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Can efflux inhibitors really counter resistance?
Abstract
Efflux, particularly that mediated by the resistance-nodulation-division (RND) family of multiple antibiotic efflux systems that are prevalent in Gram-negative pathogenic bacteria, is of increasing concern as a mechanism of resistance to clinically important antimicrobials. In considering the continued utility of existing agents and the development of new ones, then, efflux must be taken into account. We describe here a number of approaches, realized and hypothetical, for avoiding or inhibiting efflux as a way of countering efflux-mediated resistance.Section editors:Gary Woodnutt – CovX, San Diego, USAPaul-Henri Lambert – Centre of Vaccinology, University of Geneva, Switzerland
... Since antibiotic resistance due to efflux pumps is an important mechanism of drug resistance which necessitates the need to address this problem [150,151], newer fluoroquinolones and macrolides are emerging as the unconventional treatment options for Gram-negative pathogens [150][151][152]. However, in GNB, a wide allocation of RND family efflux pumps and their broad substrate activity is a matter of great concern to develop new antimicrobial agents [153]. ...
... Since antibiotic resistance due to efflux pumps is an important mechanism of drug resistance which necessitates the need to address this problem [150,151], newer fluoroquinolones and macrolides are emerging as the unconventional treatment options for Gram-negative pathogens [150][151][152]. However, in GNB, a wide allocation of RND family efflux pumps and their broad substrate activity is a matter of great concern to develop new antimicrobial agents [153]. ...
... However, in GNB, a wide allocation of RND family efflux pumps and their broad substrate activity is a matter of great concern to develop new antimicrobial agents [153]. EPIs-targeting RND family efflux systems should be taken into account to overcome resistance [150,151,154]. Efflux pumps being one of the several mechanisms of resistance, it should be kept in mind that only those pathogens get affected by EPI in which efflux pumps play a major role in developing resistance. ...
Antibiotic resistance is defined as a condition which assists any disease-causing organism to resist antibiotics of different class with distinct structure and function to eliminate the organism where the microorganism can survive on exposure to an antibiotic. Multiple resistance determinants play a major mechanism of bacterial resistance, including multidrug efflux pumps. Different types of Efflux pump mechanisms like single drug-specific or multidrug, are the significant determinants of resistance either intrinsic or acquired to the respective antibiotics. This has resulted in the development of efflux pump inhibitors (EPIs). By understanding the working mechanism of the efflux pump; various antibacterial agents, known as efflux reversals can be described as efflux pump inhibitors, membrane permeabilizers, blockers, energy poisons, etc. Over the last few years, a lot of effort was exerted to identify the new and unique EPIs however, none was found suitable.
... 3 Therefore, the susceptibility of resistant strains might be restored by coadministration of efflux pump inhibitors (EPIs) and antibacterials (efflux pump substrates); thus, efflux pumps are viable antibacterial targets and identification of potent EPIs is a promising and valid strategy to fight antibiotic resistance. 4 The most studied efflux system in S. aureus is NorA, which is able to extrude multiple structurally dissimilar substrates such as hydrophilic fluoroquinolones (ciprofloxacin, norfloxacin), biocides (acriflavine, cetrimide, benzalkonium chloride), and dyes (ethidium bromide [EtBr]) through an antiport mechanism driven by the proton-motive force. 5−7 To date, no crystal structure of NorA has been determined. ...
... a Reagents and conditions: (i) 1,3-dibromopropane or 1,2-dibromoethane, K 2 CO 3 , ethanol reflux; (ii) (1) NaI, acetonitrile reflux, (2) K 2 CO 3 , piperazine; (iii) (1) NaI, acetonitrile reflux, (2) K 2 CO 3 . 29 Scheme 3 a a Reagents and conditions: (i) (1) 1-(3-chloropropoxy)-4-fluorobenzene, NaI, acetonitrile reflux, (2) K 2 CO 3 ; 29 (ii) 21, NaBH 4 , MeOH, rt; (iii) (1) 5-(chloromethyl)-1-phenyl-1H-tetrazole, NaI, acetonitrile reflux, (2) K 2 CO 3 ; 29 (iv) (1) 4-fluoro-3-methoxybenzaldehyde, Ti(Oi-Pr) 4 ...
... Ethyl 1-Benzyl-5-(3-((4-fluoro-3-methoxybenzyl)amino)propoxy)-2-methyl-1H-indole-3-carboxylate (16). 30 A mixture of 4-fluoro-3-methoxybenzaldehyde (43 mg, 0.28 mmol), Ti(Oi-Pr) 4 (166 μL, 0.59 mmol), and amine 6c (50 mg, 0.14 mmol) in EtOH (1 mL) was stirred under nitrogen atmosphere at rt overnight. NaBH 4 (8 mg, 0.21 mmol) was then added, and the resulting mixture was stirred at room temperature for an additional 5 h. ...
The NorA efflux pump is a potential drug target for reversal of resistance to selected antibacterial agents, and recently we described indole-based inhibitor candidates. Herein we report a second class of inhibitors derived from them, but with significant differences in shape and size. In particular, compounds 13 and 14 were very potent inhibitors in that they demonstrated the lowest IC50 values (2 μM) ever observed among all indole-based compounds we have evaluated.
... Given the importance of these proteins for the maintenance of resistance and other bacterial metabolic functions, the identification of efflux pump inhibitors (EPIs) has been the target of studies [32,33]. These molecules are administered along with antibiotics so as to prevent their extrusion and increase their intracellular concentration [32,34]. ...
... Given the importance of these proteins for the maintenance of resistance and other bacterial metabolic functions, the identification of efflux pump inhibitors (EPIs) has been the target of studies [32,33]. These molecules are administered along with antibiotics so as to prevent their extrusion and increase their intracellular concentration [32,34]. Therefore, in addition to restoring antibiotic susceptibility and enhancing its action, EPIs can reduce the occurrence of persistent strains, helping to treat chronic infections [35][36][37][38]. ...
Antibiotic resistance can be characterized, in biochemical terms, as an antibiotic’s inability to reach its bacterial target at a concentration that was previously effective. Microbial resistance to different agents can be intrinsic or acquired. Intrinsic resistance occurs due to inherent functional or structural characteristics of the bacteria, such as antibiotic-inactivating enzymes, nonspecific efflux pumps, and permeability barriers. On the other hand, bacteria can acquire resistance mechanisms via horizontal gene transfer in mobile genetic elements such as plasmids. Acquired resistance mechanisms include another category of efflux pumps with more specific substrates, which are plasmid-encoded. Efflux pumps are considered one of the main mechanisms of bacterial resistance to antibiotics and biocides, presenting themselves as integral membrane transporters. They are essential in both bacterial physiology and defense and are responsible for exporting structurally diverse substrates, falling into the following main families: ATP-binding cassette (ABC), multidrug and toxic compound extrusion (MATE), major facilitator superfamily (MFS), small multidrug resistance (SMR) and resistance-nodulation-cell division (RND). The Efflux pumps NorA and Tet(K) of the MFS family, MepA of the MATE family, and MsrA of the ABC family are some examples of specific efflux pumps that act in the extrusion of antibiotics. In this review, we address bacterial efflux pump inhibitors (EPIs), including 1,8-naphthyridine sulfonamide derivatives, given the pre-existing knowledge about the chemical characteristics that favor their biological activity. The modification and emergence of resistance to new EPIs justify further research on this theme, aiming to develop efficient compounds for clinical use.
... Clearly, bacterial efflux proteins are potential biological targets for the development of new drugs; the development of efflux pump inhibitors would restore susceptibility to strains previously resistant to antimicrobial agents, and would decrease the selection of resistant mutants, thereby prolonging the useful life of antibiotics [11,14,15]. ...
... .(14). Chemical structures of sulfur-containing compounds as EPIs or NorA inhibitors ...
Background
Methicillin-resistant and vancomycin-resistant Staphylococcus aureus are pathogens causing severe infectious diseases that pose real public health threats problems worldwide. In S. aureus, the most efficient multidrugresistant system is the NorA efflux pump. For this reason, it is critical to identify efflux pump inhibitors.
Objective
In this paper we present an update of the new natural and synthetic compounds that act as modulators of antibiotic resistance through the inhibition of the S. aureus NorA efflux pump.
Results
Several classes of compounds capable to restoring the antibiotic activity have been identified against resistant-S. aureus strains, acting as NorA efflux pump inhibitors. The most promising classes of compounds were quinolines, indoles, pyridines, phenols, and sulfur-containing heterocycles. However, the substantial degree structural diversity of these compounds makes it difficult to establish good structure-activity correlations that allow design of compounds with more promising activities and properties.
Conclusion
Despite substantial efforts put forth in the search for new antibiotic adjuvants that act as efflux pump inhibitors, and despite several promising results, there are currently no efflux pump inhibitors authorized for human or veterinary use, or in clinical trials. Unfortunately, it appears that infection control strategies have remained the same since the discovery of penicillin, and that most efforts remain focused on discovering new classes of antibiotics, rather than trying to prolong the life of available antibiotics, and simultaneously fighting mechanisms of bacterial resistance.
... This may contribute to fewer strains becoming resistant and overall drug treatment regimens remaining successful [23]. Several mechanisms exist through which efflux pump inhibition can be achieved [24,25], namely the development of novel antibiotics or modified versions of existing antibiotics to bypass the efflux pumps; the disruption of efflux pump functional parts to prevent the formation of a correct complex required for efflux; the disruption of the energy sources essential for efflux pump operation; targeting the regulatory processes required for efflux gene expression and the competitive inhibition of the efflux pump substratebinding site by other compounds. Examples of well-characterized EPIs include reserpine, phenylalanine-arginyl-β-naphthylamide (PAβN) and quinolines. ...
... Although the EPI approach may seem straightforward, its clinical applications remain hindered by the specific toxicity level of each inhibitory compound [25]. Moreover, as EPIs are used in combination with antimicrobials, the pharmacokinetic properties of both types of compounds should be kept in mind for the effective application to reach the desired outcome [24]. Unfortunately, no EPIs have yet been licensed for the clinical treatment of infections in humans or animals [26]. ...
Introduction: Microbial resistance against antibiotics is a serious threat to the effective treatment of infectious diseases. Several mechanisms exist through which microorganisms can develop resistance against antimicrobial drugs, of which the over-expression of genes to produce efflux pumps is a major concern. Several efflux transporters have been identified in microorganisms, which infer resistance against specific antibiotics and even multi-drug resistance.
Areas covered: This paper focuses on microbial resistance against antibiotics by means of the mechanism of efflux and gives a critical overview of studies conducted to overcome this problem by combining efflux pump inhibitors with antibiotics. Information was obtained from a literature search done with MEDLINE, Pubmed, Scopus, ScienceDirect, OneSearch and EBSCO host.
Expert opinion: Efflux as a mechanism of multi-drug resistance has presented a platform for improved efficacy against resistant microorganisms by co-administration of efflux pump inhibitors with antimicrobial agents. Although proof of concept has been shown for this approach with in vitro experiments, further research is needed to develop more potent inhibitors with low toxicity which is clinically effective.
... The P. aeruginosa genome contains at least 10 distinct tripartite (3-protein) efflux pump system gene operons (Stover et al., 2000). Efflux pump inhibition may be due to disturbance of the regulatory pathway essential for expression of the efflux pump, alteration of the chemical structure of the antibiotic preventing its attachment to a specific substrate, hindrance of assembly of efflux pump components, inhibition of antibiotic by completive or non-completive binding, closing of the pores responsible for efflux of antimicrobial agents or disturbance of energy essential for pump activity (Poole and Lomovskaya, 2006;Pagés and Amaral, 2009). ...
... On the contrary, a certain reduction The mexA and mexB exhibited 2.93 and 3.21 fold down regulation in the gene expression with 10 mM EDTA, respectively. The down-regulation of mexA and mexB genes following treatment with EDTA disodium is probably due to chelation of divalent ions (Vaara, 1992), thereby disturbing the electrostatic interactions in the outer membrane of the bacterial cell and may be responsible for blocking of pores associated with efflux activity (Pagés and Amaral, 2009;Poole and Lomovskaya, 2006). Inhibition of the efflux pumps is promising in order to increase the intracellular drug concentration, restore the drug activity against the resistant strains, and minimize further development of resistant strains. ...
Pseudomonas aeruginosa is an opportunistic bacterium which has been shown to have multi-drug resistance against fluoroquinolones, β-lactams, and aminoglycosides. In this investigation, we studied the effect of different concentrations of ethylenediaminetetraacetic acid (EDTA) on MexA-MexB-OprM efflux pump and subsequent changes in susceptibility and expression. Next, we examined, the expression of mexAB gene following treatment with half of minimum inhibitory concentration (MIC) of drugs. Our results revealed that 10 mM EDTA significantly reduced MIC of all drugs; moreover, the higher reduction (8 fold) was observed with CSE1034. MexA and MexB expression was down regulated at 2.93 and 3.21 fold, respectively with 10 mM EDTA. When the same concentration of EDTA was incorporated with drugs, the CSE1034 down regulates 5.64 and 5.94 fold expression of mexA and mexB, respectively. Moreover, meropenem treated groups exhibited 2.63 and 3.12 fold down regulation in the expression of the genes. However, treatment with piperacillin plus tazobactam, amoxicillin plus clavulanate, cefoperazone plus sulbactam and imipenem plus cilastatin did not produce changes in the expression of MexAB-OprM. Hence, CSE1034 could be one of the best choices to treat infections caused by microorganisms that overexpressed MexAB-Opr-M as compared to other drugs. Furthermore, use of EDTA disodium at appropriated concentrations can be regarded as a safe strategy to fight against the menace provided by the efflux pumps.
... For antibacterials, this results in sublethal drug concentrations at the active site that in turn may predispose the organism to the development of high-level target-based resistance [3,7]. Therefore, efflux pumps are viable antibacterial targets and identification and development of potent efflux pump inhibitors is a promising and valid strategy [3,8] which can restore the susceptibility of resistant strains to antibacterial agents that are substrates of efflux pumps [3,9]. The combination of a resistance inhibitor with an antibiotic has already proven its efficacy with the clavulanic acid (inhibitor of beta-lactamase)/amoxicillin association [10]. ...
