Pyoverdine and proteases affect the response of pseudomonas aeruginosa to gallium in human serum

Gallium is an iron mimetic which has recently been repurposed as an antibacterial agent due to its capability to disrupt bacterial iron metabolism. In this study, the antibacterial activity of gallium nitrate [Ga(NO3)3] was investigated in complement-free human serum (HS) on 55 Pseudomonas aeruginosa clinical isolates from cystic fibrosis and non-cystic fibrosis patients. The susceptibility of P. aeruginosa to Ga(NO3)3 in HS was dependent on the bacterial ability to acquire iron from serum binding proteins (i.e., transferrin). The extent of serum protein degradation correlated well with P. aeruginosa growth in HS, while pyoverdine production did not. However, pyoverdine-deficient P. aeruginosa strains were unable to grow in HS and overcome iron restriction, albeit capable of releasing proteases. Predigestion of HS with proteinase K promoted the growth of all strains, irrespective of their ability to produce proteases and/or pyoverdine. The MICs of Ga(NO3)3 were higher in HS than in an iron-poor Casamino Acids medium, where proteolysis does not affect iron availability. Coherently, strains displaying high proteolytic activity were less susceptible to Ga(NO3)3 in HS. Our data support a model in which both pyoverdine and proteases affect the response of P. aeruginosa to Ga(NO3)3 in HS. The relatively high Ga(NO3)3 concentration required to inhibit the growth of highly proteolytic P. aeruginosa isolates in HS poses a limitation to the potential of Ga(NO3)3 in the treatment of P. aeruginosa bloodstream infections

Ferric uptake regulator Fur is conditionally essential in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the ferric uptake regulator (Fur) protein controls both metabolism and virulence in response to iron availability. Differently from other bacteria, attempts to obtain fur deletion mutants of P. aeruginosa failed, leading to the assumption that Fur is an essential protein in this bacterium. By investigating a P. aeruginosa conditional fur mutant, we demonstrate that Fur is not essential for P. aeruginosa growth in liquid media, biofilm formation, and pathogenicity in an insect model of infection. Conversely, Fur is essential for growth on solid media since Fur-depleted cells are severely impaired in colony formation. Transposon-mediated random mutagenesis experiments identified pyochelin siderophore biosynthesis as a major cause of the colony growth defect of the conditional fur mutant, and deletion mutagenesis confirmed this evidence. Impaired colony growth of pyochelin-proficient Fur-depleted cells does not depend on oxidative stress, since Fur-depleted cells do not accumulate higher levels of reactive oxygen species (ROS) and are not rescued by antioxidant agents or overexpression of ROS-detoxifying enzymes. Ectopic expression of pch genes revealed that pyochelin production has no inhibitory effects on a fur deletion mutant of Pseudomonas syringae pv. tabaci, suggesting that the toxicity of the pch locus in Fur-depleted cells involves a P. aeruginosa-specific pathway(s)

Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence

Efflux pumps of the resistance-nodulation-cell-division (RND) family increase antibiotic resistance in many bacterial pathogens, representing candidate targets for the development of antibiotic adjuvants. RND pumps have also been proposed to contribute to bacterial infection, implying that efflux pump inhibitors (EPIs) could also act as anti-virulence drugs. Nevertheless, EPIs are usually investigated only for their properties as antibiotic adjuvants, while their potential anti-virulence activity is seldom taken into account. In this study it is shown that RND efflux pumps contribute to Pseudomonas aeruginosa PAO1 pathogenicity in an insect model of infection, and that the well-characterized EPI Phe-Arg-β-naphthylamide (PAβN) is able to reduce in vivo virulence of the P. aeruginosa PAO1 laboratory strain, as well as of clinical isolates. The production of quorum sensing (QS) molecules and of QS-dependent virulence phenotypes is differentially affected by PAβN, depending on the strain. Transcriptomic and phenotypic analyses showed that the protection exerted by PAβN from P. aeruginosa PAO1 infection in vivo correlates with the down-regulation of key virulence genes (e.g. genes involved in iron and phosphate starvation). Since PAβN impacts P. aeruginosa virulence, anti-virulence properties of EPIs are worthy to be explored, taking into account possible strain-specificity of their activit

