F Lucena

Publications of F Lucena

• The persistence of bifidobacteria populations in a river measured by molecular and culture techniques.

  Authors: X Bonjoch, F Lucena, A R Blanch

  Journal of applied microbiology. 05/2009;

  Abstract Aims: To determine relative to faecal coliforms (FC) and sulfite-reducing clostridia (SRC), the environmental persistence of natural populations of Bifidobacterium spp. enumerated byAbstract Aims: To determine relative to faecal coliforms (FC) and sulfite-reducing clostridia (SRC), the environmental persistence of natural populations of Bifidobacterium spp. enumerated by culturing and quantitative polymerase chain reaction (q-PCR). Methods and Results: Dialysis tubing containing river supplemented with overnight cultures of Bifidobacterium adolescentis (BA) and Bifidobacterium dentium (BD) or urban wastewater were suspended in a river for up to 10 days. At intervals, the contents of each dialysis tube were assayed using q-PCR assays for BA and BD, and selective culture media for FC, SRC, total bifidobacteria (TB), sorbitol-fermenting bifidobacteria (SFB) and cultivable BA. Mean summer T(90) values were 251 h for SRC, 92 h for FC, 48 h for BA and BD by q-PCR, and 9 h for TB. Conclusions: Bifidobacterium spp. was the population with the lowest persistence, showing seasonal differences in T(90) when measured by culture techniques or by q-PCR. This difference in relative persistence is because of a longer persistence of molecular targets than cultivable cells. Significance and Impact of the Study: The persistence of a viable bifidobacteria cells is shorter, but the longest persistence of molecular targets. This factor could be used for origin the faecal pollution in water for the development of microbial source tracking (MST).

• 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.

• 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.

• Characterization of bacterial coliform occurrences in different zones of a drinking water distribution system.

  Authors: A R Blanch, B Galofré, F Lucena, A Terradillos, X Vilanova, F Ribas

  Journal of applied microbiology. 04/2007; 102(3):711-21.

  AIMS: To compare the bacterial coliforms detected from occurrences in three zones of a water distribution system supplied by two separate water sources. METHODS AND RESULTS: Conventional andAIMS: To compare the bacterial coliforms detected from occurrences in three zones of a water distribution system supplied by two separate water sources. METHODS AND RESULTS: Conventional and standardized protocols for identifying enterobacterial populations were applied. Additional tests to confirm isolates were included. Analyses of diversity and population similarity were performed using the Phene Plate System, a miniaturized biochemical phenotyping method. Isolates were identified by the API 20E system in tandem with biochemical phenotyping. A total of 16 576 samples were taken from the water distribution system, with 1416 isolates analysed. A low number of coliform occurrences were observed (2%). Escherichia coli was not detected in either water origin or in Zone 2 samples; however, in Zones 1 and 3 a low number of cases of E. coli were recorded. The percentages of E. coli depended on the identification criteria. Eight biochemical profiles for coliform populations were defined according to the results of the confirmative tests. There was a high diversity among these populations in the three zones studied, although no significant variations in their composition (associated with occurrences in the different zones) were observed. Klebsiella oxytoca was the most commonly detected species irrespective of zone, although seven other enterobacterial genera were also found. CONCLUSIONS: Analysis of the enzymatic activity of beta-glucuronidase or application of the criteria established in the norm ISO 9308-1, in tandem with thermotolerance was needed to evaluate the occurrence of E. coli in the distribution systems. Detected occurrences of bacterial coliforms could be associated with re-growth patterns for specific sampling points in the distribution system. Seasonal differences, independent of the studied zones, were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical phenotyping of bacterial coliforms was shown to be a useful method on the characterization of occurrences in water distribution systems.

• 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.

• 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.

• 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.

• Comparative analysis of viral pathogens and potential indicators in shellfish.

  Authors: I Muniain-Mujika, M Calvo, F Lucena, R Girones

  International journal of food microbiology. 06/2003; 83(1):75-85.

  Shellfish can be responsible of outbreaks of infectious diseases and current health measures do not guarantee the absence of viral pathogens in this product. Here we examine the presence ofShellfish can be responsible of outbreaks of infectious diseases and current health measures do not guarantee the absence of viral pathogens in this product. Here we examine the presence of pathogenic viruses and potential indicators in shellfish in a comparative analysis.Sixty shellfish samples collected in three areas with different levels of faecal contamination were analysed for Escherichia coli, total coliforms, Clostridium perfringens, somatic coliphages, F-specific phages of RNA (F-RNA), bacteriophages infecting Bacteroides fragilis RYC2056, human adenovirus, enterovirus and hepatitis A virus (HAV). Viruses were eluted in a glycine buffer at pH 10.The overall percentage of viral pathogens detected was 47% for human adenoviruses, 19% for enteroviruses and 24% for HAV. Since all the samples positive for enterovirus and HAV were also positives for human adenovirus, the latter may be considered useful as a molecular index of viral contamination in shellfish. No significant differences in the bioaccumulation of bacteria and bacteriophages for oysters or mussels were observed. It was found that the probability of detection of any of the pathogenic virus decreases as the temperature of shellfish growing waters increases. However, the probability of detecting viruses increases when phages of B. fragilis are found. Although more data are needed in order to fulfil the need of viral indicators for controlling the presence of human viruses in shellfish, the obtained results indicate that phages infecting B. fragilis RYC2056 could be a suitable group of bacteriophages to be used as an indicator of the presence of viruses in shellfish.

