Antimicrobial resistance, biofilm formation, and molecular detection of efflux pump and biofilm genes among Klebsiella pneumoniae clinical isolates from Northern Jordan.

The current study aimed to investigate the antimicrobial susceptibility profiles, biofilm production capabilities, and the prevalence of efflux pump and biofilm-associated genes among Klebsiella pneumoniae clinical isolates. One hundred sixty-seven K. pneumoniae isolates were collected from microbiology laboratories in Northern Jordan hospitals. Antimicrobial susceptibility was tested using the Kirby-Bauer method. The double-disk synergy test was used to detect the extended-spectrum beta-lactamase (ESBL) phenotype. PCR was used to detect the frequency of acrAB, tolC, and mdtk efflux pump genes and fimH-1, mrkA, and mrkD biofilm-associated genes among the isolates. The highest nonsusceptibility was observed against azithromycin (87.4 %) and nitrofurantoin (85.0 %). Among the isolates, 75.4 % and 92.2 % were multidrug resistant and produced biofilms, respectively. Efflux pump genes acrAB, tolC, and mdtK were found in 96.4 %, 95.2 %, and 90.4 % of the isolates, respectively. Biofilm-associated genes mrkD, mrkA, and fimH-1 were found in 92.2 %, 89.2 %, and 88.6 % of the isolates, respectively. The presence of the mrkA was significantly associated with biofilm formation. Overall, high percentages of multi-drug resistance, efflux pump, and biofilm-associated genes were observed among the isolates. Subsequent studies are recommended to monitor changes in the prevalence of resistance phenotypes and genotypes of isolates.

Metabolic modelling identifies mitochondrial Pi uptake and pyruvate efflux as key aspects of daytime metabolism and proton homeostasis in crassulacean acid metabolism leaves.

Crassulacean acid metabolism (CAM) leaves are characterized by nocturnal acidification and diurnal deacidification processes related with the timed actions of phosphoenolpyruvate carboxylase and Rubisco, respectively. How CAM leaves manage cytosolic proton homeostasis, particularly when facing massive diurnal proton effluxes from the vacuole, remains unclear. A 12-phase flux balance analysis (FBA) model was constructed for a mature malic enzyme-type CAM mesophyll cell in order to predict diel kinetics of intracellular proton fluxes. The charge- and proton-balanced FBA model identified the mitochondrial phosphate carrier (PiC, Pi/H+ symport), which provides Pi to the matrix to sustain ATP biosynthesis, as a major consumer of cytosolic protons during daytime (> 50%). The delivery of Pi to the mitochondrion, co-transported with protons, is required for oxidative phosphorylation and allows sufficient ATP to be synthesized to meet the high energy demand during CAM Phase III. Additionally, the model predicts that mitochondrial pyruvate originating from decarboxylation of malate is exclusively exported to the cytosol, probably via a pyruvate channel mechanism, to fuel gluconeogenesis. In this biochemical cycle, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acts as another important cytosolic proton consumer. Overall, our findings emphasize the importance of mitochondria in CAM and uncover a hitherto unappreciated role in metabolic proton homeostasis.

The vacuolar sulfate transporter PsSULTR4 is a key determinant of seed yield and protein composition in pea.

Pea is a grain legume crop with a high potential to accelerate the food transition due to its high seed protein content and relatively well-balanced amino acid composition. The critical role of external sulfur (S) supply in determining seed yield and seed quality in pea makes it essential to understand the impact of whole plant S management on the trade-off between these two traits. Here, we investigated the physiological relevance of vacuolar sulfate remobilization by targeting PsSULTR4, the only pea sulfate transporter showing substantial similarity to the vacuolar sulfate exporter AtSULTR4;1. Five mutations in PsSULTR4 were identified by TILLING (Targeting Induced Local Lesions IN Genomes), two of which, a loss of function (W78*) and a missense (E568K), significantly decreased seed yield under S deprivation. We demonstrate that PsSULTR4 triggers S distribution from source tissues, especially lower leaves, to reproductive organs to maintain seed yield under S deficiency. Under sufficient S supply, sultr4 seeds display lower levels of the S-rich storage protein PA1 at maturity. They also overaccumulate sulfate in the endosperm at the onset of seed filling. These findings uncover a role of PsSULTR4 in the remobilization of vacuolar sulfate during embryo development, allowing the efficient synthesis of S-rich proteins. Our study uncovers that PsSULTR4 functions (i) in source tissues to remobilize stored vacuolar sulfate for seed production under low S availability and (ii) in developing seeds well supplied with S to fine-tune sulfate remobilization from the endosperm as a critical control point for storage activities in the embryo.

