Detection of Only Viable Bacterial Spores Using a Live/Dead Indicator in Mixed Populations

This method uses a photoaffinity label that recognizes DNA and can be used to distinguish populations of bacterial cells from bacterial spores without the use of heat shocking during conventional culture, and live from dead bacterial spores using molecular-based methods. Biological validation of commercial sterility using traditional and alternative technologies remains challenging. Recovery of viable spores is cumbersome, as the process requires substantial incubation time, and the extended time to results limits the ability to quickly evaluate the efficacy of existing technologies. Nucleic acid amplification approaches such as PCR (polymerase chain reaction) have shown promise for improving time to detection for a wide range of applications. Recent real-time PCR methods are particularly promising, as these methods can be made at least semi-quantitative by correspondence to a standard curve. Nonetheless, PCR-based methods are rarely used for process validation, largely because the DNA from dead bacterial cells is highly stable and hence, DNA-based amplification methods fail to discriminate between live and inactivated microorganisms. Currently, no published method has been shown to effectively distinguish between live and dead bacterial spores. This technology uses a DNA binding photoaffinity label that can be used to distinguish between live and dead bacterial spores with detection limits ranging from 109 to 102 spores/mL. An environmental sample suspected of containing a mixture of live and dead vegetative cells and bacterial endospores is treated with a photoaffinity label. This step will eliminate any vegetative cells (live or dead) and dead endospores present in the sample. To further determine the bacterial spore viability, DNA is extracted from the spores and total population is quantified by real-time PCR. The current NASA standard assay takes 72 hours for results. Part of this procedure requires a heat shock step at 80 degC for 15 minutes before the sample can be plated. Using a photoaffinity label would remove this step from the current assay as the label readily penetrates both live and dead bacterial cells. Secondly, the photoaffinity label can only penetrate dead bacterial spores, leaving behind the viable spore population. This would allow for rapid bacterial spore detection in a matter of hours compared to the several days that it takes for the NASA standard assay

Methods for Detecting and Quantifying Viable Bacterial Endo-Spores

Methods and systems for detecting viable bacterial endospores in a sample and related methods to quantify viable bacterial endospores in a sample

Process to Selectively Distinguish Viable from Non-Viable Bacterial Cells

The combination of ethidium monoazide (EMA) and post-fragmentation, randomly primed DNA amplification technologies will enhance the analytical capability to discern viable from non-viable bacterial cells in spacecraft-related samples. Intercalating agents have been widely used since the inception of molecular biology to stain and visualize nucleic acids. Only recently, intercalating agents such as EMA have been exploited to selectively distinguish viable from dead bacterial cells. Intercalating dyes can only penetrate the membranes of dead cells. Once through the membrane and actually inside the cell, they intercalate DNA and, upon photolysis with visible light, produce stable DNA monoadducts. Once the DNA is crosslinked, it becomes insoluble and unable to be fragmented for post-fragmentation, randomly primed DNA library formation. Viable organisms DNA remains unaffected by the intercalating agents, allowing for amplification via post-fragmentation, randomly primed technologies. This results in the ability to carry out downstream nucleic acid-based analyses on viable microbes to the exclusion of all non-viable cells

Differences and similarities in enzymes from the neopullulanase subfamily isolated from thermophilic species

Six glycoside hydrolase (GH) family 13 members, classified under the polyspecific neopullulanase subfamily GH13_20 (also termed cyclomaltodextrinase) were analysed. They originate from thermophilic bacterial strains (Anoxybacillus flavithermus, Laceyella sacchari, and Geobacillus thermoleovorans) or from environmental DNA, collected after in situ enrichments in Icelandic hot springs. The genes were isolated following the CODEHOP consensus primer strategy, utilizing the first two of the four conserved sequence regions in GH13. The typical domain structure of GH13_20, including an N-terminal domain (classified as CBM34), the catalytic module composed of the A-and B-domains, and a C-terminal domain, was found in five of the encoded enzymes (abbreviated Amy1, 89, 92, 98 and 132). These five enzymes degraded cyclomaltodextrins (CDs) and starch, while only three, Amy92 (L. sacchari), Amy98 (A. flavithermus) and Amy132 (environmental DNA), also harboured neopullulanase activity. The L. sacchari enzyme was monomeric, but with CD as the preferred substrate, which is an unusual combination. The sixth enzyme (Amy29 from environmental DNA), was composed of the ABC-domains only. Preferred substrate for Amy29 was pullulan, which was degraded to panose, and the enzyme had no detectable activity on CDs. In addition to its different activity profile and domain composition, Amy29 also displayed a different conservation (LPKF) in the fifth conserved region (MPKL) proposed to identify the subfamily. All enzymes had apparent temperature optima in the range 50–65°C, while thermostability varied, and was highest for Amy29 with a half-life of 480 min at 80°C. Calcium dependent activity or stability was monitored in four enzymes, but could not be detected for Amy29 or 98. Tightly bound calcium can, however, not be ruled out, and putative calcium ligands were conserved in Amy98

Blood pressure in adults with cerebral palsy

OBJECTIVES: This systematic review and meta-analysis was designed to determine the overall mean blood pressure and prevalence of hypertension among a representative sample of adults living with cerebral palsy by combining individual participant data. Additional objectives included estimating variations between subgroups and investigating potential risk factors for hypertension. METHODS: Potential datasets were identified by literature searches for studies published between January 2000 and November 2017 and by experts in the field. Samples of adults with cerebral palsy (n ≥ 10, age ≥ 18 years) were included if blood pressure data, cerebral palsy-related factors (e.g. cerebral palsy subtype), and sociodemographic variables (e.g. age, sex) were available. Hypertension was defined as at least 140/90 mmHg and/or use of antihypertensive medication. RESULTS: We included data from 11 international cohorts representing 444 adults with cerebral palsy [median (IQR) age of the sample was 29.0 (23.0-38.0); 51% men; 89% spastic type; Gross Motor Function Classification System levels I-V]. Overall mean SBP was 124.9 mmHg [95% confidence interval (CI) 121.7-128.1] and overall mean DBP was 79.9 mmHg (95% CI 77.2-82.5). Overall prevalence of hypertension was 28.7% (95% CI 18.8-39.8%). Subgroup analysis indicated higher blood pressure levels or higher prevalence of hypertension in adults with cerebral palsy above 40 years of age, men, those with spastic cerebral palsy or those who lived in Africa. BMI, resting heart rate and alcohol consumption were risk factors that were associated with blood pressure or hypertension. CONCLUSION: Our findings underscore the importance of clinical screening for blood pressure in individuals with cerebral palsy beginning in young adulthood.</p

Comparison of Innovative Molecular Approaches and Standard Spore Assays for Assessment of Surface Cleanliness ▿

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces