Francesco Imperi

Whole-Genome Pyrosequencing of an Epidemic Multidrug-Resistant Acinetobacter baumannii Strain Belonging to the European Clone II Group

ABSTRACT The whole-genome sequence of an epidemic, multidrug-resistant Acinetobacter baumannii strain (strain ACICU) belonging to the European clone II group and carrying the plasmid-mediated blaOXA-58 carbapenem resistance gene was determined. The A. baumannii ACICU genome was compared with the genomes of A. baumannii ATCC 17978 and Acinetobacter baylyi ADP1, with the aim of identifying novel genes related to virulence and drug resistance. A. baumannii ACICU has a single chromosome of 3,904,116 bp (which is predicted to contain 3,758 genes) and two plasmids, pACICU1 and pACICU2, of 28,279 and 64,366 bp, respectively. Genome comparison showed 86.4% synteny with A. baumannii ATCC 17978 and 14.8% synteny with A. baylyi ADP1. A conspicuous number of transporters belonging to different superfamilies was predicted for A. baumannii ACICU. The relative number of transporters was much higher in ACICU than in ATCC 17978 and ADP1 (76.2, 57.2, and 62.5 transporters per Mb of genome, respectively). An antibiotic resistance island, AbaR2, was identified in ACICU and had plausibly evolved by reductive evolution from the AbaR1 island previously described in multiresistant strain A. baumannii AYE. Moreover, 36 putative alien islands (pAs) were detected in the ACICU genome; 24 of these had previously been described in the ATCC 17978 genome, 4 are proposed here for the first time and are present in both ATCC 17978 and ACICU, and 8 are unique to the ACICU genome. Fifteen of the pAs in the ACICU genome encode genes related to drug resistance, including membrane transporters and ex novo acquired resistance genes. These findings provide novel insight into the genetic basis of A. baumannii resistance.

Deciphering the Multifactorial Nature of Acinetobacter baumannii Pathogenicity

Background Acinetobacter baumannii is an emerging bacterial pathogen that causes a broad array of infections, particularly in hospitalized patients. Many studies have focused on the epidemiology and antibiotic resistance of A. baumannii, but little is currently known with respect to its virulence potential. Methodology/Principal Findings The aim of this work was to analyze a number of virulence-related traits of four A. baumannii strains of different origin and clinical impact for which complete genome sequences were available, in order to tentatively identify novel determinants of A. baumannii pathogenicity. Clinical strains showed comparable virulence in the Galleria mellonella model of infection, irrespective of their status as outbreak or sporadic strains, whereas a non-human isolate was avirulent. A combined approach of genomic and phenotypic analyses led to the identification of several virulence factors, including exoproducts with hemolytic, phospholipase, protease and iron-chelating activities, as well as a number of multifactorial phenotypes, such as biofilm formation, surface motility and stress resistance, which were differentially expressed and could play a role in A. baumannii pathogenicity. Conclusion/Significance This work provides evidence of the multifactorial nature of A. baumannii virulence. While A. baumannii clinical isolates could represent a selected population of strains adapted to infect the human host, subpopulations of highly genotypically and phenotypically diverse A. baumannii strains may exist outside the hospital environment, whose relevance and distribution deserve further investigation.

Molecular basis of pyoverdine siderophore recycling in Pseudomonas aeruginosa

The siderophore pyoverdine (PVD) is a primary virulence factor of the human pathogenic bacterium Pseudomonas aeruginosa, acting as both an iron carrier and a virulence-related signal molecule. By exploring a number of P. aeruginosa candidate systems for PVD secretion, we identified a tripartite ATP-binding cassette efflux transporter, here named PvdRT-OpmQ, which translocates PVD from the periplasmic space to the extracellular milieu. We show this system to be responsible for recycling of PVD upon internalization by the cognate outer-membrane receptor FpvA, thus making PVD virtually available for new cycles of iron uptake. Our data exclude the involvement of PvdRT-OpmQ in secretion of de novo synthesized PVD, indicating alternative pathways for PVD export and recycling. The PvdRT-OpmQ transporter is one of the few secretion systems for which substrate recognition and extrusion occur in the periplasm. Homologs of the PvdRT-OpmQ system are present in genomes of all fluorescent pseudomonads sequenced so far, suggesting that PVD recycling represents a general energy-saving strategy adopted by natural Pseudomonas populations.