Application of transporter assays for drug discovery and development: an update of the literature.

INTRODUCTION: Determining whether a new drug is a substrate, inhibitor or inducer of efflux or uptake membrane transporters has become a routine process during drug discovery and development. In vitro assays are utilized to establish whether a new drug has the potential to be an object (substrate) or precipitant (inhibitor, inducer) in transporter-mediated clinical drug-drug interactions. The findings from these in vitro experiments are then used to determine whether further in vivo drug interaction studies are necessary for a new drug. AREAS COVERED: This article provides an update on in vitro transporter assays, focusing on new uses of transfected cells, time-dependent inhibition, transporter induction, and complex model systems. EXPERT OPINION: The newer in vitro assays add to the toolbox in defining new drugs as transporter substrates, inhibitors, or inducers. Complex models such as spheroids, organoids, and microphysiological systems require standardization and further research with model transporter substrates and inhibitors. In drug discovery, the more traditional transporter assays may be employed as substrate and inhibitor screening assays. In drug development, more complex cell models can be employed in later drug development to better understand how transporter(s) are involved in the absorption, distribution, and excretion of new drugs.

Anti-microbial, anti-biofilm, and efflux pump inhibitory effects of ellagic acid-bonded magnetic nanoparticles against Escherichia coli isolates.

The spread of microbial resistance is a threat to public health. In this study, the anti-microbial, anti-biofilm, and efflux pump inhibitory effects of ellagic acid-loaded magnetic nanoparticles (Fe3O4NPs@EA) against beta-lactamase producing Escherichia coli isolates have been investigated. The effects of Fe3O4 NPs@EA on the growth inhibition of E. coli isolates were determined by disc diffusion method and determining the minimum inhibitory concentration was done using broth micro-dilution method. The anti-biofilm effect of nanoparticles was investigated using the microplate method. The efflux pump inhibitory effect of nanoparticles was investigated using cart-wheel method and by investigating the effect of nanoparticles on acrB and tolC genes expression levels. Fe3O4 NPs@EA showed anti-bacterial effects against test bacteria, and the MIC of these nanoparticles varied from 0.19 to 1.56 mg/mL. These nanoparticles caused a 43-62% reduction in biofilm formation of test bacteria compared to control. Furthermore, efflux pump inhibitory effect of these nanoparticles was confirmed at a concentration of 1/8 MIC, and the expression of acrB and tolC genes decreased in bacteria treated with 1/4 MIC Fe3O4 NPs@EA. According to the results, the use of nanoparticles containing ellagic acid can provide a basis for the development of new treatments against drug-resistant E. coli. This substance may improve the concentration of antibiotics in the bacterial cell and increase their effectiveness by inhibiting the efflux in E. coli isolates.

Potency and specificity of amiloride and its analogues on branchial sodium fluxes in freshwater trout and goldfish.

There is a consensus that electroneutral Na+/H+ exchangers (NHEs) are important in branchial Na+ uptake in freshwater fish. There is also widespread belief, based on mammalian data, that EIPA [5-(N-ethyl-N-isopropyl)-amiloride]], and HMA [5-(N,N-hexamethylene)-amiloride)] are more potent and specific in blocking Na+ uptake than amiloride. We evaluated this idea by testing the three drugs at 10-7 to 10-4 M, i.e. 0.1 to 100 µM in two model species, rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus), using 22Na+ to measure unidirectional Na+ influx and efflux rates. In both species, the potency order for inhibiting unidirectional Na+ influx was HMA > amiloride > EIPA (IC50 values in the 10-70 µM range), very different from in mammals. At 100 µM, all three drugs inhibited Na+ influx by >90% in both species, except for amiloride in goldfish (65%). However, at 60-100 µM, all three drugs also stimulated unidirectional Na+ efflux rates, indicating non-specific effects. In trout, HMA and EIPA caused significant increases (2.1- to 2.3-fold) in efflux rates, whereas in goldfish, significant efflux elevations were greater (3.1- to 7.2-fold) with all three drugs. We conclude that the inhibitory potency profile established in mammals does not apply to the NHEs in fish gills, that non-specific effects on Na+ efflux rates are a serious concern, and that EIPA and HMA offer no clear benefits in terms of potency or specificity. Considering its much lower cost, we recommend amiloride as the drug of choice for in vivo experiments on freshwater fishes.

