NtrC Adds a New Node to the Complex Regulatory Network of Biofilm Formation and vps Expression in Vibrio cholerae.

The biofilm growth mode is important in both the intestinal and environmental phases of the Vibrio cholerae life cycle. Regulation of biofilm formation involves several transcriptional regulators and alternative sigma factors. One such factor is the alternative sigma factor RpoN, which positively regulates biofilm formation. RpoN requires bacterial enhancer-binding proteins (bEBPs) to initiate transcription. The V. cholerae genome encodes seven bEBPs (LuxO, VC1522, VC1926 [DctD-1], FlrC, NtrC, VCA0142 [DctD-2], and PgtA) that belong to the NtrC family of response regulators (RRs) of two-component regulatory systems. The contribution of these regulators to biofilm formation is not well understood. In this study, we analyzed biofilm formation and the regulation of vpsL expression by RpoN activators. Mutants lacking NtrC had increased biofilm formation and vpsL expression. NtrC negatively regulates the expression of core regulators of biofilm formation (vpsR, vpsT, and hapR). NtrC from V. cholerae supported growth and activated glnA expression when nitrogen availability was limited. However, the repressive activity of NtrC toward vpsL expression was not affected by the nitrogen sources present. This study unveils the role of NtrC as a regulator of vps expression and biofilm formation in V. choleraeIMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, contributing to both environmental survival and transmission to a human host. Identifying key regulators of V. cholerae biofilm formation is necessary to fully understand how this important growth mode is modulated in response to various signals encountered in the environment and the host. In this study, we characterized the role of RRs that function as coactivators of RpoN in regulating biofilm formation and identified new components in the V. cholerae biofilm regulatory circuitry.

The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation.

Two-component signal transduction systems (TCSs), typically composed of a sensor histidine kinase (HK) and a response regulator (RR), are the primary mechanism by which pathogenic bacteria sense and respond to extracellular signals. The pathogenic bacterium Vibrio cholerae is no exception and harbors 52 RR genes. Using in-frame deletion mutants of each RR gene, we performed a systematic analysis of their role in V. cholerae biofilm formation. We determined that 7 RRs impacted the expression of an essential biofilm gene and found that the recently characterized RR, VxrB, regulates the expression of key structural and regulatory biofilm genes in V. choleraevxrB is part of a 5-gene operon, which contains the cognate HK vxrA and three genes of unknown function. Strains carrying ΔvxrA and ΔvxrB mutations are deficient in biofilm formation, while the ΔvxrC mutation enhances biofilm formation. The overexpression of VxrB led to a decrease in motility. We also observed a small but reproducible effect of the absence of VxrB on the levels of cyclic di-GMP (c-di-GMP). Our work reveals a new function for the Vxr TCS as a regulator of biofilm formation and suggests that this regulation may act through key biofilm regulators and the modulation of cellular c-di-GMP levels.IMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, providing protection from environmental stresses and contributing to the transmission of V. cholerae to the human host. V. cholerae can utilize two-component systems (TCS), composed of a histidine kinase (HK) and a response regulator (RR), to regulate biofilm formation in response to external cues. We performed a systematic analysis of V. cholerae RRs and identified a new regulator of biofilm formation, VxrB. We demonstrated that the VxrAB TCS is essential for robust biofilm formation and that this system may regulate biofilm formation via its regulation of key biofilm regulators and cyclic di-GMP levels. This research furthers our understanding of the role that TCSs play in the regulation of V. cholerae biofilm formation.

COMmunication with Families regarding ORgan and Tissue donation after death in intensive care (COMFORT): protocol for an intervention study.

