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. This paper reports, for the first time, H. pylori reduction in a drinking water pilot plant of two slow sand filters (SSF). Inlet water was taken from a gravel filtration system of a rural water supply in Colombia and then inoculated with viable cells of H. pylori. By determining the Genomic Units (GU) through quantitative Polymerase Chain Reaction (qPCR), the concentration of GU/sample was measured. In the inlet water amplification for SSF1 and SSF2 were 5.13 × 102 ± 4.48 × 102 and 6.59 × 102 ± 7.32 × 102, respectively, while for the treated water they were 7.0 ± 5.6 and 2.05 × 101 ± 2.9 × 101 GU/sample for SSF1 and SSF2, respectively. The SSF pilot plant reached up to 3 log reduction units of H. pylori; therefore, since there is not an H. pylori contamination indicator and its periodic monitoring is financially complicated, the SSF could guarantee the drinking water quality necessity that exists in rural areas and small municipalities in developing countries, where infection rates and prevalence of this pathogen are high.

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. Moreover, since commercial networks do not promote them, they are implemented less in everyday practice. The German standard DIN 10135 is an example of this. In this method, before PCR detection, a primary enrichment (16-20 h) followed by a secondary selective enrichment of at least 6 h is needed. Nevertheless, it allows the possibility of only applying the first step if evidence of their correct operation is provided. OBJECTIVE: To evaluate how necessary is the secondary enrichment for DIN 10135 standard. METHODS: Short and complete enrichment steps were compared in the context of the evaluation of the limit of detection for 11 types of food. Additionally, a blind assay was performed with 75 food samples. RESULTS: The data show that a simple primary enrichment may be sufficient and that the second selective enrichment with the tested matrixes would not be strictly essential. The blind study obtained a 98.6% of trueness and precision of 100%. CONCLUSIONS: At least for the end consumer products, a secondary enrichment of 6 h is not necessary for all the products tested. HIGHLIGHTS: In the context of the DIN 10135 standard, the primary enrichment (16-20 h, 37 ± 1°C) can be enough for detecting Salmonella spp.

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. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes. The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

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. Therefore, the aim of our study was to analyse the viability of H. pylori cells in urban surface waters collected at the Vallparadís public park in Terrassa, Barcelona, Spain. The water samples were analysed by viability quantitative polymerase chain reaction (qPCR) using propidium monoazide and specific primers for the H. pylori vacuolating cytotoxin (vacA gene). Viable H. pylori were found in 91.3% of the samples analysed, with an average concentration of 3.46 ± 1.06 log cell 100 mL-1. Our work proves a quick and simple procedure for evaluating viable H. pylori cells in environmental samples by qPCR. Furthermore, the results provide evidence that urban surface waters may contain considerable levels of viable H. pylori cells, thus indicating they are a potential source of infection, which represents a public health concern.

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. In the present work, the yield of dead cell DNA neutralization was enhanced by incorporating two new sample treatment strategies: tube change combined with a double light treatment. This procedure was successfully tested using artificially contaminated food samples, showing improved neutralization of dead cell DNA.

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. This pathogen has been previously detected in well water but its presence has not been confirmed in water networks. Since these bacteria have been detected in water reservoirs, it has been hypothesized that they can access artificial water systems and survive until they find appropriate conditions to proliferate. In this work, their presence in water samples from 19 non-domestic water networks was tested by quantitative polymerase chain reaction (qPCR). Approximately half of the networks (47%) were positive for W. chondrophila and the overall results revealed 20% positive samples (12/59). Furthermore, most of the samples showed low concentrations of the pathogen (<200 genomic units/L). This finding demonstrates that W. chondrophila can colonize some water networks. Therefore, they must be considered as potential infection sources in future epidemiological studies.

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. In the present work, the combination of PEMAX dye, double tube change, and double photo-activation step was established as a strategy to improve vPCR protocol. By combining these approaches, we developed an improved sample treatment protocol able to neutralize DNA signals of up to 5.0×107 dead cells/sample from both pure culture and artificially contaminated food samples. Our results indicate that vPCR can work reliable and has a potential for high throughput detection of live Salmonella cells in food samples, minimizing false-positive signals.

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. We found that if both the incubation and the photoactivation steps are carried out in different microtubes, the dead cell signal is greatly reduced than when those steps are done in the same tube. Therefore, the strategy depicted in this study presents a simple and efficient step in minimizing false-positive signal when employing viability PCR.

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. Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans collection strains were irradiated with monochromatic visible light from light emitting diodes in the presence of 5% and 0.5% eucalyptus (Eucalyptus globulus), clove (Eugenia caryophyllata), and thyme (Thymus vulgaris) essential oils. Microbial levels were measured by plate count on culture media. In this preliminary report, the results differ according to the kind and concentration of antimicrobial oils, the wavelength of light, and the prokaryotic or eukaryotic microorganism. The results support the idea that mainly blue light enhances the innate antimicrobial activity of the essential oils, especially phenols, and could offer a very efficient and natural way to combat microorganisms in several industries and medical applications (cutaneous and oral infections, medical textiles, foodstuffs and fruit surface, etc.).

