Overproduction Of Efflux Pump Research Articles

Overproduction of Efflux Pumps as a Mechanism of Metal and Antibiotic Cross-Resistance in the Natural Environment

Antibiotic and metal resistance can occur together in the environment and can be linked by the same detoxication mechanism (cross-resistance). The understanding of this linkage may be a key to further study of the spread of antibiotic resistance in the non-hospital environment worldwide. In our study, we examined the overproduction of efflux pumps as a possible mechanism of the cross-resistance of isolates originating from industrial and mine tailings. Resistance to metals (Cu, Ni, Zn and Pb) and antibiotics (ampicillin, chloramphenicol, tetracycline and kanamycin) was observed at all the sampling sites and ranged from 16 to 75%. Multiresistance (MAR index > 0.38) was recorded in 26% of the isolates and was associated with the metal selective pressure duration. Overproduction of efflux pumps has mainly been observed in multiresistant isolates. Our results may indicate that the overproduction of efflux pumps could be the mechanism of cross-resistance between metals and therefore related to metal and antibiotic multiresistance. The results also show that the importance of sustainably storing metal-containing waste lies not only in its environmental impact but also in human health via antibiotic resistance proliferation.

Occurrence of Antibiotic Resistant Bacteria in Flours and Different Plant Powders Used in Cuisine.

In recent years, several alimentary diseases have been connected with the consumption or tasting of raw flour and dough. Microbiological quality concern is also raising due to increased consumer demand for plant powders, while some of them can be consumed without prior thermal processing. In this study, we have focused on the occurrence of antibiotic-resistant coliform bacteria and enterococci in flour, plant powder and dough from Slovak retail. Our results indicated the presence of both total and antibiotic-resistant coliform bacteria and enterococci in the flour and powder samples. Lower numbers of the total, as well as resistant bacteria, were detected in flours compared to plant powders. Coliform bacteria isolates were predominantly identified as Klebsiella spp. and Enterobacter spp. Ampicillin resistance appeared in 97% of isolates followed by chloramphenicol resistance (22%) and tetracycline resistance (17%). The presence of the blaSHV gene was confirmed in 13% of isolates. The tetA and tetE genes were present in 25% of isolates of coliform bacteria. The presence of enterococci was detected only in plant powders. Antibiotic-resistant strains were identified as the following: Enterococcus casseliflavus, E. gallinarium and E. faecium. Despite the isolates showing resistance to vancomycin, the presence of the vanA gene was not detected. The majority of antibiotic-resistant isolates belonged to the group of medium biofilm producers. None of these isolates showed efflux pump overproduction. Antibiotic-resistant coliform bacteria and enterococci were not detected in the processed doughs.

New understanding of multidrug efflux and permeation in antibiotic resistance, persistence, and heteroresistance.

Antibiotics effective against Gram-negative ESKAPE pathogens are a critical area of unmet need. Infections caused by these pathogens are not only difficult to treat but finding new therapies to overcome Gram-negative resistance is also a challenge. There are not enough antibiotics in development that target the most dangerous pathogens and there are not enough novel drugs in the pipeline. The major obstacle in the antibiotic discovery pipeline is the lack of understanding of how to breach antibiotic permeability barriers of Gram-negative pathogens. These barriers are created by active efflux pumps acting across both the inner and the outer membranes. Overproduction of efflux pumps alone or together with either modification of the outer membrane or antibiotic-inactivating enzymes and target mutations contribute to clinical levels of antibiotics resistance. Recent efforts have generated significant advances in the rationalization of compound efflux and permeation across the cell envelopes of Gram-negative pathogens. Combined with earlier studies and novel mathematical models, these efforts have led to a multilevel understanding of how antibiotics permeate these barriers and how multidrug efflux and permeation contribute to the development of antibiotic resistance and heteroresistance. Here, we discuss the new developments in this area.