BACKGROUND: Discussing deceased organ donation can be difficult not only for families but for health professionals who initiate and manage the conversations. It is well recognised that the methods of communication and communication skills of health professionals are key influences on decisions made by families regarding organ donation. METHODS: This multicentre study is being performed in nine intensive care units with follow-up conducted by the Organ and Tissue Donation Service in New South Wales (NSW) Australia. The control condition is pre-intervention usual practice for at least six months before each site implements the intervention. The COMFORT intervention consists of six elements: family conversations regarding offers for organ donation to be led by a "designated requester"; family offers for donation are deferred to the designated requester; the offer of donation is separated from the end-of-life discussion that death is inevitable; it takes place within a structured family donation conversation using a "balanced" approach. Designated requesters may be intensivists, critical care nurses or social workers prepared by attending the three-day national "Family Donation Conversation" workshops, and the half-day NSW Simulation Program. The design is pre-post intervention to compare rates of family consent for organ donation six months before and under the intervention. Each ICU crosses from using the control to intervention condition after the site initiation visit. The primary endpoint is the consent rate for deceased organ donation calculated from 140 eligible next of kin families. Secondary endpoints are health professionals' adherence rates to core elements of the intervention; identification of predictors of family donation decision; and the proportion of families who regret their final donation decision at 90 days. DISCUSSION: The pragmatic design of this study may identify 'what works' in usual clinical settings when requesting organ donation in critical care areas, both in terms of changes in practice healthcare professionals are willing and able to adopt, and the effect this may have on desired outcomes. The findings of this study will be indicative of the potential benefits of the intervention and be relevant and transferrable to clinical settings in other states and countries. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12613000815763 (24 July 2013). ClinicalTrials.gov: NCT01922310 (14 August 2013) (retrospectively registered).

Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids.

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.

Vibrio cholerae Response Regulator VxrB Controls Colonization and Regulates the Type VI Secretion System.

Two-component signal transduction systems (TCS) are used by bacteria to sense and respond to their environment. TCS are typically composed of a sensor histidine kinase (HK) and a response regulator (RR). The Vibrio cholerae genome encodes 52 RR, but the role of these RRs in V. cholerae pathogenesis is largely unknown. To identify RRs that control V. cholerae colonization, in-frame deletions of each RR were generated and the resulting mutants analyzed using an infant mouse intestine colonization assay. We found that 12 of the 52 RR were involved in intestinal colonization. Mutants lacking one previously uncharacterized RR, VCA0566 (renamed VxrB), displayed a significant colonization defect. Further experiments showed that VxrB phosphorylation state on the predicted conserved aspartate contributes to intestine colonization. The VxrB regulon was determined using whole genome expression analysis. It consists of several genes, including those genes that create the type VI secretion system (T6SS). We determined that VxrB is required for T6SS expression using several in vitro assays and bacterial killing assays, and furthermore that the T6SS is required for intestinal colonization. vxrB is encoded in a four gene operon and the other vxr operon members also modulate intestinal colonization. Lastly, though ΔvxrB exhibited a defect in single-strain intestinal colonization, the ΔvxrB strain did not show any in vitro growth defect. Overall, our work revealed that a small set of RRs is required for intestinal colonization and one of these regulators, VxrB affects colonization at least in part through its regulation of T6SS genes.

Integrative epigenetic profiling analysis identifies DNA methylation changes associated with chronic alcohol consumption.

Alcoholism has always been a major public health concern in Taiwan, especially in the aboriginal communities. Emerging evidence supports the association between DNA methylation and alcoholism, though very few studies have examined the effect of chronic alcohol consumption on the epignome. Since 1986, we have been following up on the mental health conditions of four major aboriginal peoples of Taiwan. The 993 aboriginal people who underwent the phase 1 (1986) clinical interviews were followed up through phase 2 (1990-1992), and phase 3 (2003-2009). Selected individuals for the current study included 10 males from the phase 1 normal cohort who remained normal at phase 2 and became dependent on alcohol by phase 3 and 10 control subjects who have not had any drinking problems throughout the study. We profiled the DNA methylation changes in the blood samples collected at phases 2 and 3. Enrichment analyses have identified several biological processes related to immune system responses and aging in the control group. In contrast, differentially methylated genes in the case group were mostly associated with susceptibility to infections, as well as pathways related to muscular contraction and neural degeneration. The methylation levels of six genes were found to correlate with alcohol consumption. These include genes involved in neurogenesis (NPDC1) and inflammation (HERC5), as well as alcoholism-associated genes ADCY9, CKM, and PHOX2A. Given the limited sample size, our approach uncovered genes and disease pathways associated with chronic alcohol consumption at the epigenetic level. The results offer a preliminary methylome map that enhances our understanding of alcohol-induced damages and offers new targets for alcohol injury research.

