Efflux Pump Activity Research Articles

Molecular characterization of extended-spectrum beta-lactamases and carbapenemases producing Enterobacteriaceae isolated from North Eastern region of India

Objectives: This study is aimed to investigate the prevalence of genes encoding extended-spectrum β-lactamases (ESBLs) and carbapenemases production among Enterobacteriaceae isolated from North East India. Materials and Methods: A total of 210 non-duplicate multi-drug resistant Enterobacteriaceae (MDRE) strains were included in this investigation. The isolates were resistant to third-generation cephalosporins, aminoglycosides, and fluoroquinolones. First, the strains were subjected to phenotypic assays to determine ESBLs and carbapenemases production; then, multiplex polymerase chain reaction (mPCR) assays were done to detect ESBLs and carbapenemases genes. In addition, efflux pump activity was determined by phenylalanine-arginine b-naphthylamide assay. Statistical Analysis: The frequency of ESBLs and carbapenemase genes among MDRE strains was shown as percentages. The data analysis was done using Microsoft Excel computer software. Results: Among 210 MDRE clinical isolates, ESBLs production was observed in 72.86% (153) isolates. During mPCR assay, gene encoding ESBLs were detected in 55.24% (116) MDRE strains beta-lactamase Temoniera (blaTEM) (26.67%, 56), beta-lactamase Cefotaxime-Munich (blaCTX-M) (19.52%, 41), and beta-lactamase sulfhydryl reagent variable (blaSHV) (9.05%, 19)]. In addition, 55 (26.2%) and 53 (25.26%) strains were found to be meropenem and imipenem resistant, respectively. Carbapenemase nordmann-poirel (Carba-NP) test for carbapenemases activity was found to be positive in 18.58% (39) MDRE strains. The genes encoding carbapenemases production was observed in 18.58% (39) MDRE [beta-lactamase New Delhi metallo-β-lactamases-1(blaNDM-1) (8.10%, 17), beta-lactamase oxacillinase-48 (blaOXA-48) (2.86%, 6), beta-lactamase Verona imipenemase (blaVIM) (1.43%, 3), and blaOXA-48 and blaVIM (6.19%, 13)]. Efflux pump activity was observed in 5 (2.3%) of Carbapenem-resistant Enterobacteriaceae isolates. Conclusions: For the first time in this region, we have detected the presence of blaOXA-48 and blaVIM in a single MDRE isolate as high as 6.1%. Therefore, clinicians need to detect the ESBLs and carbapenemases producing Enterobacteriaceae on priority in hospital settings for therapeutic options as well as stringent infection control strategies to be adopted as precautions.

Nonlethal Molecular Nanomachines Potentiate Antibiotic Activity Against Gram-Negative Bacteria by Increasing Cell Permeability and Attenuating Efflux.

Antibiotic resistance is a pressing public health threat. Despite rising resistance, antibiotic development, especially for Gram-negative bacteria, has stagnated. As the traditional antibiotic research and development pipeline struggles to address this growing concern, alternative solutions become imperative. Synthetic molecular nanomachines (MNMs) are molecular structures that rotate unidirectionally in a controlled manner in response to a stimulus, such as light, resulting in a mechanical action that can propel molecules to drill into cell membranes, causing rapid cell death. Due to their broad destructive capabilities, clinical translation of MNMs remains challenging. Hence, here, we explore the ability of nonlethal visible-light-activated MNMs to potentiate conventional antibiotics against Gram-negative bacteria. Nonlethal MNMs enhanced the antibacterial activity of various classes of conventional antibiotics against Gram-negative bacteria, including those typically effective only against Gram-positive strains, reducing the antibiotic concentration required for bactericidal action. Our study also revealed that MNMs bind to the negatively charged phospholipids of the bacterial inner membrane, leading to permeabilization of the cell envelope and impairment of efflux pump activity following light activation of MNMs. The combined effects of MNMs on membrane permeability and efflux pumps resulted in increased antibiotic accumulation inside the cell, reversing antibiotic resistance and attenuating its development. These results identify nonlethal MNMs as pleiotropic antibiotic enhancers or adjuvants. The combination of MNMs with traditional antibiotics is a promising strategy against multidrug-resistant Gram-negative infections. This approach can reduce the amount of antibiotics needed and slow down antibiotic resistance development, thereby preserving the effectiveness of our current antibiotics.

