Multidrug Efflux System Research Articles

Multidrug Resistance and Efflux Pumps: Insights from Molecular Dynamics Simulations

Evolution of bacteria resistant to the most diverse antibiotics is posing one of the major threats to public health. Particular alarm is raised by those genetic lines that develop phenotypes simultaneously resistant to multiple drugs. Among the different mechanisms leading to multidrug resistance, multidrug efflux pumps raise particular concern. These are large macromolecular constructs localised at cell boundaries, which are able to actively bind and transport out of the cell several chemically uncorrelated substrates. In this last decade, computer modelling has proved to be a valuable tool for the investigation of multiple drug-efflux systems at the molecular level. In particular, molecular dynamics simulations unveiled several aspects of the molecular mechanisms governing the recognition and transport of drugs by these systems. Computer-aided protocols constitute a bottom-up reductionist approach that has the privilege of obtaining clean data referring intrinsically to those single parts of the efflux process explicitly taken into account. Combining computational data to the experimental determinations may therefore help in the definition of possible general criteria limiting the action of these systems against both patented and new putative antibiotic agents. Here, we review the most relevant contributions by computational scientists to the understanding of multidrug-efflux systems in the recent past. Particular care is put in the description of the dynamical features of multidrug exporters, a valuable piece of information for which computer modelling represents one of the best investigation tools available at present.

Inhibition of multidrug efflux as a strategy to prevent biofilm formation

We have recently shown that inactivation of any of the multidrug efflux systems of Salmonella results in loss of the ability to form a competent biofilm. The aim of this study was to determine the mechanism linking multidrug efflux and biofilm formation, and to determine whether inhibition of efflux is a viable antibiofilm strategy. Mutants lacking components of the AcrAB-TolC system in Salmonella enterica serovar Typhimurium were investigated for their ability to aggregate, produce biofilm matrix components and form a biofilm. The potential for export of a biofilm-relevant substrate via efflux pumps was investigated and expression of genes that regulate multidrug efflux and production of biofilm matrix components was measured. The ability of efflux inhibitors carbonyl cyanide m-chlorophenylhydrazone, chlorpromazine and phenyl-arginine-β-naphthylamide to prevent biofilm formation by Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus under static and flow conditions was assessed. Mutants of Salmonella Typhimurium that lack TolC or AcrB, but surprisingly not AcrA, were compromised in their ability to form biofilms. This defect was not related to changes in cellular hydrophobicity, aggregative ability or export of any biofilm-specific factor. The biofilm defect resulted from transcriptional repression of curli biosynthesis genes and consequent inhibition of production of curli. All three efflux inhibitors significantly reduced biofilm production in both static and flow biofilm assays, although different concentrations of each inhibitor were most active against each species. This work shows that both genetic inactivation and chemical inhibition of efflux pumps results in transcriptional repression of biofilm matrix components and a lack of biofilm formation. Therefore, inhibition of efflux is a promising antibiofilm strategy.

RamA, which controls expression of the MDR efflux pump AcrAB-TolC, is regulated by the Lon protease

ObjectivesRamA regulates the AcrAB-TolC multidrug efflux system. Using Salmonella Typhimurium, we investigated the stability of RamA and its impact on antibiotic resistance.MethodsTo detect RamA, we introduced ramA::3XFLAG::aph into plasmid pACYC184 and transformed this into Salmonella Typhimurium SL1344ramA::cat and lon::aph mutants. An N-terminus-deleted mutant [pACYC184ramA(Δ2-21)::3XFLAG::aph] in which the first 20 amino acids of RamA were deleted was also constructed. To determine the abundance and half-life of FLAG-tagged RamA, we induced RamA with chlorpromazine (50 mg/L) and carried out western blotting using anti-FLAG antibody. Susceptibility to antibiotics and phenotypic characterization of the lon mutant was also carried out.ResultsWe show that on removal of chlorpromazine, a known inducer of ramA, the abundance of RamA decreased to pre-induced levels. However, in cells lacking functional Lon, we found that the RamA protein was not degraded. We also demonstrated that the 21 amino acid residues of the RamA N-terminus are required for recognition by the Lon protease. Antimicrobial susceptibility and phenotypic tests showed that the lon mutant was more susceptible to fluoroquinolone antibiotics, was filamentous when observed by microscopy and grew poorly, but showed no difference in motility or the ability to form a biofilm. There was also no difference in the ability of the lon mutant to invade human intestinal cells (INT-407).ConclusionsIn summary, we show that the ATP-dependent Lon protease plays an important role in regulating the expression of RamA and therefore multidrug resistance via AcrAB-TolC in Salmonella Typhimurium.

