J Jofre

Publications of J Jofre

• Feasibility of methods based on nucleic acid amplification techniques to fulfil the requirements for microbiological analysis of water quality.

  Authors: J Jofre, A R Blanch

  Journal of applied microbiology. 12/2010; 109(6):1853-67.

  Molecular methods based on nucleic acid recognition and amplification are valuable tools to complement and support water management decisions. At present, these decisions are mostly supported by theMolecular methods based on nucleic acid recognition and amplification are valuable tools to complement and support water management decisions. At present, these decisions are mostly supported by the principle of end-point monitoring for indicators and a small number of selected measured by traditional methods. Nucleic acid methods show enormous potential for identifying isolates from conventional culture methods, providing data on cultivable and noncultivable micro-organisms, informing on the presence of pathogens in waters, determining the causes of waterborne outbreaks, and, in some cases, detecting emerging pathogens. However, some features of water microbiology affect the performance of nucleic acid-based molecular techniques and thus challenge their suitability for routine water quality control. These features include the variable composition of target water samples, the generally low numbers of target micro-organisms, the variable water quality required for different uses and the physiological status or condition of such micro-organisms. The standardization of these molecular techniques is also an important challenge for its routine use in terms of accuracy (trueness and precision) and robustness (reproducibility and reliability during normal usage). Most of national and international water regulations recommend the application of standard methods, and any new technique must be validated respect to established methods and procedures. Moreover, molecular methods show a high cost-effectiveness value that limits its practicability on some microbial water analyses. However, new molecular techniques could contribute with new information or at least to supplement the limitation of traditional culture-based methods. Undoubtedly, challenges for these nucleic acid-based methods need to be identified and solved to improve their feasibility for routine microbial water monitoring.

• Quantification of Shiga toxin 2-encoding bacteriophages, by real-time PCR and correlation with phage infectivity.

  Authors: L Imamovic, R Serra-Moreno, J Jofre, M Muniesa

  Journal of applied microbiology. 03/2010; 108(3):1105-14.

  To evaluate a qPCR-based protocol for the enumeration of Shiga toxin (Stx) 2 phages and to compare the results of qPCR with the number of infective Stx phage particles. An approach based on qPCR wasTo evaluate a qPCR-based protocol for the enumeration of Shiga toxin (Stx) 2 phages and to compare the results of qPCR with the number of infective Stx phage particles. An approach based on qPCR was applied to count Stx phages in five phage lysates of known titre. The number of viral particles from each phage lysate was determined by electron microscopy using latex spheres. The infectivity of the Stx phages was evaluated onto three bacterial host strains, by double agar layer assay and plaque blot hybridization. The number of phage particles detected by electron microscopy correlates with the number calculated by qPCR in all the phages assayed. The number of infectious phages was from 1 to 3 log10 units below the numbers obtained by qPCR and electron microscopy. The approach allows accurate quantification of Stx phages with a high recovery. The number of infectious phages is always below the number of phage particles detected by qPCR. The qPCR method is a good approach to enumerate Stx phages. However, these results should be carefully considered when related to the number of infectious phages for each lysate that could be applied in real samples, because values of infectious particles are always below the number of Stx phages detected by qPCR.

• Occurrence and distribution of culturable enteroviruses in wastewater and surface waters of north-eastern Spain.

  Authors: A Costán-Longares, L Mocé-Llivina, A Avellón, J Jofre, F Lucena

  Journal of applied microbiology. 01/2009; 105(6):1945-55.

  AIMS: Update information regarding occurrence and levels of culturable enteroviruses in several types of surface polluted waters in north-eastern Spain and determine the proportion of the differentAIMS: Update information regarding occurrence and levels of culturable enteroviruses in several types of surface polluted waters in north-eastern Spain and determine the proportion of the different species and serotypes. METHODS AND RESULTS: The best procedures on hand in our laboratory for concentrating and quantifying culturable enteroviruses from different water sample types were used. Sequencing was used for typing the virus isolates. Geometric means of enteroviruses densities expressed in plaque forming units per litre were 968 in raw sewage, 12.51 in secondary effluents, 0.017 in tertiary effluents, 0.4 in river water and 0.36 in seawater. Enterovirus densities in wastewater revealed certain seasonality with a maximum at the end of spring - beginning of the summer. Coxsackievirus B, and amid them serotype CB4, were the most abundant species and serotypes detected. CONCLUSIONS: Densities of enteroviruses in different north-eastern Spain surface waters are similar to those present in industrialized countries with temperate climate. No wild polioviruses were detected. Distribution of species showed a clear prevalence of coxsackieviruses. SIGNIFICANCE AND IMPACT OF THE STUDY: Information regarding enteroviruses in this geographical area provides valuable information to estimate the risk of enteroviruses transmission through water and for complementing clinical epidemiological data.