Unveiling the potential of recently FDA-approved drugs as quorum sensing inhibitors against P. Aeruginosa using high-performance computational techniques

Through cell-to-cell communication, activation of efflux pumps, formation of biofilms, and other mechanisms, pseudomonas aeruginosa’s quorum sensing systems (QSS), notably the lasl/las-r system, contribute a vital role in the development of anti-microbial resistance (AMR). Identifying potential drugs against these targets could have significant implications for combating pseudomonal infections. The current study aims to identify promising recently FDA-approved drugs against lasl/las-r proteins. The ligands were selected from the FDA-approved drug lists of the last 5 years. Out of 202, 78 drugs were checked for interaction with lasl/las-r protein and 4 drugs revealed top binding conformations characterized by favorable energetic profiles within the active site of the las-r protein which were further assigned for 250-ns molecular dynamics (MD) simulation. The MD analysis confirmed the dynamical stability of brexanolone and oteseconazole with las-r protein. The root mean square deviation (RMSD), radius of gyration (Rg) and solvent-accessible surface area (SASA) analysis have indicated less deviation, more compactness of protein and less exposure of protein ligand complex to its surroundings as compared to the reference ligand-protein complex. The hydroxyl group in the oteseconazole whereas hydroxyl and ketone group in the brexanolone were responsible for hydrogen bonds with the active site residue of las r ptotein as indicated by ligand-protein contacts diagram. The binding energies per residue analysis revealed TYR-47 as the most contributing amino acid residue for interaction with oteseconazole and brexanolone. The identified drugs may be potential repurposing candidates against pseudomonal infections through inhibition of las-r protein. Communicated by Ramaswamy H. Sarma

Tryptophan transport gene inactivation promotes the development of antibiotic resistance in Escherichia coli.

Indole serves as a signaling molecule that could regulate different bacterial physiological processes, including antibiotic resistance through biofilm formation and drug efflux pump activity. In Escherichia coli, indole is produced through the tryptophan pathway, which involves three permeases (Mtr, AroP, and TnaB) that can transport the amino acid tryptophan. Although these permeases play distinct roles in the secretion of indole biosynthesis, their impact on multidrug resistance mediated by indole remaines unclear. This study was designed to investigate the connection between the tryptophan transport system and antibiotic resistance by constructing seven gene deletion mutants from E. coli MG1655 (wild type). Our result showed that deletion of the aroP or tnaB gene led to increased antibiotic resistance as evaluated by MICs for different antibiotics. Efflux activity test results revealed that the increased antibiotic resistance was related with the AcrAB-Tolc drug efflux pump in the mutants. The transcriptome analysis further demonstrated that decreased susceptibility to kanamycin and ampicillin in E. coli was accompanied by reduced accumulation of reactive oxygen species and decreased motility. These findings highlight the substantial influence of the tryptophan transport system on antibiotic resistance in E. coli, which is crucial for developing strategies against antibiotic resistance in bacterial infections.

Transcriptome reveals the role of the htpG gene in mediating antibiotic resistance through cell envelope modulation in Vibrio mimicus SCCF01.

HtpG, a bacterial homolog of the eukaryotic 90 kDa heat-shock protein (Hsp90), represents the simplest member of the heat shock protein family. While the significance of Hsp90 in fungal and cancer drug resistance has been confirmed, the role of HtpG in bacterial antibiotic resistance remains largely unexplored. This research aims to investigate the impact of the htpG gene on antibiotic resistance in Vibrio mimicus. Through the creation of htpG gene deletion and complementation strains, we have uncovered the essential role of htpG in regulating the structural integrity of the bacterial cell envelope. Our transcriptomics analysis demonstrates that the deletion of htpG increases the sensitivity of V. mimicus to antimicrobial peptides, primarily due to upregulated lipopolysaccharide synthesis, reduced glycerophospholipid content, and weakened efflux pumps activity. Conversely, reduced sensitivity to β-lactam antibiotics in the ΔhtpG strain results from decreased peptidoglycan synthesis and dysregulated peptidoglycan recycling and regulation. Further exploration of specific pathway components is essential for a comprehensive understanding of htpG-mediated resistance mechanisms, aiding in the development of antimicrobial agents. To our knowledge, this is the first effort to explore the relationship between htpG and drug resistance in bacteria.

Bacterial efflux pumps excrete SYTO™ dyes and lead to false-negative staining results.

