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1.  Staphylococcal Phenotypes Induced by Naturally Occurring and Synthetic Membrane-Interactive Polyphenolic β-Lactam Resistance Modifiers 
PLoS ONE  2014;9(4):e93830.
Galloyl catechins, in particular (-)-epicatechin gallate (ECg), have the capacity to abrogate β-lactam resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA); they also prevent biofilm formation, reduce the secretion of a large proportion of the exoproteome and induce profound changes to cell morphology. Current evidence suggests that these reversible phenotypic traits result from their intercalation into the bacterial cytoplasmic membrane. We have endeavoured to potentiate the capacity of ECg to modify the MRSA phenotype by stepwise removal of hydroxyl groups from the B-ring pharmacophore and the A:C fused ring system of the naturally occurring molecule. ECg binds rapidly to the membrane, inducing up-regulation of genes responsible for protection against cell wall stress and maintenance of membrane integrity and function. Studies with artificial membranes modelled on the lipid composition of the staphylococcal bilayer indicated that ECg adopts a position deep within the lipid palisade, eliciting major alterations in the thermotropic behaviour of the bilayer. The non-galloylated homolog (-)-epicatechin enhanced ECg-mediated effects by facilitating entry of ECg molecules into the membrane. ECg analogs with unnatural B-ring hydroxylation patterns induced higher levels of gene expression and more profound changes to MRSA membrane fluidity than ECg but adopted a more superficial location within the bilayer. ECg possessed a high affinity for the positively charged staphylococcal membrane and induced changes to the biophysical properties of the bilayer that are likely to account for its capacity to disperse the cell wall biosynthetic machinery responsible for β-lactam resistance. The ability to enhance these properties by chemical modification of ECg raises the possibility that more potent analogs could be developed for clinical evaluation.
doi:10.1371/journal.pone.0093830
PMCID: PMC3974817  PMID: 24699700
2.  Staphylococcus aureus PSM peptides modulate dendritic cell functions and increase in vitro priming of regulatory T cells1 
The major human pathogen Staphylococcus aureus has very efficient strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant S. aureus (CA-MRSA) depends on phenol-soluble modulin (PSM) peptide toxins, which are known to attract and lyse neutrophils. However, their influences on other immune cells remain elusive. Here, we analyzed the impact of PSMs on dendritic cells (DCs) playing an essential role in linking innate and adaptive immunity. In human neutrophils, PSMs exert their function by binding to the formyl peptide receptor (FPR) 2. We show that mouse DCs express the FPR2 homologue mFPR2 as well as its paralog mFPR1 and that PSMs are chemoattractants for DCs at non-cytotoxic concentrations. PSMs reduced clathrin-mediated endocytosis and inhibited TLR2 ligand-induced secretion of the proinflammatory cytokines TNF, IL-12 and IL-6 while inducing IL-10 secretion by DCs. As a consequence, treatment with PSMs impaired the capacity of DCs to induce activation and proliferation of CD4+ T cells, characterized by reduced Th1 but increased frequency of FOXP3+ regulatory T cells (Tregs). These Tregs secreted high amounts of IL-10 and their suppression capacity was dependent on IL-10 and TGF-β. Interestingly, the induction of tolerogenic DCs by PSMs appeared to be independent of mFPRs as shown by experiments with mice lacking mFPR2 (mFPR2−/−) and the cognate G protein (p110γ−/−). Thus, PSMs from highly virulent pathogens affect DC functions thereby modulating the adaptive immune response and probably increasing the tolerance towards the pathogen.
doi:10.4049/jimmunol.1202563
PMCID: PMC3608756  PMID: 23460735
3.  Do Comorbid Anxiety Disorders Moderate the Effects of Psychotherapy for Bipolar Disorder? Results From STEP-BD 
The American journal of psychiatry  2014;171(2):178-186.
Objective
At least 50% of individuals with bipolar disorder have a lifetime anxiety disorder. Individuals with both bipolar disorder and a co-occurring anxiety disorder experience longer illness duration, greater illness severity, and poorer treatment response. The study explored whether comorbid lifetime anxiety in bipolar patients moderates psychotherapy treatment outcome.
Method
In the Systematic Treatment Enhancement Program randomized controlled trial of psychotherapy for bipolar depression, participants received up to 30 sessions of intensive psychotherapy (family-focused therapy, interpersonal and social rhythm therapy, or cognitive-behavioral therapy) or collaborative care, a three-session comparison treatment, plus pharmacotherapy. Using the number needed to treat, we computed effect sizes to analyze the relationship between lifetime anxiety disorders and rates of recovery across treatment groups after 1 year.