The Contribution of Efflux Systems to Levofloxacin Resistance in Stenotrophomonas maltophilia Clinical Strains Isolated in Warsaw, Poland

Simple SummaryFluoroquinolones, mainly levofloxacin, are considered an alternative treatment option of Stenotrophomonas maltophilia infections to trimethoprim/sulfamethoxazole. However, an increase in the number of levofloxacin-resistant strains is observed worldwide. The fluoroquinolone resistance in S. maltophilia is usually caused by an overproduction of various multidrug efflux pumps, which are able to extrude antibiotics and chemotherapeutics from the bacterial cells. The purpose of the study was to analyze the contribution of efflux systems to levofloxacin resistance in S. maltophilia clinical strains, isolated in Warsaw, by phenotypic and molecular methods. Previously, the occurrence of genes encoding various ten efflux pumps was shown in 94 studied isolates. Additionally, 44 of 94 isolates demonstrated reduction in susceptibility to levofloxacin. In this study, in the presence of efflux pump inhibitors, an increase in levofloxacin susceptibility was observed in 13 isolates. The overexpression of genes encoding two efflux pump system, such as SmeDEF and Sme VWX (in five and one isolate, respectively), was demonstrated. Sequencing analysis revealed an amino acid change in the local regulators of these efflux pump operons. Our data indicate that the overproduction of the SmeVWX efflux system, unlike SmeDEF, plays a significant role in the levofloxacin resistance of the clinical isolates.Levofloxacin is considered an alternative treatment option of Stenotrophomonas maltophilia infections to trimethoprim/sulfamethoxazole. The fluoroquinolone resistance in S. maltophilia is usually caused by an overproduction of efflux pumps. In this study, the contribution of efflux systems to levofloxacin resistance in S. maltophilia clinical isolates was demonstrated using phenotypic (minimal inhibitory concentrations, MICs, of antibiotics determination ± efflux pump inhibitors, EPIs) and molecular (real-time polymerase-chain-reaction and sequencing) methods. Previously, the occurrence of genes encoding ten efflux pumps was shown in 94 studied isolates. Additionally, 44/94 isolates demonstrated reduction in susceptibility to levofloxacin. Only 5 of 13 isolates (with ≥4-fold reduction in levofloxacin MIC) in the presence of EPIs showed an increased susceptibility to levofloxacin and other antibiotics. The overexpression of smeD and smeV genes (in five and one isolate, respectively) of 5 tested efflux pump operons was demonstrated. Sequencing analysis revealed 20–35 nucleotide mutations in local regulatory genes such as smeT and smeRv. However, mutations leading to an amino acid change were shown only in smeT (Arg123Lys, Asp182Glu, Asp204Glu) for one isolate and in smeRv (Gly266Ser) for the other isolate. Our data indicate that the overproduction of the SmeVWX efflux system, unlike SmeDEF, plays a significant role in the levofloxacin resistance.

Characterization of Antibiotic Resistant Coliform Bacteria and Resistance Genes Isolated from Samples of Smoothie Drinks and Raw Milk.

Raw foodstuffs have been marked as a healthier alternative in the context of nutrient content and are becoming more popular with consumers. Thermally untreated foods may represent a microbiological risk connected with the possible presence of antimicrobial resistance. The aim of this study was to prove that popular raw food beverages such as smoothies and raw milk may be a source of antibiotic-resistant coliform bacteria and resistant genes. The majority of antibiotic-resistant isolates (110) were identified as Enterobacter spp., Escherichia coli, and species of Klebsiella spp., predominantly β-lactam and chloramphenicol resistant. Multidrug resistance has been registered in one-third of resistants. Overproduction of efflux pumps was clarified in 8 different bacteria. The majority of resistant isolates were strong biofilm producers. Antibiotic resistance gene blaOXA was detected in 25% of isolates, especially in E. coli. Resistance genes blaTEM and blaSHV were detected in 19% and 14%, respectively. This is the first study to point out that popular raw drinks such as smoothies or raw milk, besides their nutrient benefits, could represent a reservoir of antibiotic-resistant bacteria as well as antibiotic resistance genes. According to this, raw drinks could contribute to the dissemination of antibiotic resistance in the human gastrointestinal tract and environment.

