Biofilm Formation Restrained by Subinhibitory Concentrations of Tigecyclin in Acinetobacter baumannii Is Associated with Downregulation of Efflux Pumps

Chemotherapy ◽  
2016 ◽  
Vol 62 (2) ◽  
pp. 128-133 ◽  
Author(s):  
Huale Chen ◽  
Jianming Cao ◽  
Cui Zhou ◽  
Haiyang Liu ◽  
Xiaoxiao Zhang ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Real Time ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Extracellular Dna ◽  
Proteinase K ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Subinhibitory Concentrations

Background: Tigecycline, one of the few therapeutic options against multidrug-resistant Acinetobacter baumannii, reaches subinhibitory serum concentrations only with cautious clinical dosing and pharmacokinetics. Subinhibitory concentrations of tigecycline might induce an A. baumannii biofilm. Methods: Biofilm formation was assessed via the crystal violet staining method. We further analyzed the main biofilm components with NaIO4, proteinase K, and DNase. Real-time RT-PCR was applied for quantitative detection of biofilm potential-associated genes. Results: In this study, A. baumannii proved to be a strong biofilm producer, and we found that proteins and extracellular DNA are crucial components of the A. baumannii biofilm. Quantitative real-time RT-PCR revealed positive correlations between biofilm formation restrained by subinhibitory concentrations of tigecycline and the expression of biofilm potential-associated genes, especially the AdeFGH efflux pump gene. Conclusion: Our results suggest that downregulation of efflux pumps, especially the AdeFGH efflux pump, is probably responsible for the decline in biofilm formation in A. baumannii treated with subinhibitory concentrations of tigecyclin.

scholarly journals Effect of Titanium Dioxide Nanoparticles on the Expression of Efflux Pump and Quorum-Sensing Genes in MDR Pseudomonas aeruginosa Isolates

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 625
Author(s):  
Fatma Y. Ahmed ◽  
Usama Farghaly Aly ◽  
Rehab Mahmoud Abd El-Baky ◽  
Nancy G. F. M. Waly
Keyword(s):  
Titanium Dioxide ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Titanium Dioxide Nanoparticles ◽  
Sol Gel ◽  
Efflux Pumps ◽  
Antibiofilm Activity ◽  
The Impact

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.

Rapid and quantitative detection of mumps virus RNA by one-step real-time RT-PCR

2004 ◽  
Vol 49 (2) ◽  
pp. 83-88 ◽  
Author(s):  
Ayhan Kubar ◽  
Mehmet Yapar ◽  
Bulent Besirbellioglu ◽  
I.Yasar Avci ◽  
Cakır Guney
Keyword(s):  
Real Time ◽  
Mumps Virus ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Virus Rna ◽  
One Step

scholarly journals Strain variation in Bacillus cereus biofilms and their susceptibility to extracellular matrix-degrading enzymes

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0245708
Author(s):  
Eun Seob Lim ◽  
Seung-Youb Baek ◽  
Taeyoung Oh ◽  
Minseon Koo ◽  
Joo Young Lee ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Cereus ◽  
Biofilm Formation ◽  
Dnase I ◽  
Extracellular Dna ◽  
Proteinase K ◽  
Strain Variation ◽  
Protein Ratio ◽  
Degrading Enzymes ◽  
Matrix Degrading Enzymes

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi (BC4, BC10, and BC72) and the ATCC 10987 reference strain were incubated at 30°C to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional and imaging analyses of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.

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

2015 ◽  
Vol 59 (8) ◽  
pp. 4817-4825 ◽  
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.

scholarly journals Systematic Analysis of Efflux Pump-Mediated Antiseptic Resistance in Staphylococcus aureus Suggests a Need for Greater Antiseptic Stewardship

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Patrick T. LaBreck ◽  
Audrey C. Bochi-Layec ◽  
Joshua Stanbro ◽  
Gina Dabbah-Krancher ◽  
Mark P. Simons ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Transcriptional Analysis ◽  
Systematic Analysis ◽  
Daily Lives ◽  
Single Strain ◽  
Content Type ◽  
Subinhibitory Concentrations ◽  
Different Strains