High-level tolerance to triclosan may play a role in Pseudomonas aeruginosa antibiotic resistance in immunocompromised hosts: evidence from outbreak investigation

<p>Abstract</p> <p>Background and methods</p> <p><it>Pseudomonas aeruginosa </it>is a major infectious threat to immunocompromised patients. We recently reported a fatal epidemic of multidrug-resistant <it>P. aeruginosa </it>in an onchoematology unit, linked to massive contamination of a triclosan-based disinfectant. The aim of this study is to evaluate the antimicrobial activity of triclosan and chlorhexidine digluconate against the epidemic strain of <it>P. aeruginosa</it>, to confirm the hypothesis that the soap dispenser acted as a continuous source of the infection during the outbreak, and to explore the potential role of triclosan in increasing the level of resistance to selected antibiotics.</p> <p>Susceptibility tests and time-kill assays for disinfectans were performed using two commercial formulations containing triclosan and chlorhexidine digluconate, respectively. Antibiotic susceptibility testing was performed by the broth microdilution method.</p> <p>Findings</p> <p>The <it>P. aeruginosa </it>epidemic strain exhibited an extremely high level of triclosan resistance (apparent MIC = 2,125 mg/L), while it was markedly susceptible to chlorhexidine digluconate (apparent MIC = 12.5 mg/L). Upon gradual adaptation to triclosan, the epidemic strain survived for a long period (> 120 h) in the presence of 3,400 mg/L (equivalent to 1.6 × MIC) of triclosan, concomitantly increasing the resistance to six antibiotics that are typical substrates of drug efflux pumps of the resistance nodulation division family. This effect was reversed by efflux pump inhibitors.</p> <p>Conclusions</p> <p>The epidemic <it>P. aeruginosa </it>strain was resistant to triclosan and its previous exposure to triclosan increases antibiotic resistance, likely through active efflux mechanisms. Since <it>P. aeruginosa </it>can become tolerant to elevated triclosan concentrations, the use of triclosan-based disinfectants should be avoided in those healthcare settings hosting patients at high risk for <it>P. aeruginosa </it>infection.</p

Draft Genome Sequence of the Carboxydotrophic Alphaproteobacterium Aminobacter carboxidus Type Strain DSM 1086

Aminobacter carboxidus is a soil Gram-negative alphaproteobacterium belonging to the physiological group of carboxydobacteria which aerobically oxidize CO to CO2 Here, we report the draft genome sequence of the A. carboxidus DSM 1086 type strain and the identification of both form I and form II CO dehydrogenase systems in this strain

Virulence-related traits of epidemic Acinetobacter baumannii strains belonging to the international clonal lineages I-III and to the emerging genotypes ST25 and ST78

BACKGROUND: Acinetobacter baumannii is responsible for large epidemics in hospitals, where it can persist for long time on abiotic surfaces. This study investigated some virulence-related traits of epidemic A. baumannii strains assigned to distinct MLST genotypes, including those corresponding to the international clones I-III as well as emerging genotypes responsible for recent epidemics. METHODS: Genotyping of bacteria was performed by PFGE analysis and MLST according to the Pasteur’s scheme. Biofilm formation on polystyrene plates was assessed by crystal violet staining; resistance to desiccation was evaluated on glass cover-slips when kept at room-temperature and 31% relative humidity; adherence to and invasion of A549 human alveolar epithelial cells were determined by the analysis of viable bacteria associated with or internalized by A549 human alveolar epithelial cells; Galleria mellonella killing assays were used to analyze the virulence of A. baumannii in vivo. RESULTS: The ability to form biofilm was significantly higher for A. baumannnii strains assigned to ST2 (international clone II), ST25 and ST78 compared to other STs. All A. baumannii strains survived on dry surfaces for over 16 days, and strains assigned to ST1 (international clone I) and ST78 survived for up to 89 and 96 days, respectively. Adherence to A549 pneumocytes was higher for strains assigned to ST2, ST25 and ST78 than other genotypes; a positive correlation exists between adherence and biofilm formation. Strains assigned to ST78 also showed significantly higher ability to invade A549 cells. No significant differences in the killing of G. mellonella worms were found among strains. CONCLUSIONS: Elevated resistance to desiccation, high biofilm-forming capacity on abiotic surfaces and adherence to A549 cells might have favoured the spread and persistence in the hospital environment of A. baumannii strains assigned to the international clones I and II and to the emerging genotypes ST25 and ST78

