Iron efflux by IetA enhances ß-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. OBJECTIVES: To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. METHODS: In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. RESULTS: The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the ß-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. CONCLUSION: Iron efflux is critical to alleviate oxidative stress damage and ß-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.

How a natural antibiotic uses oxidative stress to kill oxidant-resistant bacteria.

Natural products that possess antibiotic and antitumor qualities are often suspected of working through oxidative mechanisms. In this study, two quinone-based small molecules were compared. Menadione, a classic redox-cycling compound, was confirmed to generate high levels of reactive oxygen species inside Escherichia coli. It inactivated iron-cofactored enzymes and blocked growth. However, despite the substantial levels of oxidants that it produced, it was unable to generate significant DNA damage and was not lethal. Streptonigrin, in contrast, was poorer at redox cycling and did not inactivate enzymes or block growth; however, even in low doses, it damaged DNA and killed cells. Its activity required iron and oxygen, and in vitro experiments indicated that its quinone moiety transferred electrons through the adjacent iron atom to oxygen. Additionally, in vitro experiments revealed that streptonigrin was able to damage DNA without inhibition by catalase, indicating that hydrogen peroxide was not involved. We infer that streptonigrin can reduce bound oxygen directly to a ferryl species, which then oxidizes the adjacent DNA, without release of superoxide or hydrogen peroxide intermediates. This scheme allows streptonigrin to kill a bacterial cell without interference by scavenging enzymes. Moreover, its minimal redox-cycling behavior avoids alerting either the OxyR or the SoxRS systems, which otherwise would block killing. This example highlights qualities that may be important in the design of oxidative drugs. These results also cast doubt on proposals that bacteria can be killed by stressors that merely stimulate intracellular O2- and H2O2 formation.

Chemistry, Biosynthesis and Pharmacology of Streptonigrin: An Old Molecule with Future Prospects for New Drug Design, Development and Therapy.

Streptonigrin is an aminoquinone alkaloid isolated from Streptomyces flocculus and is gaining attention as a drug molecule owing to its potential antitumor and antibiotic effects. It was previously used as an anticancer drug but has been discontinued because of its toxic effects. However, according to the most recent studies, the toxicity of streptonigrin and its structurally modified derivatives has been reduced while maintaining their potential pharmacological action at lower concentrations. To date, many investigations have been conducted on this molecule and its derivatives to determine the most effective molecule with low toxicity to enable new drug discovery. Therefore, the main objective of this study is to provide a comprehensive review and to discuss the prospects for streptonigrin and its derived compounds, which may boost the molecule as a highly interesting target molecule for new drug design, development and therapy. To complete this review, relevant literature was collected from several scientific databases, including Google Scholar, PubMed, Scopus and ScienceDirect. Following a complete screening, the obtained information is summarized in the present review to provide a good reference and accelerate the development and utilization of streptonigrin and its derivatives as pharmaceuticals.

Streptonigrin Mitigates Lung Cancer-induced Cachexia by Suppressing TCF4/TWIST1-induced PTHLH Expression.

BACKGROUND/AIM: Cachexia - a wasting disorder of adipose and skeletal muscle tissue - is the most common driver of poor prognosis in patients with advanced lung cancer. Parathyroid hormone-like hormone (PTHLH) is potentially a critical factor in cancer-associated cachexia. We previously showed that streptonigrin - an aminoquinone with antitumor effects - inhibited the interaction between TCF4 and TWIST1. This study aimed to determine the anti-cachectic performance of streptonigrin in lung cancer. MATERIALS AND METHODS: We assessed the effect of streptonigrin on the interaction of TCF4 and TWIST1 using co-immunoprecipitation and a mammalian-two hybrid luciferase assay, which was confirmed by an in vitro GST pull-down assay using recombinant bHLH domain-containing TCF4 and TWIST1. We assessed the anti-cachectic effect of streptonigrin in vivo using an LLC1 cell-induced tumour-bearing mouse model. Changes in the degree of skeletal muscle and adipose tissue wasting were determined by measuring the weights of gastrocnemius and epidydimal white adipose tissue. RESULTS: Streptonigrin was found to inhibit the interaction of TCF4 with TWIST1 in a dose-dependent manner. The in vitro GST pull-down assay revealed that streptonigrin directly inhibited the interaction between TCF4 and TWIST1. The expression of PTHLH mRNA, which is transcriptionally regulated by the TCF4/TWIST1 complex in response to TGF-ß1 signalling, was decreased in streptonigrin-treated lung cancer cells. Streptonigrin significantly decreased the expression of proteolysis-related genes in skeletal muscle and browning-related genes in white adipose tissues of LLC1-induced tumour-bearing mice. CONCLUSION: Streptonigrin exerts potent therapeutic effects on lung cancer-induced cachexia by suppressing TCF4/TWIST1-mediated PTHLH expression.