Bioflavonoid Baicalein Modulates Tetracycline Resistance by Inhibiting Efflux Pump in Staphylococcus aureus.

The rise in antibiotic resistance among bacterial pathogens, particularly Staphylococcus aureus, has become a critical global health issue, necessitating the search for novel antimicrobial agents. S. aureus uses various mechanisms to resist antibiotics, including the activation of efflux pumps, biofilm formation, and enzymatic modification of drugs. This study explores the potential of baicalein, a bioflavonoid from Scutellaria baicalensis, in modulating tetracycline resistance in S. aureus by inhibiting efflux pumps. The synergistic action of baicalein and tetracycline was evaluated through various assays. The minimum inhibitory concentration (MIC) of baicalein and tetracycline against S. aureus was 256 and 1.0 µg/mL, respectively. Baicalein at 64 µg/mL reduced the MIC of tetracycline by eightfold, indicating a synergistic effect (fractional inhibitory concentration index: 0.375). Time-kill kinetics demonstrated a 1.0 log CFU/mL reduction in bacterial count after 24 hours with the combination treatment. The ethidium bromide accumulation assay showed that baicalein mediated significant inhibition of efflux pumps, with a dose-dependent increase in fluorescence. In addition, baicalein inhibited DNA synthesis by 73% alone and 92% in combination with tetracycline. It also markedly reduced biofilm formation and the invasiveness of S. aureus into HeLa cells by 52% at 64 µg/mL. These findings suggest that baicalein enhances tetracycline efficacy and could be a promising adjunct therapy to combat multidrug-resistant S. aureus infections.

From Proteome to Potential Drugs: Integration of Subtractive Proteomics and Ensemble Docking for Drug Repurposing against Pseudomonas aeruginosa RND Superfamily Proteins.

Pseudomonas aeruginosa (P. aeruginosa) poses a significant threat as a nosocomial pathogen due to its robust resistance mechanisms and virulence factors. This study integrates subtractive proteomics and ensemble docking to identify and characterize essential proteins in P. aeruginosa, aiming to discover therapeutic targets and repurpose commercial existing drugs. Using subtractive proteomics, we refined the dataset to discard redundant proteins and minimize potential cross-interactions with human proteins and the microbiome proteins. We identified 12 key proteins, including a histidine kinase and members of the RND efflux pump family, known for their roles in antibiotic resistance, virulence, and antigenicity. Predictive modeling of the three-dimensional structures of these RND proteins and subsequent molecular ensemble-docking simulations led to the identification of MK-3207, R-428, and Suramin as promising inhibitor candidates. These compounds demonstrated high binding affinities and effective inhibition across multiple metrics. Further refinement using non-covalent interaction index methods provided deeper insights into the electronic effects in protein-ligand interactions, with Suramin exhibiting superior binding energies, suggesting its broad-spectrum inhibitory potential. Our findings confirm the critical role of RND efflux pumps in antibiotic resistance and suggest that MK-3207, R-428, and Suramin could be effectively repurposed to target these proteins. This approach highlights the potential of drug repurposing as a viable strategy to combat P. aeruginosa infections.

Intestinal absorption mechanism and nutritional synergy promotion strategy of dietary flavonoids: transintestinal epithelial pathway mediated by intestinal transport proteins.

Dietary flavonoids exhibit a variety of physiological functions in regulating glucose and lipid metabolism, improving cardiovascular function, and enhancing stress resistance. However, poor intestinal absorption limits their health benefits. Previous studies on improving the absorption efficiency of flavonoids have focused on targeted release, enhanced gastrointestinal stability and prolonged retention time in digestive tract. But less attention has been paid to promoting the uptake and transport of flavonoids by intestinal epithelial cells through modulation of transporter protein-mediated pathways. Interestingly, some dietary nutrients have been found to modulate the expression or function of transporter proteins, thereby synergistically or antagonistically affecting flavonoid absorption. Therefore, this paper proposed an innovative regulatory strategy known as the "intestinal transport protein-mediated pathway" to promote intestinal absorption of dietary flavonoids. The flavonoid absorption mechanism in the intestinal epithelium, mediated by intestinal transport proteins, was summarized. The functional differences between the uptake transporter and efflux transporters during flavonoid trans-intestinal cellular transport were discussed. Finally, from the perspective of nutritional synergy promotion of absorption, the feasibility of promoting flavonoid intestinal absorption by regulating the expression/function of transport proteins through dietary nutrients was emphasized. This review provides a new perspective and developing precise dietary nutrient combinations for efficient dietary flavonoid absorption.

