Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies

Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

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Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

Multidrug resistant bacteria are a global threat for human and animal health. However, they are only part of the problem of antibiotic failure. Another bacterial strategy that contributes to their capacity to withstand antimicrobials is the formation of biofilms. Biofilms are associations of microorganisms embedded a self-produced extracellular matrix. They create particular environments that confer bacterial tolerance and resistance to antibiotics by different mechanisms that depend upon factors such as biofilm composition, architecture, the stage of biofilm development, and growth conditions. The biofilm structure hinders the penetration of antibiotics and may prevent the accumulation of bactericidal concentrations throughout the entire biofilm. In addition, gradients of dispersion of nutrients and oxygen within the biofilm generate different metabolic states of individual cells and favor the development of antibiotic tolerance and bacterial persistence. Furthermore, antimicrobial resistance may develop within biofilms through a variety of mechanisms. The expression of efflux pumps may be induced in various parts of the biofilm and the mutation frequency is induced, while the presence of extracellular DNA and the close contact between cells favor horizontal gene transfer. A deep understanding of the mechanisms by which biofilms cause tolerance/resistance to antibiotics helps to develop novel strategies to fight these infections.

https://www.mdpi.com/2079-6382/10/1/3/pdf

Biofilms as Promoters of Bacterial Antibiotic Resistance and Tolerance.

Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits spanning both the inner and the outer membrane and are composed of three principal components—the energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS- and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

The nonfermenting bacteria belonging to Acinetobacter spp. and Pseudomonas spp. are capable of colonizing both humans and animals and can also be opportunistic pathogens. More specifically, the species Acinetobacter baumannii and Pseudomonas aeruginosa have been recurrently reported as multidrug-resistant and even pandrug-resistant in clinical isolates. Both species were categorized among the ESKAPE pathogens, ESKAPE standing for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species. These six pathogens are the major cause of nosocomial infections in the United States and are a threat all over the world because of their capacity to become increasingly resistant to all available antibiotics. A. baumannii and P. aeruginosa are both intrinsically resistant to many antibiotics due to complementary mechanisms, the main ones being the low permeability of their outer membrane, the production of the AmpC beta-lactamase, and the production of several efflux systems belonging to the resistance-nodulation-cell division family. In addition, they are both capable of acquiring multiple resistance determinants, such as beta-lactamases or carbapenemases. Even if such enzymes have rarely been identified in bacteria of animal origin, they may sooner or later spread to this reservoir. The goal of this article is to give an overview of the resistance phenotypes described in these pathogens and to provide a comprehensive analysis of all data that have been reported on Acinetobacter spp. and Pseudomonas spp. from animal hosts.

Antimicrobial Resistance in Acinetobacter spp. and Pseudomonas spp.

Resistance to various antimicrobial agents is an increasing problem in Pseudomonas aeruginosa and Acinetobacter baumannii infections. In this study, the roles of integrons were examined in 101 P. aeruginosa isolates and 176 A. baumannii isolates from patients. The frequencies and characteristics of class 1, 2 and 3 integrons were investigated and the horizontal transfer of integrons was assessed. Among these isolates, class 1 integrons with a resistance gene cassette were detected in 69.3% of P. aeruginosa and 31.8% of A. baumannii isolates, but class 2 and 3 integrons were not found. Five novel gene cassette arrays were identified in P. aeruginosa: aacA7-cmlA, aadB-blaOXA,-o-aadA1, aadB-arr-2-cmlA-blaOXA,-o-aadA1, aadB-cmlA-aadA1 and aadB-cmlA-blaOXA-10-aadA15. The integrons found in A. baumannii isolates in this study were previously reported. Horizontal transfer of some class 1 integrons was detected in both P. aeruginosa (2/70) and A. baumannii (5/57). These data confirm the high prevalence of class 1 integrons with a variety of gene cassette combinations among multidrug-resistant P. aeruginosa and A. baumannii clinical isolates.

Class 1 integrons in Pseudomonas aeruginosa and Acinetobacter baumannii isolated from clinical isolates.

The purpose of this study was to examine expression and regulation of 6 multidrug efflux systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY, MexJK, and MexVW, in 13 non-cystic fibrosis (CF) clinical isolates of Pseudomonas aeruginosa. These isolates displayed a high level of resistance to many clinically important antibiotics. Some isolates simultaneously overexpressed up to 4 different Mex systems, as determined by quantitative real-time reverse transcription PCR. None of the isolates overexpressed MexCD-OprJ, and only 1 isolate overproduced MexJK. All the isolates overexpressed MexXY, while overexpression of MexEF-OprN and MexVW was common. DNA sequencing analysis of regulatory genes showed that no clear correlation could be established among (i) the presence of mutations, (ii) the type of mutations, (iii) the expression level of the Mex systems, and (iv) resistance to antibiotic substrates. The results suggest that the concomitant overexpression of some Mex systems may superimpose their antimicrobial drug efflux capabilities, contributing to the multidrug resistance phenotype in the P. aeruginosa non-CF clinical isolates. The existence of uncharacterized regulators for the Mex systems was signified.

