Transporter Efflux Pump Research Articles

Efflux-mediated Multidrug Resistance in Critical Gram-negative Bacteria and Natural Efflux Pump Inhibitors.

Multidrug Resistance mechanisms in microorganisms confer the slackness of the existing drugs, leading to added difficulty in treating infections. As a consequence, efficient novel drugs and innovative therapies to treat MDR infections are necessarily required. One of the primary contributors to the emergence of multidrug resistance in gram-negative bacteria has been identified as the efflux pumps. These transporter efflux pumps reduce the intracellular concentration of antibiotics and aid bacterial survival in suboptimal low antibiotic concentration environments that may cause treatment failure. The reversal of this resistance via inhibition of the efflux mechanism is a promising method for increasing the effectiveness of antibiotics against multidrug-resistant pathogens. Such EPI, in combination with antibiotics, can make it easier to reintroduce traditional antibiotics into clinical practice. This review mostly examines efflux-mediated multidrug resistance in critical gram-negative bacterial pathogens and EPI of plant origin that have been reported over previous decades.

Whole-genome sequencing of Klebsiella pneumoniae MDR circulating in a pediatric hospital setting: a comprehensive genome analysis of isolates from Guayaquil, Ecuador

BackgroundKlebsiella pneumoniae is the major cause of nosocomial infections worldwide and is related to a worsening increase in Multidrug-Resistant Bacteria (MDR) and virulence genes that seriously affect immunosuppressed patients, long-stay intensive care patients, elderly individuals, and children. Whole-Genome Sequencing (WGS) has resulted in a useful strategy for characterizing the genomic components of clinically important bacteria, such as K. pneumoniae, enabling them to monitor genetic changes and understand transmission, highlighting the risk of dissemination of resistance and virulence associated genes in hospitals. In this study, we report on WGS 14 clinical isolates of K. pneumoniae from a pediatric hospital biobank of Guayaquil, Ecuador.ResultsThe main findings revealed pronounced genetic heterogeneity among the isolates. Multilocus sequencing type ST45 was the predominant lineage among non-KPC isolates, whereas ST629 was found more frequently among KPC isolates. Phylogenetic analysis suggested local transmission dynamics. Comparative genomic analysis revealed a core set of 3511 conserved genes and an open pangenome in neonatal isolates. The diversity of MLSTs and capsular types, and the high genetic diversity among these isolates indicate high intraspecific variability. In terms of virulence factors, we identified genes associated with adherence, biofilm formation, immune evasion, secretion systems, multidrug efflux pump transporters, and a notably high number of genes related to iron uptake. A large number of these genes were detected in the ST45 isolate, whereas iron uptake yersiniabactin genes were found exclusively in the non-KPC isolates. We observed high resistance to commonly used antibiotics and determined that these isolates exhibited multidrug resistance including β-lactams, aminoglycosides, fluoroquinolones, quinolones, trimetropins, fosfomycin and macrolides; additionally, resistance-associated point mutations and cross-resistance genes were identified in all the isolates. We also report the first K. pneumoniae KPC-3 gene producers in Ecuador.ConclusionsOur WGS results for clinical isolates highlight the importance of MDR in neonatal K. pneumoniae infections and their genetic diversity. WGS will be an imperative strategy for the surveillance of K. pneumoniae in Ecuador, and will contribute to identifying effective treatment strategies for K. pneumoniae infections in critical units in patients at stratified risk.

The dynamic roles of intracellular vacuoles in heavy metal detoxification by Rhodotorula mucilaginosa.

Rhodotorula mucilaginosa (Rho) can develop a range of strategies to resist the toxicity of heavy metals. This study aimed to investigate the physiological responses and transcriptomic regulation of the fungus under different heavy metal stresses. This study applied transmission electron microscopy and RNA-seq to investigate the fungal resistance to Pb, Cd, and Cu stresses. Under Pb stress, the activated autophagy-related genes, vesicle-fusing ATPase, and vacuolar ATP synthase improved vacuolar sequestration. This offsets the loss of lipids. However, the metal sequestration by vacuoles was not improved under Cd stress. Vacuolar fusion was also inhibited following the interference of intravacuolar Ca2+ due to their similar ionic radii. Cu2+ showed the maximum toxic effects due to its lowest cellular sorption (as low as 7%) with respect to Pb2+ and Cd2+, although the efflux pumps and divalent metal ion transporters partially contributed to the detoxification. Divalent cation transporters and vacuolar sequestration are the critical strategies for Rho to resist Pb stress. Superoxide dismutase (SOD) is the main strategy for Cd resistance in Rho. The intracellular Cu level was decreased by efflux pump and divalent metal ion transporters.

