Local Repressor AcrR Regulates AcrAB Efflux Pump Required for Biofilm Formation and Virulence in Acinetobacter nosocomialis

Most of the infections caused by multi-drug resistant (MDR) P. aeruginosa strains are extremely difficult to be treated with conventional antibiotics. Biofilm formation and efflux pumps are recognized as the major antibiotic resistance mechanisms in MDR P. aeruginosa. Biofilm formation by P. aeruginosa depends mainly on the cell-to-cell communication quorum-sensing (QS) systems. Titanium dioxide nanoparticles (TDN) have been used as antimicrobial agents against several microorganisms but have not been reported as an anti-QS agent. This study aims to evaluate the impact of titanium dioxide nanoparticles (TDN) on QS and efflux pump genes expression in MDR P. aeruginosa isolates. The antimicrobial susceptibility of 25 P. aeruginosa isolates were performed by Kirby–Bauer disc diffusion. Titanium dioxide nanoparticles (TDN) were prepared by the sol gel method and characterized by different techniques (DLS, HR-TEM, XRD, and FTIR). The expression of efflux pumps in the MDR isolates was detected by the determination of MICs of different antibiotics in the presence and absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Biofilm formation and the antibiofilm activity of TDN were determined using the tissue culture plate method. The effects of TDN on the expression of QS genes and efflux pump genes were tested using real-time polymerase chain reaction (RT-PCR). The average size of the TDNs was 64.77 nm. It was found that TDN showed a significant reduction in biofilm formation (96%) and represented superior antibacterial activity against P. aeruginosa strains in comparison to titanium dioxide powder. In addition, the use of TDN alone or in combination with antibiotics resulted in significant downregulation of the efflux pump genes (MexY, MexB, MexA) and QS-regulated genes (lasR, lasI, rhll, rhlR, pqsA, pqsR) in comparison to the untreated isolate. TDN can increase the therapeutic efficacy of traditional antibiotics by affecting efflux pump expression and quorum-sensing genes controlling biofilm production.

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Bioactive constituents with antibacterial, resistance modulation, anti-biofilm formation and efflux pump inhibition properties from Aidia genipiflora stem bark

Clinical Phytoscience ◽

10.1186/s40816-021-00266-4 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Daniel Anokwah ◽

Evelyn Asante-Kwatia ◽

Abraham Y. Mensah ◽

Cynthia Amaning Danquah ◽

Benjamin K. Harley ◽

...

Keyword(s):

Biofilm Formation ◽

Efflux Pump ◽

Ethyl Acetate Fraction ◽

Stem Bark ◽

Resistance Mechanisms ◽

Bioactive Constituents ◽

Biofilm Inhibition ◽

E Coli ◽

Efflux Pump Inhibition ◽

Oleanonic Acid

Abstract Background Antimicrobial resistance is a global health challenge. The involvement of bacterial biofilms and efflux pumps in the development of multidrug resistance (MDR) is well established. Medicinal plants have been proposed as alternatives for combating MDR focusing on their bioactive constituents with resistance modulatory activities. This study was aimed at investigating the stem bark of Aidia genipiflora for bioactive constituents with anti-biofilm, efflux pump inhibition and resistance modulatory activities. Method The crude methanol extract was purified by column chromatography and isolated compounds characterized by mass and nuclear magnetic resonance spectrometry. Antibacterial activity was determined by the High-throughput spot culture growth inhibition and the broth micro-dilution assay. The ethidium bromide accumulation assay was used to determine efflux pump inhibition property. Biofilm inhibition was determined in a microplate crystal violet retention assay. Results Purification of the ethyl acetate fraction led to the isolation of oleanonic acid (1), 4-hydroxy cinnamic acid docosyl ester (2), β-stigmasterol/β-sitosterol (mixture 3a/b) and D-mannitol (4). The minimum inhibitory concentrations (MICs) ranged from 250 to > 500 μg/mL for extracts and fractions and from 15 to 250 μg/mL for compounds. In the presence of sub-inhibitory concentrations of the compounds, the MIC of amoxicillin against E. coli (20 μg/mL) and P. aeruginosa (320 μg/mL) was reduced by 32 and 10 folds respectively. The whole extract demonstrated anti-biofilm formation and efflux pump inhibition in E. coli, S. aureus and P. aeruginosa. The sterol mixture (3a/b) at concentration of 100 μg/mL caused the highest inhibition (73%) of biofilm formation in S. aureus. Oleanonic acid (1) demonstrated remarkable efflux pump inhibition at MIC of 7.8 μg/mL in E. coli better than the standard drugs verapamil and chlorpromazine. Conclusion This study confirms the prospects of A. genipiflora as a source of new antibacterial agents and adjuvants that could interact with some resistance mechanisms in bacteria to enhance the activity of hitherto ineffective antibiotics. “A small portion of the study has been presented in a conference in the form of poster”.

