Gene expression in Pseudomonas aeruginosa biofilms.
Whiteley M,Bangera M G,Bumgarner R E,Parsek M R,Teitzel G M,Lory S,Greenberg E P
Bacteria often adopt a sessile biofilm lifestyle that is resistant to antimicrobial treatment. Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections. To gain insights into the differences between free-living P. aeruginosa cells and those in biofilms, and into the mechanisms underlying the resistance of biofilms to antibiotics, we used DNA microarrays. Here we show that, despite the striking differences in lifestyles, only about 1% of genes showed differential expression in the two growth modes; about 0.5% of genes were activated and about 0.5% were repressed in biofilms. Some of the regulated genes are known to affect antibiotic sensitivity of free-living P. aeruginosa. Exposure of biofilms to high levels of the antibiotic tobramycin caused differential expression of 20 genes. We propose that this response is critical for the development of biofilm resistance to tobramycin. Our results show that gene expression in biofilm cells is similar to that in free-living cells but there are a small number of significant differences. Our identification of biofilm-regulated genes points to mechanisms of biofilm resistance to antibiotics.
Quantitative contributions of target alteration and decreased drug accumulation to Pseudomonas aeruginosa fluoroquinolone resistance.
Bruchmann Sebastian,Dötsch Andreas,Nouri Bianka,Chaberny Iris F,Häussler Susanne
Antimicrobial agents and chemotherapy
Quinolone antibiotics constitute a clinically successful and widely used class of broad-spectrum antibiotics; however, the emergence and spread of resistance increasingly limits the use of fluoroquinolones in the treatment and management of microbial disease. In this study, we evaluated the quantitative contributions of quinolone target alteration and efflux pump expression to fluoroquinolone resistance in Pseudomonas aeruginosa. We generated isogenic mutations in hot spots of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, and parC and inactivated the efflux regulator genes so as to overexpress the corresponding multidrug resistance (MDR) efflux pumps. We then introduced the respective mutations into the reference strain PA14 singly and in various combinations. Whereas the combined inactivation of two efflux regulator-encoding genes did not lead to resistance levels higher than those obtained by inactivation of only one efflux regulator-encoding gene, the combination of mutations leading to increased efflux and target alteration clearly exhibited an additive effect. This combination of target alteration and overexpression of efflux pumps was commonly observed in clinical P. aeruginosa isolates; however, these two mechanisms were frequently found not to be sufficient to explain the level of fluoroquinolone resistance. Our results suggest that there are additional mechanisms, independent of the expression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and/or MexXY-OprM efflux pump, that increase ciprofloxacin resistance in isolates with mutations in the QRDRs.
Aminoglycoside modifying enzymes.
Ramirez Maria S,Tolmasky Marcelo E
Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy
Aminoglycosides have been an essential component of the armamentarium in the treatment of life-threatening infections. Unfortunately, their efficacy has been reduced by the surge and dissemination of resistance. In some cases the levels of resistance reached the point that rendered them virtually useless. Among many known mechanisms of resistance to aminoglycosides, enzymatic modification is the most prevalent in the clinical setting. Aminoglycoside modifying enzymes catalyze the modification at different -OH or -NH₂ groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be nucleotidyltransferases, phosphotransferases, or acetyltransferases. The number of aminoglycoside modifying enzymes identified to date as well as the genetic environments where the coding genes are located is impressive and there is virtually no bacteria that is unable to support enzymatic resistance to aminoglycosides. Aside from the development of new aminoglycosides refractory to as many as possible modifying enzymes there are currently two main strategies being pursued to overcome the action of aminoglycoside modifying enzymes. Their successful development would extend the useful life of existing antibiotics that have proven effective in the treatment of infections. These strategies consist of the development of inhibitors of the enzymatic action or of the expression of the modifying enzymes.
Cyclic di-GMP: second messenger extraordinaire.
Jenal Urs,Reinders Alberto,Lori Christian
Nature reviews. Microbiology
Cyclic dinucleotides (CDNs) are highly versatile signalling molecules that control various important biological processes in bacteria. The best-studied example is cyclic di-GMP (c-di-GMP). Known since the late 1980s, it is now recognized as a near-ubiquitous second messenger that coordinates diverse aspects of bacterial growth and behaviour, including motility, virulence, biofilm formation and cell cycle progression. In this Review, we discuss important new insights that have been gained into the molecular principles of c-di-GMP synthesis and degradation, which are mediated by diguanylate cyclases and c-di-GMP-specific phosphodiesterases, respectively, and the cellular functions that are exerted by c-di-GMP-binding effectors and their diverse targets. Finally, we provide a short overview of the signalling versatility of other CDNs, including c-di-AMP and cGMP-AMP (cGAMP).
Transcriptional profiling of Pseudomonas aeruginosa PAO1 in response to anti-biofilm and anti-infection agent exopolysaccharide EPS273.
Wu Zuodong,Zheng Rikuan,Zhang Jing,Wu Shimei
Journal of applied microbiology
AIMS:Relative few anti-biofilm polysaccharides against Pseudomonas aeruginosa were done to investigate the underlying molecular mechanism. Exopolysaccharide EPS273 can clearly reduce biofilm formation and infection of P. aeruginosa. This study aims to investigate its anti-biofilm and anti-infection mechanism on transcriptional level. METHODS AND RESULTS:Herein we used an RNA-Seq transcriptomic approach to investigate the underlying anti-biofilm and anti-infection mechanism of EPS273. The expression levels of a large number of genes were changed after P. aeruginosa PAO1 was treated with EPS273. Especially, the genes related to biofilm formation, such as gene involved in production of extracellular matrix and virulence factor, genes involved in flagella and cell motility and genes involved in iron acquisition. Notably, the expression levels of genes involved in regulatory and signal transduction were markedly downregulated, such as two-component system PhoP-PhoQ and quorum sensing (QS) system LasI/ LasR and RhlI/ RhlR. Furthermore, when gene phoP and phoQ was disrupted respectively, the reduction of biofilm formation and cell motility in mutant △phoP or △phoQ was also detected. CONCLUSION:EPS273 may exert its anti-biofilm and anti-infection function by downregulating gene expression of two-component system PhoP-PhoQ and QS systems LasI/ LasR and RhlI/ RhlR of P. aeruginosa, which further regulated expression of genes involved in biofilm formation. SIGNIFICANCE AND IMPACT OF THE STUDY:Our data will expand understanding of anti-biofilm mechanisms of polysaccharides on transcriptomic level.
