Subtracting the background by reducing cell-free DNA's confounding effects on Mycobacterium tuberculosis quantitation and the sputum microbiome.

DNA characterisation in people with tuberculosis (TB) is critical for diagnostic and microbiome evaluations. However, extracellular DNA, more frequent in people on chemotherapy, confounds results. We evaluated whether nucleic acid dyes [propidium monoazide (PMA), PEMAX] and DNaseI could reduce this.

Reduction of Helicobacter pylori cells in rural water supply using slow sand filtration.

Helicobacter pylori is a microorganism that infects 60% of the population and is considered the main cause of atrophic gastritis, gastric and duodenal ulcers, and gastric cancer. Different emerging pathogens have been found in drinking water and their presence is considered to be an important public health problem. For this reason, it is necessary to carry out the validation of reliable technologies for this type of pathogens and evaluate their performance.

Relevance of Secondary Enrichment in the Detection of Salmonella spp. in Food Samples by qPCR According to DIN 10135.

Background: When detecting Salmonella spp. in food samples, unlike with culture-based procedures where there are solid standards, PCR techniques are generally dominated by commercial solutions, often backed by the conformity of reference organizations, and based on rigorous validation studies. The few independent standards that exist are not subject to revision and improvement to the same extent as the manufacturer's methods.

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples.

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent. At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use.

Monitoring levels of viable Helicobacter pylori in surface water by qPCR in Northeast Spain.

Helicobacter pylori infection is a risk factor for chronic active gastritis, peptic ulcers, gastric carcinoma and lymphoma. Although the infection may be acquired through different transmission routes, the presence and viability of H. pylori in water sources are not well known.

Optimization of a Viability PCR Method for the Detection of Listeria monocytogenes in Food Samples.

Rapid detection of Listeria and other microbial pathogens in food is an essential part of quality control and it is critical for ensuring the safety of consumers. Culture-based methods for detecting foodborne pathogens are time-consuming, laborious and cannot detect viable but non-culturable microorganism, whereas viability PCR methodology provides quick results; it is able to detect viable but non-culturable cells, and allows for easier handling of large amount of samples. Although the most critical point to use viability PCR technique is achieving the complete exclusion of dead cell amplification signals, many improvements are being introduced to overcome this.

Presence of Waddlia chondrophila in hot water systems from non-domestic buildings in France.

The presence of Waddlia chondrophila has been related to respiratory tract infections and human and animal fetal death. Although several sources of infection have been suggested, the actual source remains unknown and limited information exists on the prevalence of W. chondrophila in the environment.

Improved sample treatment protocol for accurate detection of live Salmonella spp. in food samples by viability PCR.

Culture-based detection is still considered as the standard way for detection of Salmonella in foods, although molecular methods, such as viability PCR (vPCR), have been introduced to overcome some disadvantages of traditional culture methods. Despite the success of the vPCR methodology, the problem of false-positive results is a major drawback, especially when applied to environmental samples, hindering the interpretation of the results. To improve the efficiency of vPCR, many approaches have been introduced by several authors during the last years.

False-Positive Viability PCR Results: An Association with Microtubes.

Currently, one of the most challenged points to expand the use of viability PCR technique is achieving the complete exclusion of dead cells amplification signals, thus avoiding the overestimation of live cells population. Considering that, and based on the hypothesis that DNA may be retained by microtube walls, the impact of the microtube was addressed on signals from live and heat-killed cells. A double-dye reagent, PEMAX™, which comprises a mix of photo-reactive azide forms of phenanthridium, was used in this work.

Visible light enhances the antimicrobial effect of some essential oils.

The photodisinfection is a topical, broad spectrum antimicrobial technology, targeting bacteria, virus, fungi, and protozoa effective for single cells as for biofilms. Natural molecules have been studied less than synthetic agents in the process but they are currently receiving great interest. Therefore, the aim of this study is to evaluate for the first time if non-coherent blue and red light enhances the antimicrobial activity of some essential oils when standard strains for antibiotic or fungicide tests are enlightened in vitro.

Specific LED-based red light photo-stimulation procedures improve overall sperm function and reproductive performance of boar ejaculates.

The present study evaluated the effects of exposing liquid-stored boar semen to different red light LED regimens on sperm quality and reproductive performance. Of all of the tested photo-stimulation procedures, the best pattern consisted of 10 min light, 10 min rest and 10 min of further light (10-10-10 pattern). This pattern induced an intense and transient increase in the majority of motility parameters, without modifying sperm viability and acrosome integrity.

Cell membrane integrity and distinguishing between metabolically active and inactive cells as a means of improving viability PCR.

Viability PCR uses cell membrane integrity to differentiate live cells from dead. Our new approach improves viability PCR by enabling it to also discriminate between cells with an intact cell membrane and the ability to actively maintain bacterial homeostasis and cells that have an intact membrane but are metabolically inactive.

