A comparison of DNA fragmentation methods - Applications for the biochip technology.

The efficiency of hybridization signal detection in a biochip is affected by the method used for test DNA preparation, such as fragmentation, amplification and fluorescent labelling. DNA fragmentation is the commonest methods used and it is recognised as a critical step in biochip analysis. Currently methods used for DNA fragmentation are based either on sonication or on the enzymatic digestion. In this study, we compared the effect of different types of enzymatic DNA fragmentations, using DNase I to generate ssDNA breaks, NEBNext dsDNA fragmentase and SaqAI restrictase, on DNA labelling. DNA from different Desulfovibrio species was used as a substrate for these enzymes. Of the methods used, DNA fragmented by NEBNext dsDNA Fragmentase digestion was subsequently labelled with the greatest efficiency. As a result of this, the use of this enzyme to fragment target DNA increases the sensitivity of biochip-based detection significantly, and this is an important consideration when determining the presence of targeted DNA in ecological and medical samples.

A novel cassette method for probe evaluation in the designed biochips.

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip.

Correlation between MMP-9 and extracellular cytokine HMGB1 in prediction of human ischemic stroke outcome.

Ischemic stroke (IS) outcome predictors include clinical features, biochemical parameters and some risk factors. The relations between two main players in the ischemic brain, MMPs and HMGB1, were estimated in the plasma of ischemic stroke patients stratified according to the Glasgow Outcome Scale and the Oxfordshire Community Stroke Project classification. IS patients exhibited higher plasma concentration of MMP-9 and the inflammatory cytokine HMGB1 compared with healthy controls. A full-blown correlation between MMP-9 activation and increased plasma MMP-9 concentration was observed in case of IS patients. A similar activity of MMP-2 and MMP-12 was characteristic of healthy volunteers and IS patients. In patients with ischemic stroke increased plasma levels of MMP-9 and HMGB1 are associated with a poor functional outcome and are significantly correlated with each other (P=0.0054). We suggest that diagnostic benefits will be obtained if plasma HMGB1 levels are measured for IS patients in addition to MMP-9.

Methicillin-resistant Staphylococcus aureus transmission in a low-prevalence healthcare setting.

BACKGROUND: The aim of this study was to assess the nosocomial transmission of methicillin-resistant Staphylococcus aureus (MRSA) and the predictive role of colonization pressure (CP) in a low-prevalence healthcare setting. METHODS: A retrospective analysis of MRSA infection rates from 2004 to 2009 at the Saudi Aramco Dhahran Health Center, Saudi Arabia, was performed. MRSA patient-days, susceptible patient-days, nosocomial incidence and CP were calculated for each month from January 2008 to December 2009. RESULTS: During the study period, 878 cases of MRSA colonization/infection were identified. Of these cases, 777 (88.4%) and 101 (11.5%) were community-acquired MRSA (CA-MRSA) and healthcare-associated MRSA (HA-MRSA) cases, respectively. A decrease in the number of HA-MRSA cases and an increase in the number of CA-MRSA cases were observed during the study period. The incidence of nosocomial infection per 1000 susceptible patient-days was 1.17 in 2008 and 0.7 in 2009. The monthly colonization pressure ranged from 0.1 to 1.62 throughout the 2-year period. Nosocomial transmission was observed in 13 months of the 24-month study period. No association between the CP of the preceding month and the incidence of nosocomial transmission in the subsequent month was observed. CONCLUSION: In a setting of low MRSA prevalence, CP does not appear to be a useful predictor of nosocomial transmission or incidence.

Using the polymerase chain reaction coupled with denaturing gradient gel electrophoresis to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted acute severe pancreatitis.