Role of a Conserved Residue R780 in Escherichia coli Multidrug Transporter AcrB

Multidrug efflux pumps play important roles in bacteria drug resistance. A major multidrug efflux system in Gram-negative bacteria is composed of the inner membrane transporter AcrB, outer membrane protein channel TolC, and membrane fusion protein AcrA. These three proteins form a large complex that spans both layers of cell membranes and the periplasmic space. AcrB exists and functions as a homotrimer. To identify residues at the trimer interface that play important roles in AcrB function, we conducted site directed mutagenesis and discovered a key residue, R780. Although R780K was partially functional, all other R780 mutants tested were completely nonfunctional. Replacement of R780 by other residues disrupted trimer association. However, a decrease of trimer stability was not the lone cause for the observed loss of activity, because the activity loss could not be restored by strengthening trimer interaction. Using both heat and chemical denaturation methods, we found that the mutation decreased protein stability. Finally, we identified a repressor mutation, M774K, through random mutagenesis. It restored the activity of AcrBR780A to a level close to that of the wild-type protein. To examine the mechanism of activity restoration, we monitored denaturation of AcrBR780A/M774K and found that the repressor mutation improved protein stability. These results suggest that R780 is critical for AcrB stability. When R780 was replaced by Ala, the protein retained the overall structure, still trimerized in the cell membrane, and interacted with AcrA. However, local structural rearrangement might have occurred and lead to the decrease of protein stability and loss of substrate efflux activity.

Salmonella enterica serovar Typhimurium BaeSR two-component system positively regulates sodA in response to ciprofloxacin

In response to antibiotics, bacteria activate regulatory systems that control the expression of genes that participate in detoxifying these compounds, like multidrug efflux systems. We previously demonstrated that the BaeSR two-component system from Salmonella enterica serovar Typhimurium (S. Typhimurium) participates in the detection of ciprofloxacin, a bactericidal antibiotic, and in the positive regulation of mdtA, an efflux pump implicated in antibiotic resistance. In the present work, we provide further evidence for a role of the S. Typhimurium BaeSR two-component system in response to ciprofloxacin treatment and show that it regulates sodA expression. We demonstrate that, in the absence of BaeSR, the transcript levels of sodA and the activity of its gene product are lower. Using electrophoretic mobility shift assays and transcriptional fusions, we demonstrate that BaeR regulates sodA by a direct interaction with the promoter region.

The Synergistic Effect of Visible Light and Gentamycin on <em>Pseudomona aeruginosa</em> Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens(1-5). The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region(4,6,7). There are also reports of biocidal effect of red and near infra red(8) as well as green light(9). In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems(10). The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's. The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The crystal structure of multidrug-resistance regulator RamR with multiple drugs

RamR is a transcriptional repressor of the gene-encoding RamA protein, which controls the expression of the multidrug efflux system genes acrAB-tolC. RamR is an important multidrug-resistance factor, however, its structure and the identity of the molecules to which it responds have been unknown. Here, we report the crystal structure of RamR in complex with multiple drugs, including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G. All compounds are found to interact with Phe155 of RamR, and each compound is surrounded by different amino acid residues. Binding of these compounds to RamR reduces its DNA-binding affinity, which results in the increased expression of ramA. Our results reveal significant flexibility in the substrate-recognition region of RamR, which regulates the bacterial efflux participating in multidrug resistance.

Global Transcriptional and Phenotypic Analyses of Escherichia coli O157:H7 Strain Xuzhou21 and Its pO157_Sal Cured Mutant