... Efflux pumps in Gram-positive bacteria belong to four unrelated families (Table 1): MFS (major facilitator superfamily), SMR (small multidrug resistance), ABC (ATP-binding cassette) and MATE (Multidrug And Toxic Compound Extrusion) [9,[39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54]. ...
The paper focuses on recent achievements in the search for new chemical compounds able to inhibit multidrug resistance (MDR) mechanisms in Gram-positive pathogens. An analysis of the results of the search for new efflux pump inhibitors (EPIs) for Gram-positive bacteria, which have been performed over the last decade, indicates that almost all efforts are focused on the NorA (MFS) efflux pump in S. aureus. Considering the chemical structures of the NorA EPIs that have been identified, it can be observed that the most active agents belong to the families of compounds possessing conjugated double bonds, e.g., chalcones, piperine-like compounds, N-cinnamoylphenalkylamides or citral amide derivatives. Indole-, dihydronaphthyl-, 2-chloro-5-bromo-phenyl-or piperidine moieties seem to be profitable for the EPI properties, as well. These results, together with an increasing knowledge about a variety of efflux pumps that are involved in MDR of Gram-positive pathogens underline that further search for new EPIs should pay more attention to develop MDR efflux protein targets, including SMR, MATE, ABC or other members of the MFS family.
... The main advantage of using the PAβN-derived (EPIs) is the difficulty to develop resistance to them, where any pump mutation leading to inhibitor resistance will lead to resistance to the antibiotic substrate. The disadvantage of those compounds is their low affinity to the target that necessitates the use of higher doses and for a longer time in addition to only substrates that share the inhibitor binding site will be affected (40). ...
... The inhibition of efflux pumps can be achieved by different mechanisms as shown in (Fig. 2): (1) interference with the regulatory steps needed for the expression of the efflux pump, (2) chemical changes in the antibiotic structure hence hindering its attachment as the specific substrate, (3) disruption of the assembly of the efflux pump-components, (4) inhibition of the substrate (antibiotic) binding by either competitive or non-competitive binding using other compounds, (5) blocking the outer most pores responsible for the efflux of antibiotic compound, (6) interference with the energy required for the pump activity (37, 40). ...
Pseudomonas aeruginosa is an opportunistic human pathogen and one of the leading causes of nosocomial infections worldwide. The difficulty in treatment of pseudomonas infections arises from being multidrug resistant (MDR) and exhibits resistance to most antimicrobial agents due to the expression of different mechanisms overcoming their effects. Of these resistance mechanisms, the active efflux pumps in Pseudomonas aeruginosa that belong to the resistance nodulation division (RND) plays a very important role in extruding the antibiotics outside the bacterial cells providing a protective means against their antibacterial activity. Beside its role against the antimicrobial agents, these pumps can extrude biocides, detergents, and other metabolic inhibitors. It is clear that efflux pumps can be targets for new antimicrobial agents. Peptidomimetic compounds such as phenylalanine arginyl β-naphthylamide (PAβN) have been introduced as efflux pump inhibitors (EPIs); their mechanism of action is through competitive inhibition with antibiotics on the efflux pump resulting in increased intracellular concentration of antibiotic, hence, restoring its antibacterial activity. The advantage of EPIs is the difficulty to develop bacterial resistance against them, but the disadvantage is their toxic property hindering their clinical application. The structure activity relationship of these compounds showed other derivatives from PAβN that are higher in their activity with higher solubility in biological fluids and decreased toxicity level. This raises further questions on how can we compact Pseudomonas infections. Of particular importance, the recent resurgence in the use of older antibiotics such as polymyxins and probably applying stricter control measures in order to prevent their spread in clinical sittings.
... Given the importance of these proteins in antibiotic resistance and bacterial metabolic functions, the identification of efflux pump inhibitors (EPIs) has been the target of several drug development studies [32,33]. These molecules are administered along with antibiotics to prevent their extrusion and increase their intracellular concentration [36,37]. Therefore, in addition to restoring antibiotic susceptibility and enhancing its action, EPIs can reduce the occurrence of persistent strains, helping to treat chronic infections [38,39]. ...
The bacterial species Staphylococcus aureus presents a variety of resistance mechanisms, among which the expression of β-lactamases and efflux pumps stand out for providing a significant degree of resistance to clinically relevant antibiotics. The 1,8-naphthyridines are nitrogen heterocycles with a broad spectrum of biological activities and are promising research targets. However, the potential roles of these compounds on bacterial resistance management remain to be better investigated. Therefore, the present study evaluates the antibacterial activity of 1,8-naphthyridine sulfonamides, addressing their ability to act as inhibitors of β-lactamases and efflux pump (QacA/B and QacC) inhibitors in the strains SA-K4414 and SA-K4100 of S. aureus. All substances were prepared at an initial concentration of 1024 μg/mL, and their minimum inhibiting concentrations (MIC) were de-termined by the broth microdilution method. Subsequently, their effects on β-lactamase- and efflux pump-mediated antibiotic resistance was evaluated from the reduction of the MIC of ethidium bromide (EtBr) and β-lactam antibiotics, respectively. The 1,8-naphthyridines did not present direct antibacterial activity against the strains SA-K4414 and SA-K4100 of S. aureus. On the other hand, when associated with antibiotics against both strains, the compounds reduced the MIC of EtBr and β-lactam antibiotics, suggesting that they may act by inhibiting β-lactamases and efflux such as QacC and QacA/B efflux pumps. However, further research is required to elucidate the molecular mechanisms underlying these observed effects.
... In other words, attainment of the inhibition of these mechanisms can be through expression interference of those changing the chemical structure of antibiotics or antibiotics efflux can be caused by blocking of outer pores. Disturbance of assembly of their components or energy interference that is necessary for the activity of the pump is considered another efflux pump inhibition mechanism [20,21]. The importance of these inhibitors is attributed to successful therapy, decreasing the resistance level and increasing the drugs concentration intracellularly. ...
According to the prevalence of multidrug resistance bacteria, especially Pseudomonas aeruginosa, in which the essential mechanism of drug resistance is the ability to possess an efflux pump by which extrusion of antimicrobial agents usually occurs, this study aims to detect the presence of mexB multidrug efflux gene in some local isolates of this bacteria that show resistance towards three antibiotics, out of five. Sensitivity test to antibiotics was performed on all isolates by using meropenem (10µg/disc), imipenem (10µg/disc), amikacin (30 μg/disc), ciprofloxacin (5µg/disc) and ceftazidime (30 µg/disc). Conventional PCR results showed the presence of mexB gene (244bp) in four isolates out of ten (40%). In addition,25, 50μg/ml of curcumin was used to detect its efficacy with the antibiotics that the bacteria showed resistance towards. Results showed the highest resistance for ciprofloxacin (80%), while all of them were sensitive to imipenem. In addition, the present results show that both concentrations of curcumin (25, 50μg/ml) were effective in increasing the zone of inhibition from zero to 10 mm for isolates towards amikacin. Same result was obtained towards ciprofloxacin, except for an increase of inhibition zone from zero to 7 mm to one isolate (38T) when treated with 50 μg/ml, and finally an increase in sensitivity to ceftazidime was found and inhibition zone was increased from 8 to 11 for the second isolate (42E), which revealed that curcumin potentiates antibiotics activity by inhibition of efflux pump mechanisms that can be related to the synergetic activity between antibiotics and curcumin.
... The NorA efflux pump that belongs to the major facilitator superfamily (MFS) is a significant contributor to antibiotic resistance as it can expel a wide range of chemical probes, such as ethidium bromide (EtBr), acriflavine, quaternary amine compounds, fluoroquinolones, rhodamine-6-G, puromycin, and chloramphenicol (CHL), and confers resistance (Markham et al., 1999). Also, NorA shares sequence homology with other MDR pumps, which led to a hypothesis that it may possess a large hydrophobic binding pocket with a wide range of substrate specificity and so an attractive novel drug target for the identification of efflux pump inhibitors (EPIs) to fight against resistance (Neyfakh, 2002;Poole and Lomovskaya, 2006;Cloete R. et al., 2018). Several non-antibiotics such as reserpine, verapamil, flavones, isoflavones, porphyrin pheophorbide A, and acylated glycosides are reported to be NorA inhibitors that act as selective small-molecule modulators rather than clinical drug candidates (Schillaci et al., 2017). ...
Efflux pumps are one of the predominant microbial resistant mechanisms leading to the development of multidrug resistance. In Staphylococcus aureus, overexpression of NorA protein enables the efflux of antibiotics belonging to the class of fluoroquinolones and, thus, makes S. aureus resistant. Hence, NorA efflux pumps are being extensively exploited as the potential drug target to evade bacterial resistance and resensitize bacteria to the existing antibiotics. Although several molecules are reported to inhibit NorA efflux pump effectively, boronic acid derivatives were shown to have promising NorA efflux pump inhibition. In this regard, the current study exploits 6-(3-phenylpropoxy)pyridine-3-boronic acid to further improve the activity and reduce cytotoxicity using the bioisostere approach, a classical medicinal chemistry concept. Using the SWISS-Bioisostere online tool, from the parent compound, 42 compounds were obtained upon the replacement of the boronic acid. The 42 compounds were docked with modeled NorA protein, and key molecular interactions of the prominent compounds were assessed. The top hit compounds were further analyzed for their drug-like properties using ADMET studies. The identified potent lead, 5-nitro-2-(3-phenylpropoxy)pyridine (5-NPPP), was synthesized, and in vitro efficacy studies have been proven to show enhanced efflux inhibition, thus acting as a potent antibiotic breaker to resensitize S. aureus without elucidating any cytotoxic effect to the host Hep-G2 cell lines.
... 25 Since, many researches focused on multidrug resistance MDR in Gram-negative pathogens due to the complex mechanisms of MDR in these bacteria because of the doublemembrane around the bacterial cell that enables the expression of a triple efflux pump system as MexA/ MexB/OprM of Pseudomonas aeruginosa or AcrA/AcrB/TolC of Enterobacteriaceae. [26][27][28][29][30][31] Here is focusing on efflux pump transporters in Gram-positive pathogens and in detail pumping of bile in S. aureus bacteria which belong to four main groups of efflux pump: major facilitator superfamily MFS, ATP-binding cassette ABC, Multidrug and Toxic Compound Extrusion MATE and small multidrug resistance SMR. [32][33][34][35] ...
Gram-positive pathogens are causing many serious infections that affect humans and result in mild to severe diseases worldwide. In order to survive and initiate infection, enteric pathogens must resist the physiochemical defence factors in the human intestinal tract. One of these defence factors is bile, a potent antibacterial like compound in the intestine. Efflux pumps are the important mechanism by which bacteria resist antibacterial agents such as bile. Efflux of antimicrobial substances outside the bacterial cell considered as a key factor for intestinal colonization and virulence of enteric pathogens. This paper will review the research conducted on efflux–mediated bile resistance in Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis and Clostridium perfringens. Theses bacteria colonize in the human & animal gastrointestinal tract and they have a multiple mechanism to resist the innate defences in the gut and antibacterial activity of bile. However, bile resistance in theses bacteria is not fully understood. The evidence from this review suggests that Gram-positive pathogens have ability to active transport of bile. Further research is needed to know how these pathogen sense bile and how bile regulate its virulence factor. In general, therefore, it seems that understanding of the specific mechanism of bile resistance in enteric bacteria including gram–positive pathogen may involve in the development of novel strategies to control and treatment of gastrointestinal infections.
... The NorA efflux pump that belongs to the major facilitator superfamily (MFS) is a significant contributor to antibiotic resistance as it can expel a wide range of chemical probes, such as ethidium bromide (EtBr), acriflavine, quaternary amine compounds, fluoroquinolones, rhodamine-6-G, puromycin, and chloramphenicol (CHL), and confers resistance (Markham et al., 1999). Also, NorA shares sequence homology with other MDR pumps, which led to a hypothesis that it may possess a large hydrophobic binding pocket with a wide range of substrate specificity and so an attractive novel drug target for the identification of efflux pump inhibitors (EPIs) to fight against resistance (Neyfakh, 2002;Poole and Lomovskaya, 2006;Cloete R. et al., 2018). Several non-antibiotics such as reserpine, verapamil, flavones, isoflavones, porphyrin pheophorbide A, and acylated glycosides are reported to be NorA inhibitors that act as selective small-molecule modulators rather than clinical drug candidates (Schillaci et al., 2017). ...
Efflux pumps are one of the predominant microbial resistant mechanisms leading to the development of multidrug resistance. In Staphylococcus aureus , overexpression of NorA protein enables the efflux of antibiotics belonging to the class of fluoroquinolones and, thus, makes S. aureus resistant. Hence, NorA efflux pumps are being extensively exploited as the potential drug target to evade bacterial resistance and resensitize bacteria to the existing antibiotics. Although several molecules are reported to inhibit NorA efflux pump effectively, boronic acid derivatives were shown to have promising NorA efflux pump inhibition. In this regard, the current study exploits 6-(3-phenylpropoxy)pyridine-3-boronic acid to further improve the activity and reduce cytotoxicity using the bioisostere approach, a classical medicinal chemistry concept. Using the SWISS-Bioisostere online tool, from the parent compound, 42 compounds were obtained upon the replacement of the boronic acid. The 42 compounds were docked with modeled NorA protein, and key molecular interactions of the prominent compounds were assessed. The top hit compounds were further analyzed for their drug-like properties using ADMET studies. The identified potent lead, 5-nitro-2-(3-phenylpropoxy)pyridine (5-NPPP), was synthesized, and in vitro efficacy studies have been proven to show enhanced efflux inhibition, thus acting as a potent antibiotic breaker to resensitize S. aureus without elucidating any cytotoxic effect to the host Hep-G2 cell lines.
... And also Chaudhary and Payasi found that inhibition of efflux pump in P. aeruginosa isolates by EDTA starts from a concentration of 5 mM-10 mM [25]. The down-regulation of mexA and mexB genes after treatment with EDTA disodium is probably due to divalent ions chelation causing electrostatic interactions disturbance in the outer membrane of the bacterial cell which leads to block pores associated with efflux activity [62,66,68]. ...
