main transporter families, which present key targets in antibiotic resistance reversion. Our study thus highlights the importance of considering indirectly related genes as targets for antibiotic resistance reversion besides ARGs sensu stricto. These results allow us to propose using eugenol as an antibiotic resistance reversing agent to be included in disinfectant solutions as an excellent alternative to limit the spread of MDR bacteria and their ARGs in the food chain and the environment.202539827501
5761120.9993The Effects of Sub-inhibitory Antibiotic Concentrations on Pseudomonas aeruginosa: Reduced Susceptibility Due to Mutations. Pseudomonas aeruginosa chronically infects in the lungs of people with cystic fibrosis and other forms of lung disease. Infections are treated with antibiotics, but over time, the bacteria acquire mutations that reduce their antibiotic susceptibility. The effects of inhibitory amounts of antibiotics in selecting for antibiotic-resistant mutants have been well studied. However, the concentrations of antibiotics that reach infecting bacteria can be sub-inhibitory and but may nonetheless promote emergence of antibiotic-resistant bacteria. Therefore, the aim of this research was to investigate the effects of sub-inhibitory concentrations of antibiotics on the antibiotic susceptibility of P. aeruginosa. Two P. aeruginosa reference strains, PAO1 and PA14, and six isolates from individuals with cystic fibrosis were studied. The bacteria were passaged in the presence of antibiotics (ceftazidime, ciprofloxacin, meropenem or tobramycin) at sub-inhibitory amounts. Fifteen populations of bacteria (up to five per strain) were exposed to each of the four antibiotics. Antibiotic susceptibility was determined following 10 passages on agar supplemented with antibiotic and compared with susceptibility prior to antibiotic exposure. Antibiotic exposure resulted in susceptibility being significantly (>2-fold) reduced for 13 of the 60 populations. Seven samples had reduced susceptibility to ciprofloxacin, three to tobramycin, two to ceftazidime and one to meropenem. Whole-genome sequencing revealed the mutations arising following antibiotic exposure. Mutants with reduced antibiotic susceptibility had mutations in genes known to affect antibiotic resistance, including regulators of efflux pumps (mexR, mexS, mexZ and nalC) and the fusA1 gene that is associated with aminoglycoside resistance. Genes not previously associated with resistance, including gacS, sigX and crfX and two genes with no known function, were also mutated in some isolates with reduced antibiotic susceptibility. Our results show that exposure to sub-inhibitory amounts of antibiotics can select for mutations that reduce the susceptibility of P. aeruginosa to antibiotics and that the profile of mutations is different from that arising during selection with inhibitory antibiotic concentrations. It is likely that exposure to sub-inhibitory amounts of antibiotics during infection contributes to P. aeruginosa becoming antibiotic-resistant.202134987489
6288130.9993Regulation of ofloxacin resistance in Escherichia coli strains causing calf diarrhea by quorum-sensing acyl-homoserine lactone signaling molecules. Escherichia coli is a major pathogen responsible for calf diarrhea. However, it has developed resistance to many antimicrobial drugs for their inappropriate usage. The bacterial quorum sensing system transmits information between bacteria, it's important in regulating bacterial virulence, drug and acid resistance and so on. This system can found in Gram-negative bacteria and operates through acyl-homoserine lactone (AHL) signaling molecules. In this study, a type I quorum sensing AHL, N-Octanoyl-L-Homoserine lactone (C8), was added to E. coli growth medium to investigate its regulatory functions in drug resistance. After screening out the strains of E. coli that showed an obvious regulatory effect to the drug ofloxacin (OFX), transcriptomic sequencing was performed on the E. coli strains from the sub-inhibitory concentration group that concentration plus C8 group, and the control group. It shows that C8 significantly influenced resistance to OFX and the minimum inhibitory concentration of OFX in the tested strain was significantly increased. To Analyze transcriptome sequencing results identified 415 differentially expressed genes between the control and sub-inhibitory concentration groups, of which 201 were up-regulated and 214 were down. There were 125 differentially expressed genes between bacteria treated with a sub-inhibitory concentration of OFX and those treated with C8, of which 102 were up-regulated and 23 were down. Finally, It found that to adding the C8 significantly increased the resistance of tested bacteria to OFX. Data from transcriptome sequencing on differently expressed genes helps to explain how the type I quorum sensing system controls drug resistance in E. coli.202539974163