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Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00456-12 ◽

2012 ◽

Vol 56 (8) ◽

pp. 4450-4458 ◽

Cited By ~ 59

Author(s):

Mark Veleba ◽

Paul G. Higgins ◽

Gerardo Gonzalez ◽

Harald Seifert ◽

Thamarai Schneiders

Keyword(s):

Multidrug Resistance ◽

Klebsiella Pneumoniae ◽

Efflux Pump ◽

Content Type ◽

Resistance Phenotype ◽

Serratia Proteamaculans ◽

Virulence Potential ◽

Article Type ◽

Acrab Efflux Pump

ABSTRACTTranscriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes.Klebsiella pneumoniaeis a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription oframAis associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the availableKlebsiellagenome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded inK. pneumoniae,Enterobactersp. 638,Serratia proteamaculans568, andEnterobacter cloacae. We show that the overexpression ofrarAresults in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show thatrarA(MGH 78578 KPN_02968) and its neighboring efflux pump operonoqxAB(KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest thatrarAoverexpression upregulates theoqxABefflux pump. Additionally, it appears thatoqxR, encoding a GntR-type regulator adjacent to theoqxABoperon, is able to downregulate the expression of theoqxABefflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

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Transporters and Efflux Pumps Are the Main Mechanisms Involved in Staphylococcus epidermidis Adaptation and Tolerance to Didecyldimethylammonium Chloride

Microorganisms ◽

10.3390/microorganisms8030344 ◽

2020 ◽

Vol 8 (3) ◽

pp. 344 ◽

Cited By ~ 1

Author(s):

Urška Ribič ◽

Jernej Jakše ◽

Nataša Toplak ◽

Simon Koren ◽

Minka Kovač ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

Efflux Pump ◽

Thioredoxin Reductase ◽

Multidrug Transporter ◽

Acid Biosynthesis ◽

Tolerance Mechanisms ◽

Down Regulation ◽

Didecyldimethylammonium Chloride ◽

First Time

Staphylococcus epidermidis cleanroom strains are often exposed to sub-inhibitory concentrations of disinfectants, including didecyldimethylammonium chloride (DDAC). Consequently, they can adapt or even become tolerant to them. RNA-sequencing was used to investigate adaptation and tolerance mechanisms of S. epidermidis cleanroom strains (SE11, SE18), with S. epidermidis SE11Ad adapted and S. epidermidis SE18To tolerant to DDAC. Adaptation to DDAC was identified with up-regulation of genes mainly involved in transport (thioredoxin reductase [pstS], the arsenic efflux pump [gene ID, SE0334], sugar phosphate antiporter [uhpT]), while down-regulation was seen for the Agr system (agrA, arC, agrD, psm, SE1543), for enhanced biofilm formation. Tolerance to DDAC revealed the up-regulation of genes associated with transporters (L-cysteine transport [tcyB]; uracil permease [SE0875]; multidrug transporter [lmrP]; arsenic efflux pump [arsB]); the down-regulation of genes involved in amino-acid biosynthesis (lysine [dapE]; histidine [hisA]; methionine [metC]), and an enzyme involved in peptidoglycan, and therefore cell wall modifications (alanine racemase [SE1079]). We show for the first time the differentially expressed genes in DDAC-adapted and DDAC-tolerant S. epidermidis strains, which highlight the complexity of the responses through the involvement of different mechanisms.

