Phase change materials for portable isothermal incubators as a solution to shorten effective analysis times in microbiological quality control of drinking water.

The microbiological quality control of water for human consumption of parameters relevant as E.coli and total coliforms does not start on the field despite the existence of test methods that could make it possible. One of the things that makes this difficult is the possibility of initiating an effective and reliable incubation at the sampling site. The appearance of isothermal media with phase change materials solves this limitation. When phase change materials combine a relatively high melting heat with a suitable melting temperature adapted to the application temperature, they become excellent materials for thermal protection and for thermal energy storage. Starting the test at the same sampling point means that the effective times to obtain a result are shorter, improving water quality control. On the other hand, operationally, it also allows longer sampling routes. Both aspects are essential for managers responsible for controlling water quality for human consumption. In this work, the evidence that demonstrates the feasibility of this approach is presented.

Understanding the reaction balances behind the viability PCR protocols based on photoreactive dyes.

Viability-PCR (vPCR) protocols are mainly based on photo-reactive dyes impermeant to intact cell membranes. The absence of cell barriers allows the reagent's interaction with the genetic material after a short incubation period. By light-induced reaction, DNA becomes the unsuitable mould for the polymerases and thus cannot be amplified and detected by PCR. General rules and consensus exist on critical aspects of successful vPCR protocol development. However, the understanding of the vPCR reaction concerning how much reagent is really effective or the proper amount of light has been poorly studied. The convenience of using 600 times more dye than bases pairs exist suggests that although these dyes are DNA intercalating reagents, many organic molecules can adsorb it. Concerning light, no exact references exist about how much energy is needed to activate the azide group of reagents such as propidium monoazide. Therefore, it cannot be calculated in terms of energy how much light needs a vPCR protocol. The general rule is to provide reagents and energy in excess. This work provides different responses (based on experimental results) to both questions, which can contribute to a better understanding of the theoretical basis of vPCR protocols.

Key Factors for Removing Bias in Viability PCR-Based Methods: A Review.

During the last decade, the viability PCR approach has been investigated intensively with the goal of detecting solely the DNA derived from intact cells by removing the bias resulting from dead or damaged cell artifacts. However, since the first published paper in 2003 and despite the various continuous improvements, the stubborn reality is that in most cases the goal has not been achieved. Complete neutralization of nucleic acids derived from dead microorganisms was not possible, at least without affecting live cells. However, recently published works in this field show how neutralization rates can be improved. In this communication, we review recent improvements that enable effective vPCR procedures.

Viability qPCR, a new tool for Legionella risk management.

BACKGROUND: Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. METHODS: The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. RESULTS: In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. CONCLUSIONS: The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy.

Quantification of relative proportions of intact cells in microbiological samples using the example of Cryptosporidium parvum oocysts.

UNLABELLED: The fast analysis of relative proportions of live and dead cells can be of great value whether for comparing inactivation efficiencies of different biocidal treatments or for monitoring organisms of interest in environmental samples. We introduce here a straightforward method to determine the percentage of intact cells based on treatment of samples with the viability dye propidium monoazide (PMA). PMA selectively enters membrane-damaged cells and suppresses their PCR detection through modification of their DNA. The study was performed using Cryptosporidium parvum oocysts as a model although the principle should be applicable to other organisms. Validation was performed with defined mixtures of live and heat-killed oocysts and by exposing oocysts to a heat stress gradient. The method correctly indicated increasingly lower proportions of intact cells with increasing temperatures. When comparing the loss of membrane integrity of UV-killed (40 mJ cm(-2) ) oocysts during storage in nonsterile tap water, results suggested that integrity declines slowly (over weeks) and at a rate comparable to non-UV-exposed oocysts. For all experiments, the amplification of longer DNA sequences was found beneficial. In the UV experiment, longer amplicons revealed not only higher sensitivity in excluding membrane-damaged oocysts, but also in excluding DNA with UV-induced damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Whether in the context of microbial ecology or in an industrial context, many questions in microbiology are linked to microbial viability. As cultivation of micro-organisms can be long or may not be possible, fast methods to assess the numbers of live cells are in great demand. We present here a straightforward strategy to determine the relative proportions of intact cells. The PCR-based rapid method is expected to be useful where relative information is sufficient (e.g. for comparing the effect of different antimicrobial treatments on known numbers of micro-organisms) or when the presence of PCR inhibitors does not allow absolute quantification.