Development of benzo[1,4]oxazines as biofilm inhibitors and dispersal agents against Vibrio cholerae.

Bacterial biofilms are estimated to be associated with over 65 percent of all nosocomial infections. However, no therapeutics have been approved by the FDA which directly mediate biofilm formation or persistence. Herein we report oxazine as a highly potent inhibitor, disperser and in the presence of the appropriate antibiotic eradicator of V. cholerae biofilms.

Vibrio cholerae utilizes direct sRNA regulation in expression of a biofilm matrix protein.

Vibrio cholerae biofilms contain exopolysaccharide and three matrix proteins RbmA, RbmC and Bap1. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. VrrA is a conserved, 140-nt sRNA of V. cholerae, whose expression is controlled by sigma factor σE. In this study, we demonstrate that VrrA negatively regulates rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation is not stringently dependent on the RNA chaperone protein Hfq. These results point to VrrA as a molecular link between the σE-regulon and biofilm formation in V. cholerae. In addition, VrrA represents the first example of direct regulation of sRNA on biofilm matrix component, by-passing global master regulators.

Image-based 384-well high-throughput screening method for the discovery of skyllamycins A to C as biofilm inhibitors and inducers of biofilm detachment in Pseudomonas aeruginosa.

To date, most antibiotics have primarily been developed to target bacteria in the planktonic state. However, biofilm formation allows bacteria to develop tolerance to antibiotics and provides a mechanism to evade innate immune systems. Therefore, there is a significant need to identify small molecules to prevent biofilm formation and, more importantly, to disperse or eradicate preattached biofilms, which are a major source of bacterial persistence in nosocomial infections. We now present a modular high-throughput 384-well image-based screening platform to identify Pseudomonas aeruginosa biofilm inhibitors and dispersal agents. Biofilm coverage measurements were accomplished using non-z-stack epifluorescence microscopy to image a constitutively expressing green fluorescent protein (GFP)-tagged strain of P. aeruginosa and quantified using an automated image analysis script. Using the redox-sensitive dye XTT, bacterial cellular metabolic activity was measured in conjunction with biofilm coverage to differentiate between classical antibiotics and nonantibiotic biofilm inhibitors/dispersers. By measuring biofilm coverage and cellular activity, this screen identifies compounds that eradicate biofilms through mechanisms that are disparate from traditional antibiotic-mediated biofilm clearance. Screening of 312 natural-product prefractions identified the cyclic depsipeptide natural products skyllamycins B and C as nonantibiotic biofilm inhibitors with 50% effective concentrations (EC50s) of 30 and 60 µM, respectively. Codosing experiments of skyllamycin B and azithromycin, an antibiotic unable to clear preattached biofilms, demonstrated that, in combination, these compounds were able to eliminate surface-associated biofilms and depress cellular metabolic activity. The skyllamycins represent the first known class of cyclic depsipeptide biofilm inhibitors/dispersers.

Discovery and biological characterization of the auromomycin chromophore as an inhibitor of biofilm formation in Vibrio cholerae.

Bacterial biofilms pose a significant challenge in clinical environments due to their inherent lack of susceptibility to antibiotic treatment. It is widely recognized that most pathogenic bacterial strains in the clinical setting persist in the biofilm state, and are the root cause of many recrudescent infections. The discovery and development of compounds capable of either inhibiting biofilm formation or initiating biofilm dispersal might provide new therapeutic avenues for reducing the number of hospital-acquired, biofilm-mediated infections. We detail here the application of our recently reported image-based, high-throughput screen to the discovery of microbially derived natural products with inhibitory activity against Vibrio cholerae biofilm. Examination of a prefractionated library of microbially derived marine natural products has led to the identification of a new biofilm inhibitor that is structurally unrelated to previously reported inhibitors and is one of the most potent inhibitors of V. cholerae reported to date. Combination of this compound with sub-MIC concentrations of a number of clinically relevant antibiotics was shown to improve the inhibitory efficacy of this new compound compared to monotherapy treatments, and provides evidence for the potential therapeutic benefit of biofilm inhibitors in treating persistent biofilm-mediated infections.

