Resistance-nodulation-division Research Articles

Molecular insight into the transport of TMMs by MmpL3 in Mtb

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) and poses a severe threat to human health. Therefore, finding new anti-TB drugs and targets is of great importance. One of the targets is Mycobacterial membrane protein Large 3 (MmpL3), a member of the resistance-nodulation-division (RND) class of transporters located in the inner membrane (IM). The MmpL3 protein is critical for transporting trehalose monomycolates (TMMs), the most abundant outer membrane lipids of Mtb, from the IM to the periplasmic space.

Metagenomic and Recombination Analyses of Antimicrobial Resistance Genes from Recreational Waters of Black Sea Coastal Areas and Other Marine Environments Unveil Extensive Evidence for Their both Intrageneric and Intergeneric Transmission across Genetically Very Diverse Microbial Communities.

Microbial communities of marine coastal recreation waters have become large reservoirs of AMR genes (ARGs), contributing to the emergence and transmission of various zoonotic, foodborne and other infections that exhibit resistance to various antibiotics. Thus, it is highly imperative to determine ARGs assemblages as well as mechanisms and trajectories of their transmission across these microbial communities for our better understanding of the evolutionary trends of AMR (AMR). In this study, using metagenomics approaches, we screened for ARGs in recreation waters of the Black Sea coastal areas of the Batumi City (Georgia). Also, a large array of the recombination detection algorithms of the SplitsTree, RDP4, and GARD was applied to elucidate genetic recombination of ARGs and trajectories of their transmission across various marine microbial communities. The metagenomics analyses of sea water samples, obtained from across the above marine sites, could identify putative ARGs encoding for multidrug resistance efflux transporters mainly from the Major Facilitator and Resistance Nodulation Division superfamilies. The data, generated by SplitsTree (fit ≥95.619; bootstrap values ≥ 95; Phi p≤0.0494), RDP4 (p≤0.0490), and GARD, provided strong statistical evidence not only for intrageneric recombination of these ARGs, but also for their intergeneric recombination across fairly large and diverse microbial communities of marine environment. These bacteria included both human pathogenic and nonpathogenic species, exhibiting collectively the genera of Vibrio, Aeromonas, Synechococcus, Citromicrobium, Rhodobacteraceae, Pseudoalteromonas, Altererythrobacter, Erythrobacter, Altererythrobacter, Marivivens, Xuhuaishuia, and Loktanella. The above nonpathogenic bacteria are strongly suggested to contribute to ARGs transmission in marine ecosystems.

Metformin Reverses tmexCD1-toprJ1- and tet(A)-Mediated High-Level Tigecycline Resistance in K. pneumoniae.

Tigecycline (TIG) is one of the last effective options against multidrug resistance bacteria. Recently, the RND (resistance–nodulation–division) efflux pump gene cluster, tmexCD1-toprJ1, and the tetracycline-efflux pump tet(A) mutation were reported to mediate high level resistance to TIG in clinically important pathogens, weakening the efficacy of TIG. In this study, we report the potent synergistic effect of the antidiabetic drug metformin in combination with TIG against tet(A) mutant and tmexCD1-toprJ1 positive K. pneumoniae. The fractional inhibitory concentration index (FICI) of TIG and metformin were less than 0.05 for all the tested isolates. The time–kill curve assay showed that the combination of TIG and metformin exhibited much better antimicrobial effect than TIG alone. The synergistic effect was also confirmed in vivo using a well-studied Galleria mellonella larvae model. Mechanistic studies demonstrated that metformin disrupted the important component of proton motive force, the electric potential (Δψ) and the function of efflux pump, thereby increasing the intracellular concentration of TIG. This finding revealed that metformin might be a possible adjuvant of TIG for combating with superbugs carrying the tet(A) mutant and tmexCD1-toprJ1 genes.

Role of AxyABM overexpression in acquired resistance in Achromobacter xylosoxidans.