Extensive use of antibiotics in clinical practice has been associated with increasing frequency of resistant microorganisms. So new strategy is needed to treat the resistant pathogens. Hence this study was conducted to determine the effect of Ethylenediaminetetraacetic acid (EDTA) in increasing the inhibition effect of some antibiotics on multi-drug resistant (MDR) gram-negative bacteria. For this purpose, 40 E. coli isolates, 40 K. pneumoniae isolates and 50 P. aeruginosa isolates were collected from different University's hospitals in Mansoura, Egypt. Antibacterial susceptibility pattern against 9 different antimicrobials were studied by disc diffusion method. Also the effect of two sub-inhibitory concentrations of EDTA (1 and 2 mM) on the inhibition zones of antibiotic discs against the highly multidrug resistant (MDR) isolates was determined. Checkerboard method was used for testing the activity of gentamicin/EDTA and cefotaxime/EDTA combinations on the highly MDR isolates. Additionally, the effect of EDTA on the expression of efflux pump genes was tested by real time-PCR. Most of the clinical isolates were found to be resistant to the tested antibiotics except imipenem and high prevalence of MDR isolates was recorded. 34 isolates were selected as those showed the highest multi-drug resistance and were tested to specify their MIC for EDTA as EDTA showed strong antibacterial activity with MIC ranging 4–8 mM. The addition of sub-MIC of EDTA (1or 2 mM) to the agar plate resulted in changing the 11 tested E. coli isolates from resistant to sensitive to ceftazidime, gentamicin, rifampin, ampicillin, erythromycin and vancomycin, the tested K. pneumoniae isolates were turned also from resistant to sensitive to gentamicin and ceftazidime, additionally the tested P. aeruginosa isolates became sensitive to gentamicin, ceftazidime and ciprofloxacin. Indifference to additive activity was observed for tested combinations and MIC value of cefotaxime or gentamicin in combination with EDTA was less than antibiotic alone in the most tested isolates. Moreover, significant reduction (P < 0.01) in the expression of all tested efflux pump genes in treated E. coli, K. pneumoniae and P. aeruginosa isolates with EDTA compared to untreated isolates was observed. In conclusion, these results suggest that the combination of antibiotic especially gentamicin with EDTA may be fruitful for management of resistant gram negative infections.
... Efflux pump inhibition can be attained by interference with the regulatory mechanisms for the efflux pump expression, blocking of outer pores causing antibiotics efflux or changing the antibiotics structure chemically. Other mechanisms including disturbance of the efflux pump-components assembly or interference with the energy required for the pump activity [20,21]. Efflux pump inhibitors (EPIs) are important for a successful therapy and are used to decrease the level of resistance and increase the intracellular concentration of the therapeutic drugs. ...
Today, one of the most important challenges for physicians is the adequate treatment of infections due to multidrug resistant organism (MDR). Pseudomonas aeruginosa is considered an opportunistic organism causing different types of healthcare associated infections (HAIs). We aimed to investigate the MDR and pandrug resistance (PDR) rate in P. aeruginosa in our region and detect efflux-pump mexAB genes and the proposed binding interactions of five different categories of antimicrobial agents with the mexB pump. A total of 180 non-duplicated P. aeruginosa strains were isolated from patients with HAIs in the Suez Canal University Hospital. Phenotypically, minimum inhibitory concentration (MIC) was done for all MDR and PDR strains before and after addition of efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Molecular detection of mexA and mexB genes was done by using polymerase chain reaction (PCR). Most of the isolated strains (126 strains) were MDR (70%); only 10 samples (5.5%) were PDR. MexA and mexB genes were detected in 88.2% (120 strains) and 70.5% (96 strains) of stains, respectively. All PDR strains (10 stains) carried both mexA and mexB genes. Efflux mexAB genes were detected in all MDR and PDR strains (136 strains). Molecular modeling studies were performed to investigate the modes of intermolecular binding interactions between the antimicrobial agents and mexB key amino acids that resulted in MDR and PDR. The current study reported high prevalence of MDR and PDR P. aeruginosa in patients with HAIs in the Suez Canal University Hospitals.
... Alternatively, these compounds can act as regulators of efflux systems, namely as efflux pump inhibitors (EPI) to block the activity of drug efflux pumps 2 . Thus, EPIs help in restoring the susceptibility of resistant strains to antibacterial agents 3 . Staphylococcus aureus is a major pathogen in both hospital and the community, responsible for a wide range of infections from uncomplicated skin and soft tissue infections to more serious illnesses like pneumonia, endocarditis, and sepsis 4 . ...
... As a consequence, they are attractive novel drug targets and identification of efflux pumps inhibitors (EPIs) is a promising and valid strategy for fighting resistance to antibiotics. 44,45 EPIs have been studied since the discovery, in 1976, of P-glycoprotein, the first known efflux transporter. 46 There are many meaningful reasons for pursuing this area of research further. ...
Bacterial infections are an increasingly serious issue worldwide. The inability of existing therapies to treat multidrug‐resistant pathogens has been recognized as an important challenge of the 21st century. Efflux pumps are important in both intrinsic and acquired bacterial resistance and identification of small molecule efflux pump inhibitors (EPIs), capable of restoring the effectiveness of available antibiotics, is an active research field. In the last two decades, much effort has been made to identify novel EPIs. However, none of them has so far been approved for therapeutic use.
In this article, we explore different structural families of currently known EPIs for multidrug resistance efflux systems in the most extensively studied pathogens (NorA in Staphylococcus aureus, AcrAB‐TolC in Escherichia coli, and MexAB‐OprM in Pseudomonas aeruginosa). Both synthetic and natural compounds are described, with structure‐activity relationship studies and optimization processes presented systematically for each family individually. In vitro activities against selected test strains are presented in a unifying manner for all the EPIs described, together with the most important toxicity, pharmacokinetic and in vivo efficacy data. A critical evaluation of lead‐likeness characteristics and the potential for clinical development of the most promising inhibitors of the three efflux systems is described. This overview of EPIs is a good starting point for the identification of novel effective antibacterial drugs.
... Molecular docking experiments reported that NMP binds to the distal pockets of the AcrB protein and competes with the antimicrobial agent for binding space within the efflux pump (Takatsuka et al., 2010;Vargiu and Nikaido, 2012;Schuster et al., 2014). Having greater affinity, NMP binds preferentially to the AcrB distal pocket than the antimicrobial agent (Poole and Lomovskaya, 2006) enabling the drug to concentrate in the bacterial cytoplasm and exert its bactericidal effect. ...
Antimicrobial efflux is one of the important mechanisms causing multi-drug resistance (MDR) in bacteria. Chemosensitizers like 1-(1-naphthylmethyl)-piperazine (NMP) can inhibit an efflux pump and therefore can overcome MDR. However, secondary effects of NMP other than efflux pump inhibition are rarely investigated. Here, using phenotypic assays, phenotypic microarray and transcriptomic assays we show that NMP creates membrane destabilization in MDR Klebsiella pneumoniae MGH 78578 strain. The NMP mediated membrane destabilization activity was measured using β-lactamase activity, membrane potential alteration studies, and transmission electron microscopy assays. Results from both β-lactamase and membrane potential alteration studies shows that both outer and inner membranes are destabilized in NMP exposed K. pneumoniae MGH 78578 cells. Phenotypic Microarray and RNA-seq were further used to elucidate the metabolic and transcriptional signals underpinning membrane destabilization. Membrane destabilization happens as early as 15 min post-NMP treatment. Our RNA-seq data shows that many genes involved in envelope stress response were differentially regulated in the NMP treated cells. Up-regulation of genes encoding the envelope stress response and repair systems show the distortion in membrane homeostasis during survival in an environment containing sub-inhibitory concentration of NMP. In addition, the lsr operon encoding the production of autoinducer-2 responsible for biofilm production was down-regulated resulting in reduced biofilm formation in NMP treated cells, a phenotype confirmed by crystal violet-based assays. We postulate that the early membrane disruption leads to destabilization of inner membrane potential, impairing ATP production and consequently resulting in efflux pump inhibition.
... In presence of noxious agents, the permeability of cell can be reduced through the porins by down-regulating its number (Stress & Response, 2007). Over-expression of efflux pumps in Gram-negative bacteria results in a multi-drug-resistant (MDR) phenotype (Poole, 2005;Poole & Lomovskaya, 2006). Previous reports indicate that the major membrane transporter genes of E. coli are overexpressed after exposure to tetracycline (Stress & Response, 2007). ...
The emergence of multidrug resistance (MDR) in Gram negative bacteria (GNB) and lack of novel classes of antibacterial agents have raised an immediate need to identify antibacterial agents, which can reverse the phenomenon of MDR. The purpose of present study was to evaluate synergy potential and understanding the drug resistance reversal mechanism of chanoclavine isolated from Ipomoea muricata against the multidrug resistant clinical isolate of Escherichia coli (MDREC). Although chanoclavine did not show antibacterial activity of its own, but in combination, it could reduce the minimum inhibitory concentration (MIC) of tetracycline (TET) up to 16 folds. Chanoclavine was found to inhibit the efflux pumps which seem to be ATPase dependent. In real time expression analysis, chanoclavine showed down regulation of different efflux pump genes and decreased the mutation prevention concentration of tetracycline. Further, in silico docking studies revealed significant binding affinity of chanoclavine with different proteins known to be involved in drug resistance. In in-silico ADME/toxicity studies, chanoclavine was found safe with good intestinal absorption, aqueous solubility, medium blood-brain barrier (BBB), no CYP 2D6 inhibition, no hepatotoxicity, no skin irritancy and non-mutagenic indicating towards drug likeliness of this molecule. Based on these observations, it is hypothesized that chanoclavine might be inhibiting the efflux of tetracycline from MDREC and thus enabling the more availability of tetracycline inside the cell for its action.
... Alternatively, these compounds can act as regulators of efflux systems, namely as EPIs to block the activity of drug efflux pumps (Pages & Amaral 2009). Thus, EPIs help in restoring the susceptibility of resistant strains to antibacterial agents (Poole & Lomovskaya 2006). ...
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for several infections in humans. The infections caused by this bacterial strain are difficult to treat due to the resistance of MRSA to clinically used antibiotics. Several medicinal plants extracts and their phytoconstituents have been reported to possess modulation and efflux pump inhibitory (EPI) activity against MRSA strains. Alpinia calcarata rhizomes have been reported to be used in Ayurveda for several ailments including fungal infections. Based on this information and in continuation with our efforts to discover EPIs from Indian medicinal plants, we describe EPI activity of flavonoids isolated from A. calcarata. Galangin and kaempferol showed ≥ 32-fold modulation in minimum inhibitory concentration (MIC) of ethidium bromide (EtBr) as well as norfloxacin in NorA-overexpressed S. aureus (SA-1199B) strain. Pinocembrin showed 32-fold modulation of EtBr MIC in SA-1199 strain, but not in SA-1199B and K1758 strains. A significant diference was not observed in the modulation of norfloxacin MIC by galangin in SA-1199 and SA-1199B strains, which may be due to non-specific nature of galangin as modulator or EPI. However, kaempferol modulated the MIC of EtBr as well as norfloxacin 64-fold and 32-fold, respectively. Also, the best modulatory effect of kaempferol was observed only in SA-1199B strain compared to two other strains. The EPI activity of kaempferol and galangin were found to be competitive with respect to verapamil. In dose-response assay, kaempferol at 31.25 μg/mL concentration was found to be better EPI by inhibiting NorA pump in SA-1199B strain and also demonstrated further confocal microscopy. © 2016 Institute of Molecular Biology, Slovak Academy of Sciences.
... For anti-bacterials, this results in sub-lethal drug concentrations at the active site that in turn may predispose the organism to the development of high-level targetbased resistance [40,42]. Therefore, efflux pumps are viable antibacterial targets and identification and development of potent efflux pump inhibitors is a promising and valid strategy [40,43] which can restore the susceptibility of resistant strains to antibacterial agents that are substrates of efflux pumps [40,44]. ...
ABSTRACT
Objective: The objective of this study was to report the hydrophobic interaction of 2-trifluoromethyl-N10
Methods: Binding of six compounds, 10-[3-substituted phenoxazines with bovine serum albumin and reversal of drug resistance in bacterial cells.
'-N-morpholinopropyl]-2-trifluoromethyl phenoxazine (1C), 10-[4'-N-morpholinobutyl]-2-trifluoromethyl phenoxazine (2C), 10-[3'-N-pyrrolidinopropyl]-2-trifluoromethyl phenoxazine (3C), 10-[4'-N-pyrrolidinobutyl]-2-trifluoromethyl phenoxazine (4C), 10-[N-piperidinoacetyl]-2-trifluoromethyl phenoxazine (5C), and 10-[N-pyrrolidinoacetyl]-2-trifluoromethyl phenoxazine (6C), to bovine serum albumin (BSA) has been measured by gel filtration and equilibrium dialysis methods. The binding of these compounds to BSA has been characterized by percentage of bound drug (β), the association constant (K), the apparent binding constant (k) and free energy (ΔFo
Results: The results of displacing experiments reveal that the phenoxazine benzene rings and tertiary amines attached to the side chain of phenoxazine moiety are bound to a hydrophobic region on the albumin molecule. Among the compounds examined the butyl series seems to possess better reversing ability, suggesting that the activity could be related to lipophilicity and the extent of binding to BSA. ). The binding of phenoxazine derivatives to BSA, a serum protein that binds and transports small molecules, is correlated with their partition coefficients. Further, the ability of the phenoxazines (1C-6C) on the antibacterial activity of five antibiotics, kanamycin, spectinomycin, gentamycin, streptomycin and benzylpenicillin was examined for their ability to reverse the resistance of E. coli K12 MG 1655 and E. coli ST 58.
Conclusions: Phenoxazines are bound to albumin by hydrophobic interactions of their benzene rings. The alkyl side chain, particularly butyl chain of phenoxazines intensifies the interaction of phenoxazines with BSA. The compound that binds to a greater extent with protein possesses more activity for reversing of drug resistance.
Keywords: Hydrophobic interaction, Phenoxazines, Bacterial drug resistance, Bovine serum albumin.
... The efflux activity has been correlated with increased resistance to antibiotics. It has been established that efflux pump inhibition might be associated with disturbance of the regulatory pathway required for efflux pump expression, modification of the chemical structure of the antibiotic inhibiting its attachment to specific substrates, hindrance of assembly of efflux pump components, inhibition of antibiotic by completive or non-completive binding, closing of the pores responsible for efflux of antimicrobial agents or disturbance of energy essential for pump activity (Poole and Lomovskaya, 2006;Pagés and Amaral, 2009). Our susceptibility data revealed that efflux pump positive isolates showed higher MIC compared to efflux pump deficient strains. ...
