Pseudodesulfovibrio thermohalotolerans sp. nov., a novel obligately anaerobic, halotolerant, thermotolerant, and sulfate-reducing bacterium isolated from a western offshore hydrocarbon reservoir in India.

OBJECTIVE: Characterization and documentation of strain MCM B-1480T, a novel sulfate-reducing bacterium isolated from produced water of India's western offshore hydrocarbon reservoir. METHOD: Strain MCM B-1480T was unequivocally identified using a polyphasic approach routinely followed in bacterial systematics. The morphological and biochemical characterization of strain MCM B-1480T was carried out using standard microbiological techniques. RESULTS: MCM B-1480T was a Gram-stain-negative, motile, non-spore-forming, curved-rod-shaped bacterium. MCM B-1480T could grow at temperatures between 20 and 60 °C (optimum 37 °C), pH 6-8 (optimum 7), and required 1-6% NaCl (optimum 3%) for growth. Strain MCM B-1480T was reducing sulfate to produce hydrogen sulfide during growth. This strain used lactate and pyruvate as prominent electron donors, whereas sulfate, sulfite, thiosulfate, and nitrate served as electron acceptors. MCM B-1480T shared maximum 16S rRNA gene sequence homology of 98.65% with the members of the genus Pseudodesulfovibrio. The G + C content of the 3.87 Mb MCM B-1480T genome was 60.39%. Digital DDH (27.7%) and average nucleotide identity (ANI 84%) with the closest phylogenetic affiliate (less than 70% and 95%, respectively) reaffirmed its distinctiveness. The major cellular fatty acids components, namely iso-C15:0, anteiso-C15:0, C16:0, and anteiso-C17:0, differentiated strain MCM B-1480T from other species of Pseudodesulfovibrio. Genome annotation revealed the presence of genes encoding dissimilatory sulfate reduction and nitrate reduction in strain MCM B-1480T. CONCLUSION: The polyphasic studies, including SSU rRNA gene sequencing, average nucleotide identity, Digital DNA-DNA hybridization, cell wall fatty acids analysis, etc., identified strain MCM B-1480T as a novel taxon and Pseudodesulfovibrio thermohalotolerans sp. nov. was proposed (= JCM 39269T = MCC 4711T).

A study to evaluate the burden of hearing loss and its correlation with risk factors among high-risk infants at a teaching institution, Jaipur.

Introduction: Hearing loss is a global issue of hearing disability and early detection and rehabilitation of hearing loss are important for the development of speech and language skills in hearing-impaired infants. There are multiple risk factors that aid in hearing loss but some are potential factors that contribute toward hearing loss in infants. The aim of this study was to assess the burden of hearing loss and its correlation with risk factors among high-risk infants at a teaching institution in Jaipur, Rajasthan. Method: This study was carried out after approval of institutional ethics committee on a total of 320 high-risk infants at RUHS College of medical sciences and associated hospitals. Hearing loss was assessed by brainstem evoked response audiometry (BERA). Statistical analysis of data was done by cross-tabulation analysis with Pearson correlation and quantile regression. Results: Out of 320 high-risk infants, 59.69% of infants had normal hearing, 9.09% Unilaterally hearing impaired, 20.31% were bilaterally mild-moderate hearing loss, and 10.94% had severe-profound deafness. The prevalence of important risk factors viz. hyperbilirubinemia, low birth weight, appearance, pulse, grimace, activity, and respiration score, meconium aspiration, respiratory distress, and ventilation greater than five days were 86%, 58.9%, 40%, 36%, 29%, and 22%, respectively. Conclusion: In high-risk infants, hearing loss is a common hearing disorder. Because of this, early diagnosis of hearing loss gives them the best chance of developing functional speech. Brainstem evoked response audiometry is a simple, reliable, and effective technique for the assessment of auditory functions in infants.

In vitro and in vivo evaluation of buccal bioadhesive films containing salbutamol sulphate.

The aim of present study was to prepare and evaluate buccal bioadhesive films of salbutamol sulphate (SS) for the treatment of asthma. The films were designed to release the drug for a prolonged period of time so as to reduce the frequency of administration of the available conventional dosage forms of SS. The different proportions of sodium carboxymethylcellulose (SCMC) and Carbopol 940P (CP 940P) were used for the preparation of films. Carbopol was used to incorporate the desired bioadhesiveness in the films. The films were prepared by solvent casting method and evaluated for bioadhesion, in vitro drug release and anti asthmatic effect (bronchoprotection) in histamine induced bronchospasm of guinea pigs. In vitro drug release from the film was determined using a modified Franz diffusion cell while bioadhesiveness was evaluated with a modified two-arm balance using guinea pig buccal mucosa as a model tissue. Films containing SCMC : CP 940P ratio of 76 : 24 was found to be the best with moderate swelling along with favorable bioadhesion force and in vitro drug release. The drug release mechanism was found to follow non-Fickian diffusion as release mechanism. The prolonged in vivo effect (bronchoprotection) obtained from the buccal bioadhesive film of SS administered via buccal route may improve the treatment of asthmatic disorders by reducing the frequency of administration which is associated with the tolerance effect of SS. Additionally for the clinical benefit, it is also expected to reduce the major adverse effects of SS such as tachycardia and arrhythmias via buccal absorption.