Acquired antimicrobial resistance among Achromobacter isolates from cystic fibrosis (CF) patients is frequent. Data concerning the mechanisms involved are scarce. The role of the AxyXY-OprZ and AxyEF-OprN Resistance Nodulation Division (RND) efflux systems has been demonstrated, but not that of AxyABM. To explore the role of efflux systems in the acquired multiresistance observed in a one-step mutant selected after ofloxacin exposure. The in vitro resistant mutant NCF-39-Bo2 and its parental strain NCF-39 (MICs of meropenem of 8 and 0.19 mg/L, of ceftazidime of 12 and 3 mg/L, of cefiderocol of 0.094 and 0.032 mg/L and of ciprofloxacin of 8 and 1.5 mg/L, respectively) were investigated by RNA-seq and WGS. Gene inactivation and reverse transcription quantitative PCR (RT-qPCR) were used to explore the role of the efflux systems of interest. RNA-seq showed that the AxyABM efflux system was overproduced (about 40-fold) in the in vitro mutant NCF-39-Bo2 versus its parental strain NCF-39. A substitution in AxyR, the putative regulator of AxyABM, was detected in NCF-39-Bo2. Gene inactivation of axyB (encoding the transporter component) in NCF-39-Bo2 led to a decrease in MICs of ciprofloxacin (5-fold), meropenem (64-fold), ceftazidime (12-fold) and cefiderocol (24-fold). Inactivation of axyB in the clinical isolate AXX-H2 harbouring a phenotype of resistance close to that of NCF-39-Bo2 enhanced the activity of the same molecules, especially meropenem. AxyABM overproduction is involved in acquired resistance of Achromobacter to ciprofloxacin, meropenem and ceftazidime, antibiotics widely used in CF patients, and increases the MIC of the new promising antibiotic cefiderocol.

Reducing bacterial antibiotic resistance by targeting bacterial metabolic pathways and disrupting RND efflux pump activity

Antibiotic resistance is a significant issue for the medical community, worldwide. Many bacteria develop drug resistance by utilizing multidrug resistant or MDR efflux pumps that can export antibiotics from bacterial cells. Antibiotics are expelled from bacteria by efflux pumps a part of the resistance nodulation division (RND) family. Types of RND efflux pumps include the AcrAB-TolC tripartite protein pump. There are an excessive number of antibiotic compounds that have been discovered; however, only a few antibiotics are effective against MDR bacteria. Many bacteria become drug resistant when sharing genes that encode MDR efflux pump expression. MDR efflux pump encoding genes are incorporated into plasmids and then shared among bacteria. As a consequence, advancements in genetic engineering can sufficiently target and edit pathogenic bacterial genomes for perturbing drug resistance mechanisms. In this perspective and review, support will be provided for utilizing genetic modifications as an antimicrobial approach and tool that may effectively combat bacterial MDR. Ayhan et al. found that deleting acrB, acrA, and tolC increased the levels of antibiotic sensitivity in Escherichia coli. Researchers also found that glucose, glutamate, and fructose all induced the absorption of antibiotics by upregulating the gene expression of maeA and maeB that is a part of the MAL-pyruvate pathway. Therefore, the current perspective and review will discuss the potential efficacy of reducing antibiotic resistance by inhibiting genes that encode efflux protein pump expression while simultaneously upregulating metabolic genes for increased antibiotic uptake.

Characterization and Molecular Determinants for β-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium.

Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.

Subinhibitory Concentrations of Clinically-Relevant Antimicrobials Affect Resistance-Nodulation-Division Family Promoter Activity in Acinetobacter baumannii.