Antimicrobial resistance caused by efflux systems is not widely studied in Staphylococcus aureus. In the present study, prevalence of NorA and NorB efflux pumps was studied in thirteen Methicillin-Resistant Staphylococcus aureus (MRSA) strains using a pair of primers with Polymerase Chain Reaction (PCR). Antibiotic susceptibility study in NorA and NorB efflux positive isolates was carried out according to the Clinical and Laboratory Standards Institute guidelines. To evaluate the effects of various drugs at half of their MIC on NorA and NorB efflux pump, expression of NorA and NorB genes was measured through semi-quantitative PCR. Our results demonstrated that out of 13 MRSA strains, 8 strains were both NorA and NorB positive and 2 strains were NorA positive. Susceptibility data revealed that Vancoplus appeared to be the most active antibacterial against NorA and NorB efflux pumps positive MRSA strains with MIC values 0.25 to 0.5 μg mL−1. The second most active antibacterial agent was teicoplanin with MIC values 0.5 to 1 μg mL−1. The MICs of vancomycin and daptomycin ranged from 32 to 64 μg mL−1. Treatment of MRSA strains possessing NorA and NorB efflux pumps with Vancoplus caused significant down-regulation of the expression of NorA and NorB genes (P<0.001) as compared to the control (without treatment) whereas other drugs such as vancomycin, linezolid, teicoplanin and daptomycin failed to produce any significant changes in the expression of these genes (P>0.05). From the above results, it can be concluded that Vancoplus (vancomycin+ceftriaxone+VRP1020) can be one of the best options to treat infections caused by MRSA possessing NorA and NorB efflux pumps. © 2014 The Manu Chaudhary, Santosh Kumar Patnaik and Anurag Payasi.
... Enhanced drug efflux has now been shown to occur in bacterial, mycobacterial, fungal and even human cancer cells. Once identified, one of the strategies is to counter efflux-mediated resistance (Poole and Lomovskaya, 2006). Therefore, renewed strategies on treatment and prevention of drug resistance in infectious organisms are now required. ...
Zimbabwe is burdened with HIV/AIDS and TB infections. Plants are often used to treat infective diseases, and a study was carried out to evaluate the basis of their use and probe them for new anti-infective constituents. Of the 5000 plant species growing in Zimbabwe, about 10% are used in traditional medicine. The potential of medicinal plants on infectious pathogens, including mycobacteria, fungi, human cancer cells and bacteria, was studied by screening plants from three provinces: Harare, Mashonaland West and Mashonaland Central. The growth inhibition parameters for microbes and pathogenic human cells, including MICs, MBCs, GI50 and GI90, were determined. Combretum species have been identified with potential antifungal, antibacterial, antimycobacterial and anti-inflammatory activities. Further work is needed to determine the cytoxicity of these compounds using normal cell lines or animal models, and further fractionation is needed to isolate the active chemical species.
... . 위험관리 개념을 항공보안에 적용하는 것은 위 협수준이나 취약성 등에 따라 항공보안 활동을 적절히 조정한다는 의미로 해석할 수 있을 것이 다. 또한, 일부 위험한 대상(risky subjects)과 대 다수의 위험하지 않은 일반 여객 (majority of low-risk travelers)을 차별화하여 보안 절차를 적용하는 것을 의미한다 (Poole, 2006 ...
The strengthened aviation security measurements caused by increased terror threat all around the world have air passengers annoyed by uneasy treatment and some delayed process. The service level of air travel and competitiveness of airlines industry has been weakened also. The aviation security process is mainly conducted at an airport. The threat level of airport is not usually same airport to airport, and the quality of aviation security activity is neither same. However, ICAO requires every international airport to conduct similar process to ensure aviation security without considering situational difference of each airport. The paper has objectives to develop reasonable security measurements based on risk management concept. It studied the ways of achieving target level of security of an airport, considering the threat situation of the airport and its ability to conduct security procedures.
... However, no phenotypic resistance was reported in those strains to the corresponding antibiotics being other mechanisms involved to enhance their susceptibility to the mentioned antibiotics. On the other hand, mRNA expression of EfrAB was found to be decreased significantly with triclosan in E. faecalis Ac 8-2 which could be occurred for several reasons such as the disturbance of the regulatory pathway essential for expression of the efflux pump, alteration of the structure of efflux pump components, or disturbance of energy essential for pump activity as occurred in Gram-negative bacteria with other antimicrobials (Poole and Lomovskaya, 2006;Pag es and Amaral, 2009). Triclosan is a phenolic compound used in many everyday products including toothpaste, soaps, and lotions (Nester et al., 2007) to inhibit bacterial growth by the inhibition of enoyl-ACPreductase involved in bacterial fatty acid synthesis. ...
... Another strategy is to supplement quinolone treatment with pump inhibitors such as MC207,110, a compound that showed early promise with fluoroquinolones against P. aeruginosa [100]. Unfortunately, therapeutic efforts using pump inhibitors have been plagued by the inability to block multiple pump systems and by toxicity due to inhibition of mammalian transporters [107][108][109]. A third strategy is to treat with fluoroquinolone concentrations that are high enough to prevent the growth of efflux mutants. ...
Fluoroquinolones trap gyrase and topoisomerase IV on DNA as ternary complexes that block the movement of replication forks and transcription complexes. Studies with resistant mutants indicate that resistance is due to alterations in drug permeability, drug efflux, gyrase-protecting proteins, drug-modifying enzymes, and target topoisomerases. Emergence (acquisition) of resistance is thought to arise when fluoroquinolone concentration falls inside a range in which resistant mutant subpopulations are selectively enriched and amplified. The lower boundary of the range (mutant selection window) is approximated by the minimal inhibitory concentration (MIC) of the bulk, susceptible population. The upper boundary is the MIC of the least susceptible resistant mutant subpopulation, a value called the mutant prevention concentration (MPC). MPC varies among fluoroquinolones and pathogens; when combined with pharmacokinetics, MPC can be used to compare compounds and dosing regimens for selective amplification of mutant subpopulations. Batteries of resistant mutants can be used to identify compounds that have a very narrow selection window and are likely to restrict the emergence of resistance. While most of the concepts behind the emergence of quinolone resistance are based on chromosomal mutations, plasmid-borne resistance also occurs. Thus studies of quinolones can address most aspects of antibiotic resistance.
... The approach of identifying molecules that can interfere with the process of efflux to enhance the potential of existing antibiotics has become the process of choice to tackle antibiotic resistance and explore genomic-driven new lead molecules (Poole & Lomovskaya, 2006). Such compounds can significantly improve antibiotic efficacy by raising physiological levels of an antibiotic and act synergistically by reducing bacterial efflux. ...
Piperine, a trans-trans-isomer of 1-piperoyl-piperidine, was tested in combination with mupirocin for antimicrobial activity against Staphylococcus aureus strains including meticillin-resistant S. aureus. The combination markedly reduced the MIC of mupirocin and also lowered the mutation frequency. Enhanced accumulation and efflux of ethidium bromide from wild-type and mutant (Mup(r)-1) strains in the presence of piperine indicated that inhibition of efflux could be a possible mechanism of potentiation of mupirocin activity by piperine. The combination of piperine with mupirocin in a dermal infection model of mice showed better in vivo efficacy when compared with the commercially available formulation of 2 % mupirocin.
Efflux proteins are transporter molecules that actively pump out a variety of substrates, including antibiotics, from cells to the environment. They are found in both Gram-positive and Gram-negative bacteria and eukaryotic cells. Based on their protein sequence homology, energy source, and overall structure, efflux proteins can be divided into seven groups. Multidrug efflux pumps are transmembrane proteins produced by microbes to enhance their survival in harsh environments and contribute to antibiotic resistance. These pumps are present in all bacterial genomes studied, indicating their ancestral origins. Many bacterial genes encoding efflux pumps are involved in transport, a significant contributor to antibiotic resistance in microbes. Efflux pumps are widely implicated in the extrusion of clinically relevant antibiotics from cells to the extracellular environment and, as such, represent a significant challenge to antimicrobial therapy. This review aims to provide an overview of the structures and mechanisms of action, substrate profiles, regulation, and possible inhibition of clinically relevant efflux pumps. Additionally, recent advances in research and the pharmacological exploitation of efflux pump inhibitors as a promising intervention for combating drug resistance will be discussed.
Antibiotic resistance is defined as a condition that assists any disease-causing organism to resist antibiotics of different class with distinct structure and function directed for its elimination, thereby enabling the survival of the microorganism. Multiple resistance determinants play a major mechanism of bacterial resistance including multidrug efflux pumps. Different types of efflux pump mechanisms, such as single drug or multidrug-specific, are the significant determinants of resistance, either intrinsic or acquired, to the respective antibiotics. This has resulted in the development of efflux pump inhibitors (EPIs). By understanding the working mechanism of the efflux pump, various antibacterial agents known as efflux reversals can be described as EPIs, membrane permeabilizers, blockers, energy poisons, etc. Over the last few years a lot of effort was exerted to identify the new and unique EPIs; however, none was found suitable.
Novel therapeutic options are needed to halt the unabated spread of multiple-drug resistance. Phage-derived proteins, especially enzymes that can degrade the bacterial cell wall and cause cell-lysis, offer an attractive new paradigm for treating serious drug-resistant infections. Recent research and clinical data on this class of anti-infective agents have revealed their novel properties. This chapter describes the development path for phage-derived lysins, starting with discovery and leading to the strategy into the clinic, with P128, a novel chimeric antistaphylococcal lysin, as example. Standard regulatory pathways that apply to chemical antibiotics need to be modified to accommodate and accelerate the development of this class. The similarities and differences between these macromolecular bactericidal agents, and the traditional small molecule antibiotics are highlighted throughout this chapter. The data-intensive exemplification of research and development of lysin P128 in this write-up is aimed at influencing all the stakeholders, research community, clinicians, patients, biopharma industry, and regulators, into capitalizing the benefits that lysins offer.
The postantibiotic effect (PAE) refers to the temporary suppression of bacterial growth following transient antibiotic treatment. This effect has been observed for decades for a wide variety of antibiotics and microbial species. However, despite empirical observations, a mechanistic understanding of this phenomenon is lacking. Using a combination of modeling and quantitative experiments, we show that the PAE can be explained by the temporal dynamics of drug detoxification in individual cells after an antibiotic is removed from the extracellular environment. These dynamics are dictated by both the export of the antibiotic and the intracellular titration of the antibiotic by its target. This mechanism is generally applicable for antibiotics with different modes of action. We further show that efflux inhibition is effective against certain antibiotic motifs, which may help explain mixed cotreatment success.
IntroductionApproaches to Antibacterial Drug Discovery and Combating the Problem of Drug ResistanceCombination Drug TherapyStrategies to Target Mechanisms of Drug ResistanceQuorum Sensing Inhibition: A Novel Anti-Infective Drug TargetPhage Therapy: An Alternative TherapyAntibiotic Use Policy: Practical Management of the Problem of Antibiotic ResistanceConclusions
AcknowledgmentsReferences
Background/aim:
Bacterial resistance to antibiotics has become a serious problem in antibacterial chemotherapy and resistance of bacteria to chemically-unrelated anti-microbial agents can be associated with the over-expression of efflux pumps. The simultaneous therapy with efflux pump inhibitors (EPIs) could be a solution to improve the effectiveness of antibiotics. The response of an organism to an EPI often depends on how that molecule fits a particular site of a protein. Because enantiomers of a given compound rotate plane-polarized light in a solution by the same angle but in opposite directions, the rational drug design should take the chirality into account if there is a difference between the racemic compound and its enantiomers.
Materials and methods:
The main goal of the present study was to elucidate the role of chirality of N-hydroxyalkyl-2-aminophenothiazines as effective EPIs by an automated method that uses the general efflux pump substrate ethidium bromide (EB) for the assessment of AcrAB-TolC system of wild-type Escherichia coli K-12 AG100. It has been shown that the most active EPIs among the N-hydroxyalkyl-2-aminophenothiazines were the compounds rac-3i, (+)-3i, and (-)-3i by modulating the AcrAB-TolC efflux pump.
Conclusion:
Comparison of effects of enantiomeric pairs revealed that their activities were similar to that of racemic derivatives. Moreover, there was no significant difference between the racemic compounds and their enantiomers related to their antibacterial and efflux pump inhibiting effects.
Quinolones are a widely used class of antibacterial agents characterized by overall favourable pharmacokinetic and pharmacodynamic characteristics. Unfortunately, both nosocomial and community acquired resistances to these agents are increasing. Thorough structure–activity relationship (SAR) studies have demonstrated that even drastic manipulation of the quinolone scaffold may lead to compounds maintaining antibacterial activity and avoiding mechanisms of resistance. We herein report the design and synthesis of a series of substituted 2,1-benzothiazine 2,2-dioxide derivatives designed as quinolone-like analogues, with the aim to further expand the SAR for this antibacterial class as well as to assay their capability of inhibiting the S. aureus NorA efflux pump. Although none of the new compounds evaluated showed any appreciable intrinsic antibacterial activity, the 2,1-benzothiazin-4-one 2,2-dioxide derivatives 17 and 18 were able to restore, in a concentration-dependent manner, the antibacterial activity of ciprofloxacin (CPX) against the norA-overexpressing S. aureus strain SA-K2378. Thus, these compounds have emerged as new hits in the search for novel efflux pump inhibitors useful for limiting the clinical impact of efflux-related quinolone resistance.
Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of antimicrobial agents that are substrates. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, biocides, and dyes resulting in a multidrug resistant (MDR) phenotype. In this work, a series of 2-phenylquinoline derivatives was synthesized by means of ligand-based pharmacophore modeling and virtual library design in an attempt to identify improved S. aureus NorA efflux pump inhibitors (EPIs). Most of the 2-phenylquinoline derivatives displayed potent EPI activity against the norA overexpressing strain SA-1199B. The antibacterial activity of ciprofloxacin, when used in combination with some of the synthesized compounds, was completely restored in SA-1199B and SA-K2378, a strain overexpressing norA from a multicopy plasmid. Compounds 3m and 3q also showed potent synergistic activity with the dye ethidium bromide for a strain overexpressing the MepA MDR efflux pump.