• Is the replication of somatic coliphages in water environments significant?

  Authors: J Jofre

  Journal of applied microbiology. 12/2008;

  Summary Somatic coliphages are amid several groups of bacteriophages that have been suggested as indicators in water quality assessment. One of the limitations frequently endorsed to somaticSummary Somatic coliphages are amid several groups of bacteriophages that have been suggested as indicators in water quality assessment. One of the limitations frequently endorsed to somatic coliphages as indicators is that they can replicate in the water environment. This review intends to evaluate the significance of this potential replication. In view of: the threshold densities of somatic coliphages and host bacteria needed for productive infection to occur, the densities of both host cells supporting somatic coliphages replication and these phages in water environments, and the poor contribution of lysogenic induction to the free somatic coliphage numbers in water, it can be concluded that replication of somatic coliphages in waters is very unlikely. Consequently, the contribution of replication in the environment of somatic coliphages is expected to have a non-noticeable influence on the numbers of somatic coliphages detected in water environments. Thus, the replication in the environment should not be argued as a limitation to the use of somatic coliphages as indicators.

• The CI repressors of Shiga toxin-converting prophages are involved in coinfection of Escherichia coli strains, which causes a down regulation in the production of Shiga toxin 2.

  Authors: R Serra-Moreno, J Jofre, M Muniesa

  Journal of bacteriology. 08/2008; 190(13):4722-35.

  Shiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as Shiga toxin-producing E. coli (STEC). They are encoded in temperate lambdoid phages located onShiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as Shiga toxin-producing E. coli (STEC). They are encoded in temperate lambdoid phages located on the chromosome of STEC. STEC strains can carry more than one prophage. Consequently, toxin and phage production might be influenced by the presence of more than one Stx prophage on the bacterial chromosome. To examine the effect of the number of prophages on Stx production, we produced E. coli K-12 strains carrying either one Stx2 prophage or two different Stx2 prophages. We used recombinant phages in which an antibiotic resistance gene (aph, cat, or tet) was incorporated in the middle of the Shiga toxin operon. Shiga toxin was quantified by immunoassay and by cytotoxicity assay on Vero cells (50% cytotoxic dose). When two prophages were inserted in the host chromosome, Shiga toxin production and the rate of lytic cycle activation fell. The cI repressor seems to be involved in incorporation of the second prophage. Incorporation and establishment of the lysogenic state of the two prophages, which lowers toxin production, could be regulated by the CI repressors of both prophages operating in trans. Although the sequences of the cI genes of the phages studied differed, the CI protein conformation was conserved. Results indicate that the presence of more than one prophage in the host chromosome could be regarded as a mechanism to allow genetic retention in the cell, by reducing the activation of lytic cycle and hence the pathogenicity of the strains.

• The combined performance of UV light and chlorine during reclaimed water disinfection.

  Authors: M Montemayor, A Costan, F Lucena, J Jofre, J Muñoz, E Dalmau, R Mujeriego, L Sala

  Water science and technology : a journal of the International Association on Water Pollution Research. 02/2008; 57(6):935-40.

  The combined effects of disinfectant agents on the microbiological quality of reclaimed water produced by two full-scale water reclamation plants in Catalonia, Spain, were examined in this work. AllThe combined effects of disinfectant agents on the microbiological quality of reclaimed water produced by two full-scale water reclamation plants in Catalonia, Spain, were examined in this work. All the disinfectant treatments tested led to the absence, or near absence, of E. coli in 100 mL samples of water, with log reductions of more than 3 log u. Hypochlorite reduced the bacterial concentrations. However, ultraviolet light was more effective than hypochlorite at reducing the concentrations of bacteriophages, viruses and pathogenic protozoa such as Cryptosporidium spp. We conclude that a combination of these two disinfectant agents is effective in protecting public health, as each agent acts to a different degree against the different groups of microorganisms studied. Further studies should investigate the combined action of disinfectant agents at water reclamation plants with ultraviolet light equipment in more favourable working conditions in order to assess their capacity to inactivate microorganisms.