Results
A total of 269 patients (113 women) with a comorbid lifetime anxiety disorder (N=177) or without a comorbid lifetime anxiety disorder (N=92) were included in the analysis. Participants with a lifetime anxiety disorder were more likely to recover with psychotherapy than with collaborative care (66% compared with 49% recovered over 1 year; number needed to treat=5.88, small to medium effect). For patients without a lifetime anxiety disorder, there was no difference between rates of recovery in psychotherapy compared with collaborative care (64% compared with 62% recovered; number needed to treat=50, small effect). Participants with one lifetime anxiety disorder were likely to benefit from intensive psychotherapy compared with collaborative care (84% compared with 53% recovered; number needed to treat=3.22, medium to large effect), whereas patients with multiple anxiety disorders exhibited no difference in response to the two treatments (54% compared with 46% recovered; number needed to treat=12.5, small effect).
Conclusions
Depressed patients with bipolar disorder and comorbid anxiety may be in particular need of additional psychotherapy for treating acute depression. These results need to be replicated in studies that stratify bipolar patients to treatments based on their anxiety comorbidity status.
doi:10.1176/appi.ajp.2013.13020225
PMCID: PMC3946300  PMID: 24077657
4.  Impulsivity as a moderator of the intention-behavior relationship for illicit drug use in patients undergoing treatment 
Addictive behaviors  2012;38(3):1651-1655.
Introduction
Evident across clinical practice and clinical trials is a divergence between stated intentions and subsequent drug-related behaviors in substance abuse treatment settings. Impulsivity, itself related to drug abuse, may be one variable which may moderate the degree of disconnect in the intention-behavior relationship. The present study examines the relationship between self-stated desire to quit, impulsivity, and drug use in a group of outpatients receiving methadone maintenance treatment. In particular, we examined the direct and moderating influence of different facets of impulsivity (urgency, lack of premeditation, sensation seeking, and lack of perseverance) on drug use in the context of a stated desire to abstain from drugs.
Method
84 opioid-dependent individuals undergoing counseling and methadone maintenance treatment completed a battery of self-report questionnaires including measures of impulsivity (UPPS Impulsivity Scale), stated desire to quit, and past 30-day drug use. We hypothesized that two facets of impulsivity, urgency and (lack of) premeditation, would moderate the relationship between desire to quit and past 30-day drug use, such that the relationship between intention and behavior would be weaker in those with high levels of these facets of impulsivity.
Results
Consistent with the disconnect between intentions and drug-use behaviors typical of treatment settings, desire to quit was not directly associated with self-reported past month drug use. However, in separate regression analyses, 2 facets of impulsivity, premeditation and sensation seeking, moderated the relationship between desire to quit and past month use. Whereas there was not a significant relationship between desire to quit and drug use in individuals high in sensation-seeking or lack of premeditation, the relationship between intention and drug use behaviors was preserved in those low in these facets of impulsivity.
Conclusion
These findings indicate that the relationship between desire to quit and self-reported past-month drug use is weak for those high in sensation seeking or low in premeditation. These results are discussed in the context of current interventions for substance dependence.
doi:10.1016/j.addbeh.2012.09.008
PMCID: PMC3558639  PMID: 23254213
5.  Establishment of a Bacterial Infection Model Using the European Honeybee, Apis mellifera L 
PLoS ONE  2014;9(2):e89917.
Injection of human pathogenic bacteria (Pseudomonas aeruginosa, Serratia marcescens, Salmonella enterica, Staphylococcus aureus, and Listeria monocytogenes) into the hemocoel of honeybee (Apis mellifera L.) workers kills the infected bees. The bee-killing effects of the pathogens were affected by temperature, and the LD50 values at 37°C were more than 100-fold lower than those at 15°C. Gene-disrupted S. aureus mutants of virulence genes such as agrA, saeS, arlR, srtA, hla, and hlb had attenuated bee-killing ability. Nurse bees were less susceptible than foragers and drones to S. aureus infection. Injection of antibiotics clinically used for humans had therapeutic effects against S. aureus infections of bees, and the ED50 values of these antibiotics were comparable with those determined in mammalian models. Moreover, the effectiveness of orally administered antibiotics was consistent between honeybees and mammals. These findings suggest that the honeybee could be a useful model for assessing the pathogenesis of human-infecting bacteria and the effectiveness of antibiotics.
doi:10.1371/journal.pone.0089917
PMCID: PMC3933694  PMID: 24587122
6.  Separately or Combined, LukG/LukH Is Functionally Unique Compared to Other Staphylococcal Bicomponent Leukotoxins 
PLoS ONE  2014;9(2):e89308.