Simultaneous overexpression of multidrug efflux pumps in Pseudomonas aeruginosa non-cystic fibrosis clinical isolates

It is generally believed that the Pseudomonas aeruginosa biofilm matrix itself acts as a molecular sieve or sink that contributes to significant levels of drug resistance, but it is becoming more apparent that multidrug efflux pumps induced during biofilm growth significantly enhance resistance levels. We present here a novel transcriptional regulator, PA3898, which controls biofilm formation and multidrug efflux pumps in P. aeruginosa A mutant of this regulator significantly reduced the ability of P. aeruginosa to produce biofilm in vitro and affected its in vivo fitness and pathogenesis in Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA3898 modulates essential virulence genes/pathways, including multidrug efflux pumps and phenazine biosynthesis. Chromatin immunoprecipitation sequencing (ChIP-seq) identified its DNA binding sequences and confirmed that PA3898 directly interacts with promoter regions of four genes/operons, two of which are mexAB-oprM and phz2 Coimmunoprecipitation revealed a regulatory partner of PA3898 as PA2100, and both are required for binding to DNA in electrophoretic mobility shift assays. PA3898 and PA2100 were given the names MdrR1 and MdrR2, respectively, as novel repressors of the mexAB-oprM multidrug efflux operon and activators for another multidrug efflux pump, EmrAB. The interaction between MdrR1 and MdrR2 at the promoter regions of their regulons was further characterized via localized surface plasmon resonance and DNA footprinting. These regulators directly repress the mexAB-oprM operon, independent of its well-established MexR regulator. Mutants of mdrR1 and mdrR2 caused increased resistance to multiple antibiotics in P. aeruginosa, validating the significance of these newly discovered regulators.

Two Regulators, PA3898 and PA2100, Modulate the Pseudomonas aeruginosa Multidrug Resistance MexAB-OprM and EmrAB Efflux Pumps and Biofilm Formation.

As study of multidrug efflux pumps is a crucial step for development of efflux pump inhibitors for treatment of Pseudomonas aeruginosa infection, the objective of this study was to examine the contribution of the MexXY multidrug efflux systems and other chromosomal mechanisms in aminoglycoside (AMG) resistance in P. aeruginosa isolated from dogs and cats. Thirteen Pseudomonas aeruginosa isolates from canine and feline infections were examined for contribution of the MexXY multidrug efflux pump and four other chromosomally-encoded genes including PA5471, galU, nuoG and rplY to AMG resistance. All the isolates were resistant to multiple AMGs and expressed mexXY. Deletion of mexXY caused 2- to 16-fold reduction in AMG MICs. Overproduction of MexXY did not fully account for the observed AMG resistance. No good correlations were detected between MexXY transcription level and AMG MICs. While no mutations were found in mexZ, PA5471 expression varied and its impact on MexXY expression and AMG resistance is diverse. No mutations were found in galU. Only two isolates carried a single base change G-367-T in rplY. Complete transcription of nuoG was detected in all the isolates. In conclusion, the MexXY multidrug efflux pump plays a role in AMG resistance in the dog and cat P. aeruginosa isolates, while disruption of nuoG, rplY and galU did not have a significant impact. These results indicate the existence of uncharacterized AMG-resistance mechanisms.

Contribution of the MexXY Multidrug Efflux Pump and Other Chromosomal Mechanisms on Aminoglycoside Resistance in Pseudomonas aeruginosa Isolates from Canine and Feline Infections

Gene regulation network in Pseudomonas aeruginosa is complex. With a relatively large genome (6.2 Mb), there is a significant portion of genes that are proven or predicted to be transcriptional regulators. Many of these regulators have been shown to play important roles in biofilm formation and maintenance. In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth. This regulator is also essential for the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay. PA1226 and PA1413 were shown to bind collaboratively to the promoter regions of their regulons. A model is proposed, summarizing our finding on this novel dual-regulation system.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/mmi.14063

Kanchana Poonsuk

Papers

Class 1 integrons in Pseudomonas aeruginosa and Acinetobacter baumannii isolated from clinical isolates.

Simultaneous overexpression of multidrug efflux pumps in Pseudomonas aeruginosa non-cystic fibrosis clinical isolates

Two Regulators, PA3898 and PA2100, Modulate the Pseudomonas aeruginosa Multidrug Resistance MexAB-OprM and EmrAB Efflux Pumps and Biofilm Formation.

Contribution of the MexXY Multidrug Efflux Pump and Other Chromosomal Mechanisms on Aminoglycoside Resistance in Pseudomonas aeruginosa Isolates from Canine and Feline Infections

Novel dual regulators of Pseudomonas aeruginosa essential for productive biofilms and virulence.