Application of genefishing discovery system on differential gene expression study for prokaryotic system

GeneFishing Discovery is a system applied on disease and gene related research for eukaryotes and the starting material for the study could be either total RNA or mRNA. In this study we demonstrate the use of this discovery system for a prokaryotic system by modifying the eukaryotic protocol using the poly (A)-tailing approach. As an example we compared the gene expression pattern of two closely related species of bacteria under the same family (Burkholderia thailandensis and Burkholderia cepacia) with satisfactory results.Keywords: Genefishing, differential expression, prokaryotic systemAfrican Journal of Biotechnology Vol. 9(35), pp. 5629-5631, 30 August, 201

Repetitive vascular occlusion stimulus (RVOS) versus standard care to prevent muscle wasting in critically ill patients (ROSProx):a study protocol for a pilot randomised controlled trial

Background Forty per cent of critically ill patients are affected by intensive care unit-acquired weakness (ICU-AW), to which skeletal muscle wasting makes a substantial contribution. This can impair outcomes in hospital, and can cause long-term physical disability after hospital discharge. No effective mitigating strategies have yet been identified. Application of a repetitive vascular occlusion stimulus (RVOS) a limb pressure cuff inducing brief repeated cycles of ischaemia and reperfusion, can limit disuse muscle atrophy in both healthy controls and bed-bound patients recovering from knee surgery. We wish to determine whether RVOS might be effective in mitigating against muscle wasting in the ICU. Given that RVOS can also improve vascular function in healthy controls, we also wish to assess such effects in the critically ill. We here describe a pilot study to assess whether RVOS application is safe, tolerable, feasible and acceptable for ICU patients. Methods This is a randomised interventional feasibility trial. Thirty-two ventilated adult ICU patients with multiorgan failure will be recruited within 48 h of admission and randomised to either the intervention arm or the control arm. Intervention participants will receive RVOS twice daily (except only once on day 1) for up to 10 days or until ICU discharge. Serious adverse events and tolerability (pain score) will be recorded; feasibility of trial procedures will be assessed against pre-specified criteria and acceptability by semi-structured interview. Together with vascular function, muscle mass and quality will be assessed using ultrasound and measures of physical function at baseline, on days 6 and 11 of study enrolment, and at ICU and hospital discharge. Blood and urine biomarkers of muscle metabolism, vascular function, inflammation and DNA damage/repair mechanism will also be analysed. The Health questionnaire will be completed 3 months after hospital discharge. Discussion If this study demonstrates feasibility, the derived data will be used to inform the design (and sample size) of an appropriately-powered prospective trial to clarify whether RVOS can help preserve muscle mass/improve vascular function in critically ill patients.</p

Antimicrobial susceptibility and virulence genes of clinical and environmental isolates of Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is ubiquitous, has intrinsic antibiotic resistance mechanisms, and is associated with serious hospital-associated infections. It has evolved from being a burn wound infection into a major nosocomial threat. In this study, we compared and correlated the antimicrobial resistance, virulence traits and clonal relatedness between clinical and fresh water environmental isolates of P. aeruginosa. Methods 219 P. aeruginosa isolates were studied: (a) 105 clinical isolates from 1977 to 1985 (n = 52) and 2015 (n = 53), and (b) 114 environmental isolates from different fresh water sources. All isolates were subjected to ERIC-PCR typing, antimicrobial susceptibility testing and virulence factor genes screening. Results Clinical and environmental isolates of P. aeruginosa were genetically heterogenous, with only four clinical isolates showing 100% identical ERIC-PCR patterns to seven environmental isolates. Most of the clinical and environmental isolates were sensitive to almost all of the antipseudomonal drugs, except for ticarcillin/clavulanic acid. Increased resistant isolates was seen in 2015 compared to that of the archived isolates; four MDR strains were detected and all were retrieved in 2015. All clinical isolates retrieved from 1977 to 1985 were susceptible to ceftazidime and ciprofloxacin; but in comparison, the clinical isolates recovered in 2015 exhibited 9.4% resistance to ceftazidime and 5.7% to ciprofloxacin; a rise in resistance to imipenem (3.8% to 7.5%), piperacillin (9.6% to 11.3%) and amikacin (1.9% to 5.7%) and a slight drop in resistance rates to piperacillin/tazobactam (7.7% to 7.5%), ticarcillin/clavulanic acid (19.2% to 18.9%), meropenem (15.4% to 7.5%), doripenem (11.5% to 7.5%), gentamicin (7.7% to 7.5%) and netilmicin (7.7% to 7.5%). Environmental isolates were resistant to piperacillin/tazobactam (1.8%), ciprofloxacin (1.8%), piperacillin (4.4%) and carbapenems (doripenem 11.4%, meropenem 8.8% and imipenem 2.6%). Both clinical and environmental isolates showed high prevalence of virulence factor genes, but none were detected in 10 (9.5%) clinical and 18 (15.8%) environmental isolates. The exoT gene was not detected in any of the clinical isolates. Resistance to carbapenems (meropenem, doripenem and imipenem), β-lactamase inhibitors (ticarcillin/clavulanic acid and piperacillin/tazobactam), piperacillin, ceftazidime and ciprofloxacin was observed in some of the isolates without virulence factor genes. Five virulence-negative isolates were susceptible to all of the antimicrobials. Only one MDR strain harbored none of the virulence factor genes. Conclusion Over a period of 30 years, a rise in antipseudomonal drug resistance particularly to ceftazidime and ciprofloxacin was observed in two hospitals in Malaysia. The occurrence of resistant environmental isolates from densely populated areas is relevant and gives rise to collective anxiety to the community at large

Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device

Background. Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity. Method. In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC nontargeted strains were tested with iiPCR and qPCR in parallel. Results. All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45– 99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity. Conclusion. This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen

Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay

BACKGROUND: In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay. METHODS: A mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. One hundred and ten Malaysian strains (1997–2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth. RESULTS: The ipaH sequence was present in all the strains, while each of the set1A, set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non-Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation. CONCLUSIONS: The mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen

Incidence, risk factors and clinical epidemiology of melioidosis: a complex socio-ecological emerging infectious disease in the Alor Setar region of Kedah, Malaysia

<p>Abstract</p> <p>Background</p> <p>Melioidosis, a severe and fatal infectious disease caused by <it>Burkholderia pseudomallei</it>, is believed to an emerging global threat. However, data on the natural history, risk factors, and geographic epidemiology of the disease are still limited.</p> <p>Methods</p> <p>We undertook a retrospective analysis of 145 confirmed cases extracted from a hospital-based Melioidosis Registry set up from 2005 in Hospital Sultanah Bahiyah, Alor Setar, Kedah state, Malaysia, in order to provide a first description of the contemporary incidence, risk factors, and clinical epidemiology of the disease in this putatively high risk region of the country.</p> <p>Results</p> <p>The incidence of melioidosis in Alor Setar is remarkably high at 16.35 per 100,000 population per year. The mean age of patients was 50.40 years, with infection varying nonlinearly with age. Males (75.2%; <it>P </it>< 0.0001) predominated and the majority of cases were Malays (88.9%). The overall, crude mortality rate among the study patients was 33.8%. The proportions of cases and deaths were significantly greater among patients involved in farming, forestry and fishing and the unemployed (χ²= 30.57, <it>P </it>< 0.0001). A majority of cases (62.75%) were culture positive, with mortality in these patients being 45.05%. A large proportion (83.0%) of culture positives was also bacteremic. Pneumonia accounted for 42.06% of primary diagnoses followed in importance by soft tissue abscess. In patients with pneumonia and who were culture positive, the mortality rate was as high as 65.00%. Diabetes mellitus constituted the major underlying risk factor for developing and dying from melioidosis, occurring in 57% of all diagnosed cases. The age distribution of diabetes paralleled that of melioidosis cases. There were linear associations between cases and deaths with monthly rainfall.</p> <p>Conclusions</p> <p>Melioidosis represents a complex socio-ecological public health problem in Kedah, being strongly related with age, occupation, rainfall and predisposing chronic diseases, such as diabetes mellitus. Among cases, bacteremic patients were associated with significantly high mortality despite provision of the recommended antibacterial therapy. The burden of this disease is likely to grow in this region unless better informed interventions targeted at high-risk groups and associated diseases are urgently implemented.</p

Metabolic phenotype of skeletal muscle in early critical illness

No commercial use is permitted unless otherwise expressly granted.ZAP was funded by the National Institute of Health Research (UK). Additional funding has been received from the European Society of Intensive Care Medicine, Guy’s & St Thomas’ and King’s College London NIHR Comprehensive Biomedical Research Centre (BRC) and the Whittington Hospital NHS Trust. SDH received support from the Research Councils UK. NH received funding from the NIHR Clinical Research Facility and BRC at Guy’s and St Thomas’ NHS Foundation Trust (GSTT) and King’s College London. HEM was funded by University College London and UCLH BRC. The Clinical Phenotyping Centre is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. MJWM is grateful to the Wellcome Trust for support in the form of a Postdoctoral Training Fellowship for part of this work. YP is grateful to Merz Pharmaceuticals for support in the form of a training fellowship award

