Emergence of a non-sporulating secondary phenotype in Clostridium (Clostridioides) difficile ribotype 078 isolated from humans and animals.

Clostridium (Clostridioides) difficile is a Gram positive, spore forming anaerobic bacterium that is a leading cause of antibiotic associated diarrhoea in the developed world. C. difficile is a genetically diverse species that can be divided into 8 phylogenetically distinct clades with clade 5 found to be genetically distant from all others. Isolates with the PCR ribotype 078 belong to clade 5, and are often associated with C. difficile infection in both humans and animals. Colonisation of animals and humans by ribotype 078 raises questions about possible zoonotic transmission, and also the diversity of reservoirs for ribotype 078 strains within the environment. One of the key factors which enables C. difficile to be a successful, highly transmissible pathogen is its ability to produce oxygen resistant spores capable of surviving harsh conditions. Here we describe the existence of a non-sporulating variant of C. difficile ribotype 078 harbouring mutations leading to premature stop codons within the master regulator, Spo0A. As sporulation is imperative to the successful transmission of C. difficile this study was undertaken to investigate phenotypic characteristics of this asporogenous phenotype with regards to growth rate, antibiotic susceptibility, toxin production and biofilm formation.

Early HIV disclosure and nondisclosure among men and women on antiretroviral treatment in Uganda.

Efforts to expand access to HIV care and treatment often stress the importance of disclosure of HIV status to aid adherence, social support, and continued resource mobilization. We argue that an examination of disclosure processes early in the process of seeking testing and treatment can illuminate individual decisions and motivations, offering insight into potentially improving engagement in care and adherence. We report on baseline data of early HIV disclosure and nondisclosure, including reasons for and responses to disclosure from a cohort of men and women (n=949) currently accessing antiretroviral treatment in two regions of Uganda. We found early disclosures at the time of suspicion or testing positive for HIV by men and women to be largely for the purposes of emotional support and friendship. Responses to these selected disclosures were overwhelmingly positive and supportive, including assistance in accessing treatment. Nonetheless, some negative responses of worry, fear, or social ostracism did occur. Individuals deliberately chose to not disclose their status to partners, relatives, and others in their network, for reasons of privacy or not wanting to cause worry from the other person. These data demonstrate the strategic choices that individuals make early in the course of suspicion, testing, and treatment for HIV to mobilize resources and gain emotional or material support, and similarly their decisions and ability to maintain privacy regarding their status.

Phosphonoacetate biosynthesis: in vitro detection of a novel NADP(+)-dependent phosphonoacetaldehyde-oxidizing activity in cell-extracts of the marine Roseovarius nubinhibens ISM.

A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.

Stimulation of phosphate uptake and polyphosphate accumulation by activated sludge microorganisms in response to sulfite addition.

Enhanced phosphate removal from wastewaters is dependent on the synthesis and intracellular accumulation of polyphosphate by sludge microorganisms. However the role played by polyphosphate in microbial metabolism and the factors that trigger its formation remain poorly-understood. Many examples of the accumulation of the biopolymer by environmental microorganisms are documented; these include a recent report of the presence of large polyphosphate inclusions in sulfur-oxidizing marine bacteria. To investigate whether any link might exist outside the marine environment between the presence of reduced sulfur compounds and enhanced levels of microbial phosphate uptake and polyphosphate accumulation, activated sludge cultures were grown under laboratory conditions in media that contained sulfite, thiosulfate, hydrosulfite or tetrathionate. Only in the presence of sulfite was there any evidence of a stimulatory effect; in medium that contained 0.5 mM sodium sulfite some 17% more phosphate was removed by the sludge, whilst there was an almost two-fold increase in intracellular polyphosphate levels. No indications of sulfite toxicity were observed.

Evidence for phosphonate usage in the coral holobiont.

Phosphonates are characterized by a stable carbon-phosphorus bond and commonly occur as lipid conjugates in invertebrate cell membranes. Phosphonoacetate hydrolase encoded by the phnA gene, catalyses the cleavage of phosphonoacetate to acetate and phosphate. In this study, we demonstrate the unusually high phnA diversity in coral-associated bacteria. The holobiont of eight coral species tested positive when screened for phnA using degenerate primers. In two soft coral species, Sinularia and Discosoma, sequencing of the phnA gene showed 13 distinct groups on the basis of 90% sequence identity across 100% of the sequence. A total of 16 bacterial taxa capable of using phosphonoacetate as the sole carbon and phosphorus source were isolated; 8 of which had a phnA+ genotype. This study enhances our understanding of the wide taxonomic and environmental distribution of phnA, and highlights the importance of phosphonates in marine ecosystems.