• 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.

• 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.

• Depuration dynamics of viruses in shellfish.

  Authors: I Muniain-Mujika, R Girones, G Tofiño-Quesada, M Calvo, F Lucena

  International journal of food microbiology. 08/2002; 77(1-2):125-33.

  The consumption of shellfish has been associated with viral infections even in cases where shellfish complied with the current regulation, which is based on bacterial analysis. In this study,The consumption of shellfish has been associated with viral infections even in cases where shellfish complied with the current regulation, which is based on bacterial analysis. In this study, depuration rates of potential indicators and human viruses have been analysed in order to study the use of complementary parameters for evaluating the microbiological quality of depurated shellfish. Depuration of naturally highly polluted mussels has been evaluated and analyses for Escherichia coli, Clostridium perfringens, somatic coliphages, F-RNA phages and bacteriophages infecting Bacteroides fragilis RYC2056 and HSP40, human adenovirus, enterovirus have been done. Seawater of the depuration tank was disinfected by UV irradiation, ozone and passed through a skimmer and a biological filter. The correct functioning of the depuration tank was monitored by the quantification of total organic carbon (TOC), NH4+ and total aerobic bacteria in the seawater. To study the relation between the bacteriophages and the human viruses analysed, a logistic regression model was applied. F-RNA phages are significantly related to human adenoviruses and enteroviruses. Thus, they can be used as a complementary parameter for evaluating the efficiency of the depuration treatment. Somatic coliphages are also significantly associated with enteroviruses. Bacteriophages infecting B. fragilis HSP40 were analysed by the double-agar-layer (DAL) method, which quantifies infectious viruses, and by nested PCR, which detects the presence of the genome of these phages. The highest sensitivity of the molecular techniques was demonstrated and the results obtained are an indicator of a close relation between positive results by PCR and the presence of infectious viral particles in shellfish. All shellfish samples were negative for human viruses by PCR after 5 days of depuration treatment and the results obtained applying a regression model also showed negative results or nearly for F-RNA phages and bacteriophages infecting B. fragilis RYC2056. Thus, in this specific depuration treatment, 5 days may be necessary to assess the sanitary quality of shellfish.

• Removal and inactivation of indicator bacteriophages in fresh waters.

  Authors: A E Durán, M Muniesa, X Méndez, F Valero, F Lucena, J Jofre

  Journal of applied microbiology. 02/2002; 92(2):338-47.

  AIMS: The removal and inactivation of faecal coliform (FC) bacteria, enterococci (ENT), sulphite-reducing clostridia (SRC), somatic coliphages, F-specific RNA bacteriophages and bacteriophagesAIMS: 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.

• Effects of different nutrients on bacterial growth in a pilot distribution system.

  Authors: J Frias, F Ribas, F Lucena

  Antonie van Leeuwenhoek. 11/2001; 80(2):129-38.

  The growth of bacterial communities in drinking water distribution systems can lead to the development of problems incompatible with water quality requirements. This study was carried out in order toThe growth of bacterial communities in drinking water distribution systems can lead to the development of problems incompatible with water quality requirements. This study was carried out in order to determine which factors promote bacterial growth in distribution networks. A pilot distribution system was used to perform these experiments. After addition of three different inorganic elements to the network (N, P and S) the results obtained show that they did not contribute to the growth or bacteria either in the circulating water or on the surface of the pipes of the distribution system. However, when organic carbon was added an increase in the number of circulating bacteria was observed though the number of bacteria in the biofilms of the network was constant. These results indicate that in the drinking water of the Barcelona distribution system the factor that controls the growth of bacteria is organic carbon. Moreover, bacteria from the biofilm growing on the surface of the pipes may cause the problems of bacterial growth.

• Optimisation and standardisation of a method for detecting and enumerating bacteriophages infecting Bacteroides fragilis.

  Authors: R Araujo, M Muniesa, J Méndez, A Puig, N Queralt, F Lucena, J Jofre

  Journal of virological methods. 05/2001; 93(1-2):127-36.

  A method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis has been standardised. The recommended host strain is RYC2056 (ATCC 700786) because of the relatively highA method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis has been standardised. The recommended host strain is RYC2056 (ATCC 700786) because of the relatively high counts (10(4)-10(5) PFU/100ml) that it recovers in sewage from very different geographical areas. The addition of 0.25% bile to the culture and assay media and the manipulation of the host strain under strict anaerobic conditions resulted in a significant increase (more than 100%) in the number of phages detected. No other changes in the media and culture conditions resulted in changes in the phage counts detected. However, these increases do not justify changing the culture conditions and media described, taking into consideration that bile renders the media cloudy making it difficult to follow the host growth and that most laboratories do not have the facilities to work under strict anaerobic conditions. Nalidixic acid (100 microg/ml) and kanamycin (100 microg/ml) in the assay medium significantly reduce the background flora from polluted samples without affecting the phage counts. Freezing cultures just before the end of the log-phage growth at (-70+/-10) degrees C with BSA-sucrose as cryoprotector, storing of 1-2 ml in glass vials at (-