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. While incubating non-photo-stimulated sperm at 37 °C for 90 min decreased all sperm quality parameters, this reduction was prevented when the previously-described light procedure was applied. This effect was concomitant with an increase in the percentage of sperm with high mitochondrial membrane potential. When sperm were subjected to 'in vitro' capacitation, photo-stimulation also increased the percentage of sperm with capacitation-like changes in membrane structure. On the other hand, treating commercial semen doses intended for artificial insemination with the 10-10-10 photo-stimulation pattern significantly increased farrowing rates and the number of both total and live-born piglets for parturition. Therefore, our results indicate that a precise photo-stimulation procedure is able to increase the fertilising ability of boar sperm via a mechanism that could be related to mitochondrial function.

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. The overall microbial inactivation ratio ranged between 1.4 and 2.9 log-units for heterotrophic plate counts (HPC), from 1.2 to 2.2 log units for total coliforms (TC) and from 1.4 to 2.3 log units for E. coli. The presence of fine granulometry strongly influenced the removal of all the bacterial groups analyzed. This effect was significant for TC (p=0.009), E. coli (p=0.004), and FE (p=0.012). Shallow HSSF constructed wetlands were more effective for removing Clostridium spores (p=0.039), and were also more efficient for removing TC (p=0.011) and E. coli (p=0.013) when fine granulometry was used. On the other hand, changes in the total bacterial community from gravel biofilm were examined by using denaturing gradient gel electrophoresis (DGGE) and sequencing of polymerase chain reaction (PCR)-amplified fragments of the 16S rRNA gene recovered from DGGE bands. Cluster analysis of the DGGE banding pattern from the different wetlands showed that microbial assemblages separated according to water depth, and sequences of different phylogenetic groups, such as Alpha, Beta and Delta-Proteobacteria, Nitrospirae, Bacteroidetes, Acidobacteria, Firmicutes, Synergistetes and Deferribacteres could be retrieved from DGGE bands.

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. Due to this heterogeneity, both dyes were used in the present study to specifically distinguish dead from live Candida albicans cells using viable quantitative PCR (qPCR). The viable qPCR was optimized, and the best results were obtained when pre-treating the cells for 10 min in the dark with 25 µM EMA followed by continuous photoactivation for 15 min. The suitability of this technique to distinguish clotrimazole- and fluconazole-treated C. albicans cells from untreated cells was then assessed. Furthermore, the antifungal properties of two commercial essential oils (Thymus vulgaris and Matricaria chamomilla) were evaluated. The viable qPCR method was determined to be a feasible technique for assessing the viability of C. albicans after drug treatment and may help to provide a rapid diagnostic and susceptibility testing method for fungal infections, especially for patients treated with antifungal therapies.

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. The presence of Waddlia chondrophila was determined in drinking and well water samples (n=70) by quantitative PCR (Q-PCR). Positive results were observed in 10 (25%) of the 40 well samples analyzed; therefore, well water could be a potential reservoir and possible infection source of Waddlia chondrophila in animals and humans.

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. The technology is based on sample treatment with the photoactivatable, and cell membrane impermeant, nucleic acid intercalating dyes ethidium monoazide (EMA) or propidium monoazide (PMA) followed by light exposure prior to extraction of DNA and amplification. Light activation of DNA-bound dye molecules results in irreversible DNA modification and subsequent inhibition of its amplification. Sample pretreatment with viability dyes has so far been mainly used in combination with PCR (leading to the term viability PCR, v-PCR), and increasingly with isothermal amplification method. The principle is not limited to bacteria, but has also successfully been applied to fungi, protozoa and viruses. Despite the success of the method, some practical limitations have been identified, especially when applied to environmental samples. In part they can be minimized by choice of experimental parameters and conditions adequate for a particular sample. This review summarizes current knowledge and presents aspects which are important when designing experiments employing viability dyes.

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. Hence, in addition to targeting total Escherichia coli (i.e., dead and alive) for the detection of fecal pollution, various amoebae may be suitable to indicate the potential presence of pathogenic amoeba-resisting microorganisms, such as Legionellae. Therefore, monitoring amoeba levels by quantitative PCR could be a useful tool for directly and indirectly evaluating health risk and could also be a complementary approach to current microbial quality control strategies for drinking water systems.

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. In the natural history of infections with amoeba-related bacteria encountered in aquatic habitats, the transmissions by environmental aerosols or contaminated water/air systems have been extensively recognized. Therefore, understanding the feasibility of Simkania infection by these or similar routes is relevant. In the present work, we investigated the prevalence of this novel disease-associated microorganism in water samples from different sources by real-time PCR (qPCR). Our results show Simkania detection in 5 of 185 water analyzed samples (2.7%: 2 of 88 cooling towers and 3 of 8 waste water samples). However, no Simkania was detected in a drinking water.