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Complete Deletion of theramRGene in anIn Vitro-Selected Mutant of Klebsiella pneumoniae Overexpressing the AcrAB Efflux Pump

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01410-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 672-673 ◽

Cited By ~ 10

Author(s):

Suzanne Bialek-Davenet ◽

Véronique Leflon-Guibout ◽

Olivier Tran Minh ◽

Estelle Marcon ◽

Richard Moreau ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Efflux Pump ◽

Acrab Efflux Pump ◽

Complete Deletion

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Biofilm Formation Caused by Clinical Acinetobacter baumannii Isolates Is Associated with Overexpression of the AdeFGH Efflux Pump

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00877-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4817-4825 ◽

Cited By ~ 58

Author(s):

Xinlong He ◽

Feng Lu ◽

Fenglai Yuan ◽

Donglin Jiang ◽

Peng Zhao ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Gene Transcription ◽

Biofilm Formation ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Content Type ◽

Potential Association ◽

Genes Encoding ◽

Clinical Environments

ABSTRACTChronic wound infections are associated with biofilm formation, which in turn has been correlated with drug resistance. However, the mechanism by which bacteria form biofilms in clinical environments is not clearly understood. This study was designed to investigate the biofilm formation potency ofAcinetobacter baumanniiand the potential association of biofilm formation with genes encoding efflux pumps, quorum-sensing regulators, and outer membrane proteins. A total of 48 clinically isolatedA. baumanniistrains, identified by enterobacterial repetitive intergenic consensus (ERIC)-PCR as types A-II, A-III, and A-IV, were analyzed. Three representative strains, which were designatedA. baumanniiABR2, ABR11, and ABS17, were used to evaluate antimicrobial susceptibility, biofilm inducibility, and gene transcription (abaI,adeB,adeG,adeJ,carO, andompA). A significant increase in the MICs of different classes of antibiotics was observed in the biofilm cells. The formation of a biofilm was significantly induced in all the representative strains exposed to levofloxacin. The levels of gene transcription varied between bacterial genotypes, antibiotics, and antibiotic concentrations. The upregulation ofadeGcorrelated with biofilm induction. The consistent upregulation ofadeGandabaIwas detected in A-III-typeA. baumanniiin response to levofloxacin and meropenem (1/8 to 1/2× the MIC), conditions which resulted in the greatest extent of biofilm induction. This study demonstrates a potential role of the AdeFGH efflux pump in the synthesis and transport of autoinducer molecules during biofilm formation, suggesting a link between low-dose antimicrobial therapy and a high risk of biofilm infections caused byA. baumannii. This study provides useful information for the development of antibiofilm strategies.

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Aspergillus fumigatusAfssn3-Afssn8Pair Reverse Regulates Azole Resistance by Conferring Extracellular Polysaccharide, Sphingolipid Pathway Intermediates, and Efflux Pumps to Biofilm

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01978-17 ◽

2018 ◽

Vol 62 (3) ◽

Cited By ~ 2

Author(s):

Nanbiao Long ◽

Liping Zeng ◽

Shanlei Qiao ◽

Lei Li ◽

Guowei Zhong

Keyword(s):

Amino Acids ◽

Aspergillus Fumigatus ◽

Biofilm Formation ◽

Efflux Pump ◽

Extracellular Polysaccharide ◽

Azole Resistance ◽

Drug Penetration ◽

Reverse Genetic ◽

Content Type ◽

Barrier Layers

ABSTRACTAntifungal treatment is often ineffectual, partly because of biofilm formation. In this study, by using a combined forward and reverse genetic strategy, we identified that nucleus-localized AfSsn3 and its partner AfSsn8, which constitute a Cdk8-cyclin pair, are required for azole resistance inAspergillus fumigatus. Deletion ofAfssn3led to increased absorption and utilization of glucose and amino acids. Interestingly, absorption and utilization of glucose accelerated the extracellular polysaccharide formation, while utilization of the amino acids serine, threonine, and glycine increased sphingolipid pathway intermediate accumulation. In addition, the absence ofAfssn3induced the activity of the efflux pump proteins. These factors indicate the mature biofilm is responsible for the major mechanisms ofA. fumigatusresistance to azoles in the ΔAfssn3mutant. Collectively, the loss ofAfssn3led to two “barrier” layers between the intracellular and extracellular spaces, which consequently decreased drug penetration into the cell.