Flavoprotein StnP2 Catalyzes the ß-Carboline Formation during the Streptonigrin Biosynthesis.

ß-Carboline (ßC) alkaloids constitute a large family of indole alkaloids that exhibit diverse pharmacological properties, such as antitumor, antiviral, antiparasitic, and antimicrobial activities. Here, we report that a flavoprotein StnP2 catalyzes the dehydrogenation at C1-N2 of a tetrahydro-ß-carboline (THßC) generating a 3,4-dihydro-ß-carboline (DHßC), and the DHßC subsequently undergoes a spontaneous dehydrogenation to ßC formation involved in the biosynthesis of the antitumor agent streptonigrin. Biochemical characterization showed that StnP2 catalyzed the highly regio- and stereo-selective dehydrogenation, and StnP2 exhibits promiscuity toward different THßCs. This study provides an alternative kind of enzyme catalyzing the biosynthesis of ßC alkaloids and enhances the importance of flavoproteins.

Hybrids of 1,4-Quinone with Quinoline Derivatives: Synthesis, Biological Activity, and Molecular Docking with DT-Diaphorase (NQO1).

Hybrids 1,4-quinone with quinoline were obtained by connecting two active structures through an oxygen atom. This strategy allows to obtain new compounds with a high biological activity and suitable bioavailability. Newly synthesized compounds were characterized by various spectroscopic methods. The enzymatic assay used showed that these compounds were a suitable DT-diaphorase (NQO1) substrates as evidenced by increasing enzymatic conversion rates relative to that of streptonigrin. Hybrids were tested in vitro against a panel of human cell lines including melanoma, breast, and lung cancers. They showed also a high cytotoxic activity depending on the type of 1,4-quinone moiety and the applied tumor cell lines. It was found that cytotoxic activity of the studied hybrids was increasing against the cell lines with higher NQO1 protein level, such as breast (MCF-7 and T47D) and lung (A549) cancers. Selected hybrids were tested for the transcriptional activity of the gene encoding a proliferation marker (H3 histone), cell cycle regulators (p53 and p21) and the apoptosis pathway (BCL-2 and BAX). The molecular docking was used to examine the probable interaction between the hybrids and NQO1 protein.

The Neisseria gonorrhoeae type IV pilus promotes resistance to hydrogen peroxide- and LL-37-mediated killing by modulating the availability of intracellular, labile iron.

The Neisseria gonorrhoeae Type IV pilus is a multifunctional, dynamic fiber involved in host cell attachment, DNA transformation, and twitching motility. We previously reported that the N. gonorrhoeae pilus is also required for resistance against hydrogen peroxide-, antimicrobial peptide LL-37-, and non-oxidative, neutrophil-mediated killing. We tested whether the hydrogen peroxide, LL-37, and neutrophil hypersensitivity phenotypes in non-piliated N. gonorrhoeae could be due to elevated iron levels. Iron chelation in the growth medium rescued a nonpiliated pilE mutant from both hydrogen peroxide- and antimicrobial peptide LL-37-mediated killing, suggesting these phenotypes are related to iron availability. We used the antibiotic streptonigrin, which depends on free cytoplasmic iron and oxidation to kill bacteria, to determine whether piliation affected intracellular iron levels. Several non-piliated, loss-of-function mutants were more sensitive to streptonigrin killing than the piliated parental strain. Consistent with the idea that higher available iron levels in the under- and non-piliated strains were responsible for the higher streptonigrin sensitivity, iron limitation by desferal chelation restored resistance to streptonigrin in these strains and the addition of iron restored the sensitivity to streptonigrin killing. The antioxidants tiron and dimethylthiourea rescued the pilE mutant from streptonigrin-mediated killing, suggesting that the elevated labile iron pool in non-piliated bacteria leads to streptonigrin-dependent reactive oxygen species production. These antioxidants did not affect LL-37-mediated killing. We confirmed that the pilE mutant is not more sensitive to other antibiotics showing that the streptonigrin phenotypes are not due to general bacterial envelope disruption. The total iron content of the cell was unaltered by piliation when measured using ICP-MS suggesting that only the labile iron pool is affected by piliation. These results support the hypothesis that piliation state affects N. gonorrhoeae iron homeostasis and influences sensitivity to various host-derived antimicrobial agents.