Multidrug efflux pumps excrete a range of small molecules from bacterial cells. In this study, we show that bacterial efflux pumps have affinity for a range of SYTO™ dyes that are commonly used to label bacteria. Efflux pump activity will there lead to false negative results from bacterial staining and SYTO™ dyes should be used with caution on live samples.

Naturally derived efflux pump inhibitor among tetracycline-resistant Acinetobacter baumannii isolates

Abstract Background: The antimicrobial resistance of Acinetobacter baumannii is of major concern. It is one of the commonly distributed nosocomial pathogens, and its strains are frequently reported to demonstrate resistance to the most routinely prescribed antibiotics in varying degrees. Objective: This study aimed to detect the inhibitory effect of chamomile extract against the efflux pump of A. baumannii. Materials and Methods: Clinical samples (125) were collected from different sites (urine, sputum, wound, and burn swabs), followed by A. baumannii isolation and identification, and detection of tetracycline susceptibility test by disk-diffusion. Later on bacterial efflux pump activity was detected by ethidium bromide cartwheel assay and water extract of chamomile was used as an inhibitor of efflux pump A. baumannii. Results: The percentage of A. baumannii isolation 23/125 (18.4%) that distributed as 25% isolates were obtained from sputum, 17.9% isolates from burns, whereas 16% and 12.5% isolates from urine and wounds respectively. It showed various resistances against different members of the three generations of tetracyclines, with the highest resistance against minocycline 47.8% followed by resistance to tetracycline and doxycycline as 43.5% and 39%, respectively. Efflux pump was detected in all cases, water extract of chamomile showed a significant inhibitory effect especially at 80 mg/mL concentration. Conclusion: Efflux pump is an important mechanism for tetracycline resistance among A. baumannii isolates, and the watery extract of chamomile can be considered a prompt inhibitor for this pump.

Prevalence of Bacteriuria, and Characterization of Their Antimicrobial Sensitivity Pattern and Efflux Pumps Activity in Diabetic and Non-diabetic Uti Patients

Rationale: Urinary tract infections (UTI) occur more frequently in patients with type 2 diabetes. In developing countries, impairments in the defense system, metabolic disorder during diabetes, and incomplete bladder emptying all contribute to the pathogenesis of UTI in diabetic patients. This comparative study is undertaken to reveal the prevalence of uropathogens and their antimicrobial resistance patterns, and to assess hematological biomarker in diabetic and non-diabetic UTI patients. Methods: The prevalence of uropathogens were tested in 40 diabetic and 40 non-diabetic UTI patients. The antimicrobial susceptibility pattern of bacterial isolates were assessed by the Kirby-Buer disc-diffusion method. Moreover, the bacterial load, Gram-staining, biochemical and serological assays were performed using appropriate methods. Results: The findings showed that diabetic patients are more likely to UTI compared to non-diabetic patients. In terms of uropathogenic prevalence, 61% Escherichia coli, 34% Klebsiella pneumoniae, 2% Staphylococcus aureus, 2% Pseudomonas aureginosa and 1% others. The antimicrobial resistance profile demonstrated that most of the bacterial isolates were resistant to amoxicillin, ampicillin, erythromycin and methicillin but susceptible to azithromycin, chloramphenicol and ciprofloxacin. Moreover, the drug efflux pumps activity and bacterial burden were significantly high among antimicrobial resistant bacterial isolates. Besides, the C-reactive protein, Erythrocyte sedimentation rate, glycosylated hemoglobin A1C, neutrophils and lymphocytes were significantly high in diabetic UTI patients. Conclusion: This study found E. coli was the most frequent bacteria isolates, and this observation is consistent others. Diabetic UTI patients are more vulnerable with high bacterial burden and systemic inflammation. Bioresearch Commu. 10(1): 1413-1419, 2024 (January)

Type I Photosensitizer Targeting Glycans: Overcoming Biofilm Resistance by Inhibiting the Two-Component System, Quorum Sensing, and Multidrug Efflux.