Synergistic Antibacterial Activity of Gallic Acid Based Chalcone Indl 2 by Inhibiting Efflux Pump Transporters.

As a part of novel discovery of drugs from natural resources, present study was undertaken to explore the antibacterial potential of chalcone Indl-2 in combination with different group of antibiotics. MIC of antibiotics was reduced up to eight folds against the different cultures of E. coli by both chalcones. Among the two compounds, the i. e. 1-(3', 4,'5'-trimethoxyphenyl)-3-(3-Indyl)-prop-2-enone (6, Indl-2), a chalcone derivative of gallic acid (Indl-2) was better along with tetracycline (TET) worked synergistically and was found to inhibit efflux transporters as obvious by ethidium bromide efflux confirmed by ATPase assays and docking studies. In combination, Indl-2 kills the MDREC-KG4 cells, post-antibiotic effect (PAE) of TET was prolonged and mutant prevention concentration (MPC) of TET was also decreased. In-vivo studies revealed that Indl-2 reduces the concentration of TNF-α. In acute oral toxicity study, Indl-2 was non-toxic and well tolerated up-to dose of 2000 mg/kg. Perhaps, the study is going to report gallic acid derived chalcone as synergistic agent acting via inhibiting the primary efflux pumps.

Myoinositol and methyl stearate increases rifampicin susceptibility among drug-resistant Mycobacterium tuberculosis expressing Rv1819c.

The alarming increase in multidrug resistance, which includes Bedaquiline and Delamanid, stumbles success in Tuberculosis treatment outcome. Mycobacterium tuberculosis gains resistance to rifampicin, which is one of the less toxic and potent anti-TB drugs, through genetic mutations predominantly besides efflux pump mediated drug resistance. In recent decades, scientific interventions are being carried out to overcome this hurdle using novel approaches to save this drug by combining it with other drugs/molecules or by use of high dose rifampicin. This study reports five small molecules namely Ellagic acid, Methyl Stearate, Myoinositol, Rutin, and Shikimic acid that exhibit synergistic inhibitory activity with rifampicin against resistant TB isolates. In-silico examinations revealed possible blocking of Rv1819c-an ABC transporter efflux pump that was known to confer resistance in M. tuberculosis to rifampicin. The synergistic anti-TB activity was assessed using a drug combination checkerboard assay. Efflux pump inhibition activity of ellagic acid, myoinositol, and methyl stearate was observed through ethidium bromide accumulation assay in the drug-resistant M. tuberculosis clinical strains and recombinant Mycobacterium smegmatis expressing Rv1819c in coherence with the significant reduction in the minimum inhibitory concentration of rifampicin. Cytotoxicity of the active efflux inhibitors was tested using in silico and ex vivo methods. Myoinositol and methyl stearate were completely non-toxic to the hematological and epithelial cells of different organs under ex vivo conditions. Based on these findings, these molecules can be considered for adjunct TB therapy; however, their impact on other drugs of anti-TB regimen needs to be tested.

Integrated toxicology, metabolomics, and transcriptomics analyses reveal the biodegradation and adaptation mechanisms to BDE-47 in Pseudomonas plecoglossicida