The incidence of clinical mastitis and infection status at calving was assessed in quarters treated with 1 of 2 internal teat sealants at the time of dry off. Two contralateral quarters per cow (n=63 cows) were treated with a sealant that contained 0.5% chlorhexidine; the other quarters were treated with a commercial teat sealant. Ten cows were untreated (controls). On d 2, 4, and 16 after dry off, cows were challenged with Streptococcus uberis S210 strain. Cows were examined daily for 34 d after drying off and cases of clinical mastitis were recorded. Milk samples were collected for culture from any quarters that developed clinical mastitis during the first 34 d after drying-off and from all quarters on d −5 and 0 relative to treatment and at the first and twentieth milking after calving. The incidence of clinical mastitis during the examination period was lower in treated quarters (n=7/252; 1.5%; lower incidence for those treated with chlorhexidine-containing teat sealant n=3/126; 1.2%) than in untreated quarters (n=13/40; 26.8%). The protection against intramammary infection after calving, adjusted for the effect of cow, was higher in quarters treated with the novel teat sealant (89/105; 15.2%; 95% CI=9.6–23.4) than in those treated with the commercial teat sealant (71/104; 31.7%; 95% CI=23.5–41.3) and untreated controls (6/28; 78.6%; 95% CI=59.8–90.0), respectively. Quarters treated with teat sealants were less likely to have an intramammary infection after calving and had a lower incidence of clinical mastitis during the early dry period than did untreated controls in this challenge study.
The increasing resistance to antibacterials commonly employed in the clinic and the growth of multidrug resistant strains suggest that the development of new therapeutic approaches should be of primary concern. In this context, EPIs may restore life to old drugs. In the present work, the EPI activity of the COX-2 inhibitor celecoxib was confirmed and a new class of pyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide analogues acting as inhibitors of the Staphylococcus aureus NorA multidrug efflux pump was identified.
Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of substrate antimicrobial agents. Inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug resistant phenotype. In this work, a series of 2-phenyl-4(1H)-quinolone and 2-phenyl-4-hydroxyquinoline derivatives, obtained by modifying the flavone nucleus of known efflux pump inhibitors (EPIs), were synthesized in an effort to identify more potent S. aureus NorA EPIs. The 2-phenyl-4-hydroxyquinoline derivatives 28f and 29f display potent EPI activity against SA-1199B, a strain that overexpresses norA, in an ethidium bromide efflux inhibition assay. The same compounds, in combination with ciprofloxacin, were able to completely restore its antibacterial activity against both S. aureus SA-K2378 and SA-1199B, norA-overexpressing strains.
Monoterpene citral and citronellal have been used as starting material for the preparation of 5,9-dimethyl-deca-2,4,8-trienoic acid amides and 9-formyl-5-methyl-deca-2,4,8-trienoic acid amides. The amides on bioevaluation as efflux pump inhibitors (EPIs) against Staphylococcus aureus 1199 and NorA overexpressing S. aureus 1199B bacteria resulted in the identification of several of these as potent EPIs. Many of these amides have been shown to possess potency higher or equivalent to known EPIs such as reserpine, verapamil, carsonic acid, and piperine. In this communication, we report a convenient synthesis of alkenyl amides, their bioevaluation and identification as efflux pump inhibitors against S. aureus.
The thiopyranopyridine moiety was synthesized as a new heterocyclic base to be inserted at the C-7 position of selected quinolone nuclei followed by a determination of antibacterial activity against strains of Staphylococcus aureus. Selected thiopyranopyridinylquinolones showed significant antimicrobial activity, including strains having mutations in gyrA and grlA as well as other strains overexpressing the NorA multidrug (MDR) efflux pump. Most derivatives did not appear to be NorA substrates. The effect of the thiopyranopyridinyl substituent on making these quinolones poor substrates for NorA was investigated further. Several quinolone ester intermediates, devoid of any intrinsic antibacterial activity, were tested for their abilities to inhibit the activities of NorA (MFS family) and MepA (MATE family) S. aureus MDR efflux pumps. Selected quinolone esters were capable of inhibiting both MDR pumps more efficiently than the reference compound reserpine. Moreover, they also were able to restore, and even enhance, the activity of ciprofloxacin toward some genetically modified resistant S. aureus strains.
Chloramphenicol, florfenicol, and thiamphenicol are used as antibacterial drugs in clinical and veterinary medicine. Two efflux
pumps of the major facilitator superfamily encoded by the cmlR1 and cmlR2 genes mediate resistance to these antibiotics in Streptomyces coelicolor, a close relative of Mycobacterium tuberculosis. The transcription of both genes was observed by reverse transcription-PCR. Disruption of cmlR1 decreased the chloramphenicol MIC 1.6-fold, while disruption of cmlR2 lowered the MIC 16-fold. The chloramphenicol MIC of wild-type S. coelicolor decreased fourfold and eightfold in the presence of reserpine and Phe-Arg-β-naphthylamide, respectively. These compounds
are known to potentiate the activity of some antibacterial drugs via efflux pump inhibition. While reserpine is known to potentiate
drug activity against gram-positive bacteria, this is the first time that Phe-Arg-β-naphthylamide has been shown to potentiate
drug activity against a gram-positive bacterium.
Gram-negative bacteria have evolved diverse secretion systems/machineries to translocate substrates across the cell envelope. These various machineries fulfil a wide variety of functions but are also essential for pathogenic bacteria to infect human or plant cells. Secretion systems, of which there are seven, utilize one of two secretion mechanisms: (i) the one-step mechanism, whereby substrates are translocated directly from the bacterial-cytoplasm to the extracellular medium or into the eukaryotic-target cell; (ii) the two-step mechanism, whereby substrates are first translocated across the bacterial-inner membrane; once in the periplasm, substrates are targeted to one of the secretion systems that mediate the transport across the outer membrane and the release outside the bacterial cell. This review describes in details the main structural features of these secretion systems. Structural biology offers the possibility to understand the molecular mechanisms at play in the various secretion systems. It also helps to design specifically drugs that can block these machineries and thus attenuate the virulence of pathogenic bacteria.
Quantitative structure activity relationship (QSAR) analysis of piperine analogs as inhibitors of efflux pump NorA from Staphylococcus aureus has been performed in order to obtain a highly accurate model enabling prediction of inhibition of S. aureus NorA of new chemical entities from natural sources as well as synthetic ones. Algorithm based on genetic function approximation method of variable selection in Cerius2 was used to generate the model. Among several types of descriptors viz., topological, spatial, thermodynamic, information content and E-state indices that were considered in generating the QSAR model, three descriptors such as partial negative surface area of the compounds, area of the molecular shadow in the XZ plane and heat of formation of the molecules resulted in a statistically significant model with r(2)=0.962 and cross-validation parameter q(2)=0.917. The validation of the QSAR models was done by cross-validation, leave-25%-out and external test set prediction. The theoretical approach indicates that the increase in the exposed partial negative surface area increases the inhibitory activity of the compound against NorA whereas the area of the molecular shadow in the XZ plane is inversely proportional to the inhibitory activity. This model also explains the relationship of the heat of formation of the compound with the inhibitory activity. The model is not only able to predict the activity of new compounds but also explains the important regions in the molecules in quantitative manner.
Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.
Antibiotic resistance continues to plague antimicrobial chemotherapy of infectious disease. And while true biocide resistance is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are common and the history of antibiotic resistance should not be ignored in the development and use of biocidal agents. Efflux mechanisms of resistance, both drug specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials, with some accommodating both antibiotics and biocides. This latter raises the spectre (as yet generally unrealized) of biocide selection of multiple antibiotic-resistant organisms. Multidrug efflux mechanisms are broadly conserved in bacteria, are almost invariably chromosome-encoded and their expression in many instances results from mutations in regulatory genes. In contrast, drug-specific efflux mechanisms are generally encoded by plasmids and/or other mobile genetic elements (transposons, integrons) that carry additional resistance genes, and so their ready acquisition is compounded by their association with multidrug resistance. While there is some support for the latter efflux systems arising from efflux determinants of self-protection in antibiotic-producing Streptomyces spp. and, thus, intended as drug exporters, increasingly, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, appear not to be intended as drug exporters but as exporters with, perhaps, a variety of other roles in bacterial cells. Still, given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents, for example, less impacted by efflux and in targeting efflux directly with efflux inhibitors.
As part of an ongoing project to identify plant natural products as efflux pump inhibitors (EPIs), bioassay-guided fractionation of the methanolic extract of Mirabilis jalapa Linn. (Nyctaginaceae) led to the isolation of an active polyphenolic amide: N-trans-feruloyl 4'-O-methyldopamine. This compound showed moderate activity as an EPI against multidrug-resistant (MDR) Staphylococcus aureus overexpressing the multidrug efflux transporter NorA, causing an 8-fold reduction of norfloxacin MIC at 292 microM (100 microg/mL). This prompted us to synthesize derivatives in order to provide structure-activity relationships and to access more potent inhibitors. Among the synthetic compounds, some were more active than the natural compound and N-trans-3,4-O-dimethylcaffeoyl tryptamine showed potentiation of norfloxacin in MDR S. aureus comparable to that of the standard reserpine.
Antibiotic resistance continues to hamper antimicrobial chemotherapy of infectious disease, and while biocide resistance outside of the laboratory is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are not uncommon. Efflux mechanisms, both drug-specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials in important human pathogens. Multidrug efflux mechanisms are generally chromosome-encoded, with their expression typically resultant from mutations in regulatory genes, while drug-specific efflux mechanisms are encoded by mobile genetic elements whose acquisition is sufficient for resistance. While it has been suggested that drug-specific efflux systems originated from efflux determinants of self-protection in antibiotic-producing Actinomycetes, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, are appreciated as having an intended housekeeping function unrelated to drug export and resistance. Thus, it will be important to elucidate the intended natural function of these efflux mechanisms in order, for example, to anticipate environmental conditions or circumstances that might promote their expression and, so, compromise antimicrobial chemotherapy. Given the clinical significance of antimicrobial exporters, it is clear that efflux must be considered in formulating strategies for treatment of drug-resistant infections, both in the development of new agents, for example, less impacted by efflux or in targeting efflux directly with efflux inhibitors.
A quantitative structure-activity relationship (QSAR) analysis has been performed on a data set of 42 aryl alkenyl amides/imines as bacterial efflux pump inhibitors. Several types of descriptors including topological, spatial, thermodynamic, information content and E-state indices have been used to derive a quantitative relationship between the efflux pump inhibiting activity and structural properties. Algorithm based on genetic function approximation method of variable selection was used to generate the model. Statistically significant model (with r(2)=0.87) was obtained with the descriptors like radius of gyration and heat of formation besides E-state indices, AlogP atom types and solvent accessible charged surface area playing an important role in determining the activity of the compounds against bacterial efflux pump. The model was also tested successfully for external validation criteria. The model is not only able to predict the activity of new compounds but also explained the important regions in the molecules in quantitative manner.
Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps
(MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI
which is active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,110, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression
of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions
in MICs in the presence of MC-207,110 were observed for strains with overexpressed efflux pumps and various target mutations
that confer resistance to levofloxacin (e.g., gyrA andparC). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times
the MIC of levofloxacin (1 μg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone,
the frequency was ∼10−7 CFU/ml. In contrast, with an EPI, the frequency was below the level of detection (<10−11). In summary, we have demonstrated that inhibition of efflux pumps (i) decreased the level of intrinsic resistance significantly,
(ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of P. aeruginosa strains that are highly resistant to fluoroquinolones.
One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.
Moxifloxacin is a C8-methoxy (C8-OMe) fluoroquinolone that is highly active against Staphylococcus aureus, including many strains resistant to older fluoroquinolones such as ciprofloxacin. Available data indicate that it is a poor substrate for the NorA multidrug efflux pump. We produced a mutant of S. aureus in vitro (SA-K2068) with a novel non-NorA-mediated multidrug resistance phenotype characterized by raised MICs of several fluoroquinolones, including the C8-OMe fluoroquinolones, moxifloxacin and gatifloxacin, and the organic cations ethidium and tetraphenylphosphonium. Reserpine reduced MIC increases by two- to eight-fold. SA-K2068 also demonstrated reduced accumulation of moxifloxacin, gatifloxacin and enoxacin, and increased efflux of ethidium, activities that were completely blocked by carbonyl cyanide m-chlorophenyl hydrazone (CCCP); competition experiments indicated that a single pump was responsible for the phenotype. The effect of CCCP and ionophores identified the proton motive force as the source of energy for efflux. These data, combined with previous work from our laboratory and genome sequence data, indicate that S. aureus possesses several multidrug efflux pump proteins and it is apparent that C8-OMe fluoroquinolones can be substrates for such pumps.
AcrAB-TolC is the major, constitutively expressed efflux protein complex that provides resistance to a variety of antimicrobial
agents in Escherichia coli. Previous studies showed that AcrA, a periplasmic protein of the membrane fusion protein family, could function with at least
two other resistance-nodulation-division family pumps, AcrD and AcrF, in addition to its cognate partner, AcrB. We found that,
among other E. coli resistance-nodulation-division pumps, YhiV, but not MdtB or MdtC, could also function with AcrA. When AcrB was assessed for
the capacity to function with AcrA homologs, only AcrE, but not YhiU or MdtA, could complement an AcrA deficiency. Since AcrA
could, but YhiU could not, function with AcrB, we engineered a series of chimeric mutants of these proteins in order to determine
the domain(s) of AcrA that is required for its support of AcrB function. The 290-residue N-terminal segment of the 398-residue
protein AcrA could be replaced with a sequence coding for the corresponding region of YhiU, but replacement of the region
between residues 290 and 357 produced a protein incapable of functioning with AcrB. In contrast, the replacement of residues
357 through 397 of AcrA still produced a functional protein. We conclude that a small region of AcrA close to, but not at,
its C terminus is involved in the interaction with its cognate pump protein, AcrB.
