Mariana Fittipaldi

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 todays 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.

Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR

The advent of quantitative PCR has improved the detection of human viral pathogens in the environment. However, a serious limitation of this method may arise from the inability to discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard. To assess whether propidium monoazide (PMA) pre-treatment is a good approach to inhibiting DNA amplification from non-infectious viruses, bacteriophage T4 survival was measured using cell culture titration and real-time PCR with and without PMA pre-treatment. Heat (85 degrees C) and proteolysis methods were carried out. After these inactivation treatments, the results indicated that the PMA pre-treatment approach is not appropriate for differentiating infectious viruses. However, when a heat treatment at 110 degrees C was undertaken, PMA pre-treatment did allow differentiation of non-infectious from infectious viruses. In this case, effective binding of PMA to bacteriophage T4 DNA could be taken to indicate capsid damage. Therefore, PMA pre-treatment may be appropriate for assessing effective disinfection treatments and for a more reliable understanding of the factors that contribute to viral inactivation through capsid damage monitoring. The PMA-PCR approach could be useful as a rapid and inexpensive analytical tool for screening and evaluation of the efficacy of disinfectants.

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.

A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms.

Aims:  It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms.

Error estimation in environmental DNA targets quantification due to PCR efficiencies differences between real samples and standards

IntroductionNowadays, as in the past, our knowledge of microbiology islimited because of the capacity of available technologytools. In recent decades, molecular techniques are playinga central role in the understanding of the microbial world. Inthis sense, a complete scientific evaluation of biologicalsamples is not possible without using molecular biology.The most influential technique has probably been the poly-merasechainreaction(PCR),whichallowsustoworkbeyondclassicalmicrobiology.WithPCR,insomecases,theneedforculture may be avoided. Nevertheless, until the developmentof real-time PCR, our evaluation of the microbial complexityhas been merely qualitative.Real-time PCR is an evolution of the conventional end-point PCR wherein the amplification of a target gene and itsfluorescence detection occurs simultaneously during eachcycle. Different strategies based on the use of nonspecificDNA intercalating dyes or specific fluorescent probes existfor linking target amplification to fluorescence detection.Regardless of the fluorescence emission mechanisms in-volved, real-time PCR enables both detection and quantifica-tion of a genetic target by a continuous fluorescencemonitoring of DNA amplification (Higuchi et al. 1993).An advantage in real-time PCR isthat target quantificationsensitivity is independent of the copy number within thesample (Freeman et al. 1999). Moreover, the key aspect thatdifferentiates real-time PCR from previous semi-quantitativemethodologies is that target DNA concentration can be deter-mined from the fractional cycle at which a threshold amountof target DNA is produced (i.e., measured signal exceeds abackground level), set at a point where amplicon DNA justbecomesdetectablebutisstillwithintheexponentialphaseoftheamplification(Higuchietal.1993;Rasmussen2001).Thisquantificationcycle(Cq)(orcyclethreshold,Ct)areinverselyproportionaltotheamountoftargetnucleicacidinthesample(i.e., the lower the Cq value, the greater the amount of targetnucleic acid in the sample). This approach ensures that inter-fering factors associated with later stages of the amplificationare minimized and considerably improves the measurementprecision (Rasmussen 2001).One of the most important points is that real-time PCRallows quantitative measurements. In order to estimate atarget concentration in environmental samples, absolutequantification can be performed using a standard curveconstructed by amplifying known amounts of target DNA(Rutledge and Cote 2003). Nowadays, this approach is themost commonly used in environmental microbiology.Real-time PCR has many advantages over conventionalendpoint PCR including speed, broad dynamic range ofquantification, reduced risk of contamination, and goodsensitivity. However, despite all these objective strengths,real-time PCR sensitivity is not better than conventionalPCR if a protocol validation is not performed (Bastien etal. 2008).

Quantification of Helicobacter pylori levels in soil samples from public playgrounds in Spain

Helicobacter pylori are ubiquitous Gram-negative bacteria with a high estimated level of infection in the world populations, but a majority of the infected persons are asymptomatic. This pathogen has been classified by the World Health Organization as a class I carcinogen and recognized as the causal agent of most peptic ulcers and chronic gastritis that might lead to stomach cancer. Although not all the transmission pathways of these bacteria into humans have been properly identified, enough data have suggested that the oral-oral or fecal-oral ones are the main infection routes. Helicobacter pylori have been detected in non-treated water and in drinking water, which suggested that water might be an important infection source. As childhood is the critical period of infection, the aim of the present work was to examine the presence of Helicobacter pylori in soil samples from public playing areas of Spanish parks.

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.