Crotamine derived from Crotalus durissus terrificus venom combined with drugs increases in vitro antibacterial and antifungal activities.

This study investigated crotamine (CTA), a peptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, known for its exceptional cell penetration potential. The objective was to explore the antibacterial and antifungal activity of CTA, its ability to inhibit efflux pumps and evaluate the effectiveness of its pharmacological combination with antibiotics and antifungals. In microbiological assays, CTA in combination with antibiotics was tested against strains of S. aureus and the inhibition of NorA, Tet(K) and MepA efflux pumps was also evaluated. CTA alone did not present clinically relevant direct antibacterial action, presenting MIC > 209.7 µM against strains S. aureus 1199B, IS-58, K2068. The standard efflux pump inhibitor CCCP showed significant effects in all negative relationships to assay reproducibility. Against the S. aureus 1199B strain, CTA (20.5 µM) associated with norfloxacin diluted 10 × (320.67 µM) showed a potentiating effect, in relation to the control. Against the S. aureus IS-58 strain, the CTA associated with tetracycline did not show a significant combinatorial effect, either with 2304 or 230.4 µM tetracycline. CTA at a concentration of 2.05 µM associated with ciprofloxacin at a concentration of 309.4 µM showed a significant potentiating effect. In association with EtBr, CTA at concentrations of 2.05 and 20.5 µM potentiated the effect in all strains tested, reducing the prevention of NorA, Tet(K) and MepA efflux pumps. In the C. albicans strain, a potentiating effect of fluconazole (334.3 µM) was observed when combined with CTA (2.05 µM). Against the C. tropicalis strain, a significant effect was also observed in the association of fluconazole 334.3 µM, where CTA 2.05 µM considerably reduced fungal growth and decreased the potentiation of fluconazole. Against the C. krusei strain, no significant potentiating effect of fluconazole was obtained by CTA. Our results indicate that CTA in pharmacological combination potentiates the effects of antibiotics and antifungal. This represents a new and promising antimicrobial strategy for treating a wide variety of infections.

Emerging and alternative strategies for the treatment of nontuberculous mycobacterial infections.

INTRODUCTION: Nontuberculous mycobacteria (NTM) infections have emerged as a significant clinical challenge due to their intrinsic multidrug resistance and the limited efficacy of existing treatments. These infections are becoming increasingly prevalent, with a need for new and effective therapeutic strategies. AREAS COVERED: This review addresses several key aspects of NTM infections: i) pathogenesis and epidemiology; ii) the limitations and challenges of current treatment options; iii) emerging and alternative therapeutic strategies; iv) advanced drug delivery systems such as nanoparticles and efflux pump inhibitors; v) innovative antibacterial alternatives like antimicrobial peptides, bacteriophage therapy, and phytochemicals; and vi) other potential treatment modalities such as inhaled nitric oxide, small molecules, surgical debridement, phototherapy, and immunomodulatory therapy. EXPERT OPINION: Personalized medicine, advanced drug delivery systems, and alternative therapies hold promise for the future of NTM treatment. Early and accurate identification of NTM species, enabled by improved diagnostic methods, is critical for tailoring treatment regimens. Emerging therapies show promise against drug-resistant NTM strains, but overcoming barriers like clinical trials, regulatory hurdles, and high production costs is crucial. Continued research and innovation are essential to improve treatment efficacy and patient outcomes.

Thiazine-derived compounds in inhibiting efflux pump in Staphylococcus aureus K2068, mepA gene expression, and membrane permeability alteration.

Staphylococcus aureus is a bacterium responsible for resistance to multiple drugs and the efflux system is widely studied among the resistance mechanisms developed by this species. The present study evaluates the inhibition of the MepA efflux pump by thiadiazine-derived compounds. For this purpose, thiadiazine-derived compounds (IJ-14 to IJ-20) were tested against S. aureus K2068 strains. Microdilution tests were initially conducted to assess the Minimum Inhibitory Concentration (MIC) of the compounds and their efflux pump inhibition activity. In addition, fluorimetry tests were performed using BrEt emission and tests were conducted to inhibit the expression of the mepA gene. This involved comparing the bacterial gene expression with the antibiotic alone to the gene expression after combining compounds (IJ-17 and IJ-20) with the antibiotic. Furthermore, membrane permeability assessment tests and in silico molecular docking tests were performed. It was observed that the IJ17 and IJ20 compounds exhibited direct activity against the tested strain. The IJ17 compound produced significant results in the gene inhibition tests, which was also evidenced through the membrane permeability alteration test. These findings suggest that thiadiazine-derived compounds have promising effects against one of the main resistance mechanisms, with the IJ17 compound presenting observable mechanisms of action.