• Occurrence and levels of indicators and selected pathogens in different sludges and biosolids.

  Authors: C Guzmán, J Jofre, M Montemayor, F Lucena

  Journal of applied microbiology. 01/2008; 103(6):2420-9.

  AIMS: Determine the occurrence and levels of pathogens and indicators in raw and treated sludges and compare their persistence after two different treatments. METHODS AND RESULTS: Helminth ova,AIMS: Determine the occurrence and levels of pathogens and indicators in raw and treated sludges and compare their persistence after two different treatments. METHODS AND RESULTS: Helminth ova, Cryptosporidium spp., Salmonella spp., enteroviruses, and bacterial and viral indicators were determined in raw sludges and biosolids produced after mesophilic and thermophilic treatments. Except Salmonella, all of the parameters were quantified. Helminth ova were found at very low concentrations even in raw sludges. Viable Cryptosporidium oocysts were still present in most samples of treated sludges. Faecal coliforms, spores of sulphite-reducing clostridia (SSRC), and somatic coliphages were the only indicators with values above their detection limits in most of the samples. CONCLUSIONS: Pathogens were still detected in some treated sludge samples. SSRC were the most resistant micro-organisms to treatments and hence may be an indicator for the reduction of protozoan oocysts. Somatic coliphages constitute an alternative as viral indicators due to their detection in sludges before and after treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the persistence of some pathogens after sludge treatments, additional indicators are needed. SSRC and somatic coliphages are good candidates. Easy and inexpensive methods for the determination of these indicators are feasible both in industrialized and developing countries.

• Occurrence of bacterial indicators and bacteriophages infecting enteric bacteria in groundwater in different geographical areas.

  Authors: F Lucena, F Ribas, A E Duran, S Skraber, C Gantzer, C Campos, A Morón, E Calderón, J Jofre

  Journal of applied microbiology. 08/2006; 101(1):96-102.

  AIMS: The aim of this research was to determine the suitability of coliphages (bacteriophages) for assessing the microbial quality of groundwater. METHODS AND RESULTS: The number of several bacterialAIMS: The aim of this research was to determine the suitability of coliphages (bacteriophages) for assessing the microbial quality of groundwater. METHODS AND RESULTS: The number of several bacterial indicators (faecal coliforms, Escherichia coli, enterococci and spores of sulfite-reducing clostridia) and bacteriophages (somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis) were determined in groundwater of aquifers in various geographical areas. Results show that the relative abundance, determined as percentages of positive detections, of the bacterial indicators and bacteriophages varies depending on the aquifer. CONCLUSIONS: A single bacterial indicator may not be enough to assess microbiological quality in certain aquifers. One bacterial indicator and a bacteriophage parameter provide more information than two bacterial indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Coliphages (CPH) provide different information from that provided by bacterial indicators on the microbial quality of groundwater in different geographical areas. Easy, fast and inexpensive methods for the detection of CPH are feasible in both industrialized and developing countries.

• Characterization of Shiga toxin-producing Escherichia coli isolated from aquatic environments.

  Authors: C García-Aljaro, M Muniesa, J E Blanco, M Blanco, J Blanco, J Jofre, A R Blanch

  FEMS microbiology letters. 06/2005; 246(1):55-65.

  This study reports the phenotypic and genotypic characterization of 144 Shiga toxin-producing Escherichia coli (STEC) strains isolated from urban sewage and animal wastewaters using a Shiga toxin 2This study reports the phenotypic and genotypic characterization of 144 Shiga toxin-producing Escherichia coli (STEC) strains isolated from urban sewage and animal wastewaters using a Shiga toxin 2 gene variant (stx(2))-specific DNA colony hybridization method. All the strains were classified as E. coli and belonged to 34 different serotypes, some of which had not been previously reported to carry the stx(2) genes (O8:H31, O89:H19, O166:H21 and O181:H20). Five stx(2) subtypes (stx(2), stx(2c), stx(2d), stx(2e) and stx(2g)) were detected. The stx(2), stx(2c), stx(2d) and stx(2e) subtypes were present in urban sewage and stx(2e) was the only stx(2) subtype found in pig wastewater samples. The stx(2c) and stx(2g) were more associated with cattle wastewater. One strain was positive for the intimin gene (eae) and five strains of serotypes were positive for the adhesin encoded by the saa gene. A total of 41 different seropathotypes were found. On the basis of occurrence of virulence genes, most non-O157 STEC strains are assumed to be low-virulence serotypes.