Novel Mechanisms of Efflux-Mediated Levofloxacin Resistance and Reduced Amikacin Susceptibility in Stenotrophomonas maltophilia

Fluoroquinolone resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant factor is overproduction of efflux pumps, particularly SmeDEF, following mutation. Here, we report that mutations in the glycosyl transferase gene smlt0622 in S. maltophilia K279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVr, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVr causing overproduction of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Overproduction of one ABC transporter, Smlt1651-4 (renamed SmaCDEF), causes levofloxacin resistance in K M6 LEVr Overproduction of the other ABC transporter, Smlt2642/3 (renamed SmaAB), and SmeYZ both contribute to the elevated amikacin MIC against K M6 LEVr Accordingly, we have identified two novel ABC transporters associated with antimicrobial drug resistance in S. maltophilia and two novel regulatory systems whose mutation causes resistance to levofloxacin, clinically important as a promising drug for monotherapy against this highly resistant pathogen.

Hospital Wastewater-Important Source of Multidrug Resistant Coliform Bacteria with ESBL-Production.

This work compares the prevalence of antibiotic resistant coliform bacteria in hospital wastewater effluents in Slovak (SR) and Czech Republic (ČR). It also describes selected antibiotic resistant isolates in view of resistance mechanism and virulence factor. The highest number of multidrug resistant bacteria was detected in samples from the hospital in Valašské Meziříčí (ČR). More than half of resistant isolates showed multidrug resistance phenotype as well as strong ability to form biofilm. In 42% of isolates efflux pump overproduction was detected together with tetA and tetE genes. The production of extended-spectrum β-lactamases in coliform isolates was encoded mainly by blaTEM, blaCTX-M-2 and blaCTX-M-8/25 genes. About 62% of resistants contained a combination of two or more extended spectrum beta-lactamases (ESBL) genes. Our results strengthen the fact that hospital effluents are a source of multidrug resistant bacteria which can spread their resistance genes to other bacteria in wastewater treatment plants (WWTPs). Accordingly, hospital wastewater should be better treated before it enters urban sewerage.

First detection of insertion sequences ISpa1635 and IS1411 among non-carbapenemase producing strains of Pseudomonas aeruginosa in Kerman, Iran

BackgroundLoss of OprD, AmpC overproduction and efflux pump over activity are the main mechanism for chromosomally resistance to carbapenems in Pseudomonas aeruginosa. In this study, we investigated the chromosomally resistance mechanism to carbapenems among 9 non-carbapenemase-producing P. aeruginosa (NCPPa). Material and methodAntibacterial resistance was determined by disc diffusion method among 170 clinical isolates of P. aeruginosa. Sixty-one isolates were multiple drug resistance and resistance to imipenem, and 9 isolates (5.3%) were negative for ten carbapenemase genes and were recognized as NCPPa. To determine the mechanism of resistance in NCPPa isolates AmpC and efflux pump overproduction were evaluated. Overexpression of MexAB-oprM efflux pump system was evaluated by qReal-time PCR. Also, oprD gene sequenced to detect any insertional and point mutations. ResultsAll NCPPa isolates shown a significant overexpression of mexA gene in qReal-time PCR experiment, while 88.8% of the isolates had overproduced AmpC enzyme. Finally, oprD gene was destructed by two insertion sequences (ISs), including IS1411 and ISpa1635. ConclusionAccording to the results of this study, overexpression of efflux pumps, oprD mutation and AmpC overproduction are the main chromosomal mechanism of resistance of P. aeruginosa to carbapenems in NCPPa isolates in this study.