Stubborn biofilm infections pose serious threats to human health due to the persistence, recurrence, and dramatically magnified antibiotic resistance. Photodynamic therapy has emerged as a promising approach to combat biofilm. Nevertheless, how to inhibit the bacterial signal transduction system and the efflux pump to conquer biofilm recurrence and resistance remains a challenging and unaddressed issue. Herein, a boric acid-functionalized lipophilic cationic type I photosensitizer, ACR-DMP, is developed, which efficiently generates •OH to overcome the hypoxic microenvironment and photodynamically eradicates methicillin-resistant Staphylococcus aureus (MRSA) and biofilms. Furthermore, it not only alters membrane potential homeostasis and osmotic pressure balance due to its strong binding ability with plasma membrane but also inhibits quorum sensing and the two-component system, reduces virulence factors, and regulates the activity of the drug efflux pump attributed to the glycan-targeting ability, helping to prevent biofilm recurrence and conquer biofilm resistance. In vivo, ACR-DMP successfully obliterates MRSA biofilms attached to implanted medical catheters, alleviates inflammation, and promotes vascularization, thereby combating infections and accelerating wound healing. This work not only provides an efficient strategy to combat stubborn biofilm infections and bacterial multidrug resistance but also offers systematic guidance for the rational design of next-generation advanced antimicrobial materials.

Assessing the Effect of Oxytetracycline on the Selection of Resistant Escherichia coli in Treated and Untreated Broiler Chickens.

Oxytetracycline (OTC) is administered in the poultry industry for the treatment of digestive and respiratory diseases. The use of OTC may contribute to the selection of resistant bacteria in the gastrointestinal tract of birds or in the environment. To determine the effect of OTC on the selection of resistant Escherichia coli strains post-treatment, bacteria were isolated from droppings and litter sampled from untreated and treated birds. Bacterial susceptibility to tetracyclines was determined by the Kirby-Bauer test. A total of 187 resistant isolates were analyzed for the presence of tet(A), (B), (C), (D), (E), and (M) genes by PCR. Fifty-four strains were analyzed by PFGE for subtyping. The proportion of tetracycline-resistant E. coli strains isolated was 42.88%. The susceptibility of the strains was treatment-dependent. A high clonal diversity was observed, with the tet(A) gene being the most prevalent, followed by tet(C). Even at therapeutic doses, there is selection pressure on resistant E. coli strains. The most prevalent resistance genes were tet(A) and tet(C), which could suggest that one of the main mechanisms of resistance of E. coli to tetracyclines is through active efflux pumps.

Isolation and identification of antimicrobial susceptibility, biofilm formation, efflux pump activity, and virulence determinants in multi-drug resistant Pseudomonas aeruginosa isolated from freshwater fishes.

The present study was undertaken to evaluate the prevalence, underlying resistance mechanism, and virulence involved in Pseudomonas aeruginosa (n = 35) isolated from freshwater fishes in Andhra Pradesh, India. Antibiogram studies revealed that 68.5, 62.8, 37.1, 11.4, 8.5, 57.1, 54.2, and 48.5% of isolates had resistance to oxytetracycline, co-trimoxazole, doxycycline, enrofloxacin, ciprofloxacin, cefotaxime, ceftazidime, and ampicillin, respectively. The resistant isolates harboured the tetA (85.7%), tetD (71.4%), tetM (91.4%), sul1 (80%), blaCTX-M (57.1%), blaTEM (42.8%), and blaSHV (48.5%) genes. In total, 50% of the isolates were altered as multi-drug resistant, and the multiple antibiotic resistance index was calculated as 0.4. Furthermore, 37.3, 48.5, and 14.2% of isolates were categorized as strong, moderate, and weak biofilm formers, possessing pslA (91.5%) and pslD (88.6%) biofilm encoding genes. In total, 82.8% of the isolates exhibited efflux pump activity and harboured the mexA (74.2%), mexB (77.1%), and oprM (37.1%) genes. Virulent genes oprL, toxA, exoS, and phzM were detected in 68.5, 68.5, 100, and 17.1% of isolates, respectively. The data suggested that P. aeruginosa harbours multiple resistance mechanisms and virulence factors that may contribute to antibiotic resistance and pathogenicity, and their distribution in fish culture facilities highlights the public health hazards of the food chain.

Molecular Characterization of Hetero-Pathogenic and Diarrheagenic Escherichia coli Pathotypes in Diarrheic Children under Five Years and Exposure Environment in Ogun State, South-West Nigeria.