Microbial degradation technology is often considered as an eco-friendly and cost-effective strategy to reduce the risks of 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47). However, the number of available BDE-47-degradating bacteria is insufficient, and there has yet to fully understand the biotransformation mechanisms of BDE-47. Here, a new strain, P. plecoglossicida, was isolated for the BDE-47 degradation. The biodegradation and adaption mechanisms of P. plecoglossicida to BDE-47 were systematically examined through an integrative approach combining toxicological assessment, degradation product analysis, metabolomic analysis, and transcriptomic analysis. Toxicological assessment and metabolomic analysis indicated that the adaptation mechanism of P. plecoglossicida to BDE-47 was mainly activating the antioxidant defense system to relieve the oxidative stress induced by BDE-47. However, the defense ability of P. plecoglossicida against BDE-47 was limited, and the high concentration of BDE-47 would result in the disturbance of amino acid and purine metabolism, and ultimately apoptosis. The analysis of degradation products and transcriptomics revealed that BDE-47 underwent debromination, hydroxylation, ether bond breaking, and ring-opening cleavage with the participation of multiple intracellular oxidoreductases. Besides, active efflux of BDE-47 and its degradation products mediated by efflux pump and multiple transporters might contribute to the bacterial tolerance against them. These results provided greater insights into the mechanisms of BDE-47 biodegradation.

Transcriptome Analysis Reveals the Algicidal Mechanism of Brevibacillus laterosporus against Microcystis aeruginosa through Multiple Metabolic Pathways.

It is widely accepted that eutrophication has played an important role in the formation of harmful cyanobacterial blooms in recent decades, which impacts water quality and ecological environment and causes huge economic losses. Algicidal bacteria have a promising application prospect in controlling cyanobacterial blooms in aquaculture water. Here, the process of the algicidal bacterium Brevibacillus laterosporus strain Bl-zj acting on Microcystis aeruginosa was explored using transcriptome analysis to elucidate the algicidal mechanism. The results of the co-culture of bacterium and alga showed a strong alga-lysing effect of B. laterosporus against M. aeruginosa with an extreme morphology deformation of the algal cells. A total of 2744 differentially expressed genes of B. laterosporus were identified, which were mainly involved in the metabolism of amino acid, carbohydrate, and lipid. In the co-cultured group, the expression of genes mainly enriched in valine, leucine and isoleucine degradation, and fatty acid degradation were significantly increased. However, the expression of the genes related to ribosome were mainly inhibited. Transcriptome analysis showed that B. laterosporus obtained ATP and energy by the degradation of valine, leucine, isoleucine, and fatty acids, and destroyed algal cells by efflux pump transporters, secretion of hydrolytic enzymes, antibiotics, proteases, and other secondary metabolites, resulting in algal death and achieving the algicidal effect.

The Transcription Factor FgAtrR Regulates Asexual and Sexual Development, Virulence, and DON Production and Contributes to Intrinsic Resistance to Azole Fungicides in Fusarium graminearum.

Simple SummaryFusarium graminearum is a devastating plant pathogen that can cause wheat head blight. Azole fungicides are commonly used chemicals for control of this disease. However, F. graminearum strains resistant to these fungicides have emerged. To better understand the azole resistance mechanism of F. graminearum, we identified and characterized the Zn(II)2-Cys6 transcription factor FgAtrR in F. graminearum. We found that FgAtrR played critical roles in vegetative growth, conidia production, perithecium formation, and virulence on wheat heads and corn silks. FgAtrR was also involved in the resistance to azole antifungals by regulating the expression of the drug target FgCYP51s and efflux pump transporters. These results broadened our understanding of the azole resistance mechanisms of F. graminearum.Fusarium graminearum is the predominant causal agent of cereal Fusarium head blight disease (FHB) worldwide. The application of chemical fungicides such as azole antifungals is still the primary method for FHB control. However, to date, our knowledge of transcriptional regulation in the azole resistance of F. graminearum is quite limited. In this study, we identified and functionally characterized a Zn(II)2-Cys6 transcription factor FgAtrR in F. graminearum. We constructed a FgAtrR deletion mutant and found that deletion of FgAtrR resulted in faster radial growth with serious pigmentation defects, significantly reduced conidial production, and an inability to form perithecia. The pathogenicity of the ΔFgAtrR mutant on wheat spikes and corn silks was severely impaired with reduced deoxynivalenol production, while the tolerance to prochloraz and propiconazole of the deletion mutant was also significantly decreased. RNA-seq indicated that many metabolic pathways were affected by the deletion of FgAtrR. Importantly, FgAtrR could regulate the expression of the FgCYP51A and ABC transporters, which are the main contributors to azole resistance. These results demonstrated that FgAtrR played essential roles in asexual and sexual development, DON production, and pathogenicity, and contributed to intrinsic resistance to azole fungicides in F. graminearum. This study will help us improve the understanding of the azole resistance mechanism in F. graminearum.