Regulation of the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24 by the PhoP/PhoQ two-component system.
Yan Qing,Gao Wei,Wu Xiao-Gang,Zhang Li-Qun
Microbiology (Reading, England)
A quorum-sensing locus, pcoI/pcoR, which is involved in the regulation of root colonization and plant disease-suppressive ability, was previously identified in Pseudomonas fluorescens 2P24. In this study, we performed random mutagenesis using mini-Tn5 in order to screen the upstream transcriptional regulators of pcoI, a biosynthase gene responsible for the synthesis of N-acylhomoserine lactone signal molecules. Two mutants, PM400 and PM410, with elevated pcoI gene promoter activity, were identified from approximately 10,000 insertion clones. The amino acid sequences of the interrupted genes in these two mutants were highly similar to PhoQ, a sensor protein of the two-component regulatory system PhoP/PhoQ, which responds to environmental Mg2+ starvation and regulates virulence in Salmonella typhimurium and antimicrobial peptide resistance in Pseudomonas aeruginosa. The promoter activity of pcoI was also induced under low-Mg2+ conditions in the 2P24 strain of P. fluorescens. Deletion mutagenesis and complementation experiments demonstrated that the transcription of pcoI was negatively regulated by the sensor PhoQ but positively regulated by the response regulator PhoP. Genetic evidence also indicated that transcription of the outer-membrane protein gene oprH was induced by Mg2+ starvation through regulation of the wild-type PhoP/PhoQ system. Additionally, PhoQ was involved in biofilm formation by 2P24 under low-Mg2+ conditions through a PhoP-independent pathway.
Differential impact on motility and biofilm dispersal of closely related phosphodiesterases in Pseudomonas aeruginosa.
Cai Yu-Ming,Hutchin Andrew,Craddock Jack,Walsh Martin A,Webb Jeremy S,Tews Ivo
In Pseudomonas aeruginosa, the transition between planktonic and biofilm lifestyles is modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP) in response to environmental conditions. Here, we used gene deletions to investigate how the environmental stimulus nitric oxide (NO) is linked to biofilm dispersal, focusing on biofilm dispersal phenotype from proteins containing putative c-di-GMP turnover and Per-Arnt-Sim (PAS) sensory domains. We document opposed physiological roles for the genes ΔrbdA and Δpa2072 that encode proteins with identical domain structure: while ΔrbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in Δpa2072, and biofilm formation was inhibited, compared to wild type. A second pair of genes, ΔfimX and ΔdipA, were selected on the basis of predicted impaired c-di-GMP turnover function: ΔfimX showed increased, ΔdipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for ΔfimX and reduced swimming for ΔdipA. For all four deletion mutants we find that NO-induced biomass reduction correlates with increased NO-driven swarming, underlining a significant role for this motility in biofilm dispersal. Hence P. aeruginosa is able to differentiate c-di-GMP output using structurally highly related proteins that can contain degenerate c-di-GMP turnover domains.
High levels of cAMP inhibit Pseudomonas aeruginosa biofilm formation through reduction of the c-di-GMP content.
Almblad Henrik,Rybtke Morten,Hendiani Saghar,Andersen Jens Bo,Givskov Michael,Tolker-Nielsen Tim
Microbiology (Reading, England)
The human pathogen Pseudomonas aeruginosa can cause both acute infections and chronic biofilm-based infections. Expression of acute virulence factors is positively regulated by cAMP, whereas biofilm formation is positively regulated by c-di-GMP. We provide evidence that increased levels of cAMP, caused by either a lack of degradation or increased production, inhibit P. aeruginosa biofilm formation. cAMP-mediated inhibition of P. aeruginosa biofilm formation required Vfr, and involved a reduction of the level of c-di-GMP, as well as reduced production of biofilm matrix components. A mutant screen and characterization of defined knockout mutants suggested that a subset of c-di-GMP-degrading phosphodiesterases is involved in cAMP-Vfr-mediated biofilm inhibition in P. aeruginosa.
Refinement of OprH-LPS Interactions by Molecular Simulations.
Lee Joonseong,Patel Dhilon S,Kucharska Iga,Tamm Lukas K,Im Wonpil
The outer membrane (OM) of Gram-negative bacteria is composed of lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The outer membrane protein H (OprH) of Pseudomonas aeruginosa provides an increased stability to the OMs by directly interacting with LPS. Here we report the influence of various P. aeruginosa and, for comparison, Escherichia coli LPS environments on the physical properties of the OMs and OprH using all-atom molecular dynamics simulations. The simulations reveal that although the P. aeruginosa OMs are thinner hydrophobic bilayers than the E. coli OMs, which is expected from the difference in the acyl chain length of their lipid A, this effect is almost imperceptible around OprH due to a dynamically adjusted hydrophobic match between OprH and the OM. The structure and dynamics of the extracellular loops of OprH show distinct behaviors in different LPS environments. Including the O-antigen greatly reduces the flexibility of the OprH loops and increases the interactions between these loops and LPS. Furthermore, our study shows that the interactions between OprH and LPS mainly depend on the secondary structure of OprH and the chemical structure of LPS, resulting in distinctive patterns in different LPS environments.