AIM: To investigate the use of PCR and DGGE to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted severe AP. METHODS: Patients with biochemical and clinical evidence of acute pancreatitis and an APACHE II score > or = 8 were enrolled. PCR and DGGE were employed to detect bacterial translocation in blood samples collected on d 1, 3, and 8 after the admission. Standard microbial blood cultures were taken when there was clinical evidence of sepsis or when felt to be clinically indicated by the supervising team. RESULTS: Six patients were included. Of all the patients investigated, only one developed septic complications; the others had uneventful illness. Bacteria were detected using PCR in 4 of the 17 collected blood samples. The patient with sepsis was PCR-positive in two samples (taken on d 1 and 3), despite three negative blood cultures. Using DGGE and specific primers, the bacteria in all blood specimens which tested positive for the presence of bacterial DNA were identified as E coli. CONCLUSION: Our study confirmed that unlike traditional microbiological techniques, PCR can detect the presence of bacteria in the blood of patients with severe AP. Therefore, this latter method in conjunction with DGGE is potentially an extremely useful tool in predicting septic morbidity and evaluating patients with the disease. Further research using increased numbers of patients, in particular those patients with necrosis and sepsis, is required to assess the reliability of PCR and DGGE in the rapid diagnosis of infection in AP.

Desulfovibrio alaskensis sp. nov., a sulphate-reducing bacterium from a soured oil reservoir.

A novel sulphate-reducing bacterium (Al1T) was recovered from a soured oil well in Purdu Bay, Alaska. Light and atomic force microscopy observations revealed that cells were Gram-negative, vibrio-shaped and motile by means of a single polar flagellum. The carbon and energy sources used by the isolate and the salinity, temperature and pH ranges facilitating its growth proved to be typical of a partial lactate-oxidizing, moderately halophilic, mesophilic, sulphate-reducing bacterium. Analysis of the fatty acid profile revealed that C(18 : 0), isoC(15 : 0) and isoC(17 : 1)omega7c were the predominant species. Fatty acid profile and complete 16S rRNA gene sequencing demonstrated the similarity between strain Al1T and members of the genus Desulfovibrio. The position of strain Al1T within the phylogenetic tree indicated that it clustered closely with Desulfovibrio vietnamensis DSM 10520T (98.9 % sequence similarity), a strain recovered from a similar habitat. However, whole-cell protein profiles, Fourier-transform infrared studies and DNA-DNA hybridization demonstrated that, in spite of the high level of 16S rRNA gene sequence similarity, there is sufficient dissimilarity at the DNA sequence level between D. vietnamensis DSM 10520T and strain Al1T (10.2 % similarity) to propose that strain Al1T belongs to a separate species within the genus Desulfovibrio. Based on the results obtained, the name Desulfovibrio alaskensis sp. nov. is therefore proposed, with Al1T (= NCIMB 13491T = DSM 16109T) as the type strain.

Chemical and structural characterization of exopolymers produced by Pseudomonas sp. NCIMB 2021 in continuous culture.

The growth of marine Pseudomonas sp. NCIMB 2021 as continuous cultures in the presence of surfaces of AISI 316 stainless steel allowed the isolation and partial chemical characterization of exopolymers released into the culture medium (free exopolymers), as well as capsular and biofilm exopolymers. Fourier-transform infrared (FTIR) spectroscopy demonstrated the presence of O- and N-acetylation within the carbohydrate moieties and a predominant 310-helical structure of the protein component, highly resistant to hydrogen/deuterium exchange. Differences between the exopolymers were apparent. Relatively less uronic acid residues were detected in the capsular exopolymers compared to either the biofilm or free exopolymers. O- and N-acetylation were greatest in the biofilm exopolymer. SDS-PAGE protein profiles confirmed differences between exopolymers. The secondary structures of proteins determined using FTIR spectroscopy indicated that the capsular exopolymer had reduced helical content and an increased aggregated strand content compared to the biofilm exopolymer. However, the free exopolymer had an increased beta-sheet component and a reduced unordered component when compared to the biofilm and capsular exopolymers. These data suggest that exopolymer chemistry varies with cellular mode of growth.