Escherichia coli O157:H7 is an important food-borne pathogen that can cause hemorrhagic colitis and hemolytic-uremic syndrome in humans. pO157_Sal, a novel conjugative plasmid is present in a Chinese O157:H7 outbreak strain Xuzhou21. Here we investigated the phenotypic and transcriptional differences between the wild type strain Xuzhou21 and the pO157_Sal cured mutant strain Xuzhou21m. RNA-Seq analysis found that all 52 ORFs encoded on pO157_Sal were transcribed. One hundred and sixty eight chromosomal and pO157 genes were differentially expressed (≥2 fold difference) between Xuzhou21 and Xuzhou21m. Sixty-seven and 101 genes were up-regulated and down-regulated respectively by pO157_Sal including genes related to stress response, adaption and virulence. The plasmid-cured mutant Xuzhou21m grew slower than wild type Xuzhou21 and pO157_Sal plasmid complemented strain Xuzhou21c in M9 medium under the condition of high NaCl or presence of sodium deoxycholate (NaDC), corroborating with the RNA-Seq data. Seven differentially expressed genes are associated with NaDC resistance, including the adenine-specific DNA-methyltransferase gene (dam), multidrug efflux system subunit gene mdtA, hyperosmotically inducible periplasmic protein gene osmY and oxidation-reduction related genes while two differentially expressed genes (osmY and pspD) are likely to be related to resistance to osmotic pressure. A number of differentially expressed genes were virulence associated including four genes encoding T3SS effectors from the chromosome and ehxD from pO157. Through complementation of Xuzhou21m with a plasmid construct carrying the pO157_Sal hha homolog we further showed that the pO157_Sal hha represses the expression of T3SS effectors. These findings demonstrated that the plasmid pO157_Sal affects the transcription of the chromosomal and pO157 plasmid genes and contributes to the enhanced ability to resist stress. We conclude that pO157_Sal plays an important role in regulating global gene expression and affects the virulence and adaptation of E. coli O157:H7.

Diversity in Structural Consequences of MexZ Mutations in Pseudomonas aeruginosa

Pseudomonas aeruginosa is the major cause of morbidity and mortality in patients with cystic fibrosis. The high level of intrinsic and acquired antibiotic resistance of P.aeruginosa is attributable to the low permeability of its outer membrane in combination with the expression of several multidrug resistance efflux systems. MexZ, the negative regulator of the MexXY efflux pump, is found to be the most frequently mutated gene in P.aeruginosa isolated from cystic fibrosis patient lungs, confirming its importance in multidrug resistance. Structural consequences of four MexZ mutations, including L25P, G46V, P151L, and S202F, have been explored based on the known structure of MexZ using both molecular modeling and molecular dynamics methods. According to obtained results, G46V mutation, which completely abolishes the ability of MexZ binding to DNA, occurs in a specific evolutionary conserved region of MexZ. In addition, the most fluctuation values occur in DNA-binding domain and Helix4. The obtained results explore details of diversity in structural consequences of MexZ mutations in P.aeruginosa.

Microbial Efflux Systems and Inhibitors: Approaches to Drug Discovery and the Challenge of Clinical Implementation

Conventional antimicrobials are increasingly ineffective due to the emergence of multidrug-resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered exploration for novel and unconventional approaches to controlling microbial infections. Multidrug efflux systems (MES) have been a profound obstacle in the successful deployment of antimicrobials. The discovery of small molecule efflux system blockers has been an active and rapidly expanding research discipline. A major theme in this platform involves efflux pump inhibitors (EPIs) from natural sources. The discovery methodologies and the available number of natural EPI-chemotypes are increasing. Advances in our understanding of microbial physiology have shed light on a series of pathways and phenotypes where the role of efflux systems is pivotal. Complementing existing antimicrobial discovery platforms such as photodynamic therapy (PDT) with efflux inhibition is a subject under investigation. This core information is a stepping stone in the challenge of highlighting an effective drug development path for EPIs since the puzzle of clinical implementation remains unsolved. This review summarizes advances in the path of EPI discovery, discusses potential avenues of EPI implementation and development, and underlines the need for highly informative and comprehensive translational approaches.

Cooperation of the multidrug efflux pump and lipopolysaccharides in the intrinsic antibiotic resistance of Salmonella enterica serovar Typhimurium