Emergence and Mechanism of Resistance of Tulathromycin Against Mycoplasma hyopneumoniae in a PK/PD Model and the Fitness Costs of 23S rRNA Mutants.

Macrolides are widely used in diseases caused by Mycoplasma spp. The new semi-synthetic macrolide antibiotic tulathromycin is currently in wide use for the treatment of respiratory diseases of livestock. The objective of this study was to evaluate the antibacterial effect of tulathromycin against Mycoplasma hyopneumoniae using an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model to reveal mechanisms of antibiotic resistance and to evaluate the fitness of drug-resistant strains. In this study, high performance liquid chromatography-tandem mass spectrometry was used to determine drug concentrations for the in vitro model after dosing. The peak concentrations were in the range 0.3125–20 μg/mL (1 × MIC-64 × MIC). The ratio of the area under the concentration-time curve (AUC) over 72 h divided by the MIC (AUC72h/MIC) had the highest correlation with the antibacterial effect of tulathromycin against M. hyopneumoniae. Tulathromycin also showed concentration-dependent antimicrobial effects and promoted the emergence of drug-resistant bacteria after being cultured for 168 h and most were mutations in 23S rRNA at site A2058G (E.coli numbering) and only a single isolate was an A2058T (E.coli numbering) mutant. In the presence of reserpine, we determined the MIC of tulathromycin, tilmicosin, tiamulin and tylosin against these drug-resistant bacteria and the strains with efflux pump mechanisms were found among the strains resistant to tilmicosin. Gene expression analysis indicated that the ABC and MATE transporter efflux pump genes RS01935, RS02670, RS01115, RS01970, RS02395 and RS03540 (MATE family efflux transporter) were up-regulated in the three strains (P < 0.05 or P < 0.01). These investigations provide guidance for clinical administration of tulathromycin and elucidate the mechanism and fitness cost of drug resistance in M. hyopneumoniae.

Neochrysosporazines: Precursor-Directed Biosynthesis Defines a Marine-Derived Fungal Natural Product P-Glycoprotein Inhibitory Pharmacophore.

Upregulation of ATP binding cassette (ABC) transporter efflux pumps (i.e. P-glycoprotein, P-gp) can impart multidrug resistance, rendering many chemotherapeutics ineffective and seriously limiting treatment regimes. While ABC transporters remain an attractive target for therapeutic intervention, the development of clinically useful small-molecule inhibitors has proved challenging. In this report, we describe the structure-activity relationship (SAR) analysis of a newly discovered P-gp inhibitory pharmacophore, phenylpropanoid piperazine chrysosporazines, produced by co-isolated marine-derived fungi. In the absence of any total syntheses, we apply an innovative precursor-directed biosynthesis strategy that successfully repurposed fungal biosynthetic output, allowing us to isolate, characterize, and identify the structurally diverse neochrysosporazines A-Q. SAR analysis utilizing all known (and new) neochrysosporazines, chrysosporazines, and azachrysosporazines, plus semi-synthetic analogues, established the key structure requirements for the P-gp inhibitory pharmacophore, and, in addition, identified non-essential sites that allow for the design of affinity and other conjugated probes.

Predicted functional and taxonomic analysis of subgingival biofilm of Grade C periodontitis in young patients under maintenance therapy.