Gram-negative bacteria have, in general, much higher intrin- sic levels of resistance to various antibiotics, antiseptics, dyes, and detergents than do gram-positive bacteria. This is, in part, due to the effectiveness of the outer membrane as a barrier. The porin channels exclude large compounds (for example, vancomycin) and drastically slow down the influx of most an- tibiotics, which are usually much larger than common nutri- ents. Most antibiotics and chemotherapeutic agents that act on targets in the cytosol must cross the inner, cytoplasmic mem- brane, usually by spontaneous diffusion, and this necessitates their being at least moderately lipophilic. These compounds can in principle diffuse across the lipid bi-layer domain of the outer membrane. Transmembrane diffusion rates across this domain, however, are about two orders of magnitude slower than through the conventional phospholipid bi-layers (29), be- cause the outer leaflet, composed exclusively of lipopolysac- charides (11), acts as an effective barrier. The outer membrane barrier alone, however, only slows down the influx of most of the noxious agents, and the gram- negative bacteria need the additional contribution of multi-
Isogenic strains containing insertional disruptions of 10 Haemophilus influenzae Rd genes were investigated for their effects on the susceptibility of the organism to various classes of antimicrobial compounds.
MIC results show that HI1462, which encodes an Escherichia coli TolC homolog, is the third component of the H. influenzae AcrAB pump.
The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P. aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking
approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction
similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding
wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the
protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91,
A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization).
In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270,
H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its RND component, AcrB (C. A. Elkins and H. Nikaido, J. Bacteriol. 185:5349-5356,
2003). Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important
for and define the MexA interaction with its RND component, MexB. Still, all but one of the MexA mutations studied compromised
MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this.
Haemophilus influenzae HMC-C with high-level macrolide resistance after multi-step selection by clarithromycin reverted spontaneously and became hypersusceptible to macrolides.
Determination of macrolide resistance mechanism(s) in hypersusceptible and hyperresistant strains.
The presence of macrolide efflux in the strains was studied by radioactive erythromycin accumulation. Ribosomal mutations were investigated by sequencing. The possible role of acrAB clusters in macrolide resistance was studied by sequencing and expression analysis.
The parent strain had no ribosomal alteration, but both high-level resistant and hypersusceptible strains had R88P mutations in ribosomal protein L22. Radioactive macrolide accumulation studies pointed to the presence of macrolide efflux in the high-level resistant and parent strains, but not in the hypersusceptible derivative. Transformation of hypersusceptible strains using total DNA from the parent strain restored the macrolide efflux system in the hypersusceptible strain, which was confirmed by MIC levels and radioactive erythromycin accumulation similar to that of the mutant resistant strain. Analysis of sequence and transcription of acrAB gene clusters showed no significant differences between resistant and hypersusceptible derivatives.
Mutation in ribosomal protein L22 alone does not confer high-level macrolide resistance unless efflux is present.
The activity of WCK 771, an experimental quinolone developed to overcome quinolone resistance in staphylococci and other bacteria,
was determined against quinolone-susceptible and -resistant Staphylococcus aureus and S. epidermidis. WCK 771 MICs for 50 and 90% of the strains tested (MIC50 and MIC90, respectively) were 0.008 and 0.015 μg/ml for S. aureus (n = 43) and 0.015 and 0.03 μg/ml for S. epidermidis (n = 44) for quinolone-susceptible isolates, compared to ciprofloxacin values of 0.12 and 0.25 μg/ml and 0.25 and 0.5 μg/ml,
respectively. Values for levofloxacin were 0.12 and 0.25 μg/ml and 0.12 and 0.25 μg/ml, those for clinafloxacin were 0.015
and 0.03 μg/ml and 0.015 and 0.03 μg/ml, those for moxifloxacin were 0.03 and 0.06 μg/ml and 0.06 and 0.12 μg/ml, and those
for gatifloxacin were 0.06 and 0.12 μg/ml and 0.12 and 0.25 μg/ml, respectively. The WCK 771 MIC50 and MIC90, respectively, were 0.5 and 1 μg/ml for both species of staphylococci (n = 73 for S. aureus, n = 70 for S. epidermidis) for isolates highly resistant to ciprofloxacin (MIC50 and MIC90, >32 and >32 μg/ml, respectively). Values for levofloxacin were 8 and 32 μg/ml and 8 and 32 μg/ml, those for clinafloxacin
were 1 and 2 μg/ml and 0.5 and 2 μg/ml, those for moxifloxacin 4 and >4 μg/ml and 4 and >4 μg/ml, and those for gatifloxacin
were 4 and >4 μg/ml and 2 and >4 μg/ml, respectively. WCK 771 and clinafloxacin demonstrated MICs of 1 μg/ml against three
vancomycin-intermediate strains. WCK 771 showed concentration-independent killing for up to 24 h at 2, 4, and 8 times the
MICs against quinolone-resistant staphylococci and was also bactericidal after 8 h for high-density inocula (108 CFU/ml) of quinolone-resistant strains at 5 μg/ml, whereas moxifloxacin at 7.5 μg/ml was bacteriostatic. WCK 771 was not
a substrate of the NorA efflux pump as evident from the similar MICs against both an efflux-positive and an efflux-negative
strain. Overall, WCK 771 was the most potent quinolone tested against the staphylococci tested, regardless of quinolone susceptibility.
Several arylpiperazines capable of reversing multidrug resistance (MDR) in Escherichia coli overexpressing acrAB and acrEF but not in pump-deficient mutant strains were identified. 1-(1-Naphthylmethyl)-piperazine, one of the more active compounds,
enhanced susceptibility to fluoroquinolones and other agents and increased the intracellular concentration of levofloxacin
and ethidium bromide, suggesting efflux pump inhibition as the mechanism of MDR reversal.
Aminoglycosides (35) are a vital component of antipseudo- monal chemotherapy implicated in the treatment of a variety of infections (9, 45), particularly pulmonary infections in cystic fibrosis (CF) patients (22). These agents are bactericidal and exhibit synergy with other antimicrobials, most notably -lac- tams, with which they are often administered for the treatment of Pseudomonas aeruginosa infections; and problems with tox- icity (aminoglycosides are oto- and nephrotoxic) appear to be ameliorated by increasing the dosing intervals (157a) and, in lung infections at least, through the use of aerosolized agents (e.g., tobramycin) (22). Resistance to aminoglycosides has, however, been known for some time, with reports from the 1960s highlighting the general insusceptibilities of P. aerugi- nosa clinical isolates to, e.g., kanamycin (50, 51). Today, resis- tance to aminoglycosides with antipseudomonal activities, in- cluding gentamicin and tobramycin, but also amikacin, is also all too common and is present in virtually all areas of the world, but particularly in Europe and Latin America (Table 1). Such resistance is seen in respiratory isolates (96), particularly isolates from CF patients (57, 85, 115, 140), as well as blood- stream (86), urinary (12), wound (65), burn (36, 166), eye (2, 20), and aural (27, 128) isolates (Table 1). Resistance typically results from drug inactivation by plasmid- or chromosome- encoded enzymes harbored by resistant strains, although en- zyme-independent resistance resultant from defects in uptake and accumulation (dubbed impermeability resistance) is also commonplace, particularly in isolates from CF patients (99- 101, 114, 121, 131) and intensive care units (ICUs) (10, 48, 54). MODIFYING ENZYMES
We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against
wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination
of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect
on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining
regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting
of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance.
Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25
to 2.5 and 1.25 μg/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin,
its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the
dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.
To investigate whether telithromycin is a substrate for efflux pumps in Streptococcus pyogenes.
The distribution of telithromycin MICs was analysed for two distinct collections of Italian (n=486) and Spanish (n=210) S. pyogenes strains. The effect of an efflux mechanism was investigated using [(3)H]telithromycin.
Telithromycin MIC ranges were < or = 0.004-0.06 mg/L (MIC(50) and MIC(90), 0.01 mg/L) in erythromycin-susceptible strains (lacking both mef and erm genes) and 0.01-1 mg/L (MIC(50) and MIC(90), 0.5 mg/L) in strains endowed with the M phenotype and expressing the mef(A) gene. A distinct telithromycin efflux was detected in the strains expressing the mef(A) gene, but not in those expressing the erm(B) gene, nor in the susceptible strains lacking mef(A) or erm genes. Efflux reversibility by addition of an inhibiting compound (sodium arsenate) was demonstrated. An msr-like sequence was also found in all strains effluxing telithromycin, but not in the others.
This study shows that telithromycin can be removed from S. pyogenes by efflux. That the efflux is related to the presence of the mef(A) gene is demonstrated, but-owing to the increasingly evident complexity of S. pyogenes efflux systems-the possibility that other genes may contribute to the efflux cannot be excluded.
In an analysis of the resistance mechanisms of an mgrA mutant, we identified two genes encoding previously undescribed transporters, NorB and Tet38. norB was 1,392 bp and encoded a predicted 49-kDa protein. When overexpressed, NorB led to an increase in resistance to hydrophilic
quinolones, ethidium bromide, and cetrimide and also to sparfloxacin, moxifloxacin, and tetracycline, a resistance phenotype
of the mgrA mutant. NorA and NorB shared 30% similarity, and NorB shared 30 and 41% similarities with the Bmr and Blt transporters of
Bacillus subtilis, respectively. The second efflux pump was a more selective transporter that we have called Tet38, which had 46% similarity
with the plasmid-encoded TetK efflux transporter of S. aureus. tet38 was 1,353 bp and encoded a predicted 49-kDa protein. Overexpression of tet38 produced resistance to tetracycline but not to minocycline and other drugs. norB and tet38 transcription was negatively regulated by MgrA. Limited binding of MgrA to the promoter regions of norB and tet38 was demonstrated by gel shift assays, suggesting that MgrA was an indirect regulator of norB and tet38 expression. The mgrA norB double mutant was reproducibly twofold more susceptible to the tested quinolones than the mgrA mutant. The mgrA tet38 double mutant became more susceptible to tetracycline than the wild-type parent strain. These data demonstrate that overexpression
of NorB and Tet38 contribute, respectively, to the hydrophobic quinolone resistance and the tetracycline resistance of the
mgrA mutant and that MgrA regulates expression of norB and tet38 in addition to its role in regulation of norA expression.
Peptides based on the amino acid sequences found at protein-protein interaction sites make excellent leads for antagonist development. A statistical picture of amino acids involved in protein-protein interactions indicates that proteins recognize and interact with one another through the restricted set of specialized interface amino acid residues, Pro, Ile, Tyr, Trp, Asp and Arg. These amino acids represent residues from each of the three classes of amino acids, hydrophobic, aromatic and charged, with one anionic and one cationic residue at neutral pH. The use of peptides as drug leads has been successfully used to search for antagonists of cell-surface receptors. Peptide, peptidomimetic, and non-peptide organic inhibitors of a class of cell surface receptors, the integrins, currently serve as therapeutic and diagnostic imaging agents. In this review, we discuss the structural features of protein-protein interactions as well as the design of peptides, peptidomimetics, and small organic molecules for the inhibition of protein-protein interactions. Information gained from studying inhibitors of integrin functions is now being applied to the design and testing of inhibitors of other protein-protein interactions. Most drug development progress in the past several decades has been made using the enzyme binding-pocket model of drug targets. Small molecules are designed to fit into the substrate-binding pockets of proteins based on a lock-and-key, induced-fit, or conformational ensemble model of the protein binding site. Traditionally, enzymes have been used as therapeutic drug targets because it was easier to develop rapid, sensitive screening assays, and to find low molecular weight inhibitors that blocked the active site. However, for proteins which interact with other proteins, rather than with small substrate molecules, the lack of binding pockets means that this approach will not generally succeed. There exist many diseases in which the inhibition of protein-protein interactions would provide therapeutic benefit, but there are no general methods available to address such problems. The focus of the first part of this review is to discuss the features of protein-protein interactions which may serve as general guidelines for the development and design of inhibitors for protein-protein interactions. In the second part we focus on the design of peptides (lead compounds) and their conversion into peptidomimetics or small organic molecules for the inhibition of protein-protein interactions. We draw examples from the important and emerging area of integrin-based cell adhesion and show how the principles of protein-protein interactions are followed in the discovery, optimization and usage of specific protein interface peptides as drug leads.
Tigecycline, an expanded-broad-spectrum glycylcycline antibiotic is not affected by the classical tetracycline resistance
determinants found in Staphylococcus aureus. The in vitro selection of mutants with reduced susceptibility to tigecycline was evaluated for two methicillin-resistant
S. aureus strains by serial passage in increasing concentrations of tigecycline. Both strains showed a stepwise elevation in tigecycline
MIC over a period of 16 days, resulting in an increase in tigecycline MIC of 16- and 32-fold for N315 and Mu3, respectively.
Transcriptional profiling revealed that both mutants exhibited over 100-fold increased expression of a gene cluster, mepRAB (multidrug export protein), encoding a MarR-like transcriptional regulator (mepR), a novel MATE family efflux pump (mepA), and a hypothetical protein of unknown function (mepB). Sequencing of the mepR gene in the mutant strains identified changes that presumably inactivated the MepR protein, which suggested that MepR functions
as a repressor of mepA. Overexpression of mepA in a wild-type background caused a decrease in susceptibility to tigecycline and other substrates for MATE-type efflux pumps,
although it was not sufficient to confer high-level resistance to tigecycline. Complementation of the mepR defect by overexpressing a wild-type mepR gene reduced mepA transcription and lowered the tigecycline MIC in the mutants. Transcription of tet(M) also increased by over 40-fold in the Mu3 mutant. This was attributed to a deletion in the promoter region of the gene
that removed a stem-loop responsible for transcriptional attenuation. However, overexpression of the tet(M) transcript in a tigecycline-susceptible strain was not enough to significantly increase the MIC of tigecycline. These
results suggest that the overexpression of mepA but not tet(M) may contribute to decreased susceptibility of tigecycline in S. aureus.
Antibiotic resistance continues to plague antimicrobial chemotherapy of infectious disease. And while true biocide resistance is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are common and the history of antibiotic resistance should not be ignored in the development and use of biocidal agents. Efflux mechanisms of resistance, both drug specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials, with some accommodating both antibiotics and biocides. This latter raises the spectre (as yet generally unrealized) of biocide selection of multiple antibiotic-resistant organisms. Multidrug efflux mechanisms are broadly conserved in bacteria, are almost invariably chromosome-encoded and their expression in many instances results from mutations in regulatory genes. In contrast, drug-specific efflux mechanisms are generally encoded by plasmids and/or other mobile genetic elements (transposons, integrons) that carry additional resistance genes, and so their ready acquisition is compounded by their association with multidrug resistance. While there is some support for the latter efflux systems arising from efflux determinants of self-protection in antibiotic-producing Streptomyces spp. and, thus, intended as drug exporters, increasingly, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, appear not to be intended as drug exporters but as exporters with, perhaps, a variety of other roles in bacterial cells. Still, given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents, for example, less impacted by efflux and in targeting efflux directly with efflux inhibitors.