• Occurrence of Cryptosporidium spp. oocysts in raw and treated sewage and river water in north-eastern Spain.

  Authors: M Montemayor, F Valero, J Jofre, F Lucena

  Journal of applied microbiology. 02/2005; 99(6):1455-62.

  AIMS: To determine the occurrence and levels of Cryptosporidium parvum oocysts in wastewater and surface waters in north-eastern Spain. METHODS AND RESULTS: Samples from five sewage treatment plantsAIMS: To determine the occurrence and levels of Cryptosporidium parvum oocysts in wastewater and surface waters in north-eastern Spain. METHODS AND RESULTS: Samples from five sewage treatment plants were taken monthly and quarterly during 2003. In addition, water was collected monthly from the River Llobregat (NE Spain) during the period from 2001 to 2003. All samples were analysed by filtration on cellulose acetate filters or through Envirocheck using EPA method 1623, followed by immunomagnetic separation and examination by laser scanning cytometry. All raw sewage, secondary effluent and river water samples tested were positive for Cryptosporidium oocysts. Of the tertiary sewage effluents tested, 71% were positive for Cryptosporidium oocysts. The proportion of viable oocysts varied according to the sample. CONCLUSIONS: Two clear maxima were observed during spring and autumn in raw sewage, showing a seasonal distribution and a correlation with the number of cryptosporidiosis cases and rainfall events. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first data on the occurrence of Cryptosporidium oocysts in natural waters in north-eastern Spain.

• Free Shiga toxin bacteriophages isolated from sewage showed diversity although the stx genes appeared conserved.

  Authors: M Muniesa, R Serra-Moreno, J Jofre

  Environmental microbiology. 08/2004; 6(7):716-25.

  Phages carrying the stx2 gene were detected in a range of sewage samples using a plaque hybridization-based method. After detection, phages were isolated and propagated with a laboratory strain ofPhages carrying the stx2 gene were detected in a range of sewage samples using a plaque hybridization-based method. After detection, phages were isolated and propagated with a laboratory strain of Escherichia coli as host for characterization purposes. Although it was not possible to conduct propagation or transduction experiments on most of the phages, 11 reached a sufficiently high titre for studies of host infectivity, electron microscopy and sequencing of the stx2 flanking regions to be performed. These phages showed a wide range of host infectivity and morphology. The genetic structure of the 5' stx flanking region appeared conserved whereas the 3' region differed from that of previously described phages. This is the first description of infectious stx-phages isolated as free particles in the environment, and as such constitutes a new contribution to the study of the ecology of these phages.

• Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments.

  Authors: F Lucena, A E Duran, A Morón, E Calderón, C Campos, C Gantzer, S Skraber, J Jofre

  Journal of applied microbiology. 02/2004; 97(5):1069-76.

  AIMS: To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro-organisms by primary and secondary wastewater treatment processes. METHODS ANDAIMS: To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro-organisms by primary and secondary wastewater treatment processes. METHODS AND RESULTS: The numbers of several bacterial indicators [faecal coliforms (FC), enterococci (ENT) and sulphite-reducing clostridia (SRC)] and bacteriophages (somatic coliphages, F-specific RNA phages and bacteriophages infecting Bacteroides fragilis strain RYC2056) were determined in incoming raw sewage and effluents from various primary and secondary wastewater treatment processes in several geographical areas. Reductions in the numbers of indicators were calculated as log10 reductions. Processes based on removal and mild disinfection, showed no significant differences in the elimination of any of the indicators tested or between geographical areas. In contrast, treatment processes that include strong microbial inactivation, such as lime-aided flocculation and lagooning, showed significant differences between the log10 reductions of the various micro-organisms studied, FC showing the highest reduction and spores of SRC and phages infecting B. fragilis the lowest. CONCLUSIONS: The microbial elimination performance of treatment processes based principally on removal and mild disinfection can be evaluated with a single indicator. In contrast, processes with additional disinfecting capabilities require more than one indicator for accurate evaluation of the treatment; bacteriophages are good candidates for use as second indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages provide additional information for the evaluation of microbial elimination in some treatment plants. The easy, fast and cheap methods available for phage determination are feasible both in industrialized and developing countries.