Amine-substituted heterocyclic thioamide Cu(I) and Ag(I) complexes as effective anticancer and antibacterial agents targeting the periplasm of E. coli bacteria.

Metal complexes showing dual activity against cancer and bacterial infections are currently the focus of significant interest for their potential in treating life-threatening diseases. Aiming to investigate the impact of ligand substituents on these bioactivity properties of Group 11 d10 metal complexes, we herein present a series of mononuclear Cu(I) and Ag(I) complexes featuring the bis-NH2-substituted heterocyclic thioamide dap2SH (=4,6-diaminopyrimidine-2-thione), namely [AgCl(dap2SH)(PPh3)2] (1), [CuBr(dap2SH)(PPh3)2] (2), [CuBr(dap2SH)(xantphos)] (3), [Ag(dap2S)(xantphos)] (4), and [Cu(dap2S)(xantphos)] (5) (xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene). Complexes were characterized by means of different physicochemical methods (i.e., single crystal X-ray diffraction as well as FTIR, NMR, UV-Vis and fluorescence spectroscopy), and studied in-vitro for their antibacterial and anticancer activity against a variety of bacterial strains and cancer cell lines. Complexes 1-3 effectively inhibited both Gram (+) and Gram (-) bacterial growth, while cellular uptake studies for the most potent complex 1 against E. coli bacteria revealed the accumulation of Ag(I) ions in the periplasm of the bacteria. A high anti-proliferative effect was observed for 1 and 5 against A549, MCF7 and PC3 cancer cell lines, with 1 being capable of inducing apoptosis in A549 cells, as suggested by flow cytometry analysis. DNA interaction studies revealed the capacity of 1 to intercalate between base-pairs of CT DNA. All complexes had a moderate-to-high capacity to scavenge free radicals preventing oxidative stress. Molecular docking calculations, in combination with the experimentally obtained data, provided insights for potential mechanisms of the bioactivity of the complexes.

Novel Approaches for the Enhancement of Bioavailability of Drugs: An Updated Review.

In medicine, bioavailability is the percentage of a drug that enters the bloodstream and can be used to treat a patient. It has proven challenging throughout time to develop techniques that allow oral administration of most drugs, regardless of their properties, to achieve therapeutic systemic availability. This will be an impressive feat, considering that over 90% of pharmaceuticals are known to have limitations on their oral bioavailability. Improving bioavailability is crucial for optimizing the efficacy and safety of drugs. This review covers a wide range of techniques, including physical, chemical, and formulation approaches, highlighting their mechanisms, advantages, and limitations. Inhibitions of efflux pumps, inhibition of presystemic metabolism, and innovative drug delivery systems that capitalize on the gastrointestinal regionality of medicines are some of the new techniques that have drawn increased interest. Nanotechnology in pharmaceuticals is also being used in this field. We have collected the literature data from 2009 to 2024 using Science Direct, PubMed/Medline, Scopus, and Google Scholar.

Discovery of clinical isolation of drug-resistant Klebsiella pneumoniae with overexpression of OqxB efflux pump as the decisive drug resistance factor.