Diarrheagenic Escherichia coli (DEC) is one of the most common etiological agents of moderate-to-severe diarrhea in Low- and Middle-Income Countries (LMICs). Therefore, determining the source(s) of DEC in index cases and exposure environment is important for developing a prevention strategy. The current study aims to investigate the prevalence of DEC among children under 5 years and their exposure environment in Ogun State, Nigeria. Samples from 228 diarrheic children and their exposure environment were collected and screened for E. coli. Bio-chemically compatible distinct colonies were molecularly characterized using a 7-virulence-gene multiplex PCR with virulence factors (VFs) indicative of four pathotypes of E. coli: enterotoxigenic (ETEC), verotoxigenic (VTEC), enteropathogenic (EPEC), and enteroinvasive (EIEC). Representative pathotypes were subjected to antimicrobial susceptibility and over-expressed efflux pump assays. One or more VFs typical of specific pathotypes were detected in 25.9% (59/228) diarrhea cases consisting of ETEC (21.5%) and EPEC (0.4%), while hetero-pathogenic pathotypes were found in 4.0% of cases. Of the food sources, 27.9% (101/362) were positive for DEC, of which ETEC accounted for 21.0%, VTEC 1.9%, EPEC 0.6%, EIEC 0.6%, and hetero-pathogenic pathotypes were 3.9%. Furthermore, ETEC was the only pathotype detected in the wastewater (4/183). Interestingly, the consumption of street-vended foods was the most significant (p = 0.04) risk factor for DEC infection in the study area. A total of 73.3% of selected DEC pathotypes showed resistance to antimicrobials, while 27.5% demonstrated over-expression of efflux pump activity. The high prevalence of ETEC across all sources and the occurrence of hetero-pathogenic DEC in diarrheic children and food sources emphasizes the importance of establishing a better strategy for the control and prevention of diarrhea among children in low- and medium-income households.

Colistin Resistance among Enterobacterales Isolates: Underlying Mechanisms and Alternative Treatment Options

Global dissemination of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria (GNB) such as carbapenemase-producing Enterobacterales has resulted in reviving colistin as a final therapeutic alternative. Colistin resistance foretold a catastrophe. We aimed to detect the rates of carbapenems and colistin resistance among hospital-acquired Enterobacterales species, verify the underlying mechanisms and provide antibiogram for colistin-resistant isolates. The collected Enterobacterales isolates were tested for their antimicrobial susceptibility by the disk diffusion method and agar dilution was utilized for both imipenem and colistin. The production of ESβLs and carbapenemases was phenotypically assessed by the combined disk (CDT) and modified carbapenem inactivation (mCIM) tests, respectively. Possible attributes for colistin resistance were explored by detection of both plasmid- and efflux pump-mediated mechanisms. By multiplex PCR assay, carbapenem resistance (blaNDM-1 & blaOXA-48) and mobilized colistin-resistant-1 (mcr-1) genes were identified. A total of 160 Enterobacterales isolates were obtained of which 68.8% were MDR, 25% were XDR and 6.3% were pandrug-resistant (PDR) isolates with no statistically significant difference among Enterobacterales species (P> 0.05). Carbapenems resistance was detected in 41.3% (66/160) while colistin resistance was detected in 22% (36/160) of isolates. Proteus mirabilis expressed the highest rate of colistin resistance (100%; 16/16), followed by Enterobacter aerogenes (23.1%; 6/26), E. coli (13%; 6/46) and K.pneumoniae (11.1%; 8/72). One hundred percent (36/36) of colistin-resistant isolates proved efflux pump activity for colistin. However; only 2% (2/100) of tested Enterobacterales carried mcr-1 gene through molecular analysis. Colistin-resistant isolates exhibited variable susceptibility to the tested antimicrobial agents of which fosfomycin was the highest (94.1%). Efflux pump activity played a major role for colistin resistance among Enterobacterales species and fosfomycin could be a promising therapeutic option.

A Review of the Resistance Mechanisms for β-Lactams, Macrolides and Fluoroquinolones among Streptococcus pneumoniae.