• Comparison of polyvinylidene fluoride and polyether sulfone membranes in filtering viral suspensions.

  Authors: L Mocé-Llivina, J Jofre, M Muniesa

  Journal of virological methods. 05/2003; 109(1):99-101.

  Low-protein-binding membranes with a pore size of 0.22 microm are used to filter aqueous solutions containing viruses. Virus adsorption to the membranes is avoided if they are made of polyvinylideneLow-protein-binding membranes with a pore size of 0.22 microm are used to filter aqueous solutions containing viruses. Virus adsorption to the membranes is avoided if they are made of polyvinylidene fluoride (PVDF) or if they are made of cellulose esters saturated with beef extract. Recently, a new kind of membrane filter made of polyether sulfone (PES) has become available commercially. The manufacturers claim that such membranes allow the filtration of greater volumes of sample than those made of PVDF. We compared the filtration rate and volume that could be filtered before clogging for these two membranes. The bacteriophage and enterovirus counts were then compared in sewage after filtration through the two membranes. There were no differences in virus recovery after filtration, but PES membranes allowed a higher filtration rate and clogged more slowly. The use of PES membranes is recommended.

• Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas.

  Authors: M Formiga-Cruz, A K Allard, A-C Conden-Hansson, K Henshilwood, B E Hernroth, J Jofre, D N Lees, F Lucena, M Papapetropoulou, R E Rangdale, A Tsibouxi, A Vantarakis, R Girones

  Applied and environmental microbiology. 04/2003; 69(3):1556-63.

  The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. TheseThe distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.

• Usefulness of different groups of bacteriophages as model micro-organisms for evaluating chlorination.

  Authors: A E Durán, M Muniesa, L Mocé-Llivina, C Campos, J Jofre, F Lucena

  Journal of applied microbiology. 02/2003; 95(1):29-37.

  AIMS: To assess the usefulness of bacterial and viral indicators in chlorination processes and to collect quantitative information necessary for risk assessment analysis in water disinfectionAIMS: To assess the usefulness of bacterial and viral indicators in chlorination processes and to collect quantitative information necessary for risk assessment analysis in water disinfection processes based on chlorination. METHODS AND RESULTS: Naturally occurring bacterial indicators, bacteriophages and enteroviruses were determined to evaluate the effect of chlorination in groundwater and secondary sewage effluents. Additionally, the effect of chlorinating on selected bacteriophages, enteroviruses and Escherichia coli was also tested in spiked samples of bottled water and sewage effluents. Results indicate that chlorination inactivates more efficiently bacteria than phages and enteroviruses. Among the human viruses, phages infecting Bacteroides fragilis and selected somatic coliphages belonging to the Siphoviridae family were the most persistent to chlorination. CONCLUSIONS: The three groups of bacteriophages studied were all more resistant to chlorination than bacteria and some of the phages were more resistant than enteroviruses. Results presented here indicate that it is very risky to generalize from information obtained with inactivation experiments done with single isolates of any phage or virus. If possible, inactivation studies should be done with naturally occurring populations. Phages offer a good opportunity for studying naturally occurring populations. Thus, the bacteriophages offer a range of resistance to chlorination that may represent most of the viruses that can be found in water. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported in this study support the inclusion of bacteriophages as additional indicators of the efficiency of water chlorination processes and water quality.

• Detection, enumeration and isolation of strains carrying the stx2 gene from urban sewage.

  Authors: A R Blanch, C García-Aljaro, M Muniesa, J Jofre

  Water science and technology : a journal of the International Association on Water Pollution Research. 02/2003; 47(3):109-16.