In Grade C periodontitis in young patients (PerioC-Y), the functional roles of the subgingival community after years of periodontal treatment are still underexplored. This study evaluated the taxonomic and predicted functional content of the subgingival microbiome of PerioC-Y patients under supportive periodontal therapy (SPT). Clinical and microbiological data from subgingival biofilm were assessed from 10 PerioC-Y patients at two time points: at baseline and after 5.7 ± 1.3 years of SPT. This was compared with 15 patients without a history of periodontitis. The V1-V3 and V4-V5 regions of the 16S rRNA were sequenced using the Illumina Miseq. Microbial composition was evaluated by the core microbiome, and alpha- and beta-diversity. The microbiome functional content was predicted using Picrust2, and the gene differential abundance was analyzed with DESeq2. Clinical improvements were seen in PerioC-Y-SPT. Differences in β-diversity between PerioC-Y and health were observed (health x PerioC-Y-baseline, P=0.02; health x PerioC-Y-SPT, P=0.05). Moreover, although β-diversity did not statistically change between baseline and SPT in PerioC-Y, the microbial correlation evidenced increased Streptococcus and decreased Treponema network contributions during SPT. Based on predicted functional data, treatment induced a reduction in genes related to flagellar protein and signal transduction in PerioC-Y. However, compared with healthy individuals, some genes remained more highly abundant in PerioC-Y-SPT, such as quorum sensing and efflux pump transporters. Despite clinical improvements and a shift in taxonomic composition, the PerioC-Y patients' periodontal treatment was not enough to reach a similar microbiome to patients without disease experience. Some functional content in this biofilm remained altered in PerioC-Y regardless of disease control.

HPLC-MS identification and expression of Candida drug-resistance proteins from African HIV-infected patients.

The objective of this study was to elucidate the proteomic mechanisms of drug resistance in HIV-infected African patients. Cell membrane fractions from forty oral Candida isolates isolated from African HIV-positive patients were analysed using HPLC-MS with the aim of identifying proteins associated with their pathogenicity and drug resistance. Heat shock proteins that mediate the fungicidal activity of salivary peptides were found in all tested Candida fractions, with pH-responsive proteins associated with increased pathogenicity only being present in the three most commonly isolated species. ABC multidrug transporter efflux pumps and estrogen binding proteins were only found in C. albicans fractions, while ergosterol biosynthesis proteins were identified in four species. The combination of various adherence, invasion, upregulation and efflux pump mechanisms appear to be instrumental for the Candida host colonization and drug resistance emergence in HIV-infected individuals.

Mifepristone as a Potential Therapy to Reduce Angiogenesis and P-Glycoprotein Associated With Glioblastoma Resistance to Temozolomide.

Glioblastoma, the most common primary central nervous system tumor, is characterized by extensive vascular neoformation and an area of necrosis generated by rapid proliferation. The standard treatment for this type of tumor is surgery followed by chemotherapy based on temozolomide and radiotherapy, resulting in poor patient survival. Glioblastoma is known for strong resistance to treatment, frequent recurrence and rapid progression. The aim of this study was to evaluate whether mifepristone, an antihormonal agent, can enhance the effect of temozolomide on C6 glioma cells orthotopically implanted in Wistar rats. The levels of the vascular endothelial growth factor (VEGF), and P-glycoprotein (P-gp) were examined, the former a promoter of angiogenesis that facilitates proliferation, and the latter an efflux pump transporter linked to drug resistance. After a 3-week treatment, the mifepristone/temozolomide regimen had decreased the level of VEGF and P-gp and significantly reduced tumor proliferation (detected by PET/CT images based on 18F-fluorothymidine uptake). Additionally, mifepristone proved to increase the intracerebral concentration of temozolomide. The lower level of O6-methylguanine-DNA-methyltransferase (MGMT) (related to DNA repair in tumors) previously reported for this combined treatment was herein confirmed. After the mifepristone/temozolomide treatment ended, however, the values of VEGF, P-gp, and MGMT increased and reached control levels by 14 weeks post-treatment. There was also tumor recurrence, as occurred when administering temozolomide alone. On the other hand, temozolomide led to 100% mortality within 26 days after beginning the drug treatment, while mifepristone/temozolomide enabled 70% survival 60–70 days and 30% survived over 100 days, suggesting that mifepristone could possibly act as a chemo-sensitizing agent for temozolomide.

Phenylpiperazine 5,5-Dimethylhydantoin Derivatives as First Synthetic Inhibitors of Msr(A) Efflux Pump in Staphylococcus epidermidis.