Efflux pumps contribute to multidrug resistance in Acinetobacter baumannii due to their ability to expel a wide variety of structurally unrelated compounds. This study aimed to characterize the effect of subinhibitory concentrations of clinically-relevant antibiotics and disinfectants on the promoter activity of members of the Resistance-Nodulation-Division (RND) family in A. baumannii. The promoter regions from three RND efflux pumps (AdeABC, AdeFGH and AdeIJK) and the AdeRS regulatory system from three different A. baumannii strains (ATCC 17961, ATCC 17978, and ATCC 19606) were cloned into a luciferase reporter system (pLPV1Z). Promoter activity was quantitatively assessed in both exponential and stationary phase cultures after exposure to subinhibitory concentrations of four antibiotics from different classes (rifampicin, meropenem, tigecycline and colistin) and two disinfectants (ethanol and chlorhexidine). Subinhibitory concentrations of the compounds tested had variable effects on promoter activity that were highly dependent on the A. baumannii strain, the compound tested and the growth phase. Fold changes in AdeABC promoter activity ranged from 1.97 to 113.7, in AdeFGH from −5.6 to 1.13, in AdeIJK from −2.5 to 2, and in AdeRS from −36.2 to −1.32. Taken together, these results indicate that subinhibitory concentrations of clinically-relevant antibiotics and disinfectants affect the promoter activity of RND family members in A. baumannii in a strain and growth phase dependent manner. These results may have important implications for the treatment of infections caused by A. baumannii.

Transport Across Two Membrane Bilayers in E. coli by Efflux Pumps of Different Dimensions

AcrAB-TolC and CusBAC are two of the most well-studied Resistance-Nodulation-Division (RND) family tripartite efflux pumps in E. coli. AcrAB-TolC is a multidrug efflux system, while CusBAC transports Cu(I), Cu(II) and Ag(I). The RND pump complexes span both the inner membrane (IM) and the outer membrane (OM). The long axis dimension of the fully assembled AcrAB-TolC is ∼3nm longer than that of CusBAC. To probe if these two efflux systems with different dimensions affect each other when they need to work simultaneously in the same cell, two real-time assays were used to monitor the efflux activities of these two pumps and their impact on each other. The results showed that the presence of AcrAB-TolC substrates accelerated the accumulation of Cu(I) in BW25113 but not in BW25113ΔcusBA or BW25113ΔtolC strains. Similarly, the presence of Ag(I) slowed down the Nile red efflux in the parent strain more significantly than in the CusBA deficient mutant. To further investigate the impact of the OM/IM distance on the function of these tripartite complexes, we experimented with strains lacking the lipoprotein Lpp or containing Lpp mutant of different lengths. Data from efflux/accumulation assays and susceptibility tests revealed that mutation of Lpp resulted in functional deficiency of both AcrAB-TolC and CusBAC. In conclusion, this study demonstrated that when AcrAB-TolC and CusBAC functioned simultaneously, it took the cell a few minutes to adjust. Furthermore, the presence of Lpp of proper length is important to support full efflux activity of transporters spanning both membrane layers in E. coli.

Profile of Bacteria with ARGs Among Real-World Samples from ICU Admission Patients with Pulmonary Infection Revealed by Metagenomic NGS.

BackgroundTreatment of pulmonary infections in the intensive care unit (ICU) represents a great challenge, especially infections caused by antibiotic resistance pathogens. A thorough and up-to-date knowledge of the local spectrum of antibiotic resistant bacteria can improve the antibiotic treatment efficiency. In this study, we aimed to reveal the profile of bacteria with antibiotic resistance genes (ARGs) in real-world samples from ICU admission patients with pulmonary infection in Mainland, China, by metagenomic next-generation sequencing (mNGS).MethodsA total of 504 different types of clinical samples from 452 ICU admission patients with pulmonary infection were detected by mNGS analysis.ResultsA total of 485 samples from 434 patients got successful mNGS results. Among 434 patients, one or more bacteria with ARGs were detected in 192 patients (44.24%, 192/434), and ≥2 bacteria with ARGs were detected in 85 (19.59%, 85/434) patients. The predominant detected bacteria were Corynebacterium striatum (C. striatum) (11.76%, 51/434), Acinetobacter baumannii (A. baumannii) (11.52%, 50/434) and Enterococcus faecium (E. faecium) (8.99%, 39/434). ermX conferred resistance to MSLB and cmx to phenicol were the only two ARGs detected in C. striatum; in A. baumannii, most of ARGs were resistance-nodulation-division (RND)-type efflux pumps genes, which conferred resistance to multi-drug; ermB conferred resistance to MSLB and efmA to multi-drug were the predominant ARGs in E. faecium. Bacteria with ARGs were detected in 50% (140/280) bronchoalveolar lavage fluid (BALF) and 50.5% (48/95) sputum samples, which were significantly higher than in blood and cerebrospinal fluid (CSF) samples.ConclusionHigh level of bacteria with ARGs was observed in clinical samples, especially BALF and sputum samples from ICU admission patients with pulmonary infection in Mainland, China. And C. striatum resistant to MSLB and/or phenicol, multi-drug resistance A. baumannii and E. faecium were the lead bacteria.