A group-based intervention to increase condom use among HIV serodiscordant couples in India, Thailand, and Uganda.

This study assessed the feasibility of a group-based couples intervention to increase condom use in HIV serodiscordant couples in three countries (India, Thailand and Uganda). The intervention focused on communication, problem solving, and negotiation skills. Forty-three couples enrolled in the intervention (15 in India, 14 in Thailand, and 14 in Uganda) and 40 couples completed all study activities. Participants were interviewed at baseline and at one and three months post- intervention. The intervention consisted of two same sex sessions and two couples sessions with 'homework' to practice skills between sessions. The same intervention modules were used at each site, tailored for local appropriateness. Participants at each site were enthusiastic about the intervention, citing information about HIV serodiscordancy and the opportunity to meet couples 'like us' as important features. Participants reported increased comfort discussing sex and condoms with their partner, although some participants remain concerned about situations when condoms might not be used (e.g. when drunk). At three-month follow up 90% of the participants reported having been able to use the skills from the intervention with their partner. Our results highlight the feasibility of this couples group-based intervention and the need for ongoing support for discordant couples.

Effect of reduced pH on inorganic polyphosphate accumulation by Burkholderia cepacia complex isolates.

AIMS: Burkholderia cepacia complex (Bcc) isolates causing pulmonary infection in cystic fibrosis (CF) patients grow within an acidic environment in the lung. As exposure to acid pH has been shown to increase intracellular inorganic polyphosphate (polyP) formation in some bacteria, we investigated the inter-relationship between acidic pH and polyP accumulation in Bcc isolates. METHODS AND RESULTS: The formation of polyP by one Burkholderia cenocepacia clinical isolate was initially examined at a range of pH values by measuring total intracellular polyP accumulation and phosphate uptake. The pattern of polyP accumulation corresponded with the pattern of phosphate uptake with the maximum for both occurring at pH 5.5. Phosphate uptake and formation of polyP by this isolate was further determined over 48 h at pH 5.5, 6.5 and 7.5; formation of polyP was maximal at pH 5.5 at all time points studied. Sixteen of 17 additional clinical and environmental Bcc isolates examined also exhibited maximum phosphate uptake at pH 5.5. CONCLUSIONS: Both clinical and environmental Bcc isolates, of five genomovars, show enhanced formation of polyP in an acidic environment. Given both the speculated role of polyP in pathogenesis, cell signalling and biofilm formation and the acidic nature of the CF lung, this may be of considerable clinical importance. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth of Bcc in an acidic environment, such as that found in the lungs of CF patients may be influenced in part by polyP accumulation.

An evaluation of three new-generation tetrazolium salts for the measurement of respiratory activity in activated sludge microorganisms.

XTT (3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), and WST-1 (4-(3-4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzenedisulfonate) are tetrazolium salts that have become commercially available only in relatively recent years; they differ from earlier such compounds in that their reduction gives rise to a formazan product that is water soluble. We have established the sites in the prokaryotic respiratory chain at which each of the dyes is reduced to its corresponding formazan and have evaluated the suitability of each for the colorimetric estimation of electron transport system activity in populations of activated sludge microorganisms. Reduction of all three tetrazolium salts was shown to be proportional to cell biomass and oxygen uptake and to be susceptible to low levels of the reference toxicant 3,5-dichlorophenol. XTT, which was not inhibitory at concentrations of up to 2 mM and was reduced by 91% of isolates from a sample of culturable activated sludge bacteria, was chosen for further assay development. XTT-formazan production was found to be stimulated by the availability of an exogenous carbon and energy source, and by the presence of the electron-coupling agent phenazine methosulfate. Less than 3% of XTT reduction by an activated sludge sample was abiotic. An assay based on this compound could be a valuable and simple tool for the routine monitoring of the performance of wastewater treatment systems.

The reliability and validity of the Modified Condom Outcome Expectancy Scale (MCOES) among an international sample of HIV-negative partners of people living with HIV/AIDS.