Structure, function and regulation of Pseudomonas aeruginosa porins.
Chevalier Sylvie,Bouffartigues Emeline,Bodilis Josselin,Maillot Olivier,Lesouhaitier Olivier,Feuilloley Marc G J,Orange Nicole,Dufour Alain,Cornelis Pierre
FEMS microbiology reviews
Pseudomonas aeruginosa is a Gram-negative bacterium belonging to the γ-proteobacteria. Like other members of the Pseudomonas genus, it is known for its metabolic versatility and its ability to colonize a wide range of ecological niches, such as rhizosphere, water environments and animal hosts, including humans where it can cause severe infections. Another particularity of P. aeruginosa is its high intrinsic resistance to antiseptics and antibiotics, which is partly due to its low outer membrane permeability. In contrast to Enterobacteria, pseudomonads do not possess general diffusion porins in their outer membrane, but rather express specific channel proteins for the uptake of different nutrients. The major outer membrane 'porin', OprF, has been extensively investigated, and displays structural, adhesion and signaling functions while its role in the diffusion of nutrients is still under discussion. Other porins include OprB and OprB2 for the diffusion of glucose, the two small outer membrane proteins OprG and OprH, and the two porins involved in phosphate/pyrophosphate uptake, OprP and OprO. The remaining nineteen porins belong to the so-called OprD (Occ) family, which is further split into two subfamilies termed OccD (8 members) and OccK (11 members). In the past years, a large amount of information concerning the structure, function and regulation of these porins has been published, justifying why an updated review is timely.
The Pseudomonas aeruginosa PilSR Two-Component System Regulates Both Twitching and Swimming Motilities.
Kilmury Sara L N,Burrows Lori L
Motility is an important virulence trait for many bacterial pathogens, allowing them to position themselves in appropriate locations at appropriate times. The motility structures type IV pili and flagella are also involved in sensing surface contact, which modulates pathogenicity. In , the PilS-PilR two-component system (TCS) regulates expression of the type IV pilus (T4P) major subunit PilA, while biosynthesis of the single polar flagellum is regulated by a hierarchical system that includes the FleSR TCS. Previous studies of and implicated PilR in regulation of non-T4P-related genes, including some involved in flagellar biosynthesis. Here we used transcriptome sequencing (RNA-seq) analysis to identify genes in addition to with changes in expression in the absence of Among the genes identified were 10 genes whose transcription increased in the mutant but decreased in the mutant, despite both mutants lacking T4P and pilus-related phenotypes. The products of these inversely dysregulated genes, many of which were hypothetical, may be important for virulence and surface-associated behaviors, as mutants had altered swarming motility, biofilm formation, type VI secretion system expression, and pathogenicity in a nematode model. Further, the PilSR TCS positively regulated transcription of , and thus many genes in the FleSR regulon. As a result, deletion mutants had defects in swimming motility that were independent of the loss of PilA. Together, these data suggest that in addition to controlling T4P expression, PilSR could have a broader role in the regulation of motility and surface sensing behaviors. Surface appendages such as type IV pili and flagella are important for establishing surface attachment and infection in a host in response to appropriate cues. The PilSR regulatory system that controls type IV pilus expression in has an established role in expression of the major pilin PilA. Here we provide evidence supporting a new role for PilSR in regulating flagellum-dependent swimming motility in addition to pilus-dependent twitching motility. Further, even though both and mutants lack PilA and pili, we identified sets of genes downregulated in the mutant and upregulated in a mutant as well as genes downregulated only in a mutant, independent of pilus expression. This finding suggests that change in the inner membrane levels of PilA is only one of the cues to which PilR responds to modulate gene expression. Identification of PilR as a regulator of multiple motility pathways may make it an interesting therapeutic target for antivirulence compounds.
Loss of the Two-Component System TctD-TctE in Affects Biofilm Formation and Aminoglycoside Susceptibility in Response to Citric Acid.
Taylor Patrick K,Zhang Li,Mah Thien-Fah
The two-component system TctD-TctE is important for regulating the uptake of tricarboxylic acids in TctD-TctE accomplishes this through derepression of the gene , which encodes a tricarboxylic acid-specific porin. Previous work from our lab revealed that TctD-TctE in also has a role in resistance to aminoglycoside antibiotics. The aim of this study was to further characterize the role of TctD-TctE in in the presence of citric acid. Here it was found that deletion of PA14 TctD-TctE (Δ) resulted in a 4-fold decrease in the biofilm bactericidal concentrations of the aminoglycosides tobramycin and gentamicin when citric acid was present in nutrient media. Tobramycin accumulation assays demonstrated that deletion of TctD-TctE resulted in an increase in the amount of tobramycin retained in biofilm cells. The PA14 wild type responded to increasing concentrations of citric acid by producing less biofilm. In contrast, the amount of Δ mutant biofilm formation remained constant or enhanced. Furthermore, the Δ strain was incapable of growing on citric acid as a sole carbon source and was highly reduced in its ability to grow in the presence of citric acid even when an additional carbon source was available. Use of phenotypic and genetic microarrays found that this growth deficiency of the Δ mutant is unique to citric acid and that multiple metabolic genes are dysregulated. This work demonstrates that TctD-TctE in has a role in biofilm development that is dependent on citric acid and that is separate from the previously characterized involvement in resistance to antibiotics. Nutrient availability is an important contributor to the ability of bacteria to establish successful infections in a host. is an opportunistic pathogen in humans causing infections that are difficult to treat. In part, its success is attributable to a high degree of metabolic versatility. is able to sense and respond to varied and limited nutrient stress in the host environment. Two-component systems are important sensors-regulators of cellular responses to environmental stresses, such as those encountered in the host. This work demonstrates that the response by the two-component system TctD-TctE to the presence of citric acid has a role in biofilm formation, aminoglycoside susceptibility, and growth in .
Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa.
Chatterjee Maitrayee,D'Morris Sharon,Paul Vinod,Warrier Sruthi,Vasudevan Anil Kumar,Vanuopadath Muralidharan,Nair Sudarslal Sadasivan,Paul-Prasanth Bindhu,Mohan C Gopi,Biswas Raja
Applied microbiology and biotechnology
Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors' production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-L-homoserine lactone (C-HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.
Role of the Outer Membrane Protein OprD2 in Carbapenem-Resistance Mechanisms of Pseudomonas aeruginosa.
Shen Jilu,Pan Yaping,Fang Yaping
We investigated the relationship between the outer membrane protein OprD2 and carbapenem-resistance in 141 clinical isolates of Pseudomonas aeruginosa collected between January and December 2013 from the First Affiliated Hospital of Anhui Medical University in China. Agar dilution methods were employed to determine the minimum inhibitory concentration of meropenem (MEM) and imipenem (IMP) for P. aeruginosa. The gene encoding OprD2 was amplified from141 P. aeruginosa isolates and analyzed by PCR and DNA sequencing. Differences between the effects of IMPR and IMPS groups on the resistance of the P. aeruginosa were observed by SDS-poly acrylamide gel electrophoresis (SDS-PAGE). Three resistance types were classified in the 141 carbapenem-resistant P. aeruginosa (CRPA) isolates tested, namely IMPRMEMR (66.7%), IMPRMEMS (32.6%), and IMPRMEMS (0.7%). DNA sequencing revealed significant diverse gene mutations in the OprD2-encoding gene in these strains. Thirty-four strains had large fragment deletions in the OprD2gene, in 6 strains the gene contained fragment inserts, and in 96 resistant strains, the gene featured small fragment deletions or multi-site mutations. Only 4 metallo-β-lactamase strains and 1 imipenem-sensitive (meropenem-resistant) strain showed a normal OprD2 gene. Using SDS-PAGE to detect the outer membrane protein in 16 CRPA isolates, it was found that 10 IMPRMEMR strains and 5 IMPRMEMS strains had lost the OprD2 protein, while the IMPSMEMR strain contained a normal 46-kDa protein. In conclusion, mutation or loss of the OprD2-encoding gene caused the loss of OprD2, which further led to carbapenem-resistance of P. aeruginosa. Our findings provide insights into the mechanism of carbapenem resistance in P. aeruginosa.
Recent perspectives on the molecular basis of biofilm formation by Pseudomonas aeruginosa and approaches for treatment and biofilm dispersal.
Skariyachan Sinosh,Sridhar Vaishnavi Sneha,Packirisamy Swathi,Kumargowda Supreetha Toplar,Challapilli Sneha Basavaraj
Pseudomonas aeruginosa, a Gram-negative, rod-shaped bacterium causes widespread diseases in humans. This bacterium is frequently related to nosocomial infections such as pneumonia, urinary tract infections (UTIs) and bacteriaemia especially in immunocompromised patients. The current review focuses on the recent perspectives on biofilms formation by these bacteria. Biofilms are communities of microorganisms in which cells stick to each other and often adhere to a surface. These adherent cells are usually embedded within a self-produced matrix of extracellular polymeric substance (EPS). Pel, psl and alg operons present in P. aeruginosa are responsible for the biosynthesis of extracellular polysaccharide which plays an important role in cell surface interactions during biofilm formation. Recent studies suggested that cAMP signalling pathway, quorum-sensing pathway, Gac/Rsm pathway and c-di-GMP signalling pathway are the main mechanism that leads to the biofilm formation. Understanding the bacterial virulence depends on a number of cell-associated and extracellular factors and is very essential for the development of potential drug targets. Thus, the review focuses on the major genes involved in the biofilm formation, the state of art update on the biofilm treatment and the dispersal approaches such as targeting adhesion and maturation, targeting virulence factors and other strategies such as small molecule-based inhibitors, phytochemicals, bacteriophage therapy, photodynamic therapy, antimicrobial peptides and natural therapies and vaccines to curtail the biofilm formation by P. aeruginosa.
Free tryptophan residues inhibit quorum sensing of Pseudomonas aeruginosa: a potential approach to inhibit the development of microbial biofilm.
Chakraborty Poulomi,Daware Akshay Vishnu,Kumari Monika,Chatterjee Ahana,Bhattacharyya Disha,Mitra Garbita,Akhter Yusuf,Bhattacharjee Surajit,Tribedi Prosun
Archives of microbiology
Microbial biofilm reveals a cluster of microbial population aggregated on a surface. Pseudomonas aeruginosa, a strong biofilm forming organism, often causes several human diseases. Microorganism-based diseases become more difficult to manage when the causative organism develops biofilm during the course of disease progression as the organism attains alarming drug resistance in biofilm form. Agents inhibiting microbial biofilm formation could be considered as a potential tool to weaken the extent of microbial pathogenesis. Tryptophan has already been reported as a promising agent against the biofilm development by P. aeruginosa. In the current study, we had focused on the underlying mechanism of microbial biofilm inhibition of P. aeruginosa under the influence of tryptophan. The expression level of the mRNA of the genes (lasR, lasB and lasI) associated with quorum sensing was compared between tryptophan treated and untreated cells under similar conditions using real time polymerase chain reaction (RT-PCR). The results showed that the tested concentrations of tryptophan considerably reduced the expression of those genes (lasR, lasB and lasI) that are required during the occurrence of quorum sensing in P. aeruginosa. Molecular docking also revealed that tryptophan can interact with the proteins responsible for the occurrence of quorum sensing in P. aeruginosa. The cytotoxicity assay was carried out wherein we observed that the tested concentration of tryptophan did not show any considerable cytotoxicity against the RAW 264.7 macrophage cell line. From this study, it may be concluded that the tryptophan-mediated inhibition of biofilm formation is associated with interference of quorum sensing in P. aeruginosa. Hence, tryptophan could be used as a potential agent against the microbial biofilm mediated pathogenesis.