Characterization of IncHI1B Plasmids Encoding Efflux Pump TmexCD2-ToprJ2 in Carbapenem-Resistant Klebsiella variicola, Klebsiella quasipneumoniae, and Klebsiella michiganensis Strains.

Tigecycline serves as one of the last-resort antibiotics to treat severe infections caused by carbapenem-resistant Enterobacterales. Recently, a novel plasmid-mediated resistance-nodulation-division (RND)-type efflux pump gene cluster, TmexCD1-ToprJ1, and its variants, TmexCD2-ToprJ2 and TmexCD3-ToprJ3, encoding tetracyclines and tigecycline resistance, were revealed. In this study, we reported three TmexCD2-ToprJ2-harboring Klebsiella species strains, collected from two teaching tertiary hospitals in China, including one K. quasipneumoniae, one K. variicola, and one K. michiganensis. The three strains were characterized by antimicrobial susceptibility testing (AST), conjugation assay, WGS, and bioinformatics analysis. AST showed that K. variicola and K. quasipneumoniae strains were resistant to tigecycline with MIC values of 4μg/ml, whereas the K. michiganensis was susceptible to tigecycline with an MIC value of 1μg/ml. The TmexCD2-ToprJ2 clusters were located on three similar IncHI1B plasmids, of which two co-harbored the metallo-β-lactamase gene blaNDM-1. Conjugation experiments showed that all three plasmids were capable of self-transfer via conjugation. Our results showed, for the first time, that this novel plasmid-mediated tigecycline resistance mechanism TmexCD2-ToprJ2 has spread into different Klebsiella species, and clinical susceptibility testing may fail to detect. The co-occurrence of blaNDM-1 and TmexCD2-ToprJ2 in the same plasmid is of particular public health concern as the convergence of “mosaic” plasmids can confer both tigecycline and carbapenem resistance. Its further spread into other clinical high-risk Klebsiella clones will likely exacerbate the antimicrobial resistance crisis. A close monitoring of the dissemination of TmexCD-ToprJ encoding resistance should be considered.

Overexpression of Resistance-Nodulation-Division Efflux Pump Genes Contributes to Multidrug Resistance in Aeromonas hydrophila Clinical Isolates.

Aeromonas hydrophila is a Gram-negative bacterium that is a critical causative agent of infections in fish and is occasionally responsible for human infections following contact with contaminated water or food. Currently, the extensive use of antibiotics in clinical practice has led to increased number of isolates of multidrug-resistant (MDR) Aeromonas and has posed a serious public health challenge. The efflux pump system is a critical mechanism of antibiotic resistance in most Gram-negative bacteria. However, the role of resistance-nodulation-division (RND)-type efflux pumps in MDR A. hydrophila is not fully understood. We aimed to evaluate the contribution of the RND efflux pump system to MDR A. hydrophila clinical isolates. PCR results indicated a considerable variation in the presence of RND efflux pump genes in clinical isolates compared to that of the environmental reference strain ATCC7966T. Compared to non-MDR clinical isolates, the expression levels of three putative RND efflux pump genes, AHA0021, AHA1320, and AheB, were significantly elevated in MDR strains. The minimal inhibitory concentrations of piperacillin/tazobactam, imipenem, erythromycin, and polymyxin B were significantly reduced by phenylalanine-arginine β-naphthylamide (PAβN), further supporting the contribution of the RND efflux system in MDR A. hydrophila. We provided evidence supporting the contribution of the RND efflux system to multidrug resistance in A. hydrophila clinical isolates. Further studies are warranted to elucidate the detailed mechanisms that confer intrinsic resistance to antimicrobials in A. hydrophila.

Molecular Characterization of Resistance-Nodulation-cell Division Efflux Pump Genes in Multidrug-Resistant Acinetobacter baumannii.

