Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Sudden Increase and Regrowth of Fecal Coliforms and Escherichia Coli in Wastewater Biosolids After High Solids Centrifugation

Treatment plants that operate either thermophilic or mesophilic anaerobic digesters with centrifugal dewatering processes have consistently observed densities of fecal coliform and Escherichia coli, both indicator bacteria, that decrease during digestion but then increase after dewatering and storage. The increases have been characterized as two separate phenomena to explain this observation: 1) “Sudden Increase,” or SI, which is defined as the increase that occurs immediately after dewatering and 2) “regrowth,” which is defined as an increase during storage of cake samples over a period of hours or days. The SI observation appears to be more prevalent with biosolids that are generated with thermophilic processes and dewatered by centrifugation. Both thermophilic and mesophilic digesters with centrifuge dewatering processes have observed the regrowth phenomena. This research hypothesizes that the SI phenomenon is due to the presence of viable nonculturable (VNC) bacteria that are reactivated during dewatering. In other words, the bacteria were always present but were not enumerated by standard culturing methods (SCM). Analysis of the E. coli density in thermally treated solids by SCMs and quantitative real-time polymerase chain reaction (qPCR) indicated that E. coli densities are often underestimated by SCM. When analyzed with qPCR, the E. coli density after digestion can be 4-5 orders of magnitude greater than the non-detect levels identified by SCMs, which supports the non-culturable hypothesis. The VNC state describes a condition where bacteria are alive but unable to sustain the metabolic process needed for cellular division. Supplements added to culturing media were investigated to determine if the resuscitation of VNC bacteria could be enhanced. The autoinducer molecules Nhexanoyl- L-Homoserine lactone (C6-HSL), 3-oxo-N-octanoyl-L-Homoserine lactone (3-oxo- C8-HSL), and norepinephrine were unable to induce the resuscitation of VNC E. coli. Additional sampling was performed to determine if autoinducer molecules, peroxides, or other as of yet unknown inhibitory agents and toxins could be removed from biosolids during SCM. Culture media supplemented with the peroxide degrading compounds catalase, α-ketoglutaric acid, and sodium pyruvate was unable to resuscitate non-culturable E. coli. The additions of bentonite and exponential growth phase E. coli cell-free supernatant to culturing media were also unable to increase the culturability of E. coli. To remove inhibitory agents and toxins, a cell washing technique was employed prior to performing SCM; however, this cell washing technique may have increased cellular stresses that inhibited resuscitation since cell densities decreased. A novel laboratory-scale dewatering process was also investigated to determine if the SI and regrowth phenomena observed in full-scale centrifugal dewatering could be mimicked in the laboratory using a lab shearing device. Fecal coliform and E. coli densities in laboratory prepared cake samples were observed to be an order of magnitude higher than full-scale dewatered cakes. Additionally, the laboratory-scale dewatering process was able to resuscitate fecal coliforms and E. coli in stored sludge such that the density increased by 4-5 orders of magnitude from nondetect values. Lastly, the addition of aluminum sulfate during centrifuge dewatering at a full-scale utility produced an increased regrowth of fecal coliforms and E. coli that was sustained for 5 days.

The Legal Protection of Fundamental Human Values in Central and Eastern Europe and Modern Biotechnology: Towards European Harmonization

The protection of the fundamental human values (life, bodily integrity, human dignity, privacy) becomes imperative with the rapid progress in modern biotechnology, which can result in major alterations in the genetic make-up of organisms. It has become possible to insert human genes into pigs so that their internal organs coated in human proteins are more suitable for transplantation into humans (xenotransplantation), and micro-organisms that cam make insulin have been created, thus changing the genetic make-up of humans. At the end of the 1980s, the Central and Eastern European (CEE) countries either initiated new legislation or started to amend existing laws in this area (clinical testing of drugs, experiments on man, prenatal genetic diagnosis, legal protection of the embryo/foetus, etc.). The analysis here indicates that the CEE countries have not sufficiently adjusted their regulations to the findings of modern biotechnology, either because of the relatively short period they have had to do so, or because there are no definite answers to the questions which modern biotechnology has raised (ethical aspects of xenotransplantation, or of the use of live-aborted embryonic or foetal tissue in neuro-transplantation, etc.). In order to harmonise the existing regulations in CEE countries with respect to the EU and supranational contexts, two critical issues should be taken into consideration. The first is the necessity for CEE countries to recognise the place of humans within the achievements of modern biotechnology (a broader affirmation of the principle of autonomy, an explicit ban on the violation of the genetic identity of either born or unborn life, etc.). The second concerns the definition of the status of different biotechnological procedures and their permissibility (gene therapy, therapeutic genomes, xenotransplantation, etc.). The road towards such answers may be more easily identified once all CEE countries become members of the Council of Europe and express their wish to join the EU, which in turn presupposes taking over the entire body of EU legislation.