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Thymol tolerance in Escherichia coli induces morphological, metabolic and genetic changes

BMC Microbiology ◽

10.1186/s12866-019-1663-8 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Fatemah Al-Kandari ◽

Rabeah Al-Temaimi ◽

Arnoud H. M. van Vliet ◽

Martin J. Woodward

Keyword(s):

Antimicrobial Activity ◽

Efflux Pump ◽

Wide Spectrum ◽

Genetic Changes ◽

E Coli ◽

Nmr Data ◽

Stop Mutation ◽

In The Wild ◽

Cellular Integrity ◽

Acrab Efflux Pump

Abstract Background Thymol is a phenolic compound used for its wide spectrum antimicrobial activity. There is a limited understanding of the antimicrobial mechanisms underlying thymol activity. To investigate this, E. coli strain JM109 was exposed to thymol at sub-lethal concentrations and after 16 rounds of exposure, isolates with a 2-fold increased minimal inhibitory concentration (MIC) were recovered (JM109-Thyr). The phenotype was stable after multiple sub-cultures without thymol. Results Cell morphology studies by scanning electron microscopy (SEM) suggest that thymol renders bacterial cell membranes permeable and disrupts cellular integrity. 1H Nuclear magnetic resonance (NMR) data showed an increase in lactate and the lactic acid family amino acids in the wild type and JM109-Thyr in the presence of thymol, indicating a shift from aerobic respiration to fermentation. Sequencing of JM109-Thyr defined multiple mutations including a stop mutation in the acrR gene resulting in a truncation of the repressor of the AcrAB efflux pump. AcrAB is a multiprotein complex traversing the cytoplasmic and outer membrane, and is involved in antibiotic clearance. Conclusions Our data suggests that thymol tolerance in E. coli induces morphological, metabolic and genetic changes to adapt to thymol antimicrobial activity.

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Control of Biofilm Formation in Healthcare: Recent Advances Exploiting Quorum-Sensing Interference Strategies and Multidrug Efflux Pump Inhibitors

Materials ◽

10.3390/ma11091676 ◽

2018 ◽

Vol 11 (9) ◽

pp. 1676 ◽

Cited By ~ 18

Author(s):

Bindu Subhadra ◽

Dong Kim ◽

Kyungho Woo ◽

Surya Surendran ◽

Chul Choi

Keyword(s):

Quorum Sensing ◽

Biofilm Formation ◽

Efflux Pump ◽

Financial Burden ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Persistent Infections ◽

Healthcare Settings ◽

Recent Advances ◽

Multidrug Efflux Pumps

Biofilm formation in healthcare is an issue of considerable concern, as it results in increased morbidity and mortality, imposing a significant financial burden on the healthcare system. Biofilms are highly resistant to conventional antimicrobial therapies and lead to persistent infections. Hence, there is a high demand for novel strategies other than conventional antibiotic therapies to control biofilm-based infections. There are two approaches which have been employed so far to control biofilm formation in healthcare settings: one is the development of biofilm inhibitors based on the understanding of the molecular mechanism of biofilm formation, and the other is to modify the biomaterials which are used in medical devices to prevent biofilm formation. This review will focus on the recent advances in anti-biofilm approaches by interrupting the quorum-sensing cellular communication system and the multidrug efflux pumps which play an important role in biofilm formation. Research efforts directed towards these promising strategies could eventually lead to the development of better anti-biofilm therapies than the conventional treatments.

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