Introduction:Multidrug-resistant Acinetobacter baumannii is galloping, posing threat to tackle, and leaving us with limited options of treatment.Methods:The purpose of this study is to find the genotypic association in drug-resistant A. baumannii isolated from different sterile body fluids. Matrix-assisted laser desorption/ionization–time of flight confirmed A. baumannii isolates were taken and minimum inhibitory concentration (MIC) was determined by VITEK-2 AST system. The presence of resistance nodulation–division (RND)-efflux pump genes AdeABC-RS was detected by multiplex polymerase chain reaction.Results:Of the total 40 A. baumannii, 32 (80%) were multidrug resistant though all isolates were susceptible to Tigecycline. Similarly, 26 (81.25%) isolates were positive for RND-efflux pump genes AdeABC-RS.Discussion:RND efflux pump AdeABC-RS system plays a significant role in emerging multi drug resistant A. baumannii. Mutation in AdeS gene deciphers the role of regulatory gene. Hence, antimicrobial stewardship should be strictly followed and efflux pump inhibiting substances should be vigorously searched to bring back the era of existing antibiotics.

Importance of twitching and surface-associated motility in the virulence of Acinetobacter baumannii

ABSTRACT Acinetobacter baumannii is a pathogen of increasing clinical importance worldwide, especially given its ability to readily acquire resistance determinants. Motile strains of this bacterium can move by either or both of two types of motility: (i) twitching, driven by type IV pili, and (ii) surface-associated motility, an appendage-independent form of movement. A. baumannii strain MAR002 possesses both twitching and surface-associated motility. In this study, we isolated spontaneous rifampin-resistant mutants of strain MAR002 in which point mutations in the rpoB gene were identified that resulted in an altered motility pattern. Transcriptomic analysis of mutants lacking twitching, surface-associated motility, or both led to the identification of deregulated genes within each motility phenotype, based on their level of expression and their biological function. Investigations of the corresponding knockout mutants revealed several genes involved in the motility of A. baumannii strain MAR002, including two involved in twitching (encoding a minor pilin subunit and an RND [resistance nodulation division] component), one in surface-associated motility (encoding an amino acid permease), and eight in both (encoding RND and ABC components, the energy transducer TonB, the porin OprD, the T6SS component TagF, an IclR transcriptional regulator, a PQQ-dependent sugar dehydrogenase, and a putative pectate lyase). Virulence assays showed the reduced pathogenicity of mutants with impairments in both types of motility or in surface-associated motility alone. By contrast, the virulence of twitching-affected mutants was not affected. These results shed light on the key role of surface-associated motility and the limited role of twitching in the pathogenicity of A. baumannii.

Dispatching plasma membrane cholesterol and Sonic Hedgehog dispatch: two sides of the same coin?

Vertebrate and invertebrate Hedgehog (Hh) morphogens signal over short and long distances to direct cell fate decisions during development and to maintain tissue homeostasis after birth. One of the most important questions in Hh biology is how such Hh signaling to distant target cells is achieved, because all Hh proteins are secreted as dually lipidated proteins that firmly tether to the outer plasma membrane leaflet of their producing cells. There, Hhs multimerize into light microscopically visible storage platforms that recruit factors required for their regulated release. One such recruited release factor is the soluble glycoprotein Scube2 (Signal sequence, cubulin domain, epidermal-growth-factor-like protein 2), and maximal Scube2 function requires concomitant activity of the resistance-nodulation-division (RND) transporter Dispatched (Disp) at the plasma membrane of Hh-producing cells. Although recently published cryo-electron microscopy-derived structures suggest possible direct modes of Scube2/Disp-regulated Hh release, the mechanism of Disp-mediated Hh deployment is still not fully understood. In this review, we discuss suggested direct modes of Disp-dependent Hh deployment and relate them to the structural similarities between Disp and the related RND transporters Patched (Ptc) and Niemann-Pick type C protein 1. We then discuss open questions and perspectives that derive from these structural similarities, with particular focus on new findings that suggest shared small molecule transporter functions of Disp to deplete the plasma membrane of cholesterol and to modulate Hh release in an indirect manner.

Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae

OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-β-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 Å resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.

Structure-function analysis of MmpL7-mediated lipid transport in mycobacteria

Mycobacterial membrane protein Large (MmpL7) is a Resistance-Nodulation-Division (RND) family transporter required for the export of the virulence lipid, phthiocerol dimycocerosate (PDIM), in Mycobacterium tuberculosis. Using a null mutant of the related, vaccine strain Mycobacterium bovis BCG, we show that MmpL7 is also involved in the transport of the structurally related phenolic glycolipid (PGL), which is also produced by the hypervirulent M. tuberculosis strain HN878, but absent in M. tuberculosis H37Rv. Furthermore, we generated an in silico model of M. tuberculosis MmpL7 that revealed MmpL7 as a functional outlier within the MmpL-family, missing a canonical proton-relay signature sequence, suggesting that it employs a yet-unidentified mechanism for energy coupling for transport. In addition, our analysis demonstrates that the periplasmic porter domain 2 insert (PD2-insert), which doesn't share any recognisable homology, is highly alpha-helical in nature, suggesting an organisation similar to that seen in the hopanoid PD3/4 domains. Using the M. bovis BCG mmpL7 mutant for functional complementation with mutated alleles of mmpL7, we were able to identify residues present in the transmembrane domains TM4 and TM10, and the PD2 domain insert that play a crucial role in PDIM transport, and in certain cases, biosynthesis of PDIM.

Conserved cholesterol-related activities of Dispatched 1 drive Sonic hedgehog shedding from the cell membrane.

ABSTRACTThe Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-standing questions about this pathway include how the dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells and how the resistance–nodulation–division transporter Dispatched 1 (Disp, also known as Disp1) regulates this process. Here, we show that inactivation of Disp in Shh-expressing human cells impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. We also show that cholesterol export from Disp-deficient cells is reduced, that these cells contain increased cholesterol amounts in the plasma membrane, and that Shh shedding from Disp-deficient cells is restored by pharmacological membrane cholesterol extraction and by overexpression of transgenic Disp or the structurally related protein Patched 1 (Ptc, also known as Ptch1; a putative cholesterol transporter). These data suggest that Disp can regulate Shh function via controlled cell surface shedding and that membrane cholesterol-related molecular mechanisms shared by Disp and Ptc exercise such sheddase control.

Evaluation of efflux pumps overexpression and β-lactamase genes among colistin resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is one of the major factors in severe opportunistic and hospital-acquired infections. Structural mechanisms such as resistance–nodulation–division (RND) efflux pumps and drug resistance genes such as extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) are factors causing drug resistance. Colistin is the prominent therapeutic strategy with an excellent inhibitory role for combating multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates. Wide usage of colistin and genetics interactions leads to the emergence of colistin-resistant (CoR) PA isolates which is a dangerous occurrence in XDR-PA isolates. The current study investigates the presence and expression of PA-related RND genes and harboring the antibiotic resistance genes such as ESBL and MBL genes in CoR-PA strains. 70 clinical isolates were obtained from different clinical specimens during 2019–2020. The antimicrobial susceptibility testing was performed by the both Kirby-Bauer disk agar diffusion method and the minimum inhibitory concentration (MIC). Presence of bla TEM-1 , bla SHV , bla CTX-M , bla IMP , bla VIM and bla NDM was measured in all CoR-PA isolates. Besides, screening of mcr-1 , aadA , aadB and efflux pump genes ( mexA , mexD , mexE and mexY ) was conducted for CoR-PA isolates. To determine the expression level of efflux pump genes, Quantitative real-time PCR (qRT-PCR) reactions were performed. According to the standard antimicrobial susceptibility methods 39 isolates, (55.7%) and 31 isolates (44.3%) were considered as MDR-PA and XDR-PA strains, respectively. 12 isolates (17.1%) displayed resistance to colistin using MIC assessment. All CoR-PA isolates were negative for mcr-1 gene. The bla SHV (n = 11, 91.6%) and bla NDM (n = 7, 58.3%) were the most prevalent ESBL and MBL genes among CoR-PA, respectively. PCR results showed all CoR-PA isolates were positive for all 4 efflux pumps genes. On the other hand, overexpression of MexA , MexD , MexE and MexY genes was observed in 5 (41.6%), 5 (41.6%), 5 (41.6%) and 6 (50%) CoR-PA isolates by qRT-PCR, respectively. 9 (75%) CoR-PA isolates overexpressed at least one of the four efflux pumps and overexpression of altogether 4 efflux pumps was shown in 2 (16.6%) CoR-PA isolates. Overexpression of MexA , MexD , MexE and MexY genes was associated to the resistance toward imipenem, ciprofloxacin, ceftazidime and aminoglycosides, respectively. The current research demonstrated the co-existence of CoR-PA with ESBL and MBL genes besides overexpression of efflux pumps. The distribution of such isolates can lead to a burden on the healthcare system by the incidence of the most crucial situation named pan-drug resistant PA strains. • --39(55.7%) and 31 (44.3%) isolates -were - MDR-PA and XDR-PA strains, respectively • 12 isolates (17.1%) displayed resistance to colistin using MIC assessment. • - bla SHV (n = 11, 91.6%) and bla NDM (n = 7, 58.3%) were the most prevalent ESBL and MBL genes -, respectively. • PCR results showed all CoR-PA isolates were positive for all 4 efflux pumps genes. • Overexpression of MexA , MexD , MexE and MexY genes was observed in 5 (41.6%), 5 (41.6%), 5 (41.6%) and 6 (50%) - isolates, respectively.

