Biomarker discovery in the biofilm-forming process of Burkholderia pseudomallei by mass-spectrometry.

A serious human infectious disease called Melioidosis is a result of Burkholderia pseudomallei infection. Treatment for infected individuals is difficult due to a wide range of ineffective antibiotics including a high level of antibiotic tolerance which has been known to be caused by biofilm production. However, biofilm forming processes of this bacterium are not well documented despite multiple-methodologies being applied. In this study, we utilized a proteomics strategy called whole cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (whole cell MALDI-TOF MS) to discover a potential biomarker relating biofilm forming in B. pseudomallei. The results presented a novel specific type of enzyme amylo-alpha-1, 6-glucosidase, which was demonstrated by a higher level of gene expression during the biofilm development. Our results also suggested a list of candidate markers that might be involved in this scenario. Eventually, this knowledge may expand valuable data to the biofilm study that may increase effective treatments for people infected with B. pseudomallei and possibly other antibiotic tolerant bacteria.

Evaluation of multiplex PCR with enhanced spore germination for detection of Clostridium difficile from stool samples of the hospitalized patients.

Clostridium difficile poses as the most common etiologic agent of nosocomial diarrhea. Although there are many diagnostic methods to detect C. difficile directly from stool samples, the nucleic acid-based approach has been largely performed in several laboratories due to its high sensitivity and specificity as well as rapid turnaround time. In this study, a multiplex PCR was newly designed with recent accumulated nucleotide sequences. The PCR testing with various C. difficile ribotypes, other Clostridium spp., and non-Clostridium strains revealed 100% specificity with the ability to detect as low as ~22 genomic copy number per PCR reaction. Different combinations of sample processing were evaluated prior to multiplex PCR for the detection of C. difficile in fecal samples from hospitalized patients. The most optimal condition was the non-selective enrichment at 37 °C for 1 h in brain heart infusion broth supplemented with taurocholate, followed by the multiplex PCR. The detection limit after sample processing was shown as being 5 spores per gram of fecal sample. Two hundred and thirty-eight fecal samples collected from the University affiliated hospital were analyzed by the enrichment multiplex PCR procedure. The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting.

A superior test for diagnosis of Clostridium difficile-associated diarrhea in resource-limited settings.

In this prospective cohort study, we investigated the prevalence of Clostridium difficile-associated diarrhea (CDAD) in adult patients with nosocomial diarrhea by performing enzyme immunoassay (EIA) for detecting toxins A and B and polymerase chain reaction (PCR) for detecting the presence of the tcdB gene in stool samples. We determined the factors associated with CDAD, and the treatment outcome of CDAD from May 2010 to January 2011. A total of 175 stool samples were tested by EIA and PCR. In total, 26.9% patients tested positive for C. difficile: 12.6% by EIA and 24.0% by PCR. The kappa coefficient and total agreement of both the tests were 0.46 and 83.2%, respectively. Onset of diarrhea after antibiotic administration for 10 days or more (OR, 2.71; 95% CI, 1.14-6.44; P = 0.024) and leukocyte count >15,000 cells/mm(3) (OR, 3.12; 95% CI, 1.24-7.88; P = 0.016) were significantly associated with occurrence of CDAD. The non-response rate to CDAD treatment was 24.1%, and the all-cause mortality rate was 31.9% in the CDAD group as against 35.9% in the non-CDAD group (P = 0.721). In our study, the performance of direct PCR of stool samples for detecting tcdB was better, with the number of positive results for stool toxins A and B being twofold higher than that in the case of EIA. Patients who have diarrhea after receiving antibiotics for 10 days or more or those who have a leukocyte count of >15,000 cells/mm(3) should be investigated for CDAD.