Cefiderocol in context

New antibiotics reaching clinical development are scarce, although bacterial plasmids encoding antibiotic resistance genes seem to be everywhere. 1 Madec JY Haenni M Ponsin C et al. Sequence type 48 Escherichia coli carrying the blaCTX-M-1 IncI1/ST3 plasmid in drinking water in France. Antimicrob Agents Chemother. 2016; 60: 6430-6432 Crossref PubMed Scopus (28) Google Scholar Thus, the first clinical trial of cefiderocol, a siderophore cephalosporin active against multidrug-resistant Gram-negative pathogens, 2 Kohira N West J Ito A et al. In vitro antimicrobial activity of a siderophore cephalosporin, S-649266, against Enterobacteriaceae clinical isolates, including carbapenem-resistant strains. Antimicrob Agents Chemother. 2016; 60: 729-734 Crossref PubMed Scopus (172) Google Scholar reported by Simon Portsmouth and colleagues in The Lancet Infectious Diseases, 3 Portsmouth S van Veenhuyzen D Echols R et al. Cefiderocol versus imipenem-cilastatin for the treatment of complicated urinary tract infections caused by Gram-negative uropathogens: a phase 2, randomised, double-blind, non-inferiority trial. Lancet Infect Diseases. 2018; (published online Oct 25.)http://dx.doi.org/10.1016/S1473-3099(18)30554-1 Summary Full Text Full Text PDF PubMed Scopus (198) Google Scholar represents an eagerly-awaited development in the field. The definitive mechanism of action of cefiderocol is not novel; once inside the bacterial cell, it interrupts the synthesis of peptidoglycan, a key component of the bacterial cell wall. What is novel is its manner of getting inside to do the job—no easy task in the era of increasing efflux pump overproduction and porin mutations. Cefiderocol versus imipenem-cilastatin for the treatment of complicated urinary tract infections caused by Gram-negative uropathogens: a phase 2, randomised, double-blind, non-inferiority trialIntravenous infusion of cefiderocol (2 g) three times daily was non-inferior compared with imipenem-cilastatin (1 g each) for the treatment of complicated urinary tract infection in people with multidrug-resistant Gram-negative infections. The results of this study will provide the basis for submission of a New Drug Application to the US Food and Drug Administration. Clinical trials of hospital-acquired pneumonia and carbapenem-resistant infections are ongoing. Full-Text PDF Cefiderocol for treatment of complicated urinary tract infections – Author's replyThe enormous efforts of investigators and trial participants in the premarket development of cefiderocol should be neither disregarded nor underestimated: thanks to their work,1 this drug might soon be accessible to patients who might benefit from it. Nonetheless, a drug tested on only a few hundred people and for a few narrow indications will always require additional, postmarket development. As I have stated,2 because we, the public, have demanded an accelerated path to approval, novel antibiotics are now entering the market on the basis of very thin clinical experience. Full-Text PDF Cefiderocol for treatment of complicated urinary tract infectionsWe were pleased to read the generally positive Comment1 regarding our Article,2 published in The Lancet Infectious Diseases. However, we would like to respond to some of the points raised. Full-Text PDF

Substrate Specificities and Efflux Efficiencies of RND Efflux Pumps of Acinetobacter baumannii.