Incidence of first onset alcohol use disorder: a 16-year follow-up in the Taiwanese aborigines.

PURPOSE: To investigate the incidence and cumulative risk of first onset alcohol use disorder (AUD) in a 16-year follow-up among Taiwanese aboriginal populations. METHODS: Participants included in this study were cohort subjects free from any AUD at phase 1 survey (n = 428 for DSM-3-R and 451 for DSM-4) of the Taiwan aboriginal study project conducted in 1986-1988. They were reassessed approximately 16 years later, with a response rate of 98.8 %. A Chinese version of the WHO schedules for clinical assessment in neuropsychiatry was employed to assess the lifetime drinking history and AUD. RESULTS: Age-standardized annual incidence rates of AUD in all groups were 2.26 and 1.75 % according to DSM-3-R and DSM-4, respectively. The overall incidence rates of AUD were comparable to most of other studies in Caucasian populations, but the sex ratios of women to men were higher in this study (1:2-3) than in the latter (1:6). The incidence of AUD was higher with DSM-3-R than with DSM-4 criteria in this study, attributable to the exclusion of physical/psychological harm in DSM-4 alcohol abuse. The cumulative risks of DSM-4 AUD in this study were very high, being 72.2 ± 19.8 for men and 48.7 ± 8.2 for women up to the age of 65 years. CONCLUSIONS: High incidence rates and cumulative risks of AUD in Taiwanese aborigines demand effective prevention strategy.

Mortality and suicide after self-harm: community cohort study in Taiwan.

BACKGROUND: Little is known about outcomes after self-harm in East Asia. AIMS: To investigate mortality after self-harm in a Taiwanese population. METHOD: Between 2000 and 2003, 1083 individuals who self-harmed were identified through a population self-harm register in Nantou County, Taiwan, and followed until 2007 for date and cause of death on a national mortality database. RESULTS: In total, 145 individuals died, 48 through suicide. The risks of all-cause and suicide mortality in the first year were 4.7% and 2.1% respectively, representing 8- and 131-fold age- and gender-standardised increases. Male gender and older age were independent risk factors for both suicide and non-suicide mortality. Use of more lethal methods in the index episode was associated with higher mortality but this was accounted for by gender. CONCLUSIONS: Results in this sample support the recommendation that people with a history of recent self-harm should be a major target for suicide prevention programmes.

Non-fatal repetition of self-harm: population-based prospective cohort study in Taiwan.

BACKGROUND: Repeated self-harm is relatively common and is linked with an elevated risk of eventual suicide. There has been no study of this involving a large sample from the Far East. AIMS: To estimate the risk over the medium term of non-fatal repetition of self-harm and identify predictive factors in those carrying out self-harm. METHOD: A total of 970 individuals who had self-harmed were recruited from a community-based suicide behaviour register system in Nantou, Taiwan from July 2000 to February 2003. Information regarding demography and suicide methods was collected. Individuals were followed-up until December 2005 to examine the risk of repeated self-harm and independent predictive factors. RESULTS: Ninety cohort members had repeated self-harm during the follow-up period (accounting for 131 repeated self-harm episodes in all). The cumulative risks were 5.7% for the first year, 7.8% for the second year and 9.5% for the fourth year. The risk was highest within the first year after the self-harm event. Independent risk factors included female gender and self-cutting as well as self-poisoning with drugs. Effect of younger age was mediated through the choice of methods. CONCLUSIONS: Individuals with self-harm have a high risk of repetition, especially within the first year. Suicide prevention strategies need to focus on intervening with this population to reduce their repetition.

Effect of media reporting of the suicide of a singer in Taiwan: the case of Ivy Li.