Staphylococcus aureus is a major human pathogen that elaborates several exotoxins. Among these are the bicomponent leukotoxins (BCLs), which include γ-hemolysin, Panton-Valentine leukocidin (PVL), and LukDE. The toxin components are classified as either F or S proteins, which are secreted individually and assemble on cell surfaces to form hetero-oligomeric pores resulting in lysis of PMNs and/or erythrocytes. F and S proteins of γ-hemolysin, PVL and LukDE have ∼70% sequence homology within the same class and several heterologous combinations of F and S members from these three bicomponent toxin groups are functional. Recently, an additional BCL pair, LukGH (also called LukAB) that has only 30% homology to γ-hemolysin, PVL and LukDE, has been characterized from S. aureus. Our results showed that LukGH was more cytotoxic to human PMNs than PVL. However, LukGH-induced calcium ion influx in PMNs was markedly attenuated and slower than that induced by PVL and other staphylococcal BCLs. In contrast to other heterologous BCL combinations, LukG in combination with heterologous S components, and LukH in combination with heterologous F components did not induce calcium ion entry or cell lysis in human PMNs or rabbit erythrocytes. Like PVL, LukGH induced IL-8 production by PMNs. While individual components LukG and LukH had no cytolytic or calcium influx activity, they each induced high levels of IL-8 transcription and secretion. IL-8 production induced by LukG or LukH was dependent on NF-κB. Therefore, our results indicate LukGH differs functionally from other staphylococcal BCLs.
doi:10.1371/journal.pone.0089308
PMCID: PMC3930693  PMID: 24586678
7.  Whole Genome Analysis of a Community-Associated Methicillin-Resistant Staphylococcus aureus ST59 Isolate from a Case of Human Sepsis and Severe Pneumonia in China 
PLoS ONE  2014;9(2):e89235.
We report a case of necrotizing pneumonia in a young patient caused by community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) in a teaching hospital in the People’s Republic of China. The patient had a typical clinical presentation and was successfully treated with antibiotics and intravenous immunoglobulin. A CA-MRSA strain, named SA268, was isolated from the blood of the patient. The isolate was susceptible to most antimicrobial agents, except cephalosporins, penicillins, and β-lactamase inhibitor combinations. Multi-locus sequence typing (MLST) assigned SA268 to ST59, a clone widely spread in eastern Asia. The strain was positive for Panton Valentine Leukocidin (PVL)-encoding genes and SCCmec type V. We sequenced the complete genome of the SA268 isolate. The genome of SA268 was almost identical to that of the Taiwanese ST59 CA-MRSA strains M013 and SA957. However, we observed several differences in gene composition, which included differences in the SCCmec element and several lipoprotein genes that were present in the Taiwanese strains but absent from SA268.
doi:10.1371/journal.pone.0089235
PMCID: PMC3930696  PMID: 24586619
8.  QsrO a Novel Regulator of Quorum-Sensing and Virulence in Pseudomonas aeruginosa 
PLoS ONE  2014;9(2):e87814.
In Pseudomonas aeruginosa, the production of many secreted virulence factors is controlled by a quorum-sensing (QS) circuit, constituted of transcriptional activators (LasR, RhlR, PqsR) and their cognate signaling molecules (3-oxo-C12-HSL, C4-HSL, PQS). QS is a cooperative behavior that is beneficial to a population but can be exploited by “QS-cheaters”, individuals which do not respond to the QS-signal, but can use public goods produced by QS-cooperators. In order to identify QS-deficient clones we designed a genetic screening based on a lasB-lacZ fusion. We isolated one clone (PT1617) deficient in QS-dependent gene expression and virulence factor production despite wild type lasR, rhlR and pqsR alleles. Whole genome sequencing of PT1617 revealed a 3,552 bp deletion encompassing ORFs PA2228-PA2229-PA2230 and the pslA gene. However, complementation of PT1617 by plasmid-encoded copies of these ORFs, did not restore QS. Unexpectedly, gene expression levels of ORFs PA2228, PA2227 (vqsM) and PA2222, located adjacent to the deletion, were 10 to 100 fold higher in mutant PT1617 than in PAO1. When expressed from a constitutive promoter on a plasmid, PA2226, alone was found to be sufficient to confer a QS-negative phenotype on PAO1 as well as on PA14. Co-expression of PA2226 and PA2225 in PAO1 further prevented induction of the type III secretion system. In summary, we have identified a novel genetic locus including ORF2226 termed qsrO (QS-repressing ORF), capable of down-regulating all three known QS-systems in P. aeruginosa.
doi:10.1371/journal.pone.0087814
PMCID: PMC3923755  PMID: 24551066
9.  Phylogenetic Analysis of Staphylococcus aureus CC398 Reveals a Sub-Lineage Epidemiologically Associated with Infections in Horses 
PLoS ONE  2014;9(2):e88083.
In the early 2000s, a particular MRSA clonal complex (CC398) was found mainly in pigs and pig farmers in Europe. Since then, CC398 has been detected among a wide variety of animal species worldwide. We investigated the population structure of CC398 through mutation discovery at 97 genetic housekeeping loci, which are distributed along the CC398 chromosome within 195 CC398 isolates, collected from various countries and host species, including humans. Most of the isolates in this collection were received from collaborating microbiologists, who had preserved them over years. We discovered 96 bi-allelic polymorphisms, and phylogenetic analyses revealed that an epidemic sub-clone within CC398 (dubbed ‘clade (C)’) has spread within and between equine hospitals, where it causes nosocomial infections in horses and colonises the personnel. While clade (C) was strongly associated with S. aureus from horses in veterinary-care settings (p = 2×10−7), it remained extremely rare among S. aureus isolates from human infections.
doi:10.1371/journal.pone.0088083
PMCID: PMC3913741  PMID: 24505386
10.  Augmentation of exposure therapy with post-session administration of d-cycloserine 
Journal of psychiatric research  2012;47(2):168-174.