Antibiotic-resistant Acinetobacter baumannii causes infections that are extremely difficult to treat. A significant role in these resistance profiles is attributed to multidrug efflux pumps, especially those belonging to the resistance-nodulation-cell division (RND) superfamily of transporters. In this study, we analyzed functions and properties of RND efflux pumps in A. baumannii ATCC 17978. This strain is susceptible to antibiotics and does not contain mutations that are commonly selected upon exposure to high concentrations of antibiotics. We constructed derivatives of ATCC 17978 lacking chromosomally encoded RND pumps and complemented these strains by the plasmid-borne genes. We analyzed the substrate selectivities and efficiencies of the individual pumps in the context of native outer membranes and their hyperporinated variants. Our results show that inactivation of AdeIJK provides the strongest potentiation of antibiotic activities, whereas inactivation of AdeFGH triggers the overexpression of AdeAB. The plasmid-borne overproduction complements the hypersusceptible phenotypes of the efflux deletion mutants to the levels of the parental ATCC 17978. Only a few antibiotics strongly benefitted from the overproduction of efflux pumps and antibacterial activities of some of those depended on the synergistic interaction with the low permeability barrier of the outer membrane. Either overproduction or inactivation of efflux pumps change dramatically the lipidome of ATCC 17978. We conclude that efflux pumps of A. baumannii are tightly integrated into physiology of this bacterium and that clinical levels of antibiotic resistance in A. baumannii isolates are unlikely to be reached solely due to the overproduction of RND efflux pumps.IMPORTANCE RND-type efflux pumps are important contributors in development of clinical antibiotic resistance in A. baumannii However, their specific roles and the extent of contribution to antibiotic resistance remain unclear. We analyzed antibacterial activities of antibiotics in strains with different permeability barriers and found that the role of active efflux in antibiotic resistance of A. baumannii is limited to a few select antibiotics. Our results further show that the impact of efflux pump overproduction on antibiotic susceptibility is significantly lower than the previously reported for clinical isolates. Additional mechanisms of resistance, in particular those that improve the permeability barriers of bacterial cells and act synergistically with active efflux pumps are likely involved in antibiotic resistance of clinical A. baumannii isolates.

Overproduction of efflux pumps caused reduced susceptibility to carbapenem under consecutive imipenem-selected stress in Acinetobacter baumannii.

PurposeAcinetobacter baumannii is an important pathogen in the nosocomial infections worldwide. Combining with carbapenemases, efflux pumps and outer membrane proteins (OMPs) have been thought to affect the development of carbapenem resistance in A. baumannii. This study aimed to investigate the contributions of different efflux pumps and OMPs in developing carbapenem resistance in a clinical isolate of A. baumannii and reveal the possible mechanism of overproduction of main efflux pumps.Patients and methodsIn this study, an imipenem-susceptible clinical isolate was identified as A. baumannii and named SZE. Several common carbapenemases were detected by polymerase chain reaction (PCR). Imipenem-selected mutants were selected from SZE by serial subcultivations on Mueller–Hinton agar, and the minimum inhibitory concentration (MIC) was detected. Gene expressions of four families of efflux pumps, five OMPs, and blaOXA-51 were determined by reverse transcription quantitative PCR, and comparisons were made between SZE strain and the imipenem-selected mutants. The adeRS system in SZE and its mutant was sequenced and aligned.ResultsUnder consecutive imipenem-selected stress, the MIC to imipenem increased gradually from 0.125 μg/mL to 8 μg/mL. The effect of resistance inducement was almost neutralized when treated with an efflux pump inhibitor. The expression of efflux pumps, adeB, adeG, and adeJ, was increased by 6.9-, 4.0-, and 2.1-fold in mutants, respectively, compared to SZE. A single mutation (G to A) at position 58 was detected in the regulatory adeRS system and possibly upregulated the adeB expression, and then affected the carbapenem resistance in A. baumannii strains.ConclusionIn conclusion, under consecutive imipenem-selected stress in vitro, A. baumannii strain evolved the ability to reduce susceptibility to a variety of antimicrobials by overproduction of efflux pumps. Especially, the resistance-nodulation-cell division super family and a nucleotide mutant in adeRS regulating system caused the overexpression of adeABC.

In Vitro Antibacterial Properties of Cefiderocol, a Novel Siderophore Cephalosporin, against Gram-Negative Bacteria.