A novel streptonigrin type alkaloid from the Streptomyces flocculus CGMCC 4.1223 mutant ΔstnA/Q2.

Streptonigrin (STN) is a highly functionalized aminoquinone alkaloid with broad and potent antitumor activities. Previously, the biosynthetic gene cluster of STN was identified in Streptomyces flocculus CGMCC 4.1223, revealing an α/ß-hydrolase (StnA) and a methyltransferase (StnQ2). In this work, a double mutant ΔstnA/Q2 was constructed by genetic manipulation and produced a novel derivative of STN, named as streptonigramide. Structure of streptonigramide was established by spectroscopic analyses. Its biosynthetic pathway has been proposed as well.

Characterization of ribonucleotide reductases of emerging pathogens Elizabethkingia anophelis and Elizabethkingia meningoseptica and streptonigrin as their inhibitor: a computational study.

Antibiotic resistance is a global concern. Two members of the bacterial genus Elizabethkingia, namely, E. anophelis and E. meningoseptica have raised much concern in recent years because of their resistance to multiple commonly used antibiotics. Identification of multidrug resistant and pan-drug resistant bacteria has propelled the search for new antibiotics that can act on unconventional targets. Researches are going on to find out the possibility of using bacterial ribonucleotide reductases as a novel target for antibiotic development. Through in silico evaluations, this study aims for characterization and functional annotation of ribonucleotide reductase enzymes of E. anophelis and E. meningoseptica. Binding affinities with these enzymes of the compounds that have shown promising results in inhibiting Pseudomonas aeruginosa growth by acting on its ribonucleotide reductase were also assessed by molecular docking and dynamics simulations. Insights from this study will help in battling these infections in the near future. Communicated by Ramaswamy H. Sarma.

Characterization of the roles of activated charcoal and Chelex in the induction of PrfA regulon expression in complex medium.

The foodborne pathogen Listeria monocytogenes is able to survive across a wide range of intra- and extra-host environments by appropriately modulating gene expression patterns in response to different stimuli. Positive Regulatory Factor A (PrfA) is the major transcriptional regulator of virulence gene expression in L. monocytogenes. It has long been known that activated charcoal is required to induce the expression of PrfA-regulated genes in complex media, such as Brain Heart Infusion (BHI), but not in chemically defined media. In this study, we show that the expression of the PrfA-regulated hly, which encodes listeriolysin O, is induced 5- and 8-fold in L. monocytogenes cells grown in Chelex-treated BHI (Ch-BHI) and in the presence of activated charcoal (AC-BHI), respectively, relative to cells grown in BHI medium. Specifically, we show that metal ions present in BHI broth plays a role in the reduced expression of the PrfA regulon. In addition, we show that expression of hly is induced when the levels of bioavailable extra- or intercellular iron are reduced. L. monocytogenes cells grown Ch-BHI and AC-BHI media showed similar levels of resistance to the iron-activated antibiotic, streptonigrin, indicating that activated charcoal reduces the intracellular labile iron pool. Metal depletion and exogenously added glutathione contributed synergistically to PrfA-regulated gene expression since glutathione further increased hly expression in metal-depleted BHI but not in BHI medium. Analyses of transcriptional reporter fusion expression patterns revealed that genes in the PrfA regulon are differentially expressed in response to metal depletion, metal excess and exogenous glutathione. Our results suggest that metal ion abundance plays a role in modulating expression of PrfA-regulated virulence genes in L. monocytogenes.

Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice.

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

Streptonigrin at low concentration promotes heterochromatin formation.