Streptococcus pneumoniae (S. pneumoniae) is a bacterial species often associated with the occurrence of community-acquired pneumonia (CAP). CAP refers to a specific kind of pneumonia that occurs in individuals who acquire the infection outside of a healthcare setting. It represents the leading cause of both death and morbidity on a global scale. Moreover, the declaration of S. pneumoniae as one of the 12 leading pathogens was made by the World Health Organization (WHO) in 2017. Antibiotics like β-lactams, macrolides, and fluoroquinolones are the primary classes of antimicrobial medicines used for the treatment of S. pneumoniae infections. Nevertheless, the efficacy of these antibiotics is diminishing as a result of the establishment of resistance in S. pneumoniae against these antimicrobial agents. In 2019, the WHO declared that antibiotic resistance was among the top 10 hazards to worldwide health. It is believed that penicillin-binding protein genetic alteration causes β-lactam antibiotic resistance. Ribosomal target site alterations and active efflux pumps cause macrolide resistance. Numerous factors, including the accumulation of mutations, enhanced efflux mechanisms, and plasmid gene acquisition, cause fluoroquinolone resistance. Furthermore, despite the advancements in pneumococcal vaccinations and artificial intelligence (AI), it is not feasible for individuals to rely on them indefinitely. The ongoing development of AI for combating antimicrobial resistance necessitates more research and development efforts. A few strategies can be performed to curb this resistance issue, including providing educational initiatives and guidelines, conducting surveillance, and establishing new antibiotics targeting another part of the bacteria. Hence, understanding the resistance mechanism of S. pneumoniae may aid researchers in developing a more efficacious antibiotic in future endeavors.

Detection of Aminoglycoside Modifying Enzyme (AME) genes in Acinetobacter baumannii isolates and the inhibitory effect of efflux pump activity on drug susceptibility pattern

IntroductionThe development of aminoglycoside modifying enzymes (AMEs) and increased efflux activity are considered important aminoglycosides resistance mechanisms. Aim.This study is focused on the detection of the AMEs gene and assessing the effect of efflux pump inhibitor on the reversal of A. baumannii drug susceptibility. MethodologyBacterial DNA was amplified using AMEs gene-specific primers. Isolates were also investigated for efflux pump activity using efflux pump inhibitor (EPI) i.e. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the impact of both mechanisms was analyzed. ResultsAmong A. baumannii isolates, 55% isolates (n ​= ​22/40) were identified to have aminoglycoside modifying enzymes genes; ant(3′)-I gene (50%, 11/22), aac(6′)-Ib gene (45.4%, 10/22), aph(3′)-I gene (18.1%, 4/22) and aac(3)-I (9.1%, 2/22). Total 70% isolates have shown MIC alteration in different classes of drugs in response to EPI-CCCP. Such alteration was found in 100% amikacin sensitive and 58.6% amikacin resistant, 93.7% and 57.1% gentamicin sensitive and resistant isolates respectively. ConclusionThe presence of aminoglycosides modifying enzymes was frequent among aminoglycosides resistant A. baumannii isolates and the coexistence of efflux pumps activity also plays an important role to increase drug resistance. RepositoriesGenbank and their accession numbers are MT903331[aac(3)-I], MT903332 MT903333 [ant(3′)-I], MT903334, MT903335 [aph(3′)-I)] and MT903336, MT940242 [ aac(6’)-Ib].

The antihelminth drug rafoxanide reverses chromosomal-mediated colistin-resistance in Klebsiella pneumoniae.

The emergence and rapid spread of multi-drug-resistant (MDR) bacteria pose a serious threat to global healthcare. Although the synergistic effect of rafoxanide and colistin was reported, little is known regarding the potential mechanism of this synergy, particularly against chromosomal-mediated colistin-resistant Klebsiella pneumoniae. In the present study, we elucidated the synergistic effect of rafoxanide and colistin against chromosomal-mediated colistin-resistant Klebsiella pneumoniae isolates from human (KP-9) and swine (KP-1) infections. Treatment with 1 mg/L rafoxanide overtly reversed the MIC max to 512-fold. Time-kill assays indicated that rafoxanide acted synergistically with colistin against the growth of KP-1 and KP-9. Mechanistically, we unexpectedly found that the combination destroys the inner-membrane integrity, and ATP synthesis was also quenched, albeit, not via F1F0-ATPase; thereby also inhibiting the activity of efflux pumps. Excessive production of reactive oxygen species (ROS) was also an underlying factor contributing to the bacterial-killing effect of the combination. Transcriptomic analysis unraveled overt heterogeneous expression as treated with both administrations compared with monotherapy. Functional analysis of these differentially expressed genes (DEGs) targeted to the plasma membrane and ATP-binding corroborated phenotypic screening results. These novel findings highlight the synergistic mechanism of rafoxanide in combination with colistin which effectively eradicates chromosomal-mediated colistin-resistant Klebsiella pneumoniae. IMPORTANCE The antimicrobial resistance of Klebsiella pneumoniae caused by the abuse of colistin has increased the difficulty of clinical treatment. A promising combination (i.e., rafoxanide+ colistin) has successfully rescued the antibacterial effect of colistin. However, we still failed to know the potential effect of this combination on chromosome-mediated Klebsiella pneumoniae. Through a series of in vitro experiments, as well as transcriptomic profiling, we confirmed that the MIC of colistin was reduced by rafoxanide by destroying the inner-membrane integrity, quenching ATP synthesis, inhibiting the activity of the efflux pump, and increasing the production of reactive oxygen species. In turn, the expression of relevant colistin resistance genes was down-regulated. Collectively, our study revealed rafoxanide as a promising colistin adjuvant against chromosome-mediated Klebsiella pneumoniae.