Repurposing metformin as a quorum sensing inhibitor in .
Abbas Hisham A,Elsherbini Ahmed M,Shaldam Moutaz A
African health sciences
BACKGROUND:Quorum sensing is a mechanism of intercellular communication that controls the production of virulence factors in . Inhibition of quorum sensing can disarm the virulence factors without exerting stress on bacterial growth that leads to emergence of antibiotic resistance. OBJECTIVES:Finding a new quorum sensing inhibitor and determining its inhibitory activities against virulence factors of PAO1 strain. METHODS:Quorum sensing was evaluated by estimation of violacein production by CV026. Molecular docking was used to investigate the possible binding of metformin to LasR and rhlR receptors. The inhibition of pyocyanin, hemolysin, protease, elastase in addition to swimming and twitching motilities, biofilm formation and resistance to oxidative stress by metformin was also assessed. RESULTS:Metformin significantly reduced the production of violacein pigment. Significant inhibition of pyocyanin, hemolysin, protease and elastase was achieved. Metformin markedly decreased biofilm formation, swimming and twitching motilities and increased the sensitivity to oxidative stress. In the molecular docking study, metformin could bind to LasR by hydrogen bonding and electrostatic interaction and to rhlR by hydrogen bonding only. CONCLUSION:Metformin can act as a quorum sensing inhibitor and virulence inhibiting agent that may be useful in the treatment of infection.
Down-regulatory effects of green coffee extract on las I and las R virulence-associated genes in Pseudomonas aeruginosa.
Jamalifar Hossein,Samadi Nasrin,Nowroozi Jamileh,Dezfulian Mehrouz,Fazeli Mohammad Reza
Daru : journal of Faculty of Pharmacy, Tehran University of Medical Sciences
BACKGROUND:Antibiotic resistant strains of Pseudomonas aeruginosa are the cause of Gram negative nosocomial infections especially among the immunosuppressed patients. The bacteria contains las I and las R genes that play very important roles in the pathogenesis and mechanisms of aggression. These genes can be influenced by the quorum sensing (QS) system and such mechanism is becoming clinically important worldwide. This study aimed to investigate the preventive effects of green coffee extract (GCE) on the expression of pathogenesis-related genes, las I and las R in P. aeruginosa. METHODS:A total of fifty four P. aeruginosa strains were isolated out of 100 clinical samples collected from the infectious wards in different hospitals (Tehran province) using conventional microscopic and biochemical methods. Susceptibility of the isolates to different antibiotics, GCE and chlorogenic acid were elucidated. Multiplex polymerase chain reaction (PCR) and real-time PCR were performed to detect and quantify the expression levels of las I and las R genes. The presence of chlorogenic acid in GCE was confirmed by HPLC. RESULTS:Antibiotic susceptibility tests revealed multidrug resistance among the clinical isolates of those 40 strains were resistant to ciprofloxacin (74.07%), 43 to ceftazidime (79.26%), 29 to amikacin (53.7%), 42 to ampicillin (77.77%), 17 to colistin (31.48%), 40 to gentamicin (74.77%), and 50 to piperacillin (92.59%). PCR outcomes exhibited that the frequency of las I and las R genes were 100% in resistant and sensitive strains isolated from clinical and standard strains of P. aeruginosa (ATCC 15449). Real-time PCR analyses revealed that GCE significantly prevented the expression of las I and las R genes in P. aeruginosa. GCE at concentration level as low as 2.5 mg/mL could prevent the expression of lasI and lasR genes in P. aeruginosa clinical isolates. CONCLUSION:The presence and expression levels of las I and las R genes in P. aeruginosa isolates were investigated when the bacteria was exposed to GCE. Our results tend to suggest that genes involved in pathogenesis of:Pseudomonas aeruginosa are down regulated by quorum sensing effect of chlorogenic acid and therefore GCE could be useful as an adjuvant in combating multidrug resistance strains of Pseudomonas aeruginosa.
Polymyxin-Induced Lipid A Deacylation in Pseudomonas aeruginosa Perturbs Polymyxin Penetration and Confers High-Level Resistance.
Han Mei-Ling,Velkov Tony,Zhu Yan,Roberts Kade D,Le Brun Anton P,Chow Seong Hoong,Gutu Alina D,Moskowitz Samuel M,Shen Hsin-Hui,Li Jian
ACS chemical biology
Polymyxins are last-line antibiotics against life-threatening multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance is increasingly reported, leaving a total lack of therapies. Using lipidomics and transcriptomics, we discovered that polymyxin B induced lipid A deacylation via pagL in both polymyxin-resistant and -susceptible Pseudomonas aeruginosa. Our results demonstrated that the deacylation of lipid A is an "innate immunity" response to polymyxins and a key compensatory mechanism to the aminoarabinose modification to confer high-level polymyxin resistance in P. aeruginosa. Furthermore, cutting-edge neutron reflectometry studies revealed that an assembled outer membrane (OM) with the less hydrophobic penta-acylated lipid A decreased polymyxin B penetration, compared to the hexa-acylated form. Polymyxin analogues with enhanced hydrophobicity displayed superior penetration into the tail regions of the penta-acylated lipid A OM. Our findings reveal a previously undiscovered mechanism of polymyxin resistance, wherein polymyxin-induced lipid A remodeling affects the OM packing and hydrophobicity, perturbs polymyxin penetration, and thereby confers high-level resistance.
Association between Biofilm Formation and Antimicrobial Resistance in Carbapenem-Resistant .