Ever-Adapting RND Efflux Pumps in Gram-Negative Multidrug-Resistant Pathogens: A Race against Time.

The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR is the over-expression of intrinsic and acquired multidrug efflux pumps, belonging to the resistance-nodulation-division (RND) superfamily, which can efflux a wide range of structurally different antibiotics. Besides over-expression, however, recent amino acid substitutions within the pumps themselves—causing an increased drug efflux efficiency—are causing additional worry. In this review, we take a closer look at clinically, environmentally and laboratory-evolved Gram-negative bacterial strains and their decreased drug sensitivity as a result of mutations directly in the RND-type pumps themselves (from Escherichia coli, Salmonella enterica, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii and Legionella pneumophila). We also focus on the evolution of the efflux pumps by comparing hundreds of efflux pumps to determine where conservation is concentrated and where differences in amino acids can shed light on the broad and even broadening drug recognition. Knowledge of conservation, as well as of novel gain-of-function efflux pump mutations, is essential for the development of novel antibiotics and efflux pump inhibitors.

Delineation of cellular stages and identification of key proteins for reduction and biotransformation of Se(IV) by Stenotrophomonas bentonitica BII-R7

The widespread use of selenium (Se) in technological applications (e.g., solar cells and electronic devices) has led to an accumulation of this metalloid in the environment to toxic levels. The newly described bacterial strain Stenotrophomonas bentonitica BII-R7 has been demonstrated to reduce mobile Se(IV) to Se(0)-nanoparticles (Se(0)NPs) and volatile species. Amorphous Se-nanospheres are reported to aggregate to form crystalline nanostructures and trigonal selenium. We investigated the molecular mechanisms underlying the biotransformation of Se(IV) to less toxic forms using differential shotgun proteomics analysis of S. bentonitica BII-R7 grown with or without sodium selenite for three different time-points. Results showed an increase in the abundance of several proteins involved in Se(IV) reduction and stabilization of Se(0)NPs, such as glutathione reductase, in bacteria grown with Se(IV), in addition to many proteins with transport functions, including RND (resistance-nodulation-division) systems, possibly facilitating Se uptake. Notably proteins involved in oxidative stress defense (e.g., catalase/peroxidase HPI) were also induced by Se exposure. Electron microscopy analyses confirmed the biotransformation of amorphous nanospheres to trigonal Se. Overall, our results highlight the potential of S. bentonitica in reducing the bioavailability of Se, which provides a basis both for the development of bioremediation strategies and the eco-friendly synthesis of biotechnological nanomaterials.