Background emergence of multidrug-resistant (MDR) bacterial strains is a public health concern that threatens global and regional security. Efflux pump-overexpressing MDR strains from clinical isolates are the best subjects for studying the mechanisms of MDR caused by bacterial efflux pumps. A Klebsiella pneumoniae strain overexpressing the OqxB-only efflux pump was screened from a clinical strain library to explore reverse OqxB-mediated bacterial resistance strategies. We identified non-repetitive clinical isolated K. pneumoniae strains using a matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry clinical TOF-II (Clin-TOF-II) and susceptibility test screening against levofloxacin and ciprofloxacin. And the polymorphism analysis was conducted using pulsed-field gel electrophoresis. Efflux pump function of resistant strains is obtained by combined drug sensitivity test of phenylalanine-arginine beta-naphthylamide (PaßN, an efflux pump inhibitor) and detection with ethidium bromide as an indicator. The quantitative reverse transcription PCR was performed to assess whether the oqxB gene was overexpressed in K. pneumoniae isolates. Additional analyses assessed whether the oqxB gene was overexpressed in K. pneumoniae isolates and gene knockout and complementation strains were constructed. The binding mode of PaßN with OqxB was determined using molecular docking modeling. Among the clinical quinolone-resistant K. pneumoniae strains, one mediates resistance almost exclusively through the overexpression of the resistance-nodulation-division efflux pump, OqxB. Crystal structure of OqxB has been reported recently by N. Bharatham, P. Bhowmik, M. Aoki, U. Okada et al. (Nat Commun 12:5400, 2021, https://doi.org/10.1038/s41467-021-25679-0). The discovery of this strain will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and builds on the foundation for addressing the threat posed by quinolone resistance.IMPORTANCEThe emergence of antimicrobial resistance is a growing and significant health concern, particularly in the context of K. pneumoniae infections. The upregulation of efflux pump systems is a key factor that contributes to this resistance. Our results indicated that the K. pneumoniae strain GN 172867 exhibited a higher oqxB gene expression compared to the reference strain ATCC 43816. Deletion of oqxB led a decrease in the minimum inhibitory concentration of levofloxacin. Complementation with oqxB rescued antibiotic resistance in the oqxB mutant strain. We demonstrated that the overexpression of the OqxB efflux pump plays an important role in quinolone resistance. The discovery of strain GN 172867 will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and promotes further study of antimicrobial resistance.

Nanocomposite system with photoactive phloxine B eradicates resistant Staphylococcus aureus.

Nanomaterials modified with hybrid films functionalized with photoactive compounds can be an effective system to prevent and eradicate biofilms on medical devices. The aim of this research was to extend current knowledge on nanomaterial comprised of polyurethane (PU) modified with a nanocomposite film of organoclay with the functionalized photosensitizer (PS) phloxine B (PhB). Particles of the clay mineral saponite were, at first modified by octadecyltrimethylammonium cations to activate the surface for PhB adsorption. The colloids were filtered to get silicate films on polytetrafluoroethylene membrane filters, which were layered with a liquid mixture of PU precursors. The penetration of PU into the silicate formed a thin nanocomposite film. This nanomaterial demonstrated excellent effectiveness against methicillin-resistant S. aureus (MRSA) resistant to fluoroquinolones (L12 and S61, respectively). It showed more than 1000- and 10 000-fold inhibition of biofilm growth after irradiation with green laser compared to the unmodified PU material. Principal component analysis and multiple linear regression showed that the effectiveness of the nanomaterial was not influenced by virulence factors such as the expression of efflux pumps of the Nor family, the adhesin PIA encoded by the icaADBC operon or the robustness of the biofilms. However, the presence of organoclay, PhB and irradiation had a significant effect on the anti-biofilm properties of the nanocomposite. The anti-microbial properties of the material were strengthened after irradiation, because of high reactive oxygen species release (more than 14-fold compared to non-irradiated sample). Materials based on photoactive molecules can represent a worthwhile pathway towards the development of more complex nanomaterials applicable in various fields of medicine.

Evaluating the Expression of Efflux Pumps in Pseudomonas aeruginosa in Exposure to Sodium Dodecyl Sulfate, Didecyldimethylammonium Chloride, and Octenidine Dihydrochloride.

Emerging resistance of Gram-negative bacteria, including Pseudomonas aeruginosa, to commonly used detergents and disinfectant is encountering us with hazard. Inappropriate use of disinfectants has forced bacteria to gain resistance. The ability of bacteria to extrude substrates from the cellular interior to the external environment has enabled them to persist in exposure to toxic compounds, which is due to existence of transport proteins. Efflux pumps, in Gram-negative bacteria, are proteins responsible for exporting molecules outside of the cell, by crossing the two membranes. In this study, 40 P. aeruginosa strains from hospitals, clinics, and burn center laundries and 40 P. aeruginosa strains from urban laundries were collected. This study evaluated the minimum inhibitory concentration (MIC) level of sodium dodecyl sulfate (SDS), didecyldimethylammonium chloride (DDAC), and octenidine dihydrochloride (Od) in P. aeruginosa strains. The real-time PCR was carried out to evaluate the expression of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux system. The obtained results indicated a higher MIC level for SDS, DDAC, and Od in medical laundries. The sub-MIC level of DDAC and Od increased the expression level of MexAB-OprM, MexCD-OprJ, and MexXY-OprM in P. aeruginosa strains, suggesting that efflux pumps contribute to disinfectant resistance in P. aeruginosa.