ObjectivesIn Gram-negative bacteria, drug susceptibility is associated with multidrug efflux systems and an outer membrane (OM) barrier. Previous studies revealed that Salmonella enterica serovar Typhimurium has 10 functional drug efflux pumps. Among them, AcrB is a major factor to maintain the intrinsic drug resistance in this organism. The lipopolysaccharide (LPS) content of OM is also important for resistance to lipophilic drugs; however, the interplay between the multidrug efflux pump and LPS in the intrinsic antibiotic resistance of Salmonella remains to be studied in detail. The aim of this study was to investigate the relationship between AcrB and LPS in the intrinsic drug resistance of this organism.MethodsThe genes encoding LPS core biosynthetic proteins and AcrB were disrupted from the wild-type S. enterica strain ATCC 14028s. The plasmid carrying acrB was transformed into these mutants and then the drug susceptibilities of the mutants and transformants were determined.ResultsOur results showed that the levels of Salmonella intrinsic antibiotic resistance were decreased when the length and branches of core oligosaccharide were lost. Furthermore, the deletion of acrB reduced multidrug resistance of all LPS mutants and AcrB production from the plasmid complemented this phenotype. However, AcrB production could not completely compensate for LPS function in intrinsic resistance.ConclusionsBoth pump inactivation and shortened LPS enhanced drug susceptibility, although the maximum susceptibility was achieved when the two were combined. Hence, these results indicated that the multidrug efflux system and OM barrier are both essential for maintaining intrinsic antibiotic resistance in Salmonella.

All-Atom Molecular Dynamics Simulations of Multidrug Efflux Transporter AcrB

The multidrug transporter AcrB, which forms a part of the tripartite multidrug efflux system (AcrB/AcrA/TolC) in E. coli., actively exports a wide variety of noxious compounds using the proton-motive force as an energy source. Recently, x-ray structures provided that AcrB adopts an asymmetric structure comprising three protomers with different conformations that are sequentially converted during drug export. These cyclic conformational changes for drug export are called the functional rotation. To investigate functional rotation driven by the proton-motive force, a series of all-atom molecular dynamics simulations were performed. Our simulations demonstrate that alternating the protonation states in the transmembrane domain induces functional rotation in the porter domain that is primarily responsible for drug transport.

Multidrug Efflux Systems in Helicobacter cinaedi.

Helicobacter cinaedi causes infections, such as bacteremia, diarrhea and cellulitis in mainly immunocompromised patients. This pathogen is often problematic to analyze, and insufficient information is available, because it grows slowly and poorly in subculture under a microaerobic atmosphere. The first-choice therapy to eradicate H. cinaedi is antimicrobial chemotherapy; however, its use is linked to the development of resistance. Although we need to understand the antimicrobial resistance mechanisms of H. cinaedi, unfortunately, sufficient genetic tools for H. cinaedi have not yet been developed. In July 2012, the complete sequence of H. cinaedi strain PAGU 611, isolated from a case of human bacteremia, was announced. This strain possesses multidrug efflux systems, intrinsic antimicrobial resistance mechanisms and typical mutations in gyrA and the 23S rRNA gene, which are involved in acquired resistance to fluoroquinolones and macrolides, respectively. Here, we compare the organization and properties of the efflux systems of H. cinaedi with the multidrug efflux systems identified in other bacteria.

Recognition of Imipenem and Meropenem by the RND-Transporter MexB Studied by Computer Simulations

Basic understanding of the means by which multidrug efflux systems can efficiently recognize and transport drugs constitutes a fundamental step toward development of compounds able to tackle the continuous outbreak of new bacterial strains resistant to traditional antibiotics. We applied a series of computational techniques, from molecular docking to molecular dynamics simulations and free energy estimate methods, to determine the differences in the binding properties of imipenem and meropenem, two potent antibiotics of the carbapenem family, to MexB, the RND transporter of the major efflux system of Pseudomonas aeruginosa. We identified and characterized two affinity sites in the periplasmic domain of the transporter, sharing strong similarities with the distal and proximal binding pockets identified in AcrB, the homologue of MexB in Escherichia coli. According to our results, meropenem has a higher affinity to the distal binding pocket than imipenem while both compounds are weakly bound to the proximal pocket. This different behavior is mainly due to the hydration properties of the nonpharmacophore part of the two compounds, being that of imipenem less bulky and hydrophobic. Our data provide for the first time a rationale at molecular level for the experimental evidence indicating meropenem as a compound strongly affected by MexB contrary to imipenem, which is apparently poorly transported by the same pump.