In Gram-negative bacteria, efflux complexes, consisting of an inner-membrane pump, a periplasmic adaptor protein and outer-membrane channel, provide an efficient means for the export of structurally unrelated drugs, causing the multidrug-resistance phenotype. Resistance due to this antibiotic efflux is an increasing problem worldwide. A new molecular challenge is to combat this transport by searching for new molecules to block efflux and thus restore drug susceptibility to resistant clinical strains. Recent data shed new light on the structure and activity of the archetypal efflux pumps AcrAB-TolC and MexAB-OprM. Here, we describe recent insights into the molecular mechanisms of bacterial efflux pumps and their inhibitors. Current progress for the clinical use of efflux-pump inhibitors and new strategies to combat the drug-efflux mechanisms will be discussed.
New antimicrobial agents are urgently needed for clinical use due to the increasing prevalence and spread of multidrug-resistant bacteria that are commonly responsible for serious and life-threatening diseases. The need to develop new agents that effectively overcome existing mechanisms of resistance displayed by bacteria resistant to currently available drugs has become paramount. Tigecycline, the first in a new class of antimicrobials, the glycylcyclines, is an analogue of minocycline with additional properties that negate most mechanisms mediating resistance to the tetracyclines. In vitro testing has revealed that tigecycline has activity against vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae and many species of multidrug-resistant Gram-negative bacteria, although resistance to tigecycline by Pseudomonas aeruginosa and reduced susceptibility among Proteus species do occur. Tigecycline is being evaluated in multicentre Phase III clinical trials for therapy of many serious and life-threatening infections in which multidrug-resistant bacterial organisms may be found. Tigecycline appears to hold promise as a novel expanded spectrum antibiotic.
Apicomplexa are unicellular, obligate intracellular parasites of great medical importance. They include human pathogens like Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease (toxoplasmosis). They alone affect several hundred million people worldwide so that new drugs, especially for plasmodial infections, are urgently needed. This review will focus on a recently emerged, potential drug target, a plant-type redox system consisting of ferredoxin-NADP(+) reductase (FNR) and its redox partner, ferredoxin (Fd). Both reside in an unique organelle of these parasites, named apicoplast, which is of algal origin. The apicoplast has been shown to be required for pathogen survival. In addition to other pathways already identified in this compartment, the FNR/Fd redox system represents a promising drug target because homologous proteins are not present in host organisms. Furthermore, a wealth of structural information exists on the closely related plant proteins, which can be exploited for structure-function studies of the apicomplexan protein pair. T. gondii and P. falciparum FNRs have been cloned, and the T. gondii enzyme was shown to be a flavoprotein active as a NADPH-dependent oxidoreductase. Both phylogenetic and biochemical analyses indicate that T. gondii FNR is similar in function to the isoform present in non-photosynthetic plastids whereby electron flow is from NADPH to oxidized Fd. The resulting reduced Fd is then presumably used as a reductant for various target enzymes whose nature is just starting to emerge. Among the likely candidates is the iron-sulfur cluster biosynthesis pathway, which is also located in the apicoplast and dependent on reducing power. Furthermore, lipoic acid synthase and enzymes of the isoprenoid biosynthetic pathway may be other conceivable targets. Since all these metabolic steps are vital for the parasite, blocking electron flow from FNR to Fd by inhibition of either FNR activity or its molecular interaction with Fd should also interfere with these pathways, ultimately killing the parasite. Although the three-dimensional structure of FNR from T. gondii is not yet known, experimental and computational evidence shows that apicomplexan and plant enzymes are very similar in structure. Furthermore, single amino acid changes can have profound effects on the enzyme activity and affinity for Fd. This knowledge may be exploited for the design of inhibitors of protein-protein interaction. On the other hand, specifically tailored NAD(P) analogues or mimetics based on previously described substances might be useful lead compounds for apicomplexan FNR inhibitors.
A T578I mutation in MexB compromised the protein's contribution to antimicrobial resistance and negatively impacted its interaction
with MexA. Mutations causing single amino acid changes in the C-terminal domain of MexA (R221H, L245F, E254K, and V259I) suppressed
the antimicrobial susceptibility of a MexBT578I-expressing Pseudomonas aeruginosa strain and restored a MexA interaction with MexBT578I. These data confirm the importance of the MexA C-terminal region in MexB binding and the likely significance of the region
surrounding T587I of MexB in MexA interaction.
DX-619, a novel des-fluoro(6) quinolone, was 16- to 32-fold, twofold, and four- to eightfold more potent than ciprofloxacin,
gemifloxacin, and garenoxacin, respectively, against wild-type Staphylococcus aureus. DX-619 manifested equal fourfold increases in MIC against a common parC mutant and a common gyrA mutant and selected for mutants at up to two- to fourfold its MIC, consistent with dual-targeting properties. Of the four
independent single-step mutants selected, two had new single mutations in parC (V87F and R17H), and two shared a new gyrA mutation (A26V), one with an additional deletion mutation in parE (Δ215-7). By allelic exchange, the ParC but not the GyrA or ParE mutation was shown to be fully responsible for the resistance
phenotypes, suggesting an as yet undefined mechanism of resistance operating in conjunction with type II topoisomerase mutations
contributed to resistance to DX-619. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that DX-619 had similar activity against topoisomerase IV and gyrase (50% stimulation of cleavage complexes concentration,
1.25 and 0.62 to 1.25 μg/ml, respectively). Susceptibility studies with DX-619 and an array of efflux pump substrates with
and without reserpine, an inhibitor of efflux pumps, suggested that resistance in DX-619-selected mutants is affected by mechanisms
other than mutations in topoisomerases or known reserpine-inhibitable pumps in S. aureus and thus are likely novel.
The world of antibiotic drug discovery and development is driven by the necessity to overcome antibiotic resistance in common Gram-positive and Gram-negative pathogens. However, the lack of Gram-negative activity among both recently approved antibiotics and compounds in the developmental pipeline is a general trend despite the fact that the plethora of covered drug targets are well-conserved across the bacterial kingdom. Such intrinsic resistance in Gram-negative bacteria is largely attributed to the activity of multidrug resistance (MDR) efflux pumps. Moreover, these pumps also play a significant role in acquired clinical resistance. Together, these considerations make efflux pumps attractive targets for inhibition in that the resultant efflux pump inhibitor (EPI)/antibiotic combination drug should exhibit increased potency, enhanced spectrum of activity and reduced propensity for acquired resistance. To date, at least one class of broad-spectrum EPI has been extensively characterized. While these efforts indicated a significant potential for developing small molecule inhibitors against efflux pumps, they did not result in a clinically useful compound. Stemming from the continued clinical pressure for novel approaches to combat drug resistant bacterial infections, second-generation programs have been initiated and show early promise to significantly improve the clinical usefulness of currently available and future antibiotics against otherwise recalcitrant Gram-negative infections. It is also apparent that some changes in regulatory decision-making regarding resistance would be very helpful in order to facilitate approval of agents aiming to reverse resistance and prevent its further development.
The majority of bacterial multidrug resistance transporters belong to the class of secondary transporters. LmrP is a proton/drug antiporter of Lactococcus lactis that extrudes positively charged lipophilic substrates from the inner leaflet of the membrane to the external medium. This study shows that LmrP is a true secondary transporter. In the absence of a proton motive force, LmrP facilitates downhill fluxes of ethidium in both directions. These fluxes are inhibited by other substrates of LmrP. The cysteine-reactive agent p-chloromercuri-benzene sulfonate inhibits these fluxes in wild type LmrP but not in the cysteine-less LmrP C270A mutant. Cysteine mutagenesis of LmrP resulted in three mutants, D68C/C270A, D128C/C270A, and E327C/C270A, with an energy-uncoupled phenotype. Asp68 is located in the conserved motif GXXX(D/E)(R/K)XGRK for the major facilitator superfamily of secondary transporters and was found to play an important role in energy coupling, whereas the negatively charged residues Asp128 and Glu327 have indirect effects on the transport process. L. lactis strains expressing these uncoupled mutants of LmrP show an increased rate of ethidium influx and an increased drug susceptibility compared with cells harboring an empty vector. The rate of influx in these mutants is enhanced by a transmembrane electrical potential, inside negative. These observations suggest a new strategy for eliminating drug-resistant microbial pathogens, i.e. the design and use of modulators of secondary multidrug resistance transporters that uncouple drug efflux from proton influx, thereby allowing transmembrane electrical potential-driven influx of cationic drugs.
Organisms such as Pseudomonas aeruginosa, Stenotrophornonas maltophilia and Burkholderia spp. are of increasing clinical importance because of their innate resistance to multiple agents and their ability to develop high-level multidrug resistance (MDR)52, 53. This resistance owes much to the presence of broadly specific efflux systems which export and, thus, provide resistance to multiple antimicrobials. Drug efflux systems have been grouped into five families, the major facilitator (MF) family196, the ATP-binding cassette (ABC) family101, 223, the resistance-nodulation-division (RND) family222, 237, the small multi drug resistance (SMR) family31 (a member of the much larger drug/metabolite transporter (DMT) superfamily80) and the multidrug and toxic compound extrusion (MATE) family22 (see refs. [175, 183] for recent reviews on bacterial multidrug efflux systems). With the exception of the ABC exporters, which utilize ATP hydrolysis to promote drug extrusion, these cytoplasmic membrane-associated efflux pumps function as secondary transporters, coupling drug efflux to ion (H+ or Na+) influx. From a clinical standpoint, efflux systems of the RND family are most important, providing as they do efflux of and resistance to a variety of clinically relevant antibiotics and biocides173, 175. Members of this family operate as part of a tripar6te efflux system that also includes an outer membrane (the channel-forming OMF, outer membrane factor165) and periplasmic (the membrane fusion protein, MFP37) component (reviewed in ref. [237]), an organization seen on occasion with MF transporters (Figure 1).
This study was undertaken in order to investigate biochemical, genetic, and epidemiologic aspects of resistance to aminoglycoside antibiotics in isolates of Pseudomonas aeruginosa. Minimal inhibitory concentrations of kanamycin, gentamicin, and tobramycin were determined for 650 isolates of P. aeruginosa collected from December 1972 through March 1973. Half of all isolates were resistant to one or more of these aminoglycosidic antibiotics, with the minimal inhibitory concentration ≥ 20 μg of kanamycin/ml or 10 μg of gentamicin or tobramycin/ml. Among the 650 isolates, 56% were resistant to kanamycin, 8.9% were resistant to gentamicin, and 1.1% were resistant to tobramycin at the above levels. High level resistance (minimal inhibitory concentration, ≥ 25 μg/ml) to gentamicin or tobramycin was found in 28 strains and to kanamycin in 364 strains. Resistance to gentamicin or tobramycin occurred only among strains that were resistant to kanamycin. Four groups could be defined by patterns of high level resistance to the three antibiotics. Selected strains were tested for pyocin production; for enzymes that modify aminoglycosides by phosphorylation, acetylation, or adenylylation, and for conjugal transfer or resistance to aminoglycosides. Phosphorylating and acetylating enzymes with several different specificities for aminoglycoside substrates are present in these strains. Isolates with similar patterns of resistance to aminoglycosides could be divided into subgroups on the basis of patterns of pyocin production. No conjugal transfer of aminoglycoside resistance from these strains of P. aeruginosa to various recipient strains was observed.
We investigated the role of chromosomal beta-lactamase and the MexAB-OprM efflux system in intrinsic resistance to beta-lactams in Pseudomonas aeruginosa. Determination of the susceptibilities of a series of isogenic mutants with impaired production of the beta-lactamase and the efflux system to 16 beta-lactams including penicillins, cephems, oxacephems, carbapenems, and a monobactam demonstrated that the intrinsic resistance of P. aeruginosa to most of the beta-lactams is due to the interplay of both factors.
Drug efflux pumps in Pseudomonas aeruginosa were evaluated as potential targets for antibacterial therapy. The potential effects of pump inhibition on susceptibility to fluoroquinolone antibiotics were studied with isogenic strains that overexpress or lack individual efflux pumps and that have various combinations of efflux- and target-mediated mutations. Deletions in three efflux pump operons were constructed. As expected, deletion of the MexAB-OprM efflux pump decreased resistance to fluoroquinolones in the wild-type P. aeruginosa (16-fold reduction for levofloxacin [LVX]) or in the strain that overexpressed mexAB-oprM operon (64-fold reduction for LVX). In addition to that, resistance to LVX was significantly reduced even for the strains carrying target mutations (64-fold for strains for which LVX MICs were >4 microg/ml). We also studied the frequencies of emergence of LVX-resistant variants from different deletion mutants and the wild-type strain. Deletion of individual pumps or pairs of the pumps did not significantly affect the frequency of emergence of resistant variants (at 4x the MIC for the wild-type strain) compared to that for the wild type (10(-6) to 10(-7)). In the case of the strain with a triple deletion, the frequency of spontaneous mutants was undetectable (<10(-11)). In summary, inhibition of drug efflux pumps would (i) significantly decrease the level of intrinsic resistance, (ii) reverse acquired resistance, and (iii) result in a decreased frequency of emergence of P. aeruginosa strains highly resistant to fluoroquinolones in clinical settings.
The chromosomal multiple antibiotic resistance (mar) locus of Escherichia coli and other members of the Enterobacteriaceae controls resistance to multiple, structurally unrelated compounds including antibiotics, household disinfectants, organic solvents and other toxic chemicals. The Mar phenotype is induced following exposure to a variety of chemicals with aromatic rings.