New Life for an Old Drug: the Anthelmintic Drug Niclosamide Inhibits Pseudomonas aeruginosa Quorum Sensing

ABSTRACT The need for novel antibacterial strategies and the awareness of the importance of quorum sensing (QS) in bacterial infections have stimulated research aimed at identifying QS inhibitors (QSIs). However, clinical application of QSIs identified so far is still distant, likely due to their unsuitability for use in humans. A promising way to overcome this problem is searching for anti-QS side activity among the thousands of drugs approved for clinical use in the treatment of different diseases. Here, we applied this strategy to the search for QSIs, by screening a library of FDA-approved compounds for their ability to inhibit the QS response in the Gram-negative pathogen Pseudomonas aeruginosa. We found that the anthelmintic drug niclosamide strongly inhibits the P. aeruginosa QS response and production of acyl-homoserine lactone QS signal molecules. Microarray analysis showed that niclosamide affects the transcription of about 250 genes, with a high degree of target specificity toward the QS-dependent regulon. Phenotypic assays demonstrated that niclosamide suppresses surface motility and production of the secreted virulence factors elastase, pyocyanin, and rhamnolipids, and it reduces biofilm formation. In accordance with the strong antivirulence activity disclosed in vitro, niclosamide prevented P. aeruginosa pathogenicity in an insect model of acute infection. Besides the finding that an FDA-approved drug has a promising antivirulence activity against one of the most antibiotic-resistant bacterial pathogens, this work provides a proof of concept that a lateral anti-QS activity can be detected among drugs already used in humans, validating a new approach to identify QSIs that could easily move into clinical applications.

The Genomics of Acinetobacter baumannii: Insights into Genome Plasticity, Antimicrobial Resistance and Pathogenicity

The genome sequences of a number of Acinetobacter baumannii strains, including representatives of the main epidemic international lineages, have now been determined, and several others are in progress. The study of A. baumannii genomics has provided an expanded view of the adaptation and virulence capacities of this bacterial species, whilst also presenting novel insights into its intraspecies diversity and genome evolution. Genomic analyses have revealed that the current A. baumannii clinical population consists of low‐grade pathogens, whose pathogenicity relies mainly on an ability to persist in the hospital setting and survive antibiotic treatment. A. baumannii has a high capacity to acquire new genetic determinants and displays an open pan genome; this feature may have played a crucial role in the evolution of this human opportunistic pathogen towards clinical success.

Repurposing the antimycotic drug flucytosine for suppression of Pseudomonas aeruginosa pathogenicity

Although antibiotic resistance represents a public health emergency, the pipeline of new antibiotics is running dry. Repurposing of old drugs for new clinical applications is an attractive strategy for drug development. We used the bacterial pathogen Pseudomonas aeruginosa as a target for the screening of antivirulence activity among marketed drugs. We found that the antimycotic agent flucytosine inhibits the expression of the iron-starvation σ-factor PvdS, thereby repressing the production of major P. aeruginosa virulence factors, namely pyoverdine, PrpL protease, and exotoxin A. Flucytosine administration at clinically meaningful dosing regimens suppressed P. aeruginosa pathogenicity in a mouse model of lung infection. The in vitro and in vivo activity of flucytosine against P. aeruginosa, combined with its desirable pharmacological properties, paves the way for clinical trials on the anti-P. aeruginosa efficacy of flucytosine in humans.

Analysis of the periplasmic proteome of Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen

The Gram‐negative bacterium Pseudomonas aeruginosa is a main cause of infection in hospitalized, burned, immunocompromised, and cystic fibrosis patients. Many processes essential for P. aeruginosa pathogenesis, e.g., nutrient uptake, antibiotic resistance, and virulence, take place in the cell envelope and depend on components residing in the periplasmic space. Recent high‐throughput studies focused on P. aeruginosa membrane compartments. However, the composition and dynamics of its periplasm remain largely uncharacterized. Here, we report a detailed description of the periplasmic proteome of the wild‐type P. aeruginosa strain PAO1 by 2‐DE and MALDI‐TOF/TOF analysis. Three extraction methods were compared at proteome level in order to achieve the most reliable and comprehensive periplasmic protein map. A total of 495 spots representing 395 different proteins were identified. Most of the high intensity spots corresponded to periplasmic proteins, while cytoplasmic contaminants were mainly detected among faint spots. The majority of the identified periplasmic proteins is involved in transport, cell‐envelope integrity, and protein folding control. Notably, more than 30% still has an unpredicted function. This work provides the first overview of the P. aeruginosa periplasm and offers the basis for future studies on periplasmic proteome changes occurring during P. aeruginosa adaptation to different environments and/or antibiotic treatments.

