Removal and inactivation of indicator bacteriophages in fresh waters A.E. Dura ´n, M. Muniesa, X. Me ´ ndez, F. Valero 1 , F. Lucena and J. Jofre Department of Microbiology. University of Barcelona and 1 Aigu ¨ es Ter-Llobregat (ATLL), Barcelona, Spain 2001/140: received 13 June 2001, revised 22 August 2001 and accepted 7 September 2001 A.E. DURA ´ N, M. MUNIESA, X. ME ´ NDEZ, F. VALERO, F. LUCENA AND J. JOFRE. 2002. Aims: The removal and inactivation of faecal coliform (FC) bacteria, enterococci (ENT), sulphite-reducing clostridia (SRC), somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis in fresh waters. Methods and Results: Removal was studied in two areas of a river. The results showed different removal of each group of microbes. Faecal coliform bacteria were removed faster than any other, whereas SRC and bacteriophages infecting Bact. fragilis were the most persistent. Inactivation was measured by ‘in situ’ experiments, which showed significant differences in survival of the different groups of bacterial and bacteriophage indicators. The SRC and bacteriophages were more resistant than faecal coliforms and enterococci, with the exception of F-specific RNA bacteriophages in the summer. Inactivation experiments with pure cultures of bacteriophages confirmed that phage B40-8 of Bact. fragilis was the most resistant. Conclusions: Bacteria and bacteriophages show different resistance to natural inactivation. The use of phages allows information to be obtained in addition to that provided by bacterial indicators. Somatic coliphages and phages infecting Bact. fragilis might supply that indicator function. Significance and Impact of the Study: Confirmation was obtained that bacteriophages provided additional information to that provided by bacterial indicators to monitor the natural inactivation of viruses and/or pathogens. INTRODUCTION Three main groups of bacteriophages infecting enteric bacteria have so far been considered as potential model micro-organisms for various aspects of water quality assess- ment: somatic coliphages (Hilton and Stotzky 1973; IAWPRC 1991), F-specific RNA bacteriophages (Havelaar et al. 1986; IAWPRC 1991) and bacteriophages infecting Bacteroides fragilis (Tartera and Jofre 1987; IAWPRC 1991). The index and indicator functions can be attributed to the model organisms (IAWPRC 1991; Armon and Kott 1996). Accordingly, an index organism is one that is related to the occurrence of the surrogate micro-organism or micro- organisms. The model organism with an indicator function has a much broader definition. An indicator is basically a model that has behavioural characteristics similar to those of the surrogate micro-organism or micro-organisms and that has the same or greater resistance to environmental stresses. Thus, an indicator is used as a marker of the efficiency of treatments or of the variation in numbers of the surrogate micro-organisms in specific environments. The indicator function requires, among other characteristics, resistance to natural inactivation factors and to treatments similar to or slightly greater than that of the surrogate micro-organism or micro-organisms. The reduction in numbers of pathogens and model micro-organisms in sewage and environmental waters on their way from human or animal faeces to man through water and food is a complex process in which many factors are involved. Sedimentation, adsorption and strain- ing only affect the compartment in which the microbes are found, whereas various physicochemical and biological factors inactivate micro-organisms. The behaviour of bacteriophages in nature has been compared with that of viruses and bacterial indicators elsewhere (Bell 1976; Payment et al. 1988; Girone ´s et al. 1989; Morin ˜igo et al. 1992; Chung and Sobsey 1993; Correspondence to: J. Jofre, Department of Microbiology, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain (e-mail: joan@bio.ub.es). ª 2002 The Society for Applied Microbiology Journal of Applied Microbiology 2002, 92, 338–347