Genotypic and phenotypic characterization of determinants that mediate antimicrobial resistance in Escherichia coli strains of clinical origin in South-Western Nigeria.

Background: Multidrug resistant bacterial pathogens employ different mechanisms in evading the action of antibiotics. Multidrug resistance is wide spread among strains of Escherichia coli implicated in several infections including urinary tract infections, gastrointestinal infections, meningitis and bacteraemia. Aim/Objective: This study investigates the antibiotic resistance profile, efflux pump activity and biofilm formation ability of E. coli strains isolated from clinical samples. Methods: A total of 32 E. coli strains isolated from clinical samples were characterized and subjected to antibiotic susceptibility testing using standard methods. Isolates were screened phenotypically for biofilm formation and efflux pump activity. While molecular detection of genes encoding curli fimbriae and efflux pump activity was done by PCR. Results: All 32 (100%) E. coli isolates were resistant to ceftazidime, cefuroxime, cefixime, amoxicillin-clavulanate, ofloxacin and ciprofloxacin. While 30 (93.8%) were resistant to gentamicin, 27 (84.4%) were resistant to cefepime and the least resistance of 15.6% was to imipenem. Efflux pump encoding gene tolC was detected in 13(40.6%) of the isolates, while 1(3.1%) harboured acrA gene. acrB gene was not detected in any of the isolates. Seven (21.9%) of the isolates were strong biofilm formers, while 5 (15.6%) and 20 (62.5%) were moderate and weak biofilm formers respectively. csgA gene was detected in all E. coli isolates. Discussion: High antibiotic resistance of E. coli strains observed in this study is of public health significance. . It is therefore important to scale up efforts in regular monitoring of antibiotic resistance in both community and hospital settings.

Investigation of the role of Cremophor RH 40 and Cremophor EL in the inhibition of efflux pump of Pseudomonas aeruginosa.

Background: There is increasing emphasis on restoring the efficacy of existing antibiotics instead of developing new ones. Objectives: This study aimed to determine the role of Cremophor EL and Cremophor RH40 in the inhibition of efflux pumps in MDR Pseudomonas aeruginosa strains. Methods: Efflux pump-active MDR strains of P. aeruginosa were identified and confirmed by flow cytometry. The identified efflux-active strains were further subjected to determination of the MIC of ciprofloxacin and the synergistic role of non-ionic surfactants (Cremophor EL and Cremophor RH40) along with ciprofloxacin. Results: Out of 30 samples, 6 strains displayed high efflux pump activity. Both Cremophor EL and Cremophor RH40 showed efflux pump inhibitory roles. A 4-fold reduction in the MIC values of ciprofloxacin was observed when Cremophor EL was used along with ciprofloxacin, while a 6-fold reduction was observed when Cremophor RH40 was used along with ciprofloxacin. Both compounds showed synergistic effects with ciprofloxacin, ticarcillin and meropenem when used in a 24-well plate efflux pump inhibitory assay. Conclusion: The inhibition of the efflux pump of MDR Pseudomonas aeruginosa by non-ionic surfactants, namely, Cremophor RH40 and Cremophor EL, provided the best strategy to restore the efficacy of ciprofloxacin.

Methyl gallate attenuates virulence and decreases antibiotic resistance in extensively drug-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa infections have become a serious threat to public health due to the increasing emergence of extensively antibiotic-resistant strains and high mortality rates. Therefore, the search for new therapeutic alternatives has become crucial. In this study, the antivirulence and antibacterial activity of methyl gallate was evaluated against six clinical isolates of extensively antibiotic-resistant P. aeruginosa. Methyl gallate exhibited minimal inhibitory concentrations of 256 to 384 µg/mL; moreover, the use of subinhibitory concentrations of the compound inhibited biofilm formation, swimming, swarming, proteolytic activity, and pyocyanin production. Methyl gallate plus antipseudomonal antibiotics showed a synergistic effect by reduced the MICs of ceftazidime, gentamicin and meropenem. Furthermore, the potential therapeutic effect of methyl gallate was demonstrated in an infection model. This study evidenced the antivirulence and antimicrobial activity of methyl gallate as a therapeutic alternative against P. aeruginosa.