ABSTRACTCefiderocol (CFDC; S-649266), a novel parenteral siderophore cephalosporin conjugated with a catechol moiety, has a characteristic antibacterial spectrum with a potent activity against a broad range of aerobic Gram-negative bacterial species, including carbapenem-resistant strains of Enterobacteriaceae and nonfermenting bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii. Cefiderocol has affinity mainly for penicillin-binding protein 3 (PBP3) of Enterobacteriaceae and nonfermenting bacteria similar to that of ceftazidime. A deficiency of the iron transporter PiuA in P. aeruginosa or both CirA and Fiu in Escherichia coli caused 16-fold increases in cefiderocol MICs, suggesting that these iron transporters contribute to the permeation of cefiderocol across the outer membrane. The deficiency of OmpK35/36 in Klebsiella pneumoniae and the overproduction of efflux pump MexA-MexB-OprM in P. aeruginosa showed no significant impact on the activity of cefiderocol.

Label-free SRM-based relative quantification of antibiotic resistance mechanisms in Pseudomonas aeruginosa clinical isolates.

Both acquired and intrinsic mechanisms play a crucial role in Pseudomonas aeruginosa antibiotic resistance. Many clinically relevant resistance mechanisms result from changes in gene expression, namely multidrug efflux pump overproduction, AmpC β-lactamase induction or derepression, and inactivation or repression of the carbapenem-specific porin OprD. Changes in gene expression are usually assessed using reverse-transcription quantitative real-time PCR (RT-qPCR) assays. Here, we evaluated label-free Selected Reaction Monitoring (SRM)-based mass spectrometry to directly quantify proteins involved in antibiotic resistance. We evaluated the label-free SRM using a defined set of P. aeruginosa isolates with known resistance mechanisms and compared it with RT-qPCR. Referring to efflux systems, we found a more robust relative quantification of antibiotic resistance mechanisms by SRM than RT-qPCR. The SRM-based approach was applied to a set of clinical P. aeruginosa isolates to detect antibiotic resistance proteins. This multiplexed SRM-based approach is a rapid and reliable method for the simultaneous detection and quantification of resistance mechanisms and we demonstrate its relevance for antibiotic resistance prediction.

Countrywide spread of OXA-48 carbapenemase in Lebanon: surveillance and genetic characterization of carbapenem-non-susceptible Enterobacteriaceae in 10 hospitals over a one-year period

To detect, characterize, and assess the genetic clonality of carbapenem-non-susceptible Enterobacteriaceae in 10 Lebanese hospitals in 2012. Selected Enterobacteriaceae isolates with reduced susceptibility to carbapenems were subject to phenotypic study including antibiotic susceptibility, cloxacillin effect, modified Hodge test, and activity of efflux pump inhibitor. Carbapenemase genes were detected using PCR; clonal relatedness was studied by pulsed field gel electrophoresis. Out of 8717 Enterobacteriaceae isolated in 2012, 102 (1.2%) showed reduced susceptibility to carbapenems. Thirty-one (70%) of the 44 studied clinical isolates harbored blaOXA-48, including 15 Klebsiella pneumoniae, eight Escherichia coli, four Serratia marcescens, three Enterobacter cloacae, and one Morganella morganii. The majority of OXA-48 producers co-secreted an extended-spectrum beta-lactamase, while one had an acquired AmpC of the ACC type. In the non-OXA-48 producers, carbapenem resistance was attributed to the production of acquired AmpC cephalosporinases of MOX or CIT type, outer membrane impermeability, and/or efflux pump overproduction. DNA fingerprints revealed that OXA-48 producers were different, except for clonal relatedness among four K. pneumoniae, two E. coli, two E. cloacae, and three S. marcescens. Nosocomial carbapenem-non-susceptible Enterobacteriaceae are moderately spread in Lebanon and the predominant mechanism is OXA-48 production.

Resistance mechanisms of multiresistant Pseudomonas aeruginosa strains from Germany and correlation with hypermutation.