Exposure to potting soils and compost material as potential sources of Legionella pneumophilia in Australia.

Legionnaires' disease is a form of atypical community-acquired pneumonia usually caused by Legionella pneumophilia, which is typically associated with exposure to tower cooling or water systems. In Australia, Legionnaires' disease is more commonly caused by Legionella longbaechae, which is typically associated with exposure to soil or compost materials, and the presence of Legionella pneumophilia is less recognized. We report a sporadic case of Legionnaires' disease caused by Legionella pneumophilia serogroup 1 that was contracted following exposure to potting mix and topsoil.

Delayed diagnosis of right-sided valve endocarditis causing recurrent pulmonary abscesses: a case report.

BACKGROUND: Pulmonary valve infective endocarditis is a rare diagnosis that is usually associated with immunocompromised states or structurally abnormal hearts. It is unusual for it to occur in structurally normal hearts or to cause recurrent symptoms after targeted antibiotics. Although guidelines suggest follow-up with repeat echocardiography and inflammatory marker surveillance, this case demonstrates that these are not always useful investigations, and instead imaging of the chest may be more appropriate. CASE PRESENTATION: We describe a case of a 74-year-old man who presented with respiratory symptoms and was originally misdiagnosed with pneumonia but later found to have a large pulmonary valve vegetation caused by Streptococcus mitis. Despite initially responding to antibiotic therapy, the vegetation continued to cause pulmonary emboli and cavitating lung abscesses months later, necessitating pulmonary valve replacement. CONCLUSIONS: This case demonstrates that pulmonary valve endocarditis can present atypically with recurrent respiratory symptoms, and in such cases, echocardiography should be considered to investigate for right-sided infective endocarditis. In addition, despite correct treatment, with normalization of inflammatory markers and improvement in vegetation size, infective endocarditis can continue to cause systemic symptoms. Finally, clinicians should consider chest computed tomography routinely as part of right-sided infective endocarditis follow-up.

Gas exchange in stable patients with moderate-to-severe lung disease from cystic fibrosis.

BACKGROUND: Studies using the multiple inert gas elimination technique (MIGET) to characterise the mechanisms of impaired gas exchange in CF, provide conflicting results on the importance of ventilation-perfusion (VA/Q) inequality over shunt. We hypothesise that the mechanisms of gas exchange abnormality have changed with changing CF management over the last two decades. METHODS: Detailed gas exchange was evaluated by MIGET with venous sampling in stable patients, age > 20 years, FEV1% predicted < or = 50. RESULTS: Fifteen (14 male) subjects were studied with a mean +/- SD age 28.1 +/- 8.4 years, FEV1% 32.6 +/- 10.3, TLC% 111.5 +/- 12.9, PaO2 9.3 +/- 1.3 kPa, (69.5 +/- 9.6 mm Hg), and PaCO2 6.2 +/- 0.7 kPa, (45.9 +/- 5.3 mm Hg). The predominant gas exchange abnormality was VA/Q inequality with a log SD of the distributions of perfusion 0.91 +/- 0.30 and of ventilation 0.60 +/- 0.14. Unimodal distributions were seen in nine subjects, a low VA/Q mode in five and one subject had a bimodal distribution, mean intrapulmonary shunt was negligible. CONCLUSIONS: Subjects had a lower FEV1% by comparison with previously published studies and demonstrated severe VA/Q inequality and negligible shunt. This suggests a low degree of complete obstruction of airways in adults with CF and severe stable pulmonary disease. The primary mechanism of hypoxaemia in CF subjects reaching adulthood today appears to have changed with modern management over the last two decades.

A new signal sequence for recombinant protein secretion in Pichia pastoris.

Pichia pastoris is one of the most widely used expression systems for the secretory expression of recombinant proteins. The secretory expression in P. pastoris usually makes use of the prepro MATα sequence from Saccharomyces cerevisiae, which has a dibasic amino acid cleavage site at the end of the signal sequence. This is efficiently processed by Kex2 protease, resulting in the secretion of high levels of proteins to the medium. However, the proteins that are having the internal accessible dibasic amino acids such as KR and RR in the coding region cannot be expressed using this signal sequence, as the protein will be fragmented. We have identified a new signal sequence of 18 amino acids from a P. pastoris protein that can secrete proteins to the medium efficiently. The PMT1-gene-inactivated P. pastoris strain secretes a ~30 kDa protein into the extracellular medium. We have identified this protein by determining its N-terminal amino acid sequence. The protein secreted has four DDDK concatameric internal repeats. This protein was not secreted in the wild-type P. pastoris under normal culture conditions. We show that the 18-amino-acid signal peptide at the N-terminal of this protein is useful for secretion of heterologous proteins in Pichia.