In vitro antimicrobial activity of natural toxins and animal venoms tested against Burkholderia pseudomallei

BACKGROUND: Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism. METHODS: Here, we investigated a group of venoms (snakes, scorpions and honey bee venoms) for antimicrobial properties against two strains of Gram-negative bacteria Burkholderia pseudomallei by using disc-diffusion assay for in vitro susceptibility testing. The antibacterial activities of the venoms were compared with that of the isolated L-amino acid oxidase (LAAO) and phospholipase A(2 )(PLA(2)s) enzymes. MICs were determined using broth dilution method. Bacterial growth was assessed by measurement of optical density at the lowest dilutions (MIC 0.25 mg/ml). The cell viability was measured using tetrazolium salts (XTT) based cytotoxic assay. RESULTS: The studied venoms showed high antimicrobial activity. The venoms of C. adamanteus, Daboia russelli russelli, A. halys, P. australis, B. candidus and P. guttata were equally as effective as Chloramphenicol and Ceftazidime (30 μg/disc). Among those tested, phospholipase A(2 )enzymes (crotoxin B and daboiatoxin) showed the most potent antibacterial activity against Gram-negative (TES) bacteria. Naturally occurring venom peptides and phospholipase A(2 )proved to possess highly potent antimicrobial activity against Burkholderia pseudomallei. The XTT-assay results showed that the cell survival decreased with increasing concentrations (0.05–10 mg/mL) of Crotalus adamanteus venom, with no effect on the cell viability evident at 0.5 mg/mL. CONCLUSION: This antibacterial profile of snake venoms reported herein will be useful in the search for potential antibacterial agents against drug resistant microorganisms like B. pseudomallei

The Epidemiology and Clinical Spectrum of Melioidosis: 540 Cases from the 20 Year Darwin Prospective Study

Melioidosis is an occupationally and recreationally acquired infection important in Southeast Asia and northern Australia. Recently cases have been reported from more diverse locations globally. The responsible bacterium, Burkholderia pseudomallei, is considered a potential biothreat agent. Risk factors predisposing to melioidosis are well recognised, most notably diabetes. The Darwin prospective melioidosis study has identified 540 cases of melioidosis over 20 years and analysis of the epidemiology and clinical findings provides important new insights into this disease. Risk factors identified in addition to diabetes, hazardous alcohol use and chronic renal disease include chronic lung disease, malignancies, rheumatic heart disease, cardiac failure and age ≥50 years. Half of patients presented with pneumonia and septic shock was common (21%). The decrease in mortality from 30% in the first 5 years of the study to 9% in the last five years is attributed to earlier diagnosis and improvements in intensive care management. Of the 77 fatal cases (14%), all had known risk factors for melioidosis. This supports the most important conclusion of the study, which is that melioidosis is very unlikely to kill a healthy person, provided the infection is diagnosed early and resources are available to provide appropriate antibiotics and critical care where required

Persistent Gastric Colonization with Burkholderia pseudomallei and Dissemination from the Gastrointestinal Tract following Mucosal Inoculation of Mice

Melioidosis is a disease of humans caused by opportunistic infection with the soil and water bacterium Burkholderia pseudomallei. Melioidosis can manifest as an acute, overwhelming infection or as a chronic, recurrent infection. At present, it is not clear where B. pseudomallei resides in the mammalian host during the chronic, recurrent phase of infection. To address this question, we developed a mouse low-dose mucosal challenge model of chronic B. pseudomallei infection and investigated sites of bacterial persistence over 60 days. Sensitive culture techniques and selective media were used to quantitate bacterial burden in major organs, including the gastrointestinal (GI) tract. We found that the GI tract was the primary site of bacterial persistence during the chronic infection phase, and was the only site from which the organism could be consistently cultured during a 60-day infection period. The organism could be repeatedly recovered from all levels of the GI tract, and chronic infection was accompanied by sustained low-level fecal shedding. The stomach was identified as the primary site of GI colonization as determined by fluorescent in situ hybridization. Organisms in the stomach were associated with the gastric mucosal surface, and the propensity to colonize the gastric mucosa was observed with 4 different B. pseudomallei isolates. In contrast, B. pseudomallei organisms were present at low numbers within luminal contents in the small and large intestine and cecum relative to the stomach. Notably, inflammatory lesions were not detected in any GI tissue examined in chronically-infected mice. Only low-dose oral or intranasal inoculation led to GI colonization and development of chronic infection of the spleen and liver. Thus, we concluded that in a mouse model of melioidosis B. pseudomallei preferentially colonizes the stomach following oral inoculation, and that the chronically colonized GI tract likely serves as a reservoir for dissemination of infection to extra-intestinal sites