Gemma Agustí

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. pylori. The aim of this study was to determine whether the quantitative PCR combined with propidium monoazide could be an alternative and good technique to determine H. pylori viability in environmental samples and, to contribute to understanding of the role of the H. pylori forms.

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

ABSTRACT. 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. Autoclave and contact lens disinfecting solutions were used to inactivate amoebae. After these inactivation treatments, the results indicated that the PMA pretreatment approach is appropriate for differentiating viable A. castellani, both trophozoites and cysts. Therefore, the PMA‐PCR approach could be useful as a rapid and sensitive analytical tool for monitoring treatment and disease control, assessing effective disinfection treatments, and for a more reliable understanding of the factors that contribute to the interaction amoeba–pathogenic bacteria.

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.

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.

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.

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.).

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.

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.