Antibacterial and Anti-Efflux Activities of Cinnamon Essential Oil against Pan and Extensive Drug-Resistant Pseudomonas aeruginosa Isolated from Human and Animal Sources.

Pseudomonas aeruginosa is notorious for its ability to develop a high level of resistance to antimicrobial agents. Resistance-nodulation-division (RND) efflux pumps could mediate drug resistance in P. aeruginosa. The present study aimed to evaluate the antibacterial and anti-efflux activities of cinnamon essential oil either alone or combined with ciprofloxacin against drug resistant P. aeruginosa originated from human and animal sources. The results revealed that 73.91% of the examined samples were positive for P. aeruginosa; among them, 77.78% were of human source and 72.73% were recovered from animal samples. According to the antimicrobial resistance profile, 48.73% of the isolates were multidrug-resistant (MDR), 9.2% were extensive drug-resistant (XDR), and 0.84% were pan drug-resistant (PDR). The antimicrobial potential of cinnamon oil against eleven XDR and one PDR P. aeruginosa isolates was assessed by the agar well diffusion assay and broth microdilution technique. The results showed strong antibacterial activity of cinnamon oil against all tested P. aeruginosa isolates with inhibition zones' diameters ranging from 34 to 50 mm. Moreover, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of cinnamon oil against P. aeruginosa isolates ranged from 0.0562-0.225 µg/mL and 0.1125-0.225 µg/mL, respectively. The cinnamon oil was further used to evaluate its anti-efflux activity against drug-resistant P. aeruginosa by phenotypic and genotypic assays. The cartwheel test revealed diminished efflux pump activity post cinnamon oil exposure by two-fold indicating its reasonable impact. Moreover, the real-time quantitative polymerase chain reaction (RT-qPCR) results demonstrated a significant (p < 0.05) decrease in the expression levels of MexA and MexB genes of P. aeruginosa isolates treated with cinnamon oil when compared to the non-treated ones (fold changes values ranged from 0.4204-0.7474 for MexA and 0.2793-0.4118 for MexB). In conclusion, we suggested the therapeutic use of cinnamon oil as a promising antibacterial and anti-efflux agent against drug-resistant P. aeruginosa.

Evaluation of the antibacterial and inhibitory activity of the MepA efflux pump of Staphylococcus aureus by riparins I, II, III, and IV

The efflux pump mechanism contributes to the antibiotic resistance of widely distributed strains of Staphylococcus aureus. Therefore, in the present work, the ability of the riparins N-(4-methoxyphenethyl)benzamide (I), 2-hydroxy-N-[2-(4-methoxyphenyl)ethyl]benzamide (II), 2, 6-dihydroxy-N-[ 2-(4-methoxyphenyl)ethyl]benzamide (III), and 3,4,5-trimethoxy-N-[2-(4-methoxyphenethyl)benzamide (IV) as potential inhibitors of the MepA efflux pump in S. aureus K2068 (fluoroquinolone-resistant). In addition, we performed checkerboard assays to obtain more information about the activity of riparins as potential inhibitors of MepA efflux and also analyzed the ability of riparins to act on the permeability of the bacterial membrane of S. aureus by the fluorescence method with SYTOX Green. A molecular coupling assay was performed to characterize the interaction between riparins and MepA, and ADMET (absorption, distribution, metabolism, and excretion) properties were analyzed. We observed that I-IV riparins did not show direct antibacterial activity against S. aureus. However, combination assays with substrates of MepA, ciprofloxacin, and ethidium bromide (EtBr) revealed a potentiation of the efficacy of these substrates by reducing the minimum inhibitory concentration (MIC). Furthermore, increased EtBr fluorescence emission was observed for all riparins. The checkerboard assay showed synergism between riparins I, II, and III, ciprofloxacin, and EtBr. Furthermore, riparins III and IV exhibited permeability in the S. aureus membrane at a concentration of 200 μg/mL. Molecular docking showed that riparins I, II, and III bound in a different region from the binding site of chlorpromazine (standard pump inhibitor), indicating a possible synergistic effect with the reference inhibitor. In contrast, riparin IV binds in the same region as the chlorpromazine binding site. From the in silico ADMET prediction based on MPO, it could be concluded that the molecules of riparin I-IV present their physicochemical properties within the ideal pharmacological spectrum allowing their preparation as an oral drug. Furthermore, the prediction of cytotoxicity in liver cell lines showed a low cytotoxic effect for riparins I-IV.