Background
Pre-session administration of d-cycloserine (DCS) has been found to augment exposure therapy outcomes in a variety of anxiety disorders. To be able to enhance learning only for successful exposure sessions, it would be beneficial to have the option of administering DCS after rather than before the session, a strategy encouraged by pre-clinical work. We believe the present study is the first published report on the efficacy of post-session administration of DCS in humans.
Method
Adults (N = 29) with a DSM-IV diagnosis of acrophobia were randomized to receive two sessions of virtual reality exposure therapy (VRE) in combination with placebo or 50 mg of DCS. Instead of administering the pill prior to each of the sessions, as has been done in extant work, we administered the pill immediately following each session. Measures of acrophobia severity were collected at baseline, at each treatment session, 1-week post-treatment, and at 1-month follow-up.
Results
Mixed-effects repeated-measures ANOVAs and GLMMs revealed significant improvement in all outcome measures over time, but no between-group differences were observed. At post-treatment, 63.5% of patients in the placebo condition vs. 60.0% of those in the DCS condition were in remission. At 1-month follow up, 63.4% of those in the placebo condition vs. 66.6% of those in the DCS condition were in remission.
Conclusions
These findings do not support the application of post-session DCS administration for augmenting the efficacy of exposure-based treatments. Possible reasons for these findings are discussed. Trial Registry: The Trial is registered at ClinicalTrials.gov (NCT01102803).
doi:10.1016/j.jpsychires.2012.09.024
PMCID: PMC3732105  PMID: 23098672
Augmentation; CBT; Cognitive behavioral therapy; d-cycloserine; Exposure treatment; Randomized controlled trial; Acrophobia
11.  Characterization of the Staphylococcal Cassette Chromosome Composite Island of Staphylococcus haemolyticus SH32, a Methicillin-Resistant Clinical Isolate from China 
PLoS ONE  2014;9(1):e87346.
Staphylococcal cassette chromosome (SCC) elements contribute considerably to virulence and resistance to antibiotic agents in staphylococci. SCC elements in coagulase-negative staphylococci (CoNS) are highly diverse and there is evidence suggesting that they serve as a reservoir for antibiotic resistance genes in methicillin-resistant Staphylococcus aureus (MRSA). However, only a small number of SCC elements have been characterized in CoNS and their exact roles in the emergence and evolution of MRSA remain to be demonstrated. Here, we determined the structure of an SCC composite island (CISH32) found in the clinical Staphylococcus haemolyticus isolate SH32 by whole-genome DNA sequencing. CISH32 was 48 kb in length and mainly composed of two imperfect SCC elements, namely (i) a ΨSCCmec(SH32) part containing a class C1 mec gene complex but lacking ccr genes and (ii) a SCCSH32 part with a ccrA5B3 gene complex but lacking mec genes. In addition, CISH32 contained a type III restriction-modification system and several resistance loci, for example genes conferring resistance to cadmium and arsenic. ΨSCCmec(SH32) is almost entirely identical to a pseudo SCCmec element found in S. haemolyticus WCH1 and shares pronounced sequence similarity to a ΨSCCmec element of S. haemolyticus JCSC1435. However, staphylococci other than S. haemolyticus, including S. aureus and S. epidermidis, contain homologs of SCCSH32 that are more similar to SCCSH32 than those elements found in S. haemolyticus, suggesting that CISH32 of S. haemolyticus SH32 was assembled in recent evolutionary events. Moreover, the composite structure of CISH32 indicates that the detection of class C1 mec and ccrA5B3 gene complexes in S. haemolyticus does not always indicate the existence of a UT9-type SCCmec element, which has remained questionable.
doi:10.1371/journal.pone.0087346
PMCID: PMC3900703  PMID: 24466348
12.  The Role of the Staphylococcal VraTSR Regulatory System on Vancomycin Resistance and vanA Operon Expression in Vancomycin-Resistant Staphylococcus aureus 
PLoS ONE  2014;9(1):e85873.