  Verotoxigenic Escherichia coli strains have been related with waterborne outbreaks. Besides 0157:H7, several serotypes of E. coli and other enterobacteria have been implicated in outbreaks andVerotoxigenic Escherichia coli strains have been related with waterborne outbreaks. Besides 0157:H7, several serotypes of E. coli and other enterobacteria have been implicated in outbreaks and reported to carry the shiga toxin genes. Shiga toxins, stx1 and stx2, are important virulence factors of these strains. These genes have been linked to bacteriophages and consequently are susceptible to lateral transmission. To better understand the ecology of these genes a study of the presence of the shiga toxin 2 gene (stx2) among coliform bacteria present in sewage samples was carried out. A procedure based on colony hybridisation was developed for the isolation of enterobacteria carrying this gene. Colony growth on Chromocult agar was transferred to a membrane and hybridised with a gene specific probe. The procedure allowed detection of about one colony carrying the gene among around 1,000 faecal coliform colonies. The numbers of bacteria carrying the gene in sewage were also estimated by PCR indicating that the numbers of bacteria carrying the stx2 gene were about 1/1,000 faecal coliforms. The detected numbers by both methods were similar. Positive colony hybridisation was detected in four sewage origins. Fifty-two colonies showing positive signal were isolated from the Chromocult agar plates, confirmed to be stx2 positive by PCR and phenotypically characterised. Results of the characterisation showed certain diversity among the isolates even in isolates from the same sample. Most of these isolates would not have been isolated with the methods regularly used for the isolation of E. coli 0157:H7 strains. The method will allow study of the numbers and characteristics of bacteria carrying the stx2 gene in different water environments and isolate them in order to determine their role in the spread of the gene.

• Occurrence and densities of bacteriophages proposed as indicators and bacterial indicators in river waters from Europe and South America.

  Authors: F Lucena, X Méndez, A Morón, E Calderón, C Campos, A Guerrero, M Cárdenas, C Gantzer, L Shwartzbrood, S Skraber, J Jofre

  Journal of applied microbiology. 02/2003; 94(5):808-15.

  AIMS: To evaluate the feasibility of bacteriophages as a complementary tool for water quality assessment in surface waters from different parts of the globe. METHODS AND RESULTS: Faecal coliformAIMS: To evaluate the feasibility of bacteriophages as a complementary tool for water quality assessment in surface waters from different parts of the globe. METHODS AND RESULTS: Faecal coliform bacteria, enterococci, spores of sulphite-reducing clostridia, somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis were determined by standardized methods in raw sewage and in 392 samples of river water from 22 sampling sites in 10 rivers in Argentina, Colombia, France and Spain, which represent very different climatic and socio-economic conditions. The results showed that the indicators studied maintained the same relative densities in the raw sewage from the different areas. Classifying the river water samples according to the content of faecal coliform bacteria, it can be observed that the relative densities of the different bacterial indicators and bacteriophages changed according to the concentration of faecal coliform bacteria. There was a relative increase in the densities of all groups of bacteriophages and sulphite-reducing clostridia with respect to faecal coliforms and enterococci in the samples with low counts of faecal coliform bacteria. CONCLUSIONS: The numbers of bacterial indicators and bacteriophages were similar in the different geographical areas studied. Once released in rivers, the persistence of the different micro-organisms differed significantly. Bacteriophages and spores of sulphite-reducing clostridia persisted longer than faecal coliforms and enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages in river water samples provide additional information to that provided by bacteria about the fate of faecal micro-organisms in river water. The easy, fast and cheap methods for phage determination are feasible both in industrialized and developing countries.

• Conservation of phage reference materials and water samples containing bacteriophages of enteric bacteria.

  Authors: J Mendez, J Jofre, F Lucena, N Contreras, K Mooijman, R Araujo

  Journal of virological methods. 01/2003; 106(2):215-24.

  The survival was determined in different conservation conditions of: somatic coliphages, F-specific RNA bacteriophages and phages infecting Bacteroides fragilis proposed as model micro-organisms forThe survival was determined in different conservation conditions of: somatic coliphages, F-specific RNA bacteriophages and phages infecting Bacteroides fragilis proposed as model micro-organisms for water quality control. Titres of phages of all groups either in pure culture phage suspensions or in naturally occurring phage suspensions were stable at (-70+/-10) degrees C and at (-20+/-5) degrees C when protected with glycerol. Moreover, phage analysis of stored suspensions demonstrated that their numbers were homogeneous, both between vials and within vials, and consequently they can be used as reference materials. Furthermore, changes in the storage temperature of the vials cause unpredictable changes in the numbers of bacteriophages. Consequently, phage reference materials and samples containing a quantitative number of phages must be maintained and dispatched at a constant temperature. Consequently, the results indicate that bacteriophages should be packed in dry ice during transport and storage. Finally, the number of phages in water samples stored at (5+/-3) degrees C in the dark does not decrease significantly during the first 72 h of storage. In addition, phage concentrates from natural samples obtained by adsorption-elution to cellulose nitrate filters and mixed with 10% glycerol were stable at least for 2 months at (-70+/-10) degrees C and at (-20+/-5) degrees C.