6289140.9992Pseudomonas aeruginosa is oxygen-deprived during infection in cystic fibrosis lungs, reducing the effectiveness of antibiotics. Pseudomonas aeruginosa infects the lungs of patients with cystic fibrosis. Sputum expectorated from the lungs of patients contains low levels of oxygen, indicating that P. aeruginosa may be oxygen-deprived during infection. During in vitro growth under oxygen-limiting conditions, a P. aeruginosa reference strain increases expression of a cytochrome oxidase with a high affinity for oxygen, and of nitrate and nitrite reductases that enable it to use nitrate instead of oxygen during respiration. Here, we quantified transcription of the genes encoding these three enzymes in sputum samples from 18 infected patients, and in bacteria isolated from the sputum samples and grown in aerobic and anaerobic culture. In culture, expression of all three genes was increased by averages of 20- to 500-fold in anaerobically grown bacteria compared with those grown aerobically, although expression levels varied greatly between isolates. Expression of the same genes in sputum was similar to that of the corresponding bacteria in anaerobic culture. The isolated bacteria were less susceptible to tobramycin and ciprofloxacin, two widely used anti-pseudomonal antibiotics, when grown anaerobically than when grown aerobically. Our findings show that P. aeruginosa experiences oxygen starvation during infection in cystic fibrosis, reducing the effectiveness of antibiotic treatment.202337516450
6292150.9992Genome-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
6226160.9992Chlorhexidine Promotes Psl Expression in Pseudomonas aeruginosa That Enhances Cell Aggregation with Preserved Pathogenicity Demonstrates an Adaptation against Antiseptic. Because Pseudomonas aeruginosa is frequently in contact with Chlorhexidine (a regular antiseptic), bacterial adaptations are possible. In comparison with the parent strain, the Chlorhexidine-adapted strain formed smaller colonies with metabolic downregulation (proteomic analysis) with the cross-resistance against colistin (an antibiotic for several antibiotic-resistant bacteria), partly through the modification of L-Ara4N in the lipopolysaccharide at the outer membrane. Chlorhexidine-adapted strain formed dense liquid-solid interface biofilms with enhanced cell aggregation partly due to the Chlorhexidine-induced overexpression of psl (exopolysaccharide-encoded gene) through the LadS/GacSA pathway (c-di-GMP-independence) in 12 h biofilms and maintained the aggregation with SiaD-mediated c-di-GMP dependence in 24 h biofilms as evaluated by polymerase chain reaction (PCR). The addition of Ca(2+) in the Chlorhexidine-adapted strain facilitated several Psl-associated genes, indicating an impact of Ca(2+) in Psl production. The activation by Chlorhexidine-treated sessile bacteria demonstrated a lower expression of IL-6 and IL-8 on fibroblasts and macrophages than the activation by the parent strain, indicating the less inflammatory reactions from Chlorhexidine-exposed bacteria. However, the 14-day severity of the wounds in mouse caused by Chlorhexidine-treated bacteria versus the parent strain was similar, as indicated by wound diameters and bacterial burdens. In conclusion, Chlorhexidine induced psl over-expression and colistin cross-resistance that might be clinically important.202235955437
4702170.9992Increased antimicrobial resistance of acid-adapted pathogenic Escherichia coli, and transcriptomic analysis of polymyxin-resistant strain. This study investigated the acid adaptation and antimicrobial resistance of seven pathogenic Escherichia coli strains and one commensal strain under nutrient-rich acidic conditions. After acid adaptation, three pathogenic E. coli survived during 100 h incubation in tryptic soy broth at pH 3.25. Acid-adapted (AA) strains showed increased resistance to antimicrobials including ampicillin, ciprofloxacin and especially polymyxins (colistin and polymyxin B), the last resort antimicrobial for multidrug-resistant Gram-negative bacteria. Enterotoxigenic E. coli strain (NCCP 13717) showed significantly increased resistance to acids and polymyxins. Transcriptome analysis of the AA NCCP 13717 revealed upregulation of genes related to the acid fitness island and the arn operon, which reduces lipopolysaccharide binding affinity at the polymyxin site of action. Genes such as eptA, tolC, and ompCF were also upregulated to alter the structure of the cell membrane, reducing the outer membrane permeability compared to the control, which is likely to be another mechanism for polymyxin resistance. This study highlights the emergence of antimicrobial resistance in AA pathogenic E. coli strains, particularly polymyxin resistance, and the mechanisms behind the increased antimicrobial resistance, providing important insights for the development of risk management strategies to effectively control the antimicrobial resistant foodborne pathogens.202439307200
6286180.9992The mRNA expression of ompF, invA and invE was associated with the ciprofloxacin-resistance in Salmonella. Salmonella developed drug-resistance under durative antibiotic pressures pressure. The widespread prevalence of Salmonella has been associated with not only drug-resistance but also pathogenicity. Outer membrane porin proteins (OMPs) are critical for the drug resistance of bacteria. Virulence genes in Salmonella pathogenicity islands (SPIs) play key roles in the virulence of bacteria. In this study, we analyzed the expression levels of three critical genes in ciprofloxacin-resistant strains and ciprofloxacin-susceptible strains of Salmonella, including outer membrane porin protein F (ompF), virulence genes invA and invE. In the clinical ciprofloxacin-resistant strains of Salmonella, the expression level of ompF was decreased. Meanwhile, the expression levels of invA and invE were decreased except for only one strain, indicating generally decreased virulence. These results were also verified with ciprofloxacin-induced resistant strains. Thus, it was informative for understanding the drug-resistance in Salmonella. Monitoring drug-resistance and virulence relevant genes would be significant in the prevention and control of salmonellosis.202032535789
6249190.9992Genome-Wide Identification of Antimicrobial Intrinsic Resistance Determinants in Staphylococcus aureus. The emergence of antimicrobial resistance severely threatens our ability to treat bacterial infections. While acquired resistance has received considerable attention, relatively little is known of intrinsic resistance that allows bacteria to naturally withstand antimicrobials. Gene products that confer intrinsic resistance to antimicrobial agents may be explored for alternative antimicrobial therapies, by potentiating the efficacy of existing antimicrobials. In this study, we identified the intrinsic resistome to a broad spectrum of antimicrobials in the human pathogen, Staphylococcus aureus. We screened the Nebraska Transposon Mutant Library of 1920 single-gene inactivations in S. aureus strain JE2, for increased susceptibility to the anti-staphylococcal antimicrobials (ciprofloxacin, oxacillin, linezolid, fosfomycin, daptomycin, mupirocin, vancomycin, and gentamicin). Sixty-eight mutants were confirmed by E-test to display at least twofold increased susceptibility to one or more antimicrobial agents. The majority of the identified genes have not previously been associated with antimicrobial susceptibility in S. aureus. For example, inactivation of genes encoding for subunits of the ATP synthase, atpA, atpB, atpG and atpH, reduced the minimum inhibitory concentration (MIC) of gentamicin 16-fold. To elucidate the potential of the screen, we examined treatment efficacy in the Galleria mellonella infection model. Gentamicin efficacy was significantly improved, when treating larvae infected with the atpA mutant compared to wild type cells with gentamicin at a clinically relevant concentration. Our results demonstrate that many gene products contribute to the intrinsic antimicrobial resistance of S. aureus. Knowledge of these intrinsic resistance determinants provides alternative targets for compounds that may potentiate the efficacy of existing antimicrobial agents against this important pathogen.201628066345