Heterochromatin is essential for regulating global gene transcription and protecting genome stability, and may play a role in tumor suppression. Drugs promoting heterochromatin are potential cancer therapeutics but very few are known. In order to identify drugs that can promote heterochromatin, we used a cell-based method and screened NCI drug libraries consisting of oncology drugs and natural compounds. Since heterochromatin is originally defined as intensely stained chromatin in the nucleus, we estimated heterochromatin contents of cells treated with different drugs by quantifying the fluorescence intensity of nuclei stained with Hoechst DNA dye. We used HeLa cells and screened 231 FDA-approved oncology and natural substance drugs included in two NCI drug libraries representing a variety of chemical structures. Among these drugs, streptonigrin most prominently caused an increase in Hoechst-stained nuclear fluorescence intensity. We further show that streptonigrin treated cells exhibit compacted DNA foci in the nucleus that co-localize with Heterochromatin Protein 1 alpha (HP1α), and exhibit an increase in total levels of the heterochromatin mark, H3K9me3. Interestingly, we found that streptonigrin promotes heterochromatin at a concentration as low as one nanomolar, and at this concentration there were no detectable effects on cell proliferation or viability. Finally, in line with a previous report, we found that streptonigrin inhibits STAT3 phosphorylation, raising the possibility that non-canonical STAT function may contribute to the effects of streptonigrin on heterochromatin. These results suggest that, at low concentrations, streptonigrin may primarily enhance heterochromatin formation with little toxic effects on cells, and therefore might be a good candidate for epigenetic cancer therapy.

5,8-Quinolinedione Scaffold as a Promising Moiety of Bioactive Agents.

Natural 5,8-quinolinedione antibiotics exhibit a broad spectrum of activities including anticancer, antibacterial, antifungal, and antimalarial activities. The structure-activity research showed that the 5,8-quinolinedione scaffold is responsible for its biological effect. The subject of this review report is a presentation of the pharmacological activity of synthetic 5,8-quinolinedione compounds containing different groups at C-6 and/or C-7 positions. The relationship between the activity and the mechanism of action is included if these data have been included in the original literature. The review mostly covers the period between 2000 and 2019. Previously published literature data were used to present historical points.

StnK2 catalysing a Pictet-Spengler reaction involved in the biosynthesis of the antitumor reagent streptonigrin.

Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid antibiotic with broad and potent antitumor activity. Previous isotope-labelling and genetic studies suggested that a ß-carboline alkaloid should be a key intermediate of STN biosynthesis and formed via a Pictet-Spengler (PS) reaction. Herein, StnK2 was biochemically characterized to be a Pictet-Spenglerase (PSase) catalysing the formation of a tetrahydro-ß-carboline (TH-ßC) scaffold from (2S,3S)-ß-methyl tryptophan and d-erythrose-4-phosphate. StnK2 can tolerate the alteration of tryptophan but only accept d-erythrose-4-phosphate as the aldehyde substrate, and StnK2 was identified to be R-specific for the newly formed chiral center. This work increases the diversities of Pictet-Spenglerase in nature and set a stage for the generation of streptonigrin derivatives by precursor-directed pathway engineering based on the flexible substrate selectivity of StnK2.

Searching for antigenotoxic properties of marine macroalgae dietary supplementation against endogenous and exogenous challenges.

The functional characterization of marine macroalgae toward their potential to strength genome protection is still scarce. Hence, the aim of this study was to assess the antigenotoxic potential of Ulva rigida, Fucus vesiculosus, and Gracilaria species in Drosophila melanogaster following dietary exposure and adopting the somatic mutation and recombination test (SMART). All macroalgae displayed a genoprotection activity, namely against an exogenous challenge (streptonigrin). The action against subtler endogenous pressures was also noted indicating that supplementation level is a critical factor. Gracilaria species provided ambivalent indications, since 10% of G. vermiculophylla inhibited the egg laying and/or larvae development, while 10% of G. gracilis promoted spontaneous genotoxicity. The effects of U. rigida were modulated (in intensity) by the growing conditions, demonstrating higher genoprotection against streptonigrin-induced damage when grown in an aquaculture-controlled system, while the effectiveness against spontaneous genotoxicity was more apparent in specimens grown under wild conditions. In contrast, F. vesiculosus did not produce significant differences in its potential under varying growing conditions. Overall, these findings shed some light on the macroalgae ability toward genome protection, contributing to the development of algaculture industry, and reinforcing the concept of functional food and its benefits.

Streptonigrin Inhibits SENP1 and Reduces the Protein Level of Hypoxia-Inducible Factor 1α (HIF1α) in Cells.