In this study, we analyzed the mechanisms of multiresistance for 22 clinical multiresistant and clonally different Pseudomonas aeruginosa strains from Germany. Twelve and 10 strains originated from cystic fibrosis (CF) and non-CF patients, respectively. Overproduction of the efflux systems MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM was studied. Furthermore, loss of OprD, alterations in type II topoisomerases, AmpC overproduction, and the presence of 25 acquired resistance determinants were investigated. The presence of a hypermutation phenotype was also taken into account. Besides modifications in GyrA (91%), the most frequent mechanisms of resistance were MexXY-OprM overproduction (82%), OprD loss (82%), and AmpC overproduction (73%). Clear differences between strains from CF and non-CF patients were found: numerous genes coding for aminoglycoside-modifying enzymes and located, partially in combination with beta-lactamase genes, in class 1 integrons were found only in strains from non-CF patients. Furthermore, multiple modifications in type II topoisomerases conferring high quinolone resistance levels and overexpression of MexAB-OprM were exclusively detected in multiresistant strains from non-CF patients. Correlations of the detected phenotypes and resistance mechanisms revealed a great impact of efflux pump overproduction on multiresistance in P. aeruginosa. Confirming previous studies, we found that additional, unknown chromosomally mediated resistance mechanisms remain to be determined. In our study, 11 out of 12 strains and 3 out of 10 strains from CF patients and non-CF patients, respectively, were hypermutable. This extremely high proportion of mutator strains should be taken into consideration for the treatment of multiresistant P. aeruginosa.

Overexpression of the Multidrug Efflux Pumps MexCD-OprJ and MexEF-OprN Is Associated with a Reduction of Type III Secretion in Pseudomonas aeruginosa

The Pseudomonas aeruginosa genome contains several different multidrug resistance (MDR) efflux pumps. Overproduction of these pumps reduces susceptibility to a variety of antibiotics. Some recently published works have analyzed the effect of the overproduction of MDR efflux pumps on bacterial virulence. Here we have studied the effect of overproduction of the efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY on type III secretion (T3S) in P. aeruginosa. The type III secretion system (T3SS) is used by P. aeruginosa to deliver toxins directly into the cytoplasm of the host cell. Our data indicate that overexpression of either MexCD-OprJ or MexEF-OprN is associated with the impairment of T3S in P. aeruginosa. No effect on overexpression of either MexAB-OprM or MexXY was detected. The observed defect in T3S was due to a lack of expression of genes belonging to the T3SS regulon. Transcription of this regulon is activated by ExsA in response to environmental signals. Overexpression of this transcriptional regulator complemented the defect in T3S observed in the MexCD-OprJ- and MexEF-OprN-overproducing strains. Taken together, these results suggest that overproduction of either MexCD-OprJ or MexEF-OprN is associated with a reduction in the transcription of the T3SS regulon due to the lack of expression of the exsA gene, encoding the master regulator of the system. The relevance of potential metabolic and quorum-sensing imbalances due to overexpression of MDR pumps associated with this phenotype is also discussed.

The Role of Multidrug Efflux Pumps in the Antibiotic Resistance of Pseudomonas aeruginosa and Other Gram‐Negative Bacteria

Gram‐negative bacteria remain clinically important pathogens in both hospital and community settings. Recent research indicates that efflux pumps play a prominent role in the multidrug resistance of Pseudomonas aeruginosa and many other gram‐negative bacteria. Four multidrug efflux pump systems have been well characterized in P. aeruginosa: MexA‐MexB‐OprM, MexC‐MexD‐OprJ, MexE‐MexF‐OprN, and MexX‐MexY‐OprM. These efflux pumps have different substrate specificities, and their production and activity can be increased by many factors commonly present in infections (e.g., high inocula of bacteria, low pH, and stationary‐phase growth). Moreover, fluoroquinolone antibiotics can commonly select mutants that constitutively overproduce Mex‐Opr efflux pump systems. Based on most recent studies, the prevalence of efflux pump overproduction in clinical strains of P. aeruginosa may range from 14–75%. The best treatment for infections caused by bacteria that overproduce efflux pumps is unknown, but pharmacodynamic optimization of antibiotics and the use of antibiotic combinations that are substrates for different pump systems may represent reasonable strategies until more data are available.