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.

Alternative indicators for monitoring Cryptosporidium oocysts in reclaimed water.

With the widespread use of reclaimed water all over the world, there is a clear need to optimise its management in order to guarantee water safety. Model microorganisms (with either indicator or index function) are commonly used to assess risks related to the presence of enteric pathogens in water. Samples from five water reclamation plants located in Northeastern Spain were analysed to validate the use of three model microorganisms (Escherichia coli, somatic coliphages and spores of sulphite-reducing clostridia) as surrogates of Cryptosporidium total or infectious oocysts (TOO and IOO, respectively) in reclaimed water. Probability plots, simple and multiple linear regression and discriminant analyses were performed to assess their relationships. Results show that the detection of E. coli alone is not useful to model either the behaviour or concentrations of Cryptosporidium. However, discriminant analyses showed a high rate of correctly classified samples (91.9%) when E. coli and somatic coliphages data were used together to predict the presence/absence of IOO. Spores of sulphite-reducing clostridia (SRC) showed parallel reduction patterns and high correlation values (r = 0.76) with reductions in TOO. Furthermore, simple regression analyses of SRC and TOO in reclaimed water showed high correlation values (r = 0.85). Therefore, at the treatment plants studied, SRC can be considered to have good indicator and index functions for TOO. From the point of view of health protection, the use of SRC together with E. coli (which is mandatory in the current Spanish regulations) would satisfy the need for improved reclaimed water management.

Direct and indirect QMRA of infectious Cryptosporidium oocysts in reclaimed water.

Water scarcity leads to an increased use of reclaimed water, which in turn calls for an improvement in water reclamation procedures to ensure adequate quality of the final effluent. The presence of infectious Cryptosporidium oocysts (IOO) in reclaimed water is a health hazard for users of this resource. Here, we gathered information on Cryptosporidium (concentrations, infectivity and genotype) in order to perform quantitative microbial risk assessment (QMRA). Moreover, data concerning the spores of sulphite-reducing clostridia (SRC) were used to undertake QMRA at a screening level. Our results show that the probability of infection (PI) by Cryptosporidium depends on the tertiary treatment type. The mean PI using the exponential dose-response model was 3.69 × 10(-6) in tertiary effluents (TE) treated with UV light, whereas it was 3 log(10) units higher, 1.89 × 10(-3), in TE not treated with this disinfection method. With the ß-Poisson model, the mean PI was 1.56 × 10(-4) in UV-treated TE and 2 log(10) units higher, 4.37 × 10(-2), in TE not treated with UV. The use of SRC to perform QMRA of Cryptosporidium showed higher PI than when using directly IOO data. This observation suggests the former technique is a conservative method of QMRA.