Epigallocatechin gallate as a modulator of Campylobacter resistance to macrolide antibiotics

Comprehensive therapeutic use of macrolides in humans and animals is important in the selection of macrolide-resistant Campylobacter isolates. This study shows high co-resistance to erythromycin, azithromycin, clarithromycin, dirithromycin and tylosin, with contributions from the 23S rRNA gene and drug efflux systems. The CmeABC efflux pump plays an important role in reduced macrolide susceptibility, accompanied by contributions from the CmeDEF efflux pump and potentially a third efflux pump. To improve clinical performance of licensed antibiotics and chemotherapeutic agents, it is important to understand the factors in Campylobacter that affect susceptibility to macrolide antibiotics. Using mutants that lack the functional genes coding for the CmeB and CmeF efflux pump proteins and the CmeR transcriptional repressor, we show that these efflux pumps are potential targets for the development of therapeutic strategies that use a combination of a macrolide with an efflux pump inhibitor (EPI) to restore macrolide efficacy. The natural phenolic compound epigallocatechin gallate (EGCG) has good modulatory activity over the extrusion across the outer membrane of the macrolides tested, both in sensitive and resistant Campylobacter isolates. Comparing EGCG with known chemical EPIs, correlations in the effects on the particular macrolide antibiotics were seen. EGCG modifies Campylobacter multidrug efflux systems and thus could have an impact on restoring macrolide efficacy in resistant strains.

Reduced Expression of the rplU-rpmA Ribosomal Protein Operon in mexXY -Expressing Pan-Aminoglycoside-Resistant Mutants of Pseudomonas aeruginosa

Pan-aminoglycoside-resistant Pseudomonas aeruginosa mutants expressing the mexXY components of the aminoglycoside-accommodating MexXY-OprM multidrug efflux system but lacking mutations in the mexZ gene encoding a repressor of this efflux system and in the mexXY promoter have been reported (S. Fraud and K. Poole, Antimicrob. Agents Chemother. 55:1068-1074, 2011). Genome sequencing of one of these mutants, K2966, revealed the presence of a mutation within the predicted promoter region of the rplU-rpmA operon encoding ribosomal proteins L21 and L27, consistent with an observed 2-fold decrease in expression of this operon in the mutant relative to wild-type P. aeruginosa PAO1. Moreover, correction of the mutation restored rplU-rpmA expression and, significantly, reversed the elevated mexXY expression and pan-aminoglycoside resistance of the mutant. Reduced rplU-rpmA expression was also observed in a second mexXY-expressing pan-aminoglycoside-resistant mutant, K2968, which, however, lacked a mutation in the rplU-rpmA promoter region. Restoration of rplU-rpmA expression in the K2968 mutant following chromosomal integration of the rplU-rpmA operon derived from wild-type P. aeruginosa failed, however, to reverse the elevated mexXY expression and pan-aminoglycoside resistance of this mutant, although it did so for K2966, suggesting that the mutation impacting rplU-rpmA expression in K2968 also impacts other mexXY-related genes. Increased mexXY expression owing to reduced rplU-rpmA expression in K2966 and K2968 was dependent on PA5471, whose expression was also elevated in these mutants. Thus, mutational disruption of ribosome function, by limiting expression of ribosomal constituents, promotes recruitment of mexXY and does so via PA5471, reminiscent of mexXY induction by ribosome-disrupting antimicrobial agents. Interestingly, reduced rplU-rpmA expression was also observed in a mexXY-expressing pan-aminoglycoside-resistant clinical isolate, suggesting that ribosome-perturbing mutations have clinical relevance in the recruitment of the MexXY-OprM aminoglycoside resistance determinant.

MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa

MexT is a global LysR transcriptional regulator known to modulate antibiotic resistance and virulence in Pseudomonas aeruginosa. In this study, a novel role for MexT in mediating intrinsic disulfide stress resistance was demonstrated, representing the first identified phenotype associated with inactivation of this regulator in wild-type cells. Disruption of mexT resulted in increased susceptibility to the disulfide stress elicitor diamide [diazenedicarboxylic acid bis(N,N,-di-methylamide)]. This compound is known to elicit a specific stress response via depletion of reduced glutathione and alteration of the cellular redox environment, implicating MexT in redox control. In support of this, MexT-regulated targets, including the MexEF-OprN multidrug efflux system, were induced by subinhibitory concentrations of diamide. A mexF insertion mutant also exhibited increased diamide susceptibility, implicating the MexEF-OprN efflux system in MexT-associated disulfide stress resistance. Purified MexT protein was observed to form an oligomeric complex in the presence of oxidized glutathione, with a calculated redox potential of -189 mV. This value far exceeds the thiol-disulfide redox potential of the bacterial cytoplasm, ensuring that MexT remains reduced under normal physiological conditions. MexT is activated by mutational disruption of the predicted quinone oxidoreductase encoded by mexS. Alterations in the cellular redox state were observed in a mexS mutant (PA14nfxC), supporting a model whereby the perception of MexS-associated redox signals by MexT leads to the induction of the MexEF-OprN efflux system, which, in turn, may mediate disulfide stress resistance via efflux of electrophilic compounds.