BACKGROUND: Suicide attempters are known to be vulnerable to the influence of media reporting of suicide events. This study investigates possible influences of media reporting of a celebrity suicide on subsequent suicide attempts and associated risk factors among suicide attempters. METHODS: Sixty-three suicide attempters registered in a surveillance system of Taipei City Suicide Prevention Center were assessed using a structured interview soon after media reporting of the suicide of a young female singing star. RESULTS: Forty-three (68%) respondents had encountered with the suicide news. Among them, 37% reported being influenced by the media reporting on their subsequent suicide attempts. Men (adjusted OR 6.36, 95% CI 1.29-31.44) and younger age groups (adjusted OR 4.93, 95% CI 1.04-23.45) were more susceptible to the media reporting. There was a positive modeling effect in method of suicide (charcoal burning) (adjusted OR 7.27, 95% CI 6.31-168.66). CONCLUSIONS: This study has provided further evidence for suicide imitation among vulnerable people encountered with media reporting of celebrity suicide, and for the need to actively restrain reporting of suicides to decrease the imitation effect.

Development of a Chinese version of the Suicide Intent Scale.

This study established the psychometric properties of the Chinese version of the Suicide Intent Scale (SIS) in a clinic- and community-based sample of 36 patients and 592 respondents, respectively. Results showed that the Chinese SIS demonstrated good inter-rater and test-retest reliability. Factor analysis generated three factors (Precautions, Planning, and Seriousness) explaining 92.9% of the total variance with high internal consistency. It was moderately correlated with depressive symptoms. Results suggest that the Chinese SIS is a reliable and valid instrument for use in assessing the extent of suicidal intention among subjects with deliberate self-harm in ethnic Chinese populations.

Psychiatric consultation for post-liver-transplantation patients.

AIM: This report describes the clinical characteristics of referral inpatients who received liver transplantation and the surgeon's concordance with the psychiatric consultation. METHODS: During a 4-year period, psychiatric consultation was arranged for 30 post-liver-transplantation inpatients at Kaohsiung Chang Gung Memorial Hospital. A psychiatrist assessed these patients and made psychiatric diagnoses according to the Diagnostic and Statistical Manual, 4th edition. The clinical data were routinely collected via modified MICRO-CARES software. At the end of the 4-year study period, all the medical records of these 30 inpatients were reviewed. RESULTS: Psychiatric diagnosis was made in 70% of the patients (n = 21) in three major categories, including delirious state (n = 8), depressive disorder (n = 5), and anxiety/dyssomnia (n = 8). All these conditions were improved by psychiatric management. We found that the consultee's concordance with recommended drug prescriptions was high with antidepressants in depressive patients and low with antipsychotics in patients with delirium. Moreover, anxiolytics were frequently prescribed in post-transplant inpatients before psychiatric consultation. CONCLUSION: These findings suggest that psychiatric consultation is beneficial to inpatients after liver transplantation, especially those with depression and delirium.

A Chinese rhesus macaque (Macaca mulatta) model for vaginal Lactobacillus colonization and live microbicide development.

BACKGROUND: We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates. METHODS: Vaginal and rectal microflora in Chinese rhesus macaques (Macaca mulatta) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization. RESULTS: Chinese rhesus macaques harbored abundant vaginal Lactobacillus, with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals. CONCLUSIONS: The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus-based topical microbicides.

A community-based study of case fatality proportion among those who carry out suicide acts.

OBJECTIVE: To investigate the case fatality proportion and associated factors in those carrying out suicide acts in Nantou, Taiwan. METHOD: Data from 1,171 suicide acts (including 973 with deliberate self harm and 198 completed suicides), identified between July 2000 and February 2003, were collected from a population suicide register in Nantou County, Taiwan. Case fatality proportion and the independent effects of demographic factors and suicide method on case fatality were investigated. RESULTS: The overall case fatality proportion was 16.9%, with higher proportions in men (26.3%) and in those aged 65 and over (37.9%). Hanging was the most lethal method (fatality proportion = 81.5%); pesticide was both commonly used and associated with a high case fatality (fatality proportion = 26.3%). Only age (increased with age) and suicide method were found to be independently associated with the risk of fatality. CONCLUSIONS: Suicide method and older age are independent predictors of the fatality of suicide acts. Suicide prevention strategy and clinical assessment ought to take into account of these two factors. Diminution of pesticide toxicity and control of access to pesticides are important considerations for suicide prevention in rural Taiwan.