EFFLUX PUMPS AS A PHARMACOLOGICAL TARGET TO COMBAT ANTIBIOTIC RESISTANCE

Backround. Antibiotic resistance is the resistance of bacteria to antimicrobial drugs to which they were previously sensitive. A particular danger is the spread of multidrug-resistant healthcare-associated infections of "ESKAPE" group. Among the mechanisms of antibiotic resistance of these bacteria, the activation of efflux pumps attracts special attention.&#x0D; Aim: to review the current data on the possibility of using efflux pumps as targets for pharmacological action to overcome antibiotic resistance.&#x0D; Materials and methods. Analysis of the data presented in PubMed by keywords "antimicrobial resistance", "efflux pumps", "efflux pump inhibitors".&#x0D; Results. Multidrug efflux pumps are transmembrane transporter proteins that are located in the bacterial cell membrane and periplasm, they are naturally expressed, removing most of the clinically relevant antibiotic from the internal environment of the cell to the external environment, reducing its intracellular concentration. It is promising to combine certain antibiotics with efflux pump inhibitors, this allows to reduce the therapeutic dose of antibiotics and prevent the formation of bacterial biofilms. Data on the study of the effectiveness of well-known drugs sertraline, trimethoprim, thioridazine, diclofenac sodium, ibuprofen, nitroglycerin, metformin, and verapamil as efflux pump inhibitors are presented. The use of compounds obtained from plants (for example, berberine, artesunate and curcumin inhibit the activity of the efflux pump of gram-negative bacilli E. coli and P. aeruginosa), vegetables (in particular, Momordica balsamina), seeds of thistle, pepper, cumin, essential oils, etc. is promising.&#x0D; Conclusion. Thus, inhibition of efflux pumps may be a potential way to improve antibiotic activity. The advantage of efflux pumps inhibitors is the difficulty of developing resistance against them in bacteria. It is advisable to conduct in vivo studies to confirm their activity and clinical effectiveness.

Polyporenic Acids from the Mushroom Buglossoporus quercinus Possess Chemosensitizing and Efflux Pump Inhibitory Activities on Colo 320 Adenocarcinoma Cells.

Polyporenic acids N-R (1-5), five novel 24-methylene lanostane triterpenes along with seven known polyporenic acids (6-12), were identified from the fruiting bodies of Buglossoporus quercinus. The isolation of compounds 1-12 was performed by a combination of multistep flash chromatography and reversed-phase high-performance liquid chromatography (HPLC). The structure determination was carried out by extensive spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) experiments. The isolated fungal metabolites were investigated for their antiproliferative activity in vitro by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on the resistant Colo 320 human colon adenocarcinoma cell line expressing P-glycoprotein (ABCB1). The lanostane triterpenes exerted moderate antiproliferative activity with IC50 values in the range of 20.7-106.2 μM. A P-glycoprotein efflux pump modulatory test on resistant Colo 320 cells highlighted that fungal metabolites 3, 5, 8, and 10-12 have the ability to inhibit the efflux pump activity of cancer cells. Moreover, the drug interactions of triterpenes with doxorubicin were studied by the checkerboard method. Compounds 3-4, and 7-12 interacted in a synergistic manner, while an outstanding potency was detected for compound 9, which was defined as strong synergism (CI = 0.276). The current study reveals that B. quercinus is a remarkable source of fungal steroids with considerable chemosensitizing activity on cancer cells.