Characterization of the Flavonoid-Responsive Regulator FrrA and Its Binding Sites

Previous microarray analyses revealed that in Bradyrhizobium japonicum, about 100 genes are induced by genistein, an isoflavonoid secreted by soybean. This includes the three genes freC, freA, and freB (systematic designations bll4319, bll4320, and bll4321), which are likely to form a genistein-, daidzein-, and coumestrol-inducible operon and to encode a multidrug efflux system. Upstream of freCAB and in the opposite orientation, FrrA (systematic designation Blr4322), which has similarity to TetR-type regulators, is encoded. A deletion of frrA leads to increased expression of freB in the absence of an inducer. We identified the correct translational start codon of frrA and showed that the gene is inducible by genistein and daidzein. The protein, which was heterologously expressed and purified from Escherichia coli, binds to two palindrome-like DNA elements (operator A and operator B), which are located in the intergenic region between freC and frrA. The replacement of several nucleotides or the insertion of additional spacer nucleotides prevented binding. Binding of FrrA was also affected by the addition of genistein. By mapping the transcription start sites, we found that operator A covers the transcriptional start site of freC and operator B is probably located between the -35 regions of the two divergently oriented genes. Operator A seems to be conserved in a few similar gene constellations in other proteobacteria. Our data indicate that in B. japonicum, besides NodD1 (the LysR family) and NodVW (a two-component response regulator), a third regulator type (a TetR family member) which responds to the plant signal molecules genistein and daidzein exists.

Accumulation and efflux of polychlorinated biphenyls in Escherichia coli

Polychlorinated biphenyls (PCBs) are environmental pollutants that have been associated with numerous adverse health effects in human and animals. Hydroxylated PCBs (HPCBs) are the product of the oxidative metabolism of PCBs. The presence of hydroxyl groups in HPCBs makes these compounds more hydrophilic than the parent PCBs. One of the best approaches to break down and remove these contaminants is bioremediation; an environmentally friendly process that uses microorganisms to degrade hazardous chemicals into non-toxic ones. In this study, we investigated the cellular accumulation and toxicity of selected PCBs and HPCBs in Gram-negative bacteria, using Escherichia coli as a model organism. We found that none of the five PCBs tested were toxic to E. coli, presumably due to their limited bioavailability. Nevertheless, different HPCBs tested showed different levels of toxicity. Furthermore, we demonstrated that the primary multidrug efflux system in E. coli, AcrAB-TolC, facilitated the efflux of HPCBs out of the cell. Since AcrAB-TolC is constitutively expressed in E. coli and is conserved in all sequenced Gram-negative bacterial genomes, our results suggest that the efflux activities of multidrug resistant pumps may affect the accumulation and degradation of PCBs in Gram-negative bacteria.

Strategies to Potentiate Antimicrobial Photoinactivation by Overcoming Resistant Phenotypes†

Conventional antimicrobial strategies have become increasingly ineffective due to the emergence of multidrug resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered the exploration of alternative treatments and unconventional approaches towards controlling microbial infections. Photodynamic therapy (PDT) was originally established as an anticancer modality and is currently used in the treatment of age-related macular degeneration. The concept of photodynamic inactivation requires cell exposure to light energy, typically wavelengths in the visible region that causes the excitation of photosensitizer molecules either exogenous or endogenous, which results in the production of reactive oxygen species (ROS). ROS produce cell inactivation and death through modification of intracellular components. The versatile characteristics of PDT prompted its investigation as an anti-infective discovery platform. Advances in understanding of microbial physiology have shed light on a series of pathways, and phenotypes that serve as putative targets for antimicrobial drug discovery. Investigations of these phenotypic elements in concert with PDT have been reported focused on multidrug efflux systems, biofilms, virulence and pathogenesis determinants. In many instances the results are promising but only preliminary and require further investigation. This review discusses the different antimicrobial PDT strategies and highlights the need for highly informative and comprehensive discovery approaches.