Vancomycin is often the preferred treatment for invasive methicillin-resistant Staphylococcus aureus (MRSA) infection. With the increase in incidence of MRSA infections, the use of vancomycin has increased and, as feared, isolates of vancomycin-resistant Staphylococcus aureus (VRSA) have emerged. VRSA isolates have acquired the entercoccal vanA operon contained on transposon (Tn) 1546 residing on a conjugal plasmid. VraTSR is a vancomycin and β-lactam-inducible three-component regulatory system encoded on the S. aureus chromosome that modulates the cell-wall stress response to cell-wall acting antibiotics. Mutation in vraTSR has shown to increase susceptibility to β-lactams and vancomycin in clinical VISA strains and in recombinant strain COLVA-200 which expresses a plasmid borne vanA operon. To date, the role of VraTSR in vanA operon expression in VRSA has not been demonstrated. In this study, the vraTSR operon was deleted from the first clinical VRSA strain (VRS1) by transduction with phage harvested from a USA300 vraTSR operon deletion strain. The absence of the vraTSR operon and presence of the vanA operon were confirmed in the transductant (VRS1Δvra) by PCR. Broth MIC determinations, demonstrated that the vancomycin MIC of VRS1Δvra (64 µg/ml) decreased by 16-fold compared with VRS1 (1024 µg/ml). The effect of the vraTSR operon deletion on expression of the van gene cluster (vanA, vanX and vanR) was examined by quantitative RT-PCR using relative quantification. A 2–5-fold decreased expression of the vanA operon genes occured in strain VRS1Δvra at stationary growth phase compared with the parent strain, VRS1. Both vancomycin resistance and vancomycin-induced expression of vanA and vanR were restored by complementation with a plasmid harboring the vraTSR operon. These findings demonstrate that expression in S. aureus of the horizontally acquired enterococcal vanA gene cluster is enhanced by the staphylococcal three-component cell wall stress regulatory system VraTSR, that is present in all S. aureus strains.
doi:10.1371/journal.pone.0085873
PMCID: PMC3893269  PMID: 24454941
13.  Predictors of Dropout from Psychosocial Treatment in Opioid-Dependent Outpatients 
Background and Objectives
Early dropout is common in substance abuse treatment settings and may lead to poorer outcomes relative to those completing a full course of treatment. Attempts to identify predictors of dropout have yielded mixed results, highlighting the need for additional research in this area to clarify risk and protective factors to guide intervention and retention efforts. This study evaluated predictors of dropout from psychosocial treatment among opioid-dependent patients on methadone maintenance therapy.
Methods
Participants included 78 patients who had failed to respond to at least 4 months of methadone maintenance plus group counseling with clinic substance abuse counselors, and were enrolled in a study of randomized psychosocial treatment in addition to treatment-as-usual. Several factors that have been implicated in previous studies as well as two affective variables (distress intolerance and coping motives for drug use) were examined.
Results
Results indicated that when controlling for various risk factors, age was the only significant predictor of dropout, with younger patients more likely to discontinue treatment early.
Conclusions
This study replicates previous findings in opioid-dependent samples that younger patients are at an increased risk of early treatment dropout.
Conclusions and Significance
Targeted intervention may be needed to retain young patients in drug abuse treatment.
doi:10.1111/j.1521-0391.2013.00317.x
PMCID: PMC3651588  PMID: 23398222
14.  Coagulase-negative staphylococci as reservoirs of genes facilitating MRSA infection 
Recent research has suggested that Staphylococcus epidermidis is a reservoir of genes that, after horizontal transfer, facilitate the potential of Staphylococcus aureus to colonize, survive during infection, or resist antibiotic treatment, traits that are notably manifest in methicillin-resistant S. aureus (MRSA). S. aureus is a dangerous human pathogen and notorious for acquiring antibiotic resistance. MRSA in particular is one of the most frequent causes of morbidity and death in hospitalized patients. S. aureus is an extremely versatile pathogen with a multitude of mechanisms to cause disease and circumvent immune defenses. In contrast, most other staphylococci, such as S. epidermidis, are commonly benign commensals and only occasionally cause disease. Recent findings highlight the key importance of efforts to better understand how genes of staphylococci other than S. aureus contribute to survival in the human host, how they are transferred to S. aureus, and why this exchange appears to be uni-directional.
doi:10.1002/bies.201200112
PMCID: PMC3755491  PMID: 23165978
Staphylococcus aureus; MRSA; Staphylococcus epidermidis; horizontal gene transfer; CRISPR
15.  Staphylococcus aureus sarA Regulates Inflammation and Colonization during Central Nervous System Biofilm Formation 
PLoS ONE  2013;8(12):e84089.
Infection is a frequent and serious complication following the treatment of hydrocephalus with CSF shunts, with limited therapeutic options because of biofilm formation along the catheter surface. Here we evaluated the possibility that the sarA regulatory locus engenders S. aureus more resistant to immune recognition in the central nervous system (CNS) based on its reported ability to regulate biofilm formation. We utilized our established model of CNS catheter-associated infection, similar to CSF shunt infections seen in humans, to compare the kinetics of bacterial titers, cytokine production and inflammatory cell influx elicited by wild type S. aureus versus an isogenic sarA mutant. The sarA mutant was more rapidly cleared from infected catheters compared to its isogenic wild type strain. Consistent with this finding, several pro-inflammatory cytokines and chemokines, including IL-17, CXCL1, and IL-1β were significantly increased in the brain following infection with the sarA mutant versus wild type S. aureus, in agreement with the fact that the sarA mutant displayed impaired biofilm growth and favored a planktonic state. Neutrophil influx into the infected hemisphere was also increased in the animals infected with the sarA mutant compared to wild type bacteria. These changes were not attributable to extracellular protease activity, which is increased in the context of SarA mutation, since similar responses were observed between sarA and a sarA/protease mutant. Overall, these results demonstrate that sarA plays an important role in attenuating the inflammatory response during staphylococcal biofilm infection in the CNS via a mechanism that remains to be determined.