Cho Hye Hyun,Kwon Kye Chul,Kim Semi,Park Yumi,Koo Sun Hoe
Annals of clinical and laboratory science
Recently, carbapenem resistance in is an increasingly important problem globally. Biofilm formation is a well-known pathogenic mechanism of , and the gene, , plays an important role in its primary stages. We studied the association between biofilm formation and in carbapenem-resistant isolates, along with antimicrobial resistance and the prevalence of metallo-β-lactamase (MBL) genes, based on the presence of 82 carbapenem-resistant isolates were collected from a tertiary hospital in Daejeon, Korea, between March 2008 and June 2014. Minimum inhibitory concentrations (MICs) of nine antimicrobial agents were determined using the agar dilution method. Biofilm formation was measured by microtiter plate assay. PCR and sequencing were used to identify and the MBL gene. 76 (92.7%) carbapenem-resistant isolates were biofilm producers. These biofilm producers showed higher levels of amikacin, ceftazidime, and cefepime resistance than non-producers. was detected in 71 (93.4%) biofilm-producing isolates and these results were statically significant (<0.01). 11 isolates carrying and were extremely resistant to all antimicrobials tested. In this study, biofilm formation was significantly associated with Furthermore, the coexistence of and the MBL gene in carbapenem-resistant isolates likely contributed to the increase in antimicrobial resistance.
Investigating of four main carbapenem-resistance mechanisms in high-level carbapenem resistant Pseudomonas aeruginosa isolated from burn patients.
Rostami Soodabeh,Farajzadeh Sheikh Ahmad,Shoja Saeed,Farahani Abbas,Tabatabaiefar Mohammad Amin,Jolodar Abbas,Sheikhi Raheleh
Journal of the Chinese Medical Association : JCMA
BACKGROUND:Pseudomonas aeruginosa is an opportunistic pathogen involved in many infections. Carbapenem-resistant P.aeruginosa has emerged as an important cause of infection in different hospitals worldwide. We aimed to determine frequencies of the four main resistance mechanisms [metallo-beta lactamase (MBL) production (bla, bla, bla and bla), overproduction of the MexAB-OprM and MexXY efflux pumps, overproduction of chromosome-encoded AmpC β-lactamase, and reduced OprD expression] in high-level carbapenem-resistant P.aeruginosa isolated from patients with burns. METHODS:In a descriptive study, 107 P. aeruginosa isolates were collected from patients with burn injuries and tested for antibiotic susceptibility, by an E-test for carbapenems, an E-test for metallo-β-lactamase producer isolates, and PCR to detect MBL genes. Furthermore, high-level carbapenem-resistant isolates were tested by real-time PCR for the expression levels of the mexB, mexY, ampC, and oprD genes. RESULTS:Amongst all P. aeruginosa isolates, 78.5%, 46.7%, and 15% were imipenem-, meropenem-, and doripenem-resistant, respectively; 72% of isolates were multidrug-resistant. The bla and bla genes were detected in 17.9% and 1.2% of isolates; respectively. The bla and bla genes were not observed. Among the resistant isolates, mexB overexpression (63.2%) was the most frequent mechanism, followed by mexY overexpression (52.6%), ampC overexpression (36.8%), and reduced oprD expression (21.1%). CONCLUSION:Emerging antimicrobial resistance in burn wound bacterial pathogens is a serious therapeutic challenge for clinicians. In the present study, most of the isolates were MDR. This finding indicated an alarming spread of resistant isolates and suggested that infection control strategies should be considered. Resistance to carbapenems is influenced by several factors, not all of which were evaluated in our study; however, the results showed that production of MBLs and overexpression of the mexB gene were the most frequent mechanisms in carbapenem-resistant isolates.
Pseudomonas aeruginosa Biofilm Antibiotic Resistance Gene Expression Requires the RpoS Stationary-Phase Sigma Factor.
Hall Clayton W,Hinz Aaron J,Gagnon Luke B-P,Zhang Li,Nadeau Jean-Paul,Copeland Sarah,Saha Bratati,Mah Thien-Fah
Applied and environmental microbiology
Chronic, biofilm-based bacterial infections are exceptionally difficult to eradicate due to the high degree of antibiotic recalcitrance exhibited by cells in biofilm communities. In the opportunistic pathogen , biofilm recalcitrance is multifactorial and arises in part from the preferential expression of resistance genes in biofilms compared to exponential-phase planktonic cells. One such mechanism involves , which we have previously shown to be expressed specifically in biofilms. In this study, we investigated the regulatory basis of this lifestyle-specific expression by developing an unstable green fluorescent protein (GFP) transcriptional reporter to observe the expression pattern of We found that in addition to its expression in biofilms, was upregulated in planktonic cells as they enter stationary phase. The transcription of in both growth phases was shown to be dependent on the stationary-phase sigma factor RpoS, and mutation of a putative RpoS binding site in the promoter abolished the activity of the promoter in stationary-phase cells. Overall, we have expanded our understanding of the temporal expression of in and have uncovered a regulatory basis for its growth phase-dependent expression. Bacterial biofilms are more resistant to antibiotics than free-living planktonic cells, and understanding the mechanistic basis of this resistance can inform treatments of biofilm-based infections. In addition to chemical and structural barriers that can inhibit antibiotic entry, the upregulation of specific genes in biofilms contributes to the resistance. We investigated this biofilm-specific gene induction by examining expression patterns of , a gene involved in biofilm resistance of the opportunistic pathogen We characterized expression in planktonic and biofilm growth conditions with an unstable green fluorescent protein (GFP) reporter and found that the expression of in biofilms is dependent on the stationary-phase sigma factor RpoS. Overall, our results support the physiological similarity between biofilms and stationary-phase cells and suggest that the induction of some stationary-phase genes in biofilms may contribute to their increased antibiotic resistance.