Key design factors affecting microbial community composition and pathogenic organism removal in horizontal subsurface flow constructed wetlands.

Constructed wetlands constitute an interesting option for wastewater reuse since high concentrations of contaminants and pathogenic microorganisms can be removed with these natural treatment systems. In this work, the role of key design factors which could affect microbial removal and wetland performance, such as granular media, water depth and season effect was evaluated in a pilot system consisting of eight parallel horizontal subsurface flow (HSSF) constructed wetlands treating urban wastewater from Les Franqueses del Vallès (Barcelona, Spain). Gravel biofilm as well as influent and effluent water samples of these systems were taken in order to detect the presence of bacterial indicators such as total coliforms (TC), Escherichia coli, fecal enterococci (FE), Clostridium perfringens, and other microbial groups such as Pseudomonas and Aeromonas.

Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment.

Propidium monoazide (PMA) or ethidium bromide monoazide (EMA) treatment has been used before nucleic acid detection methods, such as PCR, to distinguish between live and dead cells using membrane integrity as viability criterion. The performance of these DNA intercalating dyes was compared in many studies utilizing different microorganisms. These studies demonstrated that EMA and PMA differ in their abilities to identify nonviable cells from mixed cell populations, depending on the microorganism and the nature of the sample.

Well water as a possible source of Waddlia chondrophila infections.

Waddlia chondrophila is an emerging pathogen considered as a potential agent of abortion in humans and bovines, and is related with human respiratory disease. Despite these findings, the infection source and transmission pathways have not been identified. The evidence of growth into amoeba suggests water as a possible environmental source.

Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification.

The ideal scenario in most applications of microbial diagnostics is that only viable cells are detected. Bacteria were traditionally considered viable when they could be cultured, whereas today's viability concept tends to be alternatively based on the presence of some form of metabolic activity, a positive energy status, responsiveness, detection of RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion, although conservative, was the focus of one of the most successful recent approaches to detect viable cells in combination with DNA amplification techniques.

Amoeba-related health risk in drinking water systems: could monitoring of amoebae be a complementary approach to current quality control strategies?

Culture-based methods for fecal indicator microorganisms are the standard protocol to assess potential health risk from drinking water systems. However, these traditional fecal indicators are inappropriate surrogates for disinfection-resistant fecal pathogens and the indigenous pathogens that grow in drinking water systems. There is now a range of molecular-based methods, such as quantitative PCR, which allow detection of a variety of pathogens and alternative indicators.

Searching Simkania negevensis in environmental waters.

Simkania negevensis is an obligate intracellular bacterium grouped into the order Chlamydiales. This new amoeba-resistant bacterium represents a novel aetiologic agent of bronchiolitis and community-acquired pneumonia in both adults and children. It has been suggested that Simkania could be an ubiquitous microorganism presented in water environments.

Prevalence study of Simkania negevensis in cooling towers in Spain.

Simkania negevensis is an obligate intracellular bacterium grouped into the order Chlamydiales. This new amoeba-resistant intracellular bacterium might represent a novel etiologic agent of bronchiolitis and community-acquired pneumonia and occurs in aquatic habitats such as drinking water and reclaimed wastewater. Another amoeba-related bacterium, Legionella pneumophila, is an etiologic agent of pneumonia transmitted by environmental aerosols or contaminated water/air cooling systems.

Discrimination of viable Acanthamoeba castellani trophozoites and cysts by propidium monoazide real-time polymerase chain reaction.

Even though the advent of quantitative polymerase chain reaction (PCR) has improved the detection of pathogen microorganisms in most of areas of microbiology, a serious limitation of this method may arise from the inability to discriminate between viable and nonviable pathogens. To overcome it, the use of real-time PCR and selective nucleic acid intercalating dyes like propidium monoazide (PMA) have been effectively evaluated for different microorganisms. To assess whether PMA pretreatment can inhibit PCR amplification of nonviable amoeba DNA, Acanthamoeba castellani survival was measured using cell culture and real-time PCR with and without PMA pretreatment.

Viability determination of Helicobacter pylori using propidium monoazide quantitative PCR.

Background: While Helicobacter pylori exists in a bacillary form in both the natural habitat and the human host, detrimental environmental circumstances have been observed to lead to the conversion of H. pylori from the bacillary to the coccoid form. However, the viability or nonviability of coccoid forms remains to be established in H.

Prevalence and concentration of non-tuberculous mycobacteria in cooling towers by means of quantitative PCR: a prospective study.

There is an increasing level of interest in non-tuberculous mycobacteria (NTM) due to the increasing reported rates of diseases caused by them. Although it is well known that NTM are widely distributed in the environment it is necessary to identify its reservoirs to prevent possible infections. In this study, we aimed to investigate the occurrence and levels of NTM in cooling towers to provide evidences for considering these settings as possible sources of respiratory infections.