A new quorum-sensing inhibitor attenuates virulence and decreases antibiotic resistance in Pseudomonas aeruginosa

Quorum sensing (QS) has been a novel target for the treatment of infectious diseases. Here structural analogs of Pseudomonas aeruginosa autoinducer N-acyl homoserine lactone (AHL) were investigated for QS inhibitor (QSI) activity and a novel QSI was discovered, N-decanoyl-L-homoserine benzyl ester (C2). Virulence assays showed that C2 down-regulated total protease and elastase activities, as well as the production of rhamnolipid, that are controlled by QS in P. aeruginosa wild-type strain PAO1 without affecting growth. C2 was also shown to inhibit swarming motility of PAO1. Using a microdilution checkerboard method, we identified synergistic interactions between C2 and several antibiotics, tobramycin, gentamycin, cefepime, and meropenem. Data from real-time RT-PCR suggested that C2 inhibited the expression of lasR (29.67%), lasI (21.57%), rhlR (28.20%), and rhlI (29.03%).     

The transcriptional regulator CzcR modulates antibiotic resistance and quorum sensing in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa responds to zinc, cadmium and cobalt by way of the CzcRS two-component system. In presence of these metals the regulatory protein CzcR induces the expression of the CzcCBA efflux pump, expelling and thereby inducing resistance to Zn, Cd and Co. Importantly, CzcR co-regulates carbapenem antibiotic resistance by repressing the expression of the OprD porin, the route of entry for these antibiotics. This unexpected co-regulation led us to address the role of CzcR in other cellular processes unrelated to the metal response. We found that CzcR affected the expression of numerous genes directly involved in the virulence of P. aeruginosa even in the absence of the inducible metals. Notably the full expression of quorum sensing 3-oxo-C12-HSL and C4-HSL autoinducer molecules is impaired in the absence of CzcR. In agreement with this, the virulence of the czcRS deletion mutant is affected in a C. elegans animal killing assay. Additionally, chromosome immunoprecipitation experiments allowed us to localize CzcR on the promoter of several regulated genes, suggesting a direct control of target genes such as oprD, phzA1 and lasI. All together our data identify CzcR as a novel regulator involved in the control of several key genes for P. aeruginosa virulence processes.     

Identification of a novel regulator of the quorum-sensing systems in Pseudomonas aeruginosa

Quorum sensing is a global gene-regulatory mechanism in bacteria that enables individual bacterial cells to communicate and coordinate their population behaviors. Quorum sensing is central to the pathogenesis of many bacterial pathogens including Pseudomonas aeruginosa and therefore has been exploited as a target for developing novel antipathogenic drugs. In P. aeruginosa , three intertwined quorum-sensing systems, las, rhl , and the 2-alkyl-4(1 H )-quinolone system, which includes the Pseudomonas quinolone signal (PQS), control virulence factor production, and pathogenesis processes. Previously, we obtained a mutant with diminished expression of the phzA1B1C1D1E1F1G1 operon that is involved in the production of virulence factor phenazine compounds. In this study, the mutant was further characterized, and evidence indicating that the disrupted gene PA1196 in the mutant is a potential regulator of the rhl and PQS systems is presented. PA1196 positively controls the expression of the rhl and PQS systems and affects bacterial motility and multiple virulence factor expression via the quorum-sensing systems. This adds an important new player in the complex quorum-sensing network in P. aeruginosa .     

The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter

A mutation in the rsaL gene of Pseudomonas aeruginosa produces dramatically higher amounts of N-acyl homoserine lactone with respect to the wild type, highlighting the key role of this negative regulator in controlling quorum sensing (QS) in this opportunistic pathogen. The DNA binding site of the RsaL protein on the rsaL-lasI bidirectional promoter partially overlaps the binding site of the LasR protein, consistent with the hypothesis that RsaL and LasR could be in binding competition on this promoter. This is the first direct demonstration that RsaL acts as a QS negative regulator by binding to the lasI promoter.     

Anaerobic transfer of antibiotic resistance from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 x 10(-9) and 5.0 x 10(-8) cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 x 10(-7) and 6.5 x 10(-8) cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.     

绿脓杆菌pprA与pprB基因的克隆及其抗药性研究

从绿脓杆菌(Pseudomonas aeruginosa)PAK菌株的染色体DNA构建的基因文库中筛选到一 基因片段与绿脓杆菌的抗性相关。经测序证明该片段包含了绿脓杆菌基因组中PA4293所编码 序列,进一步实验证明与它相邻的另一段827bp的基因也与绿脓杆菌的抗药性有关。上述2基 因分别命名为pprA和pprB。以绿脓杆菌PAK菌株为出发菌株,经过新霉素耐受培养得到抗生素 抗性菌株PAK1-3,该菌株对氨基糖苷类抗生素的抗性明显增强。经PCR方法扩增上述基因并克 隆至PAK1-3菌株中,可引起该菌株对氨基糖苷类抗生素的敏感。又经pprB基因转入临床分离 得到的致病性绿脓杆菌中,导致部分绿脓杆菌对氨基糖苷类抗生素的敏感性增强。pprA和pprB 很可能是一组与绿脓杆菌抗药性相关的双分子调节系统,并与细胞膜的通透性有关。     