ABSTRACT Staphylococcus aureus-associated infections can be difficult to treat due to multidrug resistance. Thus, infection prevention is critical. Cationic antiseptics, such as chlorhexidine (CHX) and benzalkonium chloride (BKC), are liberally used in health care and community settings to prevent infection. However, increased administration of antiseptics has selected for S. aureus strains that show reduced susceptibilities to cationic antiseptics. This increased resistance has been associated with carriage of specific efflux pumps (QacA, QacC, and NorA). Since prior published studies focused on different strains and on strains carrying only a single efflux gene, the relative importance of these various systems to antiseptic resistance is difficult to ascertain. To overcome this, we engineered a collection of isogenic S. aureus strains that harbored norA, qacA, and qacC, individually or in combination. MIC assays showed that qacA was associated with increased resistance to CHX, cetrimide (CT), and BKC, qacC was associated with resistance to CT and BKC, and norA was necessary for basal-level resistance to the majority of tested antiseptics. When all three pumps were present in a single strain, an additive effect was observed in the MIC for CT. Transcriptional analysis revealed that expression of qacA and norA was significantly induced following exposure to BKC. Alarmingly, in a strain carrying qacA and norA, preexposure to BKC increased CHX tolerance. Overall, our results reveal increased antiseptic resistance in strains carrying multiple efflux pumps and indicate that preexposure to BKC, which is found in numerous daily-use products, can increase CHX tolerance. IMPORTANCE S. aureus remains a significant cause of disease within hospitals and communities. To reduce the burden of S. aureus infections, antiseptics are ubiquitously used in our daily lives. Furthermore, many antiseptic compounds are dual purpose and are found in household products. The increased abundance of antiseptic compounds has selected for S. aureus strains that carry efflux pumps that increase resistance to antiseptic compounds; however, the effect of carrying multiple pumps within S. aureus is unclear. We demonstrated that an isogenic strain carrying multiple efflux pumps had an additive resistance phenotype to cetrimide. Moreover, in a strain carrying qacA and norA, increased chlorhexidine tolerance was observed after the strain was preexposed to subinhibitory concentrations of a different common-use antiseptic. Taken together, our findings demonstrate cooperation between antiseptic resistance efflux pumps and suggest that their protective phenotype may be exacerbated by priming with subinhibitory concentrations of household antiseptics.

Tigecycline efflux in Acinetobacter baumannii is mediated by TetA in synergy with RND-type efflux transporters

2020 ◽  
Vol 75 (5) ◽  
pp. 1135-1139 ◽  
Author(s):  
Wuen Ee Foong ◽  
Jochen Wilhelm ◽  
Heng-Keat Tam ◽  
Klaas M Pos
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Gene Knockout ◽  
Drug Susceptibility ◽  
Major Facilitator Superfamily ◽  
Rt Pcr ◽  
E Coli ◽  
Rnd Efflux Pump ◽  
Major Facilitator

Abstract Objectives To investigate the role of Major Facilitator Superfamily (MFS)-type transporters from Acinetobacter baumannii AYE in tigecycline efflux. Methods Two putative tetracycline transporter genes of A. baumannii AYE (tetA and tetG) were heterologously expressed in Escherichia coli and drug susceptibility assays were conducted with tigecycline and three other tetracycline derivatives. The importance of TetA in tigecycline transport in A. baumannii was determined by complementation of tetA in WT and Resistance Nodulation cell Division (RND) gene knockout strains of A. baumannii ATCC 19606. Gene expression of the MFS-type tetA gene and RND efflux pump genes adeB, adeG and adeJ in A. baumannii AYE in the presence of tigecycline was analysed by quantitative real-time RT–PCR. Results Overproduction of TetA or TetG conferred resistance to doxycycline, minocycline and tetracycline in E. coli. Cells expressing tetA, but not those expressing tetG, conferred resistance to tigecycline, implying that TetA is a determinant for tigecycline transport. A. baumannii WT and RND-knockout strains complemented with plasmid-encoded tetA are significantly less susceptible to tigecycline compared with non-complemented strains. Efflux pump genes tetA and adeG are up-regulated in A. baumannii AYE in the presence of subinhibitory tigecycline concentrations. Conclusions TetA plays an important role in tigecycline efflux of A. baumannii by removing the drug from cytoplasm to periplasm and, subsequently, the RND-type transporters AdeABC and AdeIJK extrude tigecycline across the outer membrane. When challenged with tigecycline, tetA is up-regulated in A. baumannii AYE. Synergy between TetA and the RND-type transporters AdeABC and/or AdeIJK appears necessary for A. baumannii to confer higher tigecycline resistance via drug efflux.

Quantitative Detection of AML1-ETO Rearrangement by Real-Time RT-PCR Using Fluorescently Labeled Probes

2001 ◽  
Vol 42 (4) ◽  
pp. 747-756 ◽  
Author(s):  
E. Barragán ◽  
P. Bolufer ◽  
I. Moreno ◽  
G. Martín ◽  
J. Nomdedeu ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Detection ◽  
Rt Pcr

scholarly journals Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli

2015 ◽  
Vol 197 (15) ◽  
pp. 2479-2488 ◽  
Author(s):  
Rajeev Misra ◽  
Keith D. Morrison ◽  
Hyun Jae Cho ◽  
Thanh Khuu
Keyword(s):  
Escherichia Coli ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Real Time ◽  
Efflux Pump ◽  
Membrane Integrity ◽  
Efflux Pumps ◽  
Permeability Barrier ◽  
Multidrug Efflux ◽  
Outer Membrane Permeability

ABSTRACTThe constitutively expressed AcrAB multidrug efflux system ofEscherichia colishows a high degree of homology with the normally silent AcrEF system. Exposure of a strain withacrABdeleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAβN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAβN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAβN. Strong action of PAβN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAβN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are compounded in cells unable to extrude PAβN.IMPORTANCEThe increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds.

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