Safer sexual behaviour is vital in curbing the HlV epidemic in many developing countries where the epidemic is primarily transmitted through heterosexual sex. Reliable and valid assessment of factors related to HIV risk behaviours are important in testing behavioural theories as well as informing effective AIDS prevention programmes. The current study tests the reliability and validity of a modified version of the Condom Outcome Expectancy Scale (COES) among a sample of HI'-negative sexual partners (n = 100) of HIV-infected individuals in Pune, India, Chang Mai,Thailand and Kampala, Uganda. Internal consistency reliability was measured using Cronbach's alpha coefficient, which had a value of 0.80 for the entire sample, with site-specific values of. 0.78 for India; 0.75 for Thailand; and 0.79 for Uganda. Test-retest reliability was conducted to test the scale's stability over time with 60% of the sample, resulting in a Cronbach's alpha coefficient of 0.70. The scale's structure was explored by analyzing response scores on the items using principal components analysis, which yielded a two-factor solution. The study indicates the utility of a modified version of the widely tested COES across international settings. Such research is necessary in understanding intervention targets across international settings.

Enhanced phosphate uptake and polyphosphate accumulation in Burkholderia cepacia grown under low pH conditions.

Of bacterial cells in a sample of activated sludge, 34% contained detectable intracellular polyphosphate inclusions following Neisser staining, when grown on glucose/mineral salts medium at pH 5.5; at pH 7.5 only 7% of cells visibly accumulated polyphosphate. In a sludge isolate of Burkholderia cepacia chosen for further study, maximal removal of phosphate and accumulation of polyphosphate occurred at pH 5.5; levels were up to 220% and 330% higher, respectively, than in cells grown at pH 7.5. During the early stationary phase of growth at pH 5.5 a maximum level of intracellular polyphosphate that comprised 13.6% of cellular dry weight was reached. Polyphosphate kinase activity was detected in actively growing cells only when cultured at pH 5.5. The phenomenon of acid-stimulated phosphate uptake and polyphosphate accumulation in this environmental bacterial population parallels observations previously made by us in the yeast Candida humicola and may thus represent a widespread microbial response to low external pH values.

Knowledge about vaccine trials and willingness to participate in an HIV/AIDS vaccine study in the Ugandan military.

In preparation for HIV vaccine trials, knowledge about vaccines, willingness to participate in a vaccine study, and motivations for participation must be assessed. The Preparation for AIDS Vaccine Evaluation study assessed knowledge about vaccines and vaccine trials and willingness to participate in a hypothetical trial in 1,182 Ugandan military men (aged 18-30 years). Participants received education about vaccine trials and were interviewed during 24 months of follow-up observation. Its key findings are that: 1) throughout follow-up, most participants expressed willingness to participate in a hypothetical HIV vaccine trial; 2) participants are familiar with vaccines but do not clearly distinguish the use of vaccines for prevention or curing; 3) the most common reason given for being interested in participating in a vaccine trial was to be protected from HIV/AIDS; 4) trials' procedures (e.g., placebos, randomization, and blinding) were unfamiliar; and 5) knowledge about trials' procedures increased incrementally over follow-up, but at different rates for different concepts. These data demonstrate that potential vaccine trials' participants may benefit from vaccine trial education if adequate time is allowed to ensure that participants are able to master the complex information required for trial participation.

Organized sports for children and preadolescents.

Participation in organized sports provides an opportunity for young people to increase their physical activity and develop physical and social skills. However, when the demands and expectations of organized sports exceed the maturation and readiness of the participant, the positive aspects of participation can be negated. The nature of parental or adult involvement can also influence the degree to which participation in organized sports is a positive experience for preadolescents. This updates a previous policy statement on athletics for preadolescents and incorporates guidelines for sports participation for preschool children. Recommendations are offered on how pediatricians can help determine a child's readiness to participate, how risks can be minimized, and how child-oriented goals can be maximized.

Prevalence of acanthosis nigricans and its association with hyperinsulinemia in New Mexico adolescents.

PURPOSE: To determine whether the association between acanthosis nigricans (AN) and hyperinsulinemia is independent of obesity and other variables. METHODS: A convenience sample of 675 New Mexico middle school students was screened to determine the presence of AN, obesity, and other variables, including: ethnicity, parental history of diabetes, and level of physical activity. Fasting glucose and insulin levels were drawn on 233 students to determine the association between risk factors and hyperinsulinemia. RESULTS: Acanthosis nigricans (AN) was present in 18.9% of students screened. Twenty-one percent of the subsample had hyperinsulinemia. Based on these results, the estimated prevalence of hyperinsulinemia among all middle school students in New Mexico was 8.9%. Forty-seven percent (47.2%) of students who had AN and were obese had hyperinsulinemia, compared with 2.4% of students who did not have either of these conditions. In multiple logistic regression analysis, AN and obesity were independently and positively associated with hyperinsulinemia, whereas physical activity was protective. CONCLUSIONS: The high prevalence of risk factors in this population makes diabetes prevention a priority for public health action. AN screening is an easily performed, noninvasive method for identifying adolescents at risk for type 2 diabetes.