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. METHODS AND RESULTS: It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]Bis(4-methoxy)-6-nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. CONCLUSIONS: We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.

Assessment of bismuth thiols and conventional disinfectants on drinking water biofilms.

AIMS: Biofilms in water distribution systems represent a far more significant reservoir of micro-organisms than the water phase. Biofilms are (i) resistant to disinfectants, (ii) nuclei for microbial regrowth, (iii) a refuge for pathogens, (iv) accompanied by taste and odour problems, and (v) corrode surfaces. The effects of the current strategies for disinfection of drinking water systems in large buildings (chlorination, copper and silver ionization, and hyper-heating) were compared with a new generation of bismuth thiol (BT) biocides. METHODS AND RESULTS: Multispecies biofilms were treated with 0.8 mg l(-1) of free chlorine, 400 and 40 microg l(-1) of copper and silver ions, respectively, at 55 and 70 degrees C, and bismuth-2,3-dimercaptopropanol (BisBAL). Furthermore, the effect of combined heat and BisBAL on planktonic cell viability was examined in monoculture using Escherichia coli suspensions. Inactivation rates for BisBAL were similar to copper-silver ions, where the effects were slower than for free chlorine or temperature. The BisBAL effect on E. coli monocultures was augmented greatly by increasing temperatures. CONCLUSIONS: Like copper-silver ions, BTs show more persistent residual effects than chlorine and hyper-heating in water systems. BT efficiency increased with temperature. Like copper-silver ions, BT action is relatively slow. SIGNIFICANCE AND IMPACT OF THE STUDY: BT presents a new approach to containing water biofilms. BT action is not as rapid, but is more thorough than chlorine, and less caustic. BTs may also be more efficacious in hot water systems. At sub-minimum inhibition concentration levels, BTs uniquely inhibit bacterial exopolysaccharide, thereby retarding biofilm formation. Thus, the combination of bactericidal and residual effects may prevent slime build-up in hot water systems.

Factors promoting colonization by legionellae in residential water distribution systems: an environmental case-control survey.

As part of a case-control study of community-acquired Legionnaires' disease, several factors related to residential water distribution systems and public drinking water systems were studied in the homes of 124 patients with community-acquired Legionnaire's disease and in the homes of 354 controls. The presence of water reservoirs and hot water tanks was studied in residential systems. Factors such as deficient chlorine levels, pipe repairs and other work, water flow interruptions, the use of alternative water sources, inadequate cleaning operations in public water reservoirs, and the position of the home within the public network (and whether this location constituted an endpoint) were studied in public water supply systems. Levels of legionellae in domestic water samples were also measured. Although the use of water reservoirs and hot water tanks promotes colonization by legionellae in residential systems, none of the variables studied seems to increase the incidence of community-acquired Legionnaires' disease.

[Community-acquired legionellosis in the Barcelona region between 1992 and 1999: epidemiological characteristics and diagnostic methods]. / Legionelosis comunitaria en las comarcas de Barcelona entre 1992 y 1999: características epidemiológicas y métodos diagnósticos.

BACKGROUND: The epidemiological characteristics of community-acquired legionellosis are not well known. We present here legionellosis cases appearing in Barcelona, Spain, districts during 1992-1999. PATIENTS AND METHOD: Questionnaire on socio-demographic, epidemiological, clinical and diagnostic data. 285 community-acquired cases of legionellosis were notified (67% of total). Incidence values increased from 0.03/100,000 in 1992 to 3.14 in 1999. Mostly used diagnostic method since 1996 was the urinary antigen test. CONCLUSIONS: The incidence of legionellosis increased sharply during the studied period. We believe that use of urinary antigen-based diagnosis allows for a better knowledge of community-acquired legionellosis.