Identification of FDA-Approved Drugs as Antivirulence Agents Targeting the pqs Quorum-Sensing System of Pseudomonas aeruginosa

Copyright © 2018 American Society for Microbiology. All Rights Reserved. The long-term use of antibiotics has led to the emergence of multidrug-resistant bacteria. A promising strategy to combat bacterial infections aims at hampering their adaptability to the host environment without affecting growth. In this context, the intercellular communication system quorum sensing (QS), which controls virulence factor production and biofilm formation in diverse human pathogens, is considered an ideal target. Here, we describe the identification of new inhibitors of the pqs QS system of the human pathogen Pseudomonas aeruginosa by screening a library of 1,600 U.S. Food and Drug Administration-approved drugs. Phenotypic characterization of ad hoc engineered strains and in silico molecular docking demonstrated that the antifungal drugs clotrimazole and miconazole, as well as an antibacterial compound active against Gram-positive pathogens, clofoctol, inhibit the pqs system, probably by targeting the transcriptional regulator PqsR. The most active inhibitor, clofoctol, specifically inhibited the expression of pqs-controlled virulence traits in P. aeruginosa, such as pyocyanin production, swarming motility, biofilm formation, and expression of genes involved in siderophore production. Moreover, clofoctol protected Galleria mellonella larvae from P. aeruginosa infection and inhibited the pqs QS system in P. aeruginosa isolates from cystic fibrosis patients. Notably, clofoctol is already approved for clinical treatment of pulmonary infections caused by Gram-positive bacterial pathogens; hence, this drug has considerable clinical potential as an antivirulence agent for the treatment of P. aeruginosa lung infections

Management of Urinary Tract Infections: Problems and Possible Solutions

In clinically suspected urinary tract infections (UTIs), empirical antibiotic treatment is usually started long before the laboratory results of urine culture and antibiogram are available. Although molecular diagnostic approaches are being applied to the diagnosis of many infections, UTIs are generally diagnosed by traditional culture methods. Patient care could greatly benefit from the development of a rapid, accurate, inexpensive test that could be done at patient’s bedside, allowing the practitioner to plan targeted, more effective therapy. Such a test would potentially reduce incorrect or unnecessary use of antibacterial drugs and reduce the emergence of bacterial resistance. In response to this pressing and unmet clinical need, several methods have been developed in the last few years. Among these, the new point-of-care test (POCT) for detecting UTIs named Micro Biological Survey (MBS) UTI CHECK holds promise, as it allows semi-quantitative determination of bacterial load in urine leading to a fast detection of UTIs and to evaluation of bacterial antibiotic susceptibility. This new technology operates through a colorimetric survey performed in low-cost, ready-to-use, disposable vials, in which 1 ml of urine is inoculated without any preliminary treatment and requiring neither specialized personnel nor a specialized equipment

Genomics of Acinetobacter baumannii iron uptake

Iron is essential for growth in most bacteria due to its redox activity and its role in essential metabolic reactions; it is a cofactor for many bacterial enzymes. The bacterium Acinetobacter baumannii is a multidrug-resistant nosocomial pathogen. A. baumannii responds to low iron availability imposed by the host through the exploitation of multiple iron-acquisition strategies, which are likely to deliver iron to the cell under a variety of environmental conditions, including human and animal infection. To date, six different gene clusters for active iron uptake have been described in A. baumannii , encoding protein systems involved in (i) ferrous iron uptake (feo); (ii) haem uptake (hemT and hemO); and (iii) synthesis and transport of the baumannoferrin(s) (bfn), acinetobactin (bas/bau) and fimsbactin(s) (fbs) siderophores. Here we describe the structure, distribution and phylogeny of iron-uptake gene clusters among >1000 genotypically diverse A. baumannii isolates, showing that feo, hemT, bfn and bas/bau clusters are very prevalent across the dataset, whereas the additional haem-uptake system hemO is only present in a portion of the dataset and the fbs gene cluster is very rare. Since the expression of multiple iron-uptake clusters can be linked to virulence, the presence of the additional haem-uptake system hemO may have contributed to the success of some A. baumannii clones