Developing AIDS vaccine trials educational programs in Uganda.

In preparation for HIV vaccine trials, data on a cohort's knowledge about vaccines and vaccine studies are required so as to tailor educational materials to adequately meet local needs. Interviews (n = 1,182) conducted as part of a 3-year prospective study of Ugandan military men aged 18 to 30 years determined what information, in addition to standard trials information, would be required to ensure comprehension of trial procedures. The interviews highlighted four points: (1) the cohort has a lot of knowledge about vaccines but conflates whether vaccines cure or prevent disease; (2) there is a general lack of knowledge about clinical trials procedures; (3) the desire to be protected from HIV/AIDS is a common reason for being willing to participate in a hypothetical vaccine trial; and (4) concern about side effects is a common reason for being unwilling to participate in a trial. These four points guided the focus of the vaccine trials education, which used locally appropriate analogies to introduce complex unfamiliar concepts such as placebos and blinding. This case study highlights the value of incorporating baseline interviews to assess the educational needs of study populations.

Acid-stimulated phosphate uptake by activated sludge microorganisms under aerobic laboratory conditions.

Activated sludge inocula taken from five different wastewater treatment plants were grown aerobically under laboratory conditions on mineral salts medium containing either glucose or skimmed milk powder as carbon source. Cultures showed increases of between 50% and 143% in levels of phosphate uptake from the medium when the growth pH was 5.5 rather than 7.5. Of 100 individual sludge microbial isolates studied, 34 demonstrated such acid-stimulated luxury phosphate uptake; the optimum pH for the process was shown to lie between 5.0 and 6.5. Enhanced phosphate removal by these isolates was accompanied by increases of between 2 and 10.5-fold in their polyphosphate content; this was visualised as intracellular inclusions. Acid-stimulated luxury phosphate uptake by environmental microorganisms is a previously-unrecognised phenomenon that may have application in novel technologies for nutrient removal from wastewaters.

Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH.

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.

Biodegradation of phosphonomycin by Rhizobium huakuii PMY1.

The biodegradation by Rhizobium huakuii PMY1 of up to 10 mM phosphonomycin as a carbon, energy, and phosphorus source with accompanying P(i) release is described. This biodegradation represents a further mechanism of resistance to this antibiotic and a novel, phosphate-deregulated route for organophosphonate metabolism by Rhizobium spp.

Common bile duct ligation in the rat: a model of intrapulmonary vasodilatation and hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) causes impaired oxygenation due to intrapulmonary vasodilatation in patients with cirrhosis. Chronic common bile duct ligation (CBDL) in the rat results in gas-exchange abnormalities similar to HPS, but intrapulmonary vasodilatation has not been evaluated. We assess intrapulmonary vasodilatation, measured in vivo, after CBDL. Sham, 2- and 5-wk CBDL, and 3-wk partial portal vein ligated (PVL) rats had hepatic and lung injury, portal pressure, and arterial blood gases assessed. The pulmonary microcirculation was evaluated by injecting microspheres (size range 5.5-10 microm) intravenously and measuring the size and number of microspheres bypassing the lungs in arterial blood. CBDL animals developed progressive hepatic injury and portal hypertension accompanied by gas-exchange abnormalities and intrapulmonary vasodilatation. PVL animals, with a similar degree of portal hypertension, did not develop intrapulmonary vasodilatation or abnormal gas exchange. No lung injury was observed. CBDL, but not PVL, causes progressive intrapulmonary vasodilatation, which accompanies worsening arterial gas exchange. These findings validate CBDL as a model to study HPS.

The purification and properties of phosphonoacetate hydrolase, a novel carbon-phosphorus bond-cleavage enzyme from Pseudomonas fluorescens 23F.

A novel, inducible, carbon-phosphorus bond-cleavage enzyme, phosphonoacetate hydrolase, was purified from cells of Pseudomonas fluorescens 23F grown on phosphonoacetate. The native enzyme had a molecular mass of approximately 80 kDa and, upon SDS/PAGE, yielded a homogenous protein band with an apparent molecular mass of about 38 kDa. Activity of purified phosphonoacetate hydrolase was Zn2+ dependent and showed pH and temperature optima of approximately 7.8 and 37 degrees C, respectively. The purified enzyme had an apparent Km of 1.25 mM for its sole substrate phosphonoacetate, and was inhibited by the structural analogues 3-phosphonopropionate and phosphonoformate. The NH2-terminal sequence of the first 19 amino acids displayed no significant similarity to other databank sequences.