Herein, 15 phenylpiperazine 3-benzyl-5,5-dimethylhydantoin derivatives (1–15) were screened for modulatory activity towards Msr(A) efflux pump present in S. epidermidis bacteria. Synthesis, crystallographic analysis, biological studies in vitro and structure–activity relationship (SAR) analysis were performed. The efflux pump inhibitory (EPI) potency was determined by employing ethidium bromide accumulation assay in both Msr(A) efflux pump overexpressed (K/14/1345) and deficient (ATCC 12228) S. epidermidis strains. The series of compounds was also evaluated for the capacity to reduce the resistance of K/14/1345 strain to erythromycin, a known substrate of Msr(A). The study identified five strong modulators for Msr(A) in S. epidermidis. The 2,4-dichlorobenzyl-hydantoin derivative 9 was found as the most potent EPI, inhibiting the efflux activity in K/14/1345 at a concentration as low as 15.63 µM. Crystallography-supported SAR analysis indicated structural properties that may be responsible for the activity found. This study identified the first synthetic compounds able to inhibit Msr(A) efflux pump transporter in S. epidermidis. Thus, the hydantoin-derived molecules found can be an attractive group in search for antibiotic adjuvants acting via Msr(A) transporter.

Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast.

Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.

Elucidating the multi-drug resistance mechanism of Enterococcus faecalis V583: A gene interaction network analysis

Resistance to antibiotics have created havoc around the globe due to the emergence of multi-drug resistant (MDR) pathogenic bacterial strains. To decipher this problem, a detailed understanding of the antimicrobial resistance (AMR) genes and their resistant mechanisms are obligatory. The present study is mainly focused on an opportunistic, nosocomial bacterial strain Enterococcus faecalis V583, which possess acquired exogenous AMR genes portraying resistance against Chloramphenicol, Tetracycline, Vancomycin, Linezolid, Ampicillin and other antibiotics. An interaction network of eight AMR genes along with 40 functional partners have been constructed and analysed. Functional enrichment analysis highlighted 20, 21 and 22 genes having significant roles in Cellular Component (CC), Molecular Functions (MF) and Biological Process (BP) respectively. Clustering analysis resulted in four densely interconnected clusters (C1-C4) which were associated with three AMR mechanisms that include the alteration in drug target (pbps, mur and van genes), complete replacement/bypass of target sites (van genes) and ATP Binding Cassette (ABC) transporter efflux pump mechanisms (msrA, EF_1680, EF_1682 and pbps). Our results showed that the genes responsible for β-lactams resistance (pbp1A, 1C, 2A, 2B); glycopeptide resistance (ddl, vanBHBRBSBWXYB); Erythromycin, Macrolides, Lincosamide and Streptogramin-B (MLSB) resistance (msrA, EF_1680, EF_1682) along with mur genes (murABBCDEFG) played an important role in MDR mechanisms. Network analysis has shown the genes mraY, pbpC, murE, murG and murD possessed 26, 24, 23, 22 and 22 interactions respectively. With more number of direct interactions, these genes can be considered as hub genes that could be exploited as potential drug targets for new drug discovery.

Role of Two Sets of RND-Type Multidrug Efflux Pump Transporter Genes, mexAB-oprM and mexEF-oprN, in Virulence of Pseudomonas syringae pv. tabaci 6605.

Pseudomonas syringae pv. tabaci 6605 has two multidrug resistance (MDR) efflux pump transporters, MexAB-OprM and MexEF-OprN. To understand the role of these MDR efflux pumps in virulence, we generated deletion mutants, ∆mexB, ∆mexF, and ∆mexB∆mexF, and investigated their sensitivity to plant-derived antimicrobial compounds, antibiotics, and virulence. Growth inhibition assays with KB soft agar plate showed that growth of the wild-type (WT) was inhibited by 5 µl of 1 M catechol and 1 M coumarin but not by other plant-derived potential antimicrobial compounds tested including phytoalexins. The sensitivity to these compounds tended to increase in ∆mexB and ∆mexB∆mexF mutants. The ∆mexB∆mexF mutant was also sensitive to 2 M acetovanillone. The mexAB-oprM was constitutively expressed, and activated in the ∆mexF and ∆mexB∆mexF mutant strains. The swarming and swimming motilities were impaired in ∆mexF and ∆mexB∆mexF mutants. The flood inoculation test indicated that bacterial populations in all mutant strains were significantly lower than that of WT, although all mutants and WT caused similar disease symptoms. These results indicate that MexAB-OprM extrudes plant-derived catechol, acetovanillone, or coumarin, and contributes to bacterial virulence. Furthermore, MexAB-OprM and MexEF-OprN complemented each other’s functions to some extent.