Detection of drug-resistance mechanism of Pseudomonas aeruginosa developing from a sensitive strain to a persister during carbapenem treatment.
Shen J L,Fang Y P
Genetics and molecular research : GMR
We explored the mechanism of the development from sensitivity to resistance to carbapenem in Pseudomonas aeruginosa. Two P. aeruginosa strains were collected during treatment with carbapenem. Strain homology was investigated using pulsed-field gel electrophoresis. Porin oprD2 expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The minimum inhibitory concentrations (MICs) of imipenem and meropenem with or without MC207110 were determined using the agar dilution method. The expression level of efflux pump mRNA was tested using real-time polymerase chain reaction. Metallo-lactamases (MBLs) were screened using the EDTA-disk synergy test. Genes encoding MBLs were amplified and then analyzed by DNA sequencing. The two treated strains belonged to the same pulsed-field gel electrophoresis type. The SDS-PAGE profile of the P. aeruginosa strains revealed that the 46-kDa membrane protein OprD2 of IMP(R)MEM(R) type strains was lost, whereas OprD2 of 1 IMP(S)MEM(S) strain was normal. With or without MC207110 treatment, the MIC of carbapenem-resistant P. aeruginosa decreased by 4-fold, while the MIC of carbapenem-sensitive P. aeruginosa did not. Compared with the carbapenem-sensitive strain, MexX mRNA expression in the carbapenem-resistant strain increased by 102.5-fold, while the mRNA expression of other efflux pumps did not markedly increase. Neither carbapenem-resistant nor carbapenem-sensitive P. aeruginosa produced MBL. The mechanism of development from sensitivity to resistance of P. aeruginosa to carbapenem during carbapenem treatment is due to porin oprD2 loss and an increased expression level of MexXY-OprM.
An α-Lipoic acid derivative, and anti-ROS agent, prevents the acquisition of multi-drug resistance in clinical isolates of Pseudomonas aeruginosa.
Hayakawa Sachiko,Kawamura Masato,Sato Takumi,Hirano Taizou,Kikuchi Toshiaki,Watanabe Akira,Fujimura Shigeru
Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy
Pseudomonas aeruginosa is one of the most common causes of nosocomial infections, and its multi-drug resistance has been a serious problem worldwide. The aim of this study was to evaluate whether exposure to piperacillin and reactive oxygen species (ROS) could lead to multi-drug resistance for clinical isolates of P. aeruginosa. The inhibition of this acquired resistance by the anti-ROS agent was also examined. In vitro inducement of multi-drug resistance was performed against 20 clinical isolates. These strains were incubated for 24 h and transferred 5 times after being exposed to 1 mM HO (ROS) in addition to a sub-MIC of piperacillin by the agar dilution method. Each MIC of piperacillin and levofloxacin was determined. As the mechanism of levofloxacin resistance, mutation of QRDR was investigated. The expression level of genes encoding efflux pumps; mexA, mexY, mexC, and D2 porin; oprD were determined by real-time PCR. Multi-resistance to both piperacillin and levofloxacin was induced with 4 of 20 strains (20%). No amino acid change was confirmed in QRDR. These strains showed overexpression of mexA, mexY, mexC, and another one showed decrease of oprD expression. Resistance development in 4 strains was inhibited by the same method including the anti-ROS agent, sodium zinc histidine dithiooctanamide (DHL-His-Zn). In conclusion, stimulation by ROS promoted acquisition of multi-drug resistance in 20% of isolates of P. aeruginosa, and DHL-His-Zn completely inhibited this acquisition of resistance. Therefore, this anti-ROS agent may be useful to assist antimicrobial chemotherapy by preventing multi-drug resistance.
The Pseudomonas aeruginosa lectin LecB binds to the exopolysaccharide Psl and stabilizes the biofilm matrix.
Passos da Silva Daniel,Matwichuk Michael L,Townsend Delaney O,Reichhardt Courtney,Lamba Doriano,Wozniak Daniel J,Parsek Matthew R
Pseudomonas aeruginosa biofilms are composed of exopolysaccharides (EPS), exogenous DNA, and proteins that hold these communities together. P. aeruginosa produces lectins LecA and LecB, which possess affinities towards sugars found in matrix EPS and mediate adherence of P. aeruginosa to target host cells. Here, we demonstrate that LecB binds to Psl, a key matrix EPS, and this leads to increased retention of both cells and EPS in a growing biofilm. This interaction is predicted to occur between the lectin and the branched side chains present on Psl. Finally, we show that LecB coordinates Psl localization in the biofilm. This constitutes a unique function for LecB and identifies it as a matrix protein that contributes to biofilm structure through EPS interactions.
Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa.
Bonomo Robert A,Szabo Dora
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
Acinetobacter species and Pseudomonas aeruginosa are noted for their intrinsic resistance to antibiotics and for their ability to acquire genes encoding resistance determinants. Foremost among the mechanisms of resistance in both of these pathogens is the production of beta -lactamases and aminoglycoside-modifying enzymes. Additionally, diminished expression of outer membrane proteins, mutations in topoisomerases, and up-regulation of efflux pumps play an important part in antibiotic resistance. Unfortunately, the accumulation of multiple mechanisms of resistance leads to the development of multiply resistant or even "panresistant" strains.
Prevention of drug access to bacterial targets: permeability barriers and active efflux.
Science (New York, N.Y.)