• Occurrence and levels of indicator bacteriophages in bathing waters throughout Europe.

  Authors: N Contreras-Coll, F Lucena, K Mooijman, A Havelaar, V Pierz, M Boque, A Gawler, C Höller, M Lambiri, G Mirolo, B Moreno, M Niemi, R Sommer, B Valentin, A Wiedenmann, V Young, J Jofre

  Water research. 12/2002; 36(20):4963-74.

  Somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, Escherichia coli and enterococci were counted in bathing waters in the late spring and summer. WeSomatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, Escherichia coli and enterococci were counted in bathing waters in the late spring and summer. We tested fresh and marine bathing waters from North, South, East and West Europe expected to contain between 100 and 500 E. coli per 100 ml, although wider ranges were sometimes found. Bacteriophages were counted after concentration, since a preliminary study proved that this step was necessary to obtain positive counts. During monitoring, a first-line quality control with reference materials for bacteria and bacteriophages was performed by all the laboratories participating in the study. The same microbes were also counted in raw sewage samples from various areas in Europe, where the bacterial indicators and the three groups of bacteriophages were detected in roughly the same numbers. All groups of bacteriophages were detected in both fresh and marine bathing waters throughout Europe. Reliable and complete results from 147 samples showed that for log-transformed values, E. coli and bacteriophages were slightly correlated. However, the slope of the regression line changed according to E. coli concentration and the correlation diminished when this concentration was close to zero per 100 ml. The ratios between E. coli and phages in bathing waters differed significantly from those in sewage. The relative amounts of bacteriophages, mainly somatic coliphages and phages infecting Bact. fragilis RYC2056, increased in bathing waters with low E. coli concentration, especially in seawater samples containing < 100 E. coli per 100 ml. The relationship of bacteriophages with respect to enterococci paralleled that of bacteriophages with respect to E. coli. Somatic coliphages and bacteriophages infecting Bact. fragilis are useful to predict the presence of some pathogens with the same origin as present bacterial indicators but with higher survival rates.

• Comparative resistance of phage isolates of four genotypes of f-specific RNA bacteriophages to various inactivation processes.

  Authors: M Schaper, A E Durán, J Jofre

  Applied and environmental microbiology. 09/2002; 68(8):3702-7.

  The effect of natural inactivation in freshwater, chlorination, ammonia, extreme pHs, temperature, and salt content on phage inactivation was evaluated on mixtures of F-specific RNA bacteriophageThe effect of natural inactivation in freshwater, chlorination, ammonia, extreme pHs, temperature, and salt content on phage inactivation was evaluated on mixtures of F-specific RNA bacteriophage isolates belonging to genotypes I, II, III, and IV. The bacteriophages studied were previously but recently isolated from natural samples, characterized as F-specific RNA bacteriophages and genotyped by plaque hybridization with genotype-specific probes. Natural inactivation in river water was modeled by in situ incubation of bacteriophages inside submerged dialysis tubes. After several days bacteriophages of genotype I showed the highest persistence, which was significantly different from that of bacteriophages of genotype II, IV, or III. The pattern of resistance of phages belonging to the various genotypes to extreme pHs, ammonia, temperature, salt concentration, and chlorination was similar. In all cases, phages of genotype I showed the highest persistence, followed by the phages of genotypes II, III, and IV. The phages of genotypes III and IV were the least resistant to all treatments, and resistance of genotypes III and IV to the treatments was similar. Bacteriophages of genotype II showed intermediate resistance to some of the treatments. The resistance of four phages of genotype I to natural inactivation and chlorination did not differ significantly. These results indicate that genotypes III and IV are much more sensitive to environmental stresses and to treatments than the other genotypes, especially than genotype I. This should be taken into consideration in future studies aimed at using genotypes of F-specific RNA bacteriophages to fingerprint the origin of fecal pollution.