Enzymatic Detoxification of Cyanide: Clues from Pseudomonas aeruginosa Rhodanese

Cyanide is a dreaded chemical because of its toxic properties. Although cyanide acts as a general metabolic inhibitor, it is synthesized, excreted and metabolized by hundreds of organisms, including bacteria, algae, fungi, plants, and insects, as a mean to avoid predation or competition. Several cyanide compounds are also produced by industrial activities, resulting in serious environmental pollution. Bioremediation has been exploited as a possible alternative to chemical detoxification of cyanide compounds, and various microbial systems allowing cyanide degradation have been described. Enzymatic pathways involving hydrolytic, oxidative, reductive, and substitution/transfer reactions are implicated in detoxification of cyanide by bacteria and fungi. Amongst enzymes involved in transfer reactions, rhodanese catalyzes sulfane sulfur transfer from thiosulfate to cyanide, leading to the formation of the less toxic thiocyanate. Mitochondrial rhodanese has been associated with protection of aerobic respiration from cyanide poisoning. Here, the biochemical and physiological properties of microbial sulfurtransferases are reviewed in the light of the importance of rhodanese in cyanide detoxification by the cyanogenic bacterium Pseudomonas aeruginosa. Critical issues limiting the application of a rhodanese-based cellular system to cyanide bioremediation are also discussed.

Cell-surface signaling in Pseudomonas: stress responses, iron transport, and pathogenicity

Membrane-spanning signaling pathways enable bacteria to alter gene expression in response to extracytoplasmic stimuli. Many such pathways are cell-surface signaling (CSS) systems, which are tripartite molecular devices that allow Gram-negative bacteria to transduce an extracellular stimulus into a coordinated transcriptional response. Typically, CSS systems are composed of the following: (1) an outer membrane receptor, which senses the extracellular stimulus; (2) a cytoplasmic membrane-spanning protein involved in signal transduction from the periplasm to the cytoplasm; and (3) an extracytoplasmic function (ECF) sigma factor that initiates expression of the stimulus-responsive gene(s). Members of genus Pseudomonas provide a paradigmatic example of how CSS systems contribute to the global control of gene expression. Most CSS systems enable self-regulated uptake of iron via endogenous (pyoverdine) or exogenous (xenosiderophores, heme, and citrate) carriers. Some are also implicated in virulence, biofilm formation, and cell-cell interactions. Incorporating insights from the well-characterized alginate regulatory circuitry, this review will illustrate common themes and variations at the level of structural and functional properties of Pseudomonas CSS systems. Control of the expression and activity of ECF sigma factors are central to gene regulation via CSS, and the variety of intrinsic and extrinsic factors influencing these processes will be discussed.

Molecular epidemiology of a Pseudomonas aeruginosa hospital outbreak driven by a contaminated disinfectant-soap dispenser.

Background and Objective Pseudomonas aeruginosa infection represents a main cause of morbidity and mortality among immunocompromised patients. This study describes a fatal epidemic of P. aeruginosa that occurred in a hematology unit in Italy. Methods Retrospective cohort study, prospective surveillance, auditing, extensive testing on healthcare workers and environmental investigation were performed to define the dynamics and potential causes of transmission. RAPD, macrorestriction analyses and sequence typing were used to define relationships between P. aeruginosa isolates. Results Eighteen cases of infection were identified in the different phases of the investigation. Of these, five constitute a significant molecular cluster of infection. A P. aeruginosa strain with the same genetic fingerprint and sequence type (ST175) as clinical isolates strain was also isolated from a heavily contaminated triclosan soap dispenser. Discussion and Conclusions Our results are consistent with the hypothesis that patients became indirectly infected, e.g., during central venous catheter handling through contaminated items, and that the triclosan soap dispenser acted as a common continuous source of P. aeruginosa infection. Since P. aeruginosa is intrinsically unsusceptible to triclosan, the use of triclosan-based disinfectant formulations should be avoided in those healthcare settings hosting patients at high risk of P. aeruginosa infection.