The TolC protein of Escherichia coli comprises an outer membrane beta-barrel channel and a contiguous alpha-helical tunnel spanning the periplasm, providing an exit duct for protein export and multidrug efflux. It forms a single transmembrane pore that is open to the outside of the cell but constricted at the peri-plasmic tunnel entrance. This sole constriction is lined by a ring of six aspartate residues, two in each of the three identical monomers. When these were replaced by alanines, the resulting TolC(DADA) protein reconstituted normally in black lipid membranes but showed altered electrophysiological characteristics. In particular, it had lost the strong pH dependence of the wild type and had switched ion selectivity from cations to anions. The function of wild-type TolC as a membrane pore was severely inhibited by divalent and trivalent cations entering the channel tunnel from the channel ("extracurricular") side. Divalent cations bound reversibly to effect complete blocking of the transmembrane ion flux. Trivalent cations were more potent. Hexamminecobalt bound at nanomolar concentrations allowed visualization of single blocking events, whereas the smaller Cr(3+) cation bound irreversibly and could also access the cation binding site via the tunnel entrance. The inhibitory cations had no effect on the mutant TolC(DADA), supporting the view that the aspartate ring is the cation binding site. The electronegative entrance is widely conserved throughout the TolC family, which is essential for efflux and export my Gram-negative bacteria, suggesting that it could present a general target for drugs.
Significant levels of fluoroquinolone resistance were obtained in Campylobacterjejuni isolates after an unique step of selection using enrofloxacin. An Asp90-to-Asn and a Thr86-to-Ile change in the gyrase subunit GyrA were found associated with a low (MIC < or = 8 /microg/ml) or a high (MIC > or = 16 microg/ml) level of resistance to ciprofloxacin, respectively. An association of both mutations conferred a higher level of resistance (MIC > or = 128 microg/ml). Further steps of selection increased the MICs of fluoroquinolones but did not result in a multiple antibiotic resistance phenotype. The Thr86-to-Ile change was found to confer different levels of resistance, pointing out other mechanisms of resistance. However, sequencing revealed no mutation in gyrB, and several attempts did not enable any amplification of the parC gene coding for topoisomerase IV, suggesting an absence of this secondary target in C. jejuni. In addition, no difference in the major outer membrane protein expression was found among the isolates. Furthermore, the use of the recently identified efflux pump inhibitor Phe-Arg-beta-naphthylamide did not result in a significant decrease of fluoroquinolone MICs or change in the frequency of isolation of enrofloxacin-resistant mutants, and thus appears ineffective against fluoroquinolone-resistant C. jejuni isolates. Results obtained during ciprofloxacin accumulation studies confirmed that efflux probably plays a minor role in fluoroquinolone resistance of C. jejuni.
Conformational restriction of the ornithine residue of the efflux pump inhibitor D-ornithine-D-homophenylalanine-3-aminoquinoline (MC-02,595, 2) furnished bioisosteric proline derivatives that were less toxic in vivo and as active as the lead in potentiating the activity of the fluoroquinolone levofloxacin via the inhibition of efflux pumps in Pseudomonas aeruginosa.
Efflux mechanisms that account for resistance to a variety of antimicrobial agents are commonly found in a wide range of bacteria. Two major groups of efflux systems are known, specific exporters and transporters conferring multidrug resistance (MDR). The MDR systems are able to remove antimicrobials of different classes from the bacterial cell and occasionally play a role in the intrinsic resistance of some bacteria to certain antimicrobials. Their genes are commonly located on the bacterial chromosome. In contrast, the genes coding for specific efflux systems are often associated with mobile genetic elements which can easily be interchanged between bacteria. Specific efflux systems have mainly been identified with resistances to macrolides, lincosamides and/or streptogramins, tetracyclines, as well as chloramphenicol/florfenicol in Gram-positive and Gram-negative bacteria. In this review, we focus on the molecular biology of antimicrobial resistance mediated by specific efflux systems and highlight the association of the respective resistance genes with mobile genetic elements and their distribution across species and genus borders.
Following the optimization of diamine-containing efflux pump inhibitors with respect to in vitro potentiation activity, in vivo stability and acute toxicity, we addressed the question of how to control the pharmacokinetic properties of the series. Upon intravenous administration in the rat, tissue levels of MC-04,124 (the lead compound) were high and prolonged compared to those in the serum. The lipophilicity and basicity of analogues of this compound were systematically varied, and effects on potency and pharmacokinetics explored. The ratio of drug levels in tissue versus serum was not significantly reduced in any of the active analogues examined.
The identification of a series of compounds that specifically inhibit efflux by the MexAB-OprM pump system in Pseudomonas aeruginosa is described. Synthesis and in vitro structure-activity relationships (SARs) are outlined. Early leads lacked activity in animal models, and efforts to improve solubility and reduce serum protein binding by the introduction of polar groups are discussed.
Resistance to antibiotics in community acquired respiratory infections is increasing worldwide. Resistance to the macrolides can be class-specific, as in efflux or ribosomal mutations, or, in the case of erythromycin ribosomal methylase (erm)-mediated resistance, may generate cross-resistance to other related classes. The ketolides are a new subclass of macrolides specifically designed to combat macrolide-resistant respiratory pathogens. X-ray crystallography indicates that ketolides bind to a secondary region in domain II of the 23S rRNA subunit, resulting in an improved structure-activity relationship. Telithromycin and cethromycin (formerly ABT-773) are the two most clinically advanced ketolides, exhibiting greater activity towards both typical and atypical respiratory pathogens. As a subclass of macrolides, ketolides demonstrate potent activity against most macrolide-resistant streptococci, including ermB- and macrolide efflux (mef)A-positive Streptococcus pneumoniae. Their pharmacokinetics display a long half-life as well as extensive tissue distribution and uptake into respiratory tissues and fluids, allowing for once-daily dosing. Clinical trials focusing on respiratory infections indicate bacteriological and clinical cure rates similar to comparators, even in patients infected with macrolide-resistant strains.
The major Escherichia coli multidrug efflux pump AcrAB-TolC expels a wide range of antibacterial agents. Using in vivo cross-linking, we show for the first time that the antiporter AcrB and the adaptor AcrA, which form a translocase in the inner membrane, interact with the outer membrane TolC exit duct to form a contiguous proteinaceous complex spanning the bacterial cell envelope. Assembly of the pump appeared to be constitutive, occurring in the presence and absence of drug efflux substrate. This contrasts with substrate-induced assembly of the closely related TolC-dependent protein export machinery, possibly reflecting different assembly dynamics and degrees of substrate responsiveness in the two systems. TolC could be cross-linked independently to AcrB, showing that their large periplasmic domains are in close proximity. However, isothermal titration calorimetry detected no interaction between the purified AcrB and TolC proteins, suggesting that the adaptor protein is required for their stable association in vivo. Confirming this view, AcrA could be cross-linked independently to AcrB and TolC in vivo, and calorimetry demonstrated energetically favourable interactions of AcrA with both AcrB and TolC proteins. AcrB was bound by a polypeptide spanning the C-terminal half of AcrA, but binding to TolC required interaction of N- and C-terminal polypeptides spanning the lipoyl-like domains predicted to present the intervening coiled-coil to the periplasmic coils of TolC. These in vivo and in vitro analyses establish the central role of the AcrA adaptor in drug-independent assembly of the tripartite drug efflux pump, specifically in coupling the inner membrane transporter and the outer membrane exit duct.
The active efflux of cytotoxic drugs mediated by multidrug transporters is the basis of multidrug resistance in prokaryotic and eukaryotic cells. Individual multidrug transporters can be extremely versatile, often exhibiting a staggering range of substrate specificity that can negate the effects of clinically relevant therapies. The effective treatment of bacterial, fungal and protozoan infections, along with certain cancer treatments, has been compromised by the presence of multidrug transporters. Traditionally, advances in the understanding of multidrug transporters have been made through biochemical analyses; more recently, however, fundamental advances have been made with the elucidation of several three dimensional structures of representative multidrug pumps. Biochemical and structural analysis of multidrug pumps could lead to the development of novel 'anti-efflux' therapies.
Gene dosage has frequently been exploited to select for genetic interactions between a particular mutant and clones from a random genomic library at high copy. We report here the first use of multicopy suppression as a forward genetic method to determine cellular targets and potential resistance mechanisms for novel antibacterial compounds identified through high-throughput screening. A screen of 8640 small molecules for growth inhibition of a hyperpermeable strain of Escherichia coli led to the identification of 49 leads for suppressor selection from clones harboring an E. coli genomic library. The majority of suppressors were found to encode the multidrug efflux pump AcrB, indicating that those compounds were substrates for efflux. Two leads, which produced clones containing the gene folA, encoding dihydrofolate reductase (DHFR), proved to target DHFR in vivo and were competitive inhibitors in vitro.
Bacterial multidrug resistance is a serious clinical problem and is commonly conferred by tripartite efflux 'pumps' in the prokaryotic cell envelope. Crystal structures of the three components of a drug efflux pump have now been solved: the outer membrane TolC exit duct in the year 2000, the inner membrane AcrB antiporter in 2002 and the periplasmic adaptor MexA in 2004. These structures have enhanced our understanding of the principles underlying pump assembly and operation, and present pumps as new drug targets.
We have developed a multiwell assay for the detection of modulators of prokaryotic transcription based on the quantification of protein-protein interaction. This assay consists of three steps: (a) the immobilization of the Escherichia coli protein sigma70 in the well, (b) the incubation of the immobilized protein with core RNA polymerase and a potential inhibitor, and (c) washing and quantification of the binding of core to sigma70 with a monoclonal antibody conjugated to horseradish peroxidase. We show that this assay is sensitive, reproducible, and robust, and is able to discriminate between control competitors with different affinities. We demonstrate the usefulness of the assay to screen for microbial RNA polymerase inhibitors as potential new drugs for the treatment of emerging antibiotic-resistant bacteria.
Quinolones are one of the largest classes of antimicrobial agents used worldwide. This review considers the quinolones that are available currently and used widely in Europe (norfoxacin, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin) within their historical perspective, while trying to position them in the context of recent and possible future advances based on an understanding of: (1) their chemical structures and how these impact on activity and toxicity; (2) resistance mechanisms (mutations in target genes, efflux pumps); (3) their pharmacodynamic properties (AUC/MIC and Cmax/MIC ratios; mutant prevention concentration and mutant selection window); and (4) epidemiological considerations (risk of emergence of resistance, clonal spread). Their main indications are examined in relation to their advantages and drawbacks. Overall, it is concluded that these important agents should be used in an educated fashion, based on a careful balance between their ease of use and efficacy vs. the risk of emerging resistance and toxicity. However, there is now substantial evidence to support use of the most potent drug at the appropriate dose whenever this is required.
Telithromycin is the first of the ketolide antibacterials to receive US Food and Drug Administration (FDA) approval for clinical use. It is approved for the treatment of community-acquired pneumonia (CAP), acute exacerbations of chronic bronchitis (AECB), and acute maxillary sinusitis (AMS) in adults.
This article reviews the mechanism of action, in vitro antimicrobial activity, pharmacokinetics and pharmacodynamics, clinical efficacy, safety, and drug-interaction profile of telithromycin.
Relevant studies were identified through a search of the English-language literature indexed on MEDLINE (1990-March 2005) using the terms telithromycin and HMR 3647, a review of the reference lists of identified articles, and a review of the briefing document prepared by the manufacturer of telithromycin for presentation to the FDA Anti-infective Drugs Advisory Committee. A search of abstracts from the Interscience Conference on Antimicrobial Agents and Chemotherapy (2001-2004) also was performed.
The results of in vitro susceptibility studies suggest that telithromycin provides coverage against the key respiratory pathogens, both typical and atypical. In addition, telithromycin may be useful against multidrug-resistant strains of Streptococcus pneumoniae and against Haemophilus influenzae, irrespective of beta-lactamase production. In randomized, double-blind, comparative trials (against amoxicillin, amoxicillin/clavulanate, cefuroxime axetil, clarithromycin, moxifloxacin, or trovafloxacin), telithromycin had comparable efficacy to its comparators in the empiric treatment of CAP (4 studies), AECB (3 studies), and AMS (3 studies). Telithromycin is dosed at 800 mg (two 400-mg tablets) QD in community-acquired respiratory tract infections (RTIs). No dose adjustment is required in the elderly, patients with mild to moderate renal insufficiency, or patients with hepatic insufficiency. The majority of adverse events associated with telithromycin were mild to moderate, with gastrointestinal effects (diarrhea, nausea, vomiting) being the most commonly reported, followed by headache and dizziness. Telithromycin has been associated with elevations in hepatic transaminases and prolongation of the electrocardiographic QTc interval, although the significance of these findings is not known. Telithromycin is also a strong inhibitor of and substrate for the cytochrome P450 (CYP) 3A4 isozyme. Therefore, it is important to monitor for potential drug interactions with medications that prolong the QTc interval or are metabolized by the CYP system.
Telithromycin appears to be a useful option for the empiric treatment of community-acquired RTIs in adults. It may be particularly useful in the outpatient setting in areas with high rates of penicillin- and macrolide-resistant S pneumoniae; it may also be an alternative agent for patients who are allergic to beta-lactams and live in areas with a high prevalence of multidrug-resistant S pneumoniae or for those who have failed to respond to beta-lactam- or macrolide-based therapy.
The efflux systems of bacteria protect cells from antibiotics and biocides by actively transporting compounds out of the cytoplasm and/or periplasm and thereby limit their steady-state accumulation at their site(s) of action. The impact of efflux systems on the efficacy of antibiotics used in human medicine and animal husbandry is becoming increasingly apparent from the characterization of drug-resistant strains with altered drug efflux properties. In most instances, efflux-mediated antibiotic resistance arises from mutational events that result in their elevated expression and, in the case of efflux pumps with broad substrate specificity, can confer multi-drug resistance (MDR) to structurally unrelated antibiotics. Knowledge of the role of efflux systems in conferring antibiotic resistance has now been successfully exploited in the pharmaceutical industry and contributed, in part, to the development of new members of the macrolide and tetracycline classes of antibiotics that circumvent the efflux-based resistance mechanisms that have limited the clinical utility of their progenitors. The therapeutic utility of compounds that inhibit bacterial drug efflux pumps and therein potentiate the activity of a co-administered antibiotic agent remains to be validated in the clinical setting, but the approach holds promise for the future in improving the efficacy and/or extending the clinical utility of existing antibiotics. This review discusses the potential of further exploiting the knowledge of efflux-mediated antibiotic resistance in bacteria toward the discovery and development of new chemotherapeutic agents.
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