Development of a novel reporter gene vector for cell based angiogenic studies.

Angiogenesis is a promising area of research that targets key therapeutic areas like cancer; wound healing, inflammatory diseases, etc. There is an increasing demand for screening of potential angiogenic and anti-angiogenic agents using sensitive, robust cell-based assays. We have developed a reporter vector containing cis-acting elements that respond to growth factors/angiogenic ligands for use in a cell-based luciferase reporter assay. We performed transient transfection of our reporter gene vector in MCF-7 cells to establish its application for screening of potential pro/anti-angiogenic agents. Reporter gene transactivation studies with different concentrations of fetal bovine serum clearly indicated that the vector is functionally responsive to the angiogenic signals mediated by serum growth factors. We also used endostatin to inhibit transactivation and prove responsiveness to the anti-angiogenic agent. This vector is a promising tool for studying angiogenesis using cell-based reporter gene assays.

Co-expression of recombinant human CYP2C9 with human cytochrome P450 reductase in protease deficient S. cerevisiae strain at a higher scale yields an enzyme of higher specific activity.

Cytochrome P450 (CYP450) isozymes play an important role in the study of drug metabolism and drug discovery. A number of reports are available that describe recombinant expression of CYP450 isozymes. In this paper, human CYP2C9 and human cytochrome P450 reductase cDNAs were cloned and expressed in Premas proprietary yeast episomal and integrative vectors respectively under the influence of GAL1 promoter. Yeast cells were grown and induced at optimal parameters to make microsomal membranes. Isolated microsomal membranes were analyzed for CYP2C9 and cytochrome P450 reductase activity, CYP2C9 content and inhibition properties. We report heterologous expression of human CYP2C9 along with human cytochrome P450 reductase in protease deficient S. cerevisiae at a 5 litre scale resulting in high yields (8-10 nmols/litre) of enzyme with higher specific activity (2-3 fold higher). This yields a superior enzyme and makes it amenable to miniaturization of screening assays with concomitant lowering of costs.

Gas exchange and exercise tolerance following bullectomy.

This study evaluates the physiological responses to giant bullectomy. A 42-year-old female with bilateral giant bullae presented with dyspnoea and exercise limitation. At baseline and 3 months after bullectomy she had tests of lung function; exercise capacity via a symptom-limited cycle test and a 6-min walk test (6MWT). Quality of life (QoL) and gas exchange using the multiple inert gas elimination technique (MIGET) were also assessed. There were significant improvements in pulmonary function following surgery with the FEF(25-75%) predicted increasing from 16 to 96. The 6MWT increased by 10% and the peak leg work capacity by 48%. A MIGET measure of the distribution of perfusion (Log SDQ) fell from 0.52 to 0.36. There was also radiological improvement in hyperinflation and diaphragmatic configuration. The QoL total score decreased from 56 to 25. This patient demonstrated significant improvements in exercise tolerance, gas exchange and QoL following bullectomy.

Effect of Burkholderia cepacia infection in the clinical course of patients with cystic fibrosis: a pilot study in a Sydney clinic.

BACKGROUND: Colonisation with Burkholderia cepacia complex in patients with cystic fibrosis (CF) has been associated with adverse outcomes. The aim of the present study was to determine the actuarial survival of CF patients colonized with B. cepacia and to evaluate the efficacy of the Royal Prince Alfred Hospital segregation policy. A secondary aim was to characterize the specific genomovars and strains of B. cepacia isolated in an Australian clinic. METHODS: Retrospective review of spirometric and microbiological data on all patients colonized with B. cepacia. Each B. cepacia-colonized subject was matched with three case-control subjects. Phenotype and genomovar typing, random amplified polymorphic DNA strain type and B. cepacia epidemic strain marker analyses were performed. The effect of B. cepacia colonization on transplant-free survival was estimated by Cox's proportional hazards regression using the entire clinic population. RESULTS: Fifteen patients were colonized with B. cepacia, of whom six (40%) had died from CF-related disease by August 1998, compared with 30 of 173 (17.3%) of the entire clinic population. Cepacia status had a significant adverse effect on survival, with a hazard ratio of 2.16 (95% confidence interval 1.0-4.69; P = 0.05). The outcome was variable in subgroups of B. cepacia. DISCUSSION: Colonization with B. cepacia had a significant adverse effect on survival within the study population. Genomovar and strain typing contributed usefully in accessing the effectiveness of the hospital's segregation policy in preventing cross-colonization.