耐喹诺酮类铜绿假单胞菌的耐药性分析及血清学分型

目的了解广州地区喹诺酮类耐药铜绿假单胞菌的耐药性及泵抑制剂对其耐药水平降低的作用,并调查血清型分布情况。方法用法国生物梅里埃公司的微生物鉴定和药敏分析系统VITEK-2对127株铜绿假单胞菌进行鉴定和药敏检测,并采用羰酰氰基-对-氯苯胺(CCCP)与环丙沙星共同作用,以琼脂稀释法测定耐药菌的最低抑菌浓度(M IC)的变化,同时用玻片凝集法对耐药株进行血清学分型。结果环丙沙星耐药菌对哌拉西林/他唑巴坦(65.5%)的敏感率最高,只有阿米卡星(64.4%)、哌拉西林(51.7%)和妥布霉素(50.6%)的敏感率大于50.0%,而敏感菌对美罗培南(97.5%)及左氧氟沙星(97.5%)的敏感率最高,妥布霉素(95.0%)次之,对临床常用的13种抗生素,耐药菌较敏感菌的敏感性明显降低(P值均<0.001);耐药菌受泵抑制CCCP作用,M IC降低1~4个稀释度;血清分型率为93.1%,耐药菌的血清型以B型(20.7%)和L型(19.5%)为主。结论耐喹诺酮类铜绿假单胞菌对临床常用抗生素的敏感性降低,并呈多重耐药,使用抗生素 泵抑制剂可提高药物对铜绿假单胞菌的敏感性;血清学分型可以快速简单地监测铜绿假单胞菌在医院内的流行情况。     

Furanone derivatives as quorum-sensing antagonists of Pseudomonas aeruginosa

The biofilm formation of Pseudomonas aeruginosa, an opportunistic human pathogen, is developed by cell-to-cell signaling, so-called quorum sensing (QS). To control the biofilm formation, we designed and synthesized new QS inhibitors of P. aeruginosa based on the structure of the previously known QS inhibitor, furanone. Newly synthesized compounds were a series of analogs of (5-oxo-2,5-dihydrofuran-3-yl)methyl alkanoate, and the structures of all six synthesized compounds was confirmed by NMR and GC/MS analyses. These new QS inhibitor candidates could remarkably inhibit both Pseudomonas QS signaling and biofilm formation, which were assayed by using the recombinant reporter system and flow cell confocal microscopy. The degree of QS inhibition by these new inhibitors varied from 20% to 90%. For the profound understanding about inhibition mechanism, we tried to estimate the binding energy between QS receptor, LasR, and our inhibitors from the in silico modeling system. The predicted binding pattern from the modeling system and our experimental data about QS inhibition were in good agreement. From these results, we suggest a new approach to develop the QS inhibitors and biofilm control agents based on structural modeling.     

Crosstalk between Pseudomonas aeruginosa antibiotic resistance and virulence mediated by phenylethylamine

Multidrug efflux pumps are among the main Pseudomonas aeruginosa antibiotic-resistance determinants. Besides, efflux pumps are also involved in other relevant activities of bacterial physiology, including the quorum sensing-mediated regulation of bacterial virulence. Nevertheless, despite the relevance of efflux pumps in bacterial physiology, their interconnection with bacterial metabolism remains obscure. The effect of several metabolites on the expression of P. aeruginosa efflux pumps, and on the virulence and antibiotic resistance of this bacterium, was studied. Phenylethylamine was found to be both inducer and substrate of MexCD-OprJ, an efflux pump involved in P. aeruginosa antibiotic resistance and in extrusion of precursors of quorum-sensing signals. Phenylethylamine did not increase antibiotic resistance; however, the production of the toxin pyocyanin, the tissue-damaging protease LasB and swarming motility were reduced in the presence of this metabolite. This decrease in virulence potential was mediated by a reduction of lasI and pqsABCDE expression, which encode the proteins that synthesise the signalling molecules of two quorum-sensing regulatory pathways. This work sheds light on the interconnection between virulence and antibiotic-resistance determinants, mediated by bacterial metabolism, and points to phenylethylamine as an anti-virulence metabolite to be considered in the study of therapies against P. aeruginosa infections.     

The sensor kinase CbrA is a global regulator that modulates metabolism, virulence, and antibiotic resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that possesses a large arsenal of virulence factors enabling the pathogen to cause serious infections in immunocompromised patients, burn victims, and cystic fibrosis patients. CbrA is a sensor kinase that has previously been implied to play a role with its cognate response regulator CbrB in the metabolic regulation of carbon and nitrogen utilization in P. aeruginosa. Here it is demonstrated that CbrA and CbrB play an important role in various virulence and virulence-related processes of the bacteria, including swarming, biofilm formation, cytotoxicity, and antibiotic resistance. The cbrA deletion mutant was completely unable to swarm while exhibiting an increase in biofilm formation, supporting the inverse regulation of swarming and biofilm formation in P. aeruginosa. The cbrA mutant also exhibited increased cytotoxicity to human lung epithelial cells as early as 4 and 6 h postinfection. Furthermore, the cbrA mutant demonstrated increased resistance toward a variety of clinically important antibiotics, including polymyxin B, ciprofloxacin, and tobramycin. Microarray analysis revealed that under swarming conditions, CbrA regulated the expression of many genes, including phoPQ, pmrAB, arnBCADTEF, dnaK, and pvdQ, consistent with the antibiotic resistance and swarming impairment phenotypes of the cbrA mutant. Phenotypic and real-time quantitative PCR (RT-qPCR) analyses of a PA14 cbrB mutant suggested that CbrA may be modulating swarming, biofilm formation, and cytotoxicity via CbrB and that the CrcZ small RNA is likely downstream of this two-component regulator. However, as CbrB did not have a resistance phenotype, CbrA likely modulates antibiotic resistance in a manner independent of CbrB.     

Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies

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.