Some species of bacteria have low-permeability membrane barriers and are thereby "intrinsically" resistant to many antibiotics; they are selected out in the multitude of antibiotics present in the hospital environment and thus cause many hospital-acquired infections. Some strains of originally antibiotic-susceptible species may also acquire resistance through decreases in the permeability of membrane barriers. Another mechanism for preventing access of drugs to targets is the membrane-associated energy-driven efflux, which plays a major role in drug resistance, especially in combination with the permeation barrier. Recent results indicate the existence of bacterial efflux systems of extremely broad substrate specificity, in many ways reminiscent of the multidrug resistance pump of mammalian cells. One such system seems to play a major role in the intrinsic resistance of Pseudomonas aeruginosa, a common opportunistic pathogen. As the pharmaceutical industry succeeds in producing agents that can overcome specific mechanisms of bacterial resistance, less specific resistance mechanisms such as permeability barriers and multidrug active efflux may become increasingly significant in the clinical setting.
Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria.
Nguyen Dao,Joshi-Datar Amruta,Lepine Francois,Bauerle Elizabeth,Olakanmi Oyebode,Beer Karlyn,McKay Geoffrey,Siehnel Richard,Schafhauser James,Wang Yun,Britigan Bradley E,Singh Pradeep K
Science (New York, N.Y.)
Bacteria become highly tolerant to antibiotics when nutrients are limited. The inactivity of antibiotic targets caused by starvation-induced growth arrest is thought to be a key mechanism producing tolerance. Here we show that the antibiotic tolerance of nutrient-limited and biofilm Pseudomonas aeruginosa is mediated by active responses to starvation, rather than by the passive effects of growth arrest. The protective mechanism is controlled by the starvation-signaling stringent response (SR), and our experiments link SR-mediated tolerance to reduced levels of oxidant stress in bacterial cells. Furthermore, inactivating this protective mechanism sensitized biofilms by several orders of magnitude to four different classes of antibiotics and markedly enhanced the efficacy of antibiotic treatment in experimental infections.
Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients.
Ciofu Oana,Tolker-Nielsen Tim,Jensen Peter Østrup,Wang Hengzhuang,Høiby Niels
Advanced drug delivery reviews
Lung infection is the main cause of morbidity and mortality in patients with cystic fibrosis and is mainly dominated by Pseudomonas aeruginosa. The biofilm mode of growth makes eradication of the infection impossible, and it causes a chronic inflammation in the airways. The general mechanisms of biofilm formation and antimicrobial tolerance and resistance are reviewed. Potential anti-biofilm therapeutic targets such as weakening of biofilms by quorum-sensing inhibitors or antibiotic killing guided by pharmacokinetics and pharmacodynamics of antibiotics are presented. The vicious circle of adaptive evolution of the persisting bacteria imposes important therapeutic challenges and requires development of new drug delivery systems able to reach the different niches occupied by the bacteria in the lung of cystic fibrosis patients.
Structures of the wild-type MexAB-OprM tripartite pump reveal its complex formation and drug efflux mechanism.
Tsutsumi Kenta,Yonehara Ryo,Ishizaka-Ikeda Etsuko,Miyazaki Naoyuki,Maeda Shintaro,Iwasaki Kenji,Nakagawa Atsushi,Yamashita Eiki
In Pseudomonas aeruginosa, MexAB-OprM plays a central role in multidrug resistance by ejecting various drug compounds, which is one of the causes of serious nosocomial infections. Although the structures of the components of MexAB-OprM have been solved individually by X-ray crystallography, no structural information for fully assembled pumps from P. aeruginosa were previously available. In this study, we present the structure of wild-type MexAB-OprM in the presence or absence of drugs at near-atomic resolution. The structure reveals that OprM does not interact with MexB directly, and that it opens its periplasmic gate by forming a complex. Furthermore, we confirm the residues essential for complex formation and observed a movement of the drug entrance gate. Based on these results, we propose mechanisms for complex formation and drug efflux.
Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms.
Lister Philip D,Wolter Daniel J,Hanson Nancy D
Clinical microbiology reviews
Treatment of infectious diseases becomes more challenging with each passing year. This is especially true for infections caused by the opportunistic pathogen Pseudomonas aeruginosa, with its ability to rapidly develop resistance to multiple classes of antibiotics. Although the import of resistance mechanisms on mobile genetic elements is always a concern, the most difficult challenge we face with P. aeruginosa is its ability to rapidly develop resistance during the course of treating an infection. The chromosomally encoded AmpC cephalosporinase, the outer membrane porin OprD, and the multidrug efflux pumps are particularly relevant to this therapeutic challenge. The discussion presented in this review highlights the clinical significance of these chromosomally encoded resistance mechanisms, as well as the complex mechanisms/pathways by which P. aeruginosa regulates their expression. Although a great deal of knowledge has been gained toward understanding the regulation of AmpC, OprD, and efflux pumps in P. aeruginosa, it is clear that we have much to learn about how this resourceful pathogen coregulates different resistance mechanisms to overcome the antibacterial challenges it faces.
Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies.
Pang Zheng,Raudonis Renee,Glick Bernard R,Lin Tong-Jun,Cheng Zhenyu
Pseudomonas aeruginosa is an opportunistic pathogen that is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals. Eradication of P. aeruginosa has become increasingly difficult due to its remarkable capacity to resist antibiotics. Strains of Pseudomonas aeruginosa are known to utilize their high levels of intrinsic and acquired resistance mechanisms to counter most antibiotics. In addition, adaptive antibiotic resistance of P. aeruginosa is a recently characterized mechanism, which includes biofilm-mediated resistance and formation of multidrug-tolerant persister cells, and is responsible for recalcitrance and relapse of infections. The discovery and development of alternative therapeutic strategies that present novel avenues against P. aeruginosa infections are increasingly demanded and gaining more and more attention. Although mostly at the preclinical stages, many recent studies have reported several innovative therapeutic technologies that have demonstrated pronounced effectiveness in fighting against drug-resistant P. aeruginosa strains. This review highlights the mechanisms of antibiotic resistance in P. aeruginosa and discusses the current state of some novel therapeutic approaches for treatment of P. aeruginosa infections that can be further explored in clinical practice.