Overexpression of efflux pump transporter genes and mutations in ERG11 pave the way to fluconazole resistance in Candida tropicalis: A study from a North India region

IntroductionFluconazole resistance in Candida tropicalis health care–associated infections is increasing. We investigated the role of efflux pump and mutations in ERG11p conferring fluconazole resistance in C. tropicalis. Materials and methodsA total of 17 C. tropicalis clinical isolates, including fluconazole-resistant and fluconazole-susceptible/fluconazole-susceptible dose-dependent isolates, were collected from a tertiary care centre in a North India region between 2015 and 2018. Antifungal susceptibility, reversal of fluconazole resistance by tacrolimus, ERG11 amplification and its sequencing and a quantitative polymerase chain reaction (PCR) assay for expression analysis of ERG11, MDR1 and CDR1 genes were performed. Results and discussionSynergism between fluconazole and tacrolimus was observed in all resistant C. tropicalis isolates. Overexpression of all the three genes, MDR1, ERG11 and CDR1, was observed in resistant isolates (P = 0.05). Among resistant isolates, mutations leading to amino acid substitution were seen in two, Ct10 (glysine464serine) and Ct16 (tyrosine132phenylalanine; serine154phenylalanine). ConclusionOverexpression in efflux pump transporter genes, together with mutations in ERG11, lead to fluconazole resistance among C. tropicalis. To the best of our knowledge, this is the first study on the C. tropicalis fluconazole resistance mechanism from the North India region.

Overexpression of Efflux Pump Genes is an Alternative Mechanism in Voriconazole Resistant Aspergillus fumigatus isolates Without Relative Mutations in CYP5A.

The overexpression of the efflux transporter genes is one of the important mechanisms of resistance in fungal pathogens such as Candida and Aspergillus species. Here, the expression alterations of drug efflux transporter genes were evaluated in non- Cyp51A voriconazole-resistant Aspergillus fumigatus isolates. Six A. fumigatus isolates including four voriconazole-resistant isolates with and without azole-resistance-related mutations in addition to two susceptible A. fumigatus isolates were selected from 300 previously characterized A. fumigatus clinical and environmental isolates, received during 2013-2015. In order to extract RNA, the minimum inhibitory concentrations (MICs) for the isolates were determined according to the broth microdilution protocol regarding the Clinical and Laboratory Standards Institute document M38-A2 (CLSI, 2008). Alteration in the expression of AfuMDR1, AfuMDR2, AfuMDR3, AfuMDR4, Cyp51A, and atrF was studied using the real-time polymerase chain reaction assay. Based on REST® output, significant overexpression of atrF, AfuMDR1, AfuMDR3, and AfuMDR4/Cyp51A, atrF, AfuMDR2, AfuMDR4 genes was observed in the isolates without azoleresistance- related mutations, respectively. No significant overexpression was seen in the isolates with T34/L98H except for the AfuMDR3 and AfuMDR4(P<0.05). Our results support the hypothesis that efflux pump transporters can contribute to voriconazole resistance in A. fumigatus.

Involvement of the RND efflux pump transporter SmeH in the acquisition of resistance to ceftazidime in Stenotrophomonas maltophilia

The emergence of antibiotic resistant Gram-negative bacteria has become a serious global health issue. In this study, we have employed the intrinsically resistant opportunistic pathogen Stenotrophomonas maltophilia as a model to study the mechanisms involved in the acquisition of mutation-driven resistance to antibiotics. To this aim, laboratory experimental evolution studies, followed by whole-genome sequencing, were performed in the presence of the third-generation cephalosporin ceftazidime. Using this approach, we determined that exposure to increasing concentrations of ceftazidime selects high-level resistance in S. maltophilia through a novel mechanism: amino acid substitutions in SmeH, the transporter protein of the SmeGH RND efflux pump. The recreation of these mutants in a wild-type background demonstrated that, in addition to ceftazidime, the existence of these substitutions provides bacteria with cross-resistance to other beta-lactam drugs. This acquired resistance does not impose relevant fitness costs when bacteria grow in the absence of antibiotics. Structural prediction of both amino acid residues points that the observed resistance phenotype could be driven by changes in substrate access and recognition.