Prevalence and associated factors of viral hepatitis and transferrin elevations in 5036 patients admitted to the emergency room of a Swiss university hospital: cross-sectional study

BACKGROUND: The epidemiology of liver disease in patients admitted to emergency rooms is largely unknown. The current study aimed to measure the prevalence of viral hepatitis B and C infection and pathological laboratory values of liver disease in such a population, and to study factors associated with these measurements. METHODS: Cross-sectional study in patients admitted to the emergency room of a university hospital. No formal exclusion criteria. Determination of anti-HBs, anti-HCV, transferrin saturation, alanine aminotransferase, and obtaining answers from a study-specific questionnaire. RESULTS: The study included 5'036 patients, representing a 14.9% sample of the target population during the study period. Prevalence of anti-HBc and anti-HCV was 6.7% (95%CI 6.0% to 7.4%) and 2.7% (2.3% to 3.2%), respectively. Factors independently associated with positive anti-HBc were intravenous drug abuse (OR 18.3; 11.3 to 29.7), foreign country of birth (3.4; 2.6 to 4.4), non-white ethnicity (2.7; 1.9 to 3.8) and age > or =60 (2.0; 1.5 to 2.8). Positive anti-HCV was associated with intravenous drug abuse (78.9; 43.4 to 143.6), blood transfusion (1.7; 1.1 to 2.8) and abdominal pain (2.7; 1.5 to 4.8). 75% of all participants were not vaccinated against hepatitis B or did not know their vaccination status. Among anti-HCV positive patients only 49% knew about their infection and 51% reported regular alcohol consumption. Transferrin saturation was elevated in 3.3% and was associated with fatigue (prevalence ratio 1.9; 1.2 to 2.8). CONCLUSION: Emergency rooms should be considered as targets for public health programs that encourage vaccination, patient education and screening of high-risk patients for liver disease with subsequent referral for treatment if indicated.

Design of a lab scale direct-filtration system and its application in process lab

The Environmental Process and Simulation Center (EPSC) at Michigan Technological University started accommodating laboratories for an Environmental Engineering senior level class CEE 4509 Environmental Process and Simulation Laboratory since 2004. Even though the five units that exist in EPSC provide the students opportunities to have hands-on experiences with a wide range of water/wastewater treatment technologies, a key module was still missing for the student to experience a full cycle of treatment. This project fabricated a direct-filtration pilot system in EPSC and generated a laboratory manual for education purpose. Engineering applications such as clean bed head loss calculation, backwash flowrate determination, multimedia density calculation and run length prediction are included in the laboratory manual. The system was tested for one semester and modifications have been made both to the direct filtration unit and the laboratory manual. Future work is also proposed to further refine the module.

Comparison of the effects of inquiry-based cooperative learning and demonstrations in science education

The reported research project involved studying how teaching science using demonstrations, inquiry-based cooperative learning groups, or a combination of the two methods affected sixth grade students’ understanding of air pressure and density. Three different groups of students were each taught the two units using different teaching methods. Group one learned about the topics through both demonstrations and inquirybased cooperative learning, whereas group two only viewed demonstrations, and group three only participated in inquiry-based learning in cooperative learning groups. The study was designed to answer the following two questions: 1. Which teaching strategy works best for supporting student understanding of air pressure and density: demonstrations, inquirybased labs in cooperative learning groups, or a combination of the two? 2. And what effect does the time spent engaging in a particular learning experience (demonstrations or labs) have on student learning? Overall, the data did not provide sufficient evidence that one method of learning was more effective than the others. The results also suggested that spending more time on a unit does not necessarily equate to a better understanding of the concepts by the students. Implications for science instruction are discussed.