doi:10.1371/journal.pone.0084089
PMCID: PMC3875531  PMID: 24386336
16.  Molecular basis of in-vivo biofilm formation by bacterial pathogens 
Chemistry & biology  2012;19(12):1503-1513.
Summary
Bacterial biofilms are involved in a multitude of serious chronic infections. In recent years, modeling biofilm infection in vitro led to the identification of microbial determinants governing biofilm development. However, we lack information as to whether biofilm formation mechanisms identified in vitro have relevance for biofilm-associated infection. Here, we discuss the molecular basis of biofilm formation using staphylococci and Pseudomonas aeruginosa to illustrate key points, as their biofilm development process is well-studied. We will focus on in-vivo findings such as obtained in animal infection models, and critically evaluate in-vivo relevance of in-vitro findings. Although results on the role of quorum-sensing in biofilm formation have been conflicting, we now argue that integration of in-vitro and in-vivo studies allows a differentiated view of this mechanism as it relates to biofilm infection.
doi:10.1016/j.chembiol.2012.10.022
PMCID: PMC3530155  PMID: 23261595
Biofilms; Staphylococcus aureus; Staphylococcus epidermidis; Pseudomonas aeruginosa; quorum-sensing
17.  Basic Rules of Hygiene Protect Health Care and Lab Workers from Nasal Colonization by Staphylococcus aureus: An International Cross-Sectional Study 
PLoS ONE  2013;8(12):e82851.
Acquisition of nasal Staphylococcus aureus (S. aureus) colonization by contaminated hands is likely an important determinant of its nasal carriage rate in health care and lab setting. The objective of our cross-sectional study was to assess the prevalence of nasal methicillin-sensitive (MSSA) or -resistant Staphylococcus aureus (MRSA) carriage among health care professionals (HCPs) attending an international symposium and to study the association between compliance with hygiene rules, individual-related parameters, and medical conditions with nasal S. aureus carriage in this population. After obtaining consent, two nasal swabs were collected. Nasal MSSA and MRSA carriage was measured by the: i) molecular approach targeting spa, mecA and mecA-orfX junction sequences, and ii) culture on selective S. aureus media combined with mecA molecular detection of isolated strains. Information on compliance with hygiene rules, demographic variables, sector of activity and long-term medication was collected by anonymous questionnaire. The participation rate was 32.3%. In total, 176 subjects from 34 countries were included in the analysis. S. aureus was isolated from the nasal swabs of 57 (32.4%) subjects, of whom 3 (5.3%) harbored MRSA strains. Overall, 123 subjects reported working in microbiology laboratories with direct manipulation of S. aureus, and 29 acknowledged regular contacts with patients. In this exposed population, hydro-alcoholic solutions appeared to have a significant protective effect against nasal S. aureus carriage (OR = 0.36; 95% CI: 0.15–0.85). Hospital work was associated with increased risk of nasal S. aureus carriage (OR = 2.38; 95% CI: 1.07–5.29). The results of this study showed that compliance with basic rules of hygiene, such as the use of hydro-alcoholic solutions, could reduce the risk of nasal S. aureus colonization. Hydro-alcoholic solution could interrupt auto-transmission of the pathogen, consequently decreasing the overall nasal carriage rate, specifically in transient carriers.
doi:10.1371/journal.pone.0082851
PMCID: PMC3867406  PMID: 24367562
18.  Cognitive Therapy Alone and in Combination with Antidepressants for Anxious Depression: A STAR*D Report 
Journal of affective disorders  2012;142(1-3):213-218.
Background
Anxious depression, defined as MDD with high levels of anxiety, has been associated with lower rates of antidepressant response and remission as well as greater chronicity, suicidality and antidepressant side-effect burden. The primary aim of this study was to assess the effectiveness of cognitive therapy (CT) alone or in combination with medications for anxious versus non-anxious depression.
Methods
We assessed the STAR*D study participants who were partial or non-responders to citalopram. Subjects were then either switched (n = 696) to a new antidepressant or to CT alone, or they were kept on citalopram and augmented (n = 577) with another antidepressant or CT. We compared response and remission rates of those who met criteria for anxious depression to those who did not across treatment conditions.
Results
Those with anxious depression had significantly lower remission rates based on the QIDS, whether assigned to switch or augmentation, compared to those with non-anxious depression. Those with anxious depression, compared to those without, had significantly lower response rates based on the QIDS only in the switch group. There was no significant interaction between anxious depression and treatment assignment.
Limitations
Limitations include the use of citalopram as the only Level 1 pharmacotherapy and medication augmentation option, depression-focused CT rather than anxiety-focused CT, and focus on acute treatment outcomes.
Conclusions
Individuals with anxious depression appear to experience higher risk of poorer outcome following pharmacotherapy and/or CT after an initial course of SSRI, and continued efforts to target this challenging form of depression are needed.
doi:10.1016/j.jad.2012.04.029
PMCID: PMC3483355  PMID: 22877961
anxious depression; MDD; CT; psychosocial interventions; STAR*D
19.  The Mechanism of Heterogeneous Beta-Lactam Resistance in MRSA: Key Role of the Stringent Stress Response 
PLoS ONE  2013;8(12):e82814.
All methicillin resistant S. aureus (MRSA) strains carry an acquired genetic determinant – mecA or mecC - which encode for a low affinity penicillin binding protein –PBP2A or PBP2A′ – that can continue the catalysis of peptidoglycan transpeptidation in the presence of high concentrations of beta-lactam antibiotics which would inhibit the native PBPs normally involved with the synthesis of staphylococcal cell wall peptidoglycan. In contrast to this common genetic and biochemical mechanism carried by all MRSA strains, the level of beta-lactam antibiotic resistance shows a very wide strain to strain variation, the mechanism of which has remained poorly understood. The overwhelming majority of MRSA strains produce a unique – heterogeneous – phenotype in which the great majority of the bacteria exhibit very poor resistance often close to the MIC value of susceptible S. aureus strains. However, cultures of such heterogeneously resistant MRSA strains also contain subpopulations of bacteria with extremely high beta-lactam MIC values and the resistance level and frequency of the highly resistant cells in such strain is a characteristic of the particular MRSA clone. In the study described in this communication, we used a variety of experimental models to understand the mechanism of heterogeneous beta-lactam resistance. Methicillin-susceptible S. aureus (MSSA) that received the mecA determinant in the laboratory either on a plasmid or in the form of a chromosomal SCCmec cassette, generated heterogeneously resistant cultures and the highly resistant subpopulations that emerged in these models had increased levels of PBP2A and were composed of bacteria in which the stringent stress response was induced. Each of the major heterogeneously resistant clones of MRSA clinical isolates could be converted to express high level and homogeneous resistance if the growth medium contained an inducer of the stringent stress response.
doi:10.1371/journal.pone.0082814
PMCID: PMC3857269  PMID: 24349368
20.  Rapid Differentiation between Livestock-Associated and Livestock-Independent Staphylococcus aureus CC398 Clades 
PLoS ONE  2013;8(11):e79645.
Staphylococcus aureus clonal complex 398 (CC398) isolates cluster into two distinct phylogenetic clades based on single-nucleotide polymorphisms (SNPs) revealing a basal human clade and a more derived livestock clade. The scn and tet(M) genes are strongly associated with the human and the livestock clade, respectively, due to loss and acquisition of mobile genetic elements. We present canonical single-nucleotide polymorphism (canSNP) assays that differentiate the two major host-associated S. aureus CC398 clades and a duplex PCR assay for detection of scn and tet(M). The canSNP assays correctly placed 88 S. aureus CC398 isolates from a reference collection into the human and livestock clades and the duplex PCR assay correctly identified scn and tet(M). The assays were successfully applied to a geographically diverse collection of 272 human S. aureus CC398 isolates. The simple assays described here generate signals comparable to a whole-genome phylogeny for major clade assignment and are easily integrated into S. aureus CC398 surveillance programs and epidemiological studies.
doi:10.1371/journal.pone.0079645
PMCID: PMC3828327  PMID: 24244535
21.  MRSA virulence and spread 
Cellular microbiology  2012;14(10):1513-1521.
Summary
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of hospital- and community-associated infections. Resistance to the entire class of β-lactam antibiotics, such as methicillin and penicillin, makes MRSA infections difficult to treat. Hospital-associated MRSA strains are often multi-drug resistant, leaving only lower efficiency drugs such as vancomycin as treatments options. Like many other S. aureus strains, MRSA strains produce a series of virulence factors, such as toxins and adhesion proteins. Recent findings have shed some new light on the molecular events that underlie MRSA epidemic waves. Newly emerging MRSA clones appear to have acquired phenotypic traits that render them more virulent or able to colonize better, either via mobile genetic elements or adaptation of gene expression. Acquisition of Panton-Valentine leukocidin genes and increased expression of core genome-encoded toxins are being discussed as potentially contributing to the success of the recently emerged community-associated MRSA strains. However, the molecular factors underlying the spread of hospital- and community-associated MRSA strains are still far from being completely understood, a situation calling for enhanced research efforts in that area.
doi:10.1111/j.1462-5822.2012.01832.x
PMCID: PMC3443268  PMID: 22747834
22.  Genetic Variation in the Staphylococcus aureus 8325 Strain Lineage Revealed by Whole-Genome Sequencing 
PLoS ONE  2013;8(9):e77122.
Staphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47) required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP) arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected Φ11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (Δ63bp) in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75%) steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs) near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin α3 (PSMα3), a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation.
doi:10.1371/journal.pone.0077122
PMCID: PMC3786944  PMID: 24098817
23.  Rates of mood and anxiety disorders and contributors to continued heroin use in methadone maintenance patients: A comparison by HIV status 
Neurobehavioral HIV medicine  2010;2010(2):49-57.
The frequency of mood and anxiety disorders is elevated among individuals with a history of intravenous drug abuse and among those with human immunodeficiency virus (HIV), and these disorders are associated with continued substance use despite treatment. The present study examined rates of mood and anxiety disorders, and recent heroin use, among HIV-infected and HIV-noninfected patients receiving methadone maintenance therapy. Participants were 160 (80 HIV-infected, 80 HIV-noninfected) methadone patients. Clinician-administered, semistructured interviews were used to identify unipolar and bipolar depression, and four major anxiety disorders (panic disorder with agoraphobia [PDA], generalized anxiety disorder [GAD], post-traumatic stress disorder [PTSD], and social anxiety disorder [SAD]). Toxicology screens and self-reporting were used to assess heroin, cocaine, marijuana, and alcohol use over the past month. The entire sample met criteria for at least one psychiatric disorder other than substance dependence. Substantial proportions of participants met criteria for major depressive disorder (55.6%), bipolar I, bipolar II, or cyclothymia (6.4%), PDA (34.4%), GAD (22.5%), SAD (16.9%), and PTSD (34.4%). A greater proportion of HIV-infected participants met criteria for SAD (χ2 = 5.03), and a greater proportion of HIV-noninfected participants met criteria for GAD (χ2 = 5.39, P < 0.01). About 14% of participants continued to use heroin over the past month, a significantly greater proportion of whom were HIV-infected. In adjusted analyses, none of the mood or anxiety disorders emerged as significant predictors of recent heroin use, but being HIV-infected did. This study highlights the high rate of psychopathology and continued heroin use despite substance abuse treatment, and underscores the need for interventions that help mitigate these problems among methadone patients.
doi:10.2147/NBHIV.S12371
PMCID: PMC3779458  PMID: 24062619
psychopathology; substance dependence; human immunodeficiency virus; methadone
24.  Essential Staphylococcus aureus toxin export system 
Nature medicine  2013;19(3):364-367.
Widespread antibiotic resistance among important bacterial pathogens such as Staphylococcus aureus1 calls for alternative routes of drug development. Interfering with critical virulence determinants is considered a promising novel approach to control bacterial infection2. Phenol-soluble modulins (PSMs) are peptide toxins with multiple key roles in pathogenesis3–5 and a major impact on the ability of highly virulent S. aureus to cause disease3,6. However, targeting PSMs for therapeutic intervention is hampered by their multitude and diversity. Here, we report that an ABC transporter with previously unknown function is responsible for the export of all PSM classes, thus representing a single target to interfere simultaneously with the production of all PSMs. The transporter had a strong effect on virulence phenotypes, such as neutrophil lysis, and the development of S. aureus infection, similar in extent to the sum of all PSMs. Furthermore, it proved essential for bacterial growth. Moreover, it protected the producer from the antimicrobial activity of secreted PSMs and contributed to defense against PSM-mediated bacterial interference. Our study reveals a non-canonical, dedicated secretion mechanism for an important toxin class and identifies this mechanism as a comprehensive potential target for the development of drugs efficiently inhibiting growth and virulence of pathogenic staphylococci.
doi:10.1038/nm.3047
PMCID: PMC3594369  PMID: 23396209
25.  Refining the Measurement of Distress Intolerance 
Behavior therapy  2011;43(3):641-651.
Distress intolerance is an important transdiagnostic variable that has long been implicated in the development and maintenance of psychological disorders. Self-report measurement strategies for distress intolerance have emerged from several different models of psychopathology and these measures have been applied inconsistently in the literature in the absence of a clear gold standard. The absence of a consistent assessment strategy has limited the ability to compare across studies and samples, thus hampering the advancement of this research agenda. This study evaluated the latent factor structure of existing measures of DI to examine the degree to which they are capturing the same construct. Results of confirmatory factor analysis in 3 samples totaling 400 participants provided support for a single factor latent structure. Individual items of these four scales were then correlated with this factor to identify those that best capture the core construct. Results provided consistent supported for 10 items that demonstrated the strongest concordance with this factor. The use of these 10 items as a unifying measure in the study of DI and future directions for the evaluation of its utility are discussed.
doi:10.1016/j.beth.2011.12.001
PMCID: PMC3483633  PMID: 22697451
distress tolerance; distress intolerance; anxiety sensitivity; discomfort intolerance; assessment

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