Streptonigrin (CAS no. 3930-19-6) is a natural product shown to have antitumor activities in clinical trials conducted in the 1960s-1970s. However, its use in clinical studies eventually faded, and the molecular mechanisms of streptonigrin antitumor effects remain poorly defined. Despite its lack of current clinical use, efforts on its total synthesis have continued. Here, we show that streptonigrin binds and inhibits the SUMO-specific protease SENP1. NMR studies identified that streptonigrin binds to SENP1 on the surface where SUMO binds and disrupts SENP1-SUMO1 interaction. Site-directed mutations in combination with NMR chemical shift perturbation suggest key roles of aromatic π stacking interactions in binding streptonigrin. Treatment of cells with streptonigrin resulted in increased global SUMOylation levels and reduced level of hypoxia inducible factor alpha (HIF1α). These findings inform both the design of SENP1 targeting strategy and the modification of streptonigrin to improve its efficacy for possible future clinical use.

The PerR-Regulated P1B-4-Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (PerR-regulated metal transporter A), a P1B-4-type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448ΔpmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448ΔpmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.

Crystal Structure of StnA for the Biosynthesis of Antitumor Drug Streptonigrin Reveals a Unique Substrate Binding Mode.

Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although StnA has no significant sequence homology with the reported α/ß-fold hydrolases, it shows typical hydrolytic activity in vivo and in vitro. In order to reveal its functional characteristics, the crystal structures of the selenomethionine substituted StnA (SeMet-StnA) and the complex (S185A mutant) with its substrate were resolved to the resolution of 2.71 Å and 2.90 Å, respectively. The overall structure of StnA can be described as an α-helix cap domain on top of a common α/ß hydrolase domain. The substrate methyl ester of 10'-demethoxystreptonigrin binds in a hydrophobic pocket that mainly consists of cap domain residues and is close to the catalytic triad Ser185-His349-Asp308. The transition state is stabilized by an oxyanion hole formed by the backbone amides of Ala102 and Leu186. The substrate binding appears to be dominated by interactions with several specific hydrophobic contacts and hydrogen bonds in the cap domain. The molecular dynamics simulation and site-directed mutagenesis confirmed the important roles of the key interacting residues in the cap domain. Structural alignment and phylogenetic tree analysis indicate that StnA represents a new subfamily of lipolytic enzymes with the specific binding pocket located at the cap domain instead of the interface between the two domains.

Evaluation of the New Centers for Disease Control and Prevention Ventilator-Associated Event Module and Criteria in Critically Ill Children in Greece.

OBJECTIVE To evaluate the new adult Centers for Disease Control and Prevention (CDC) ventilator-associated event (VAE) module in critically ill children and compare with the traditionally used CDC definition for ventilator-associated pneumonia (VAP). DESIGN Retrospective observational study of mechanically ventilated children in a pediatric intensive care unit in Greece January 1-December 31, 2011. METHODS Assessment of new adult CDC VAE module including 3 definition tiers: ventilator-associated condition (VAC), infection-related VAC, and possible/probable ventilator-associated pneumonia (VAE-VAP); comparison with traditional CDC criteria for clinically defined pneumonia in mechanically ventilated children (PNEU-VAP). We recorded Pediatric Risk of Mortality score at admission (PRISM III), number of ventilator-days, and outcome. RESULTS Among 119 patients with mechanical ventilation (median [range] number of ventilator-days, 7 [1-183]), 19 patients experienced VAC. Criteria for VAE-VAP were fulfilled in 12 of 19 patients with VAC (63%). Children with either VAC or VAE-VAP were on ventilation more days than patients without these conditions (16.5 vs 5 d, P=.0006 and 18 vs 5 d, P<.001, respectively), whereas PRISM-III score was similar between them. Mortality was significant higher in patients with new VAE-VAP definition (50%), but not in patients with VAC (31.6%), than the patients without new VAE-VAP (14%, P=.007) or VAC (15%, P=.1), respectively. No significant association was found between PNEU-VAP and death. Incidences of PNEU-VAP and VAE-VAP were similar, but the agreement was poor. CONCLUSIONS VAE-VAP and PNEU-VAP found similar prevalence in critically ill children but with poor agreement. However, excess of death was significantly associated only with VAE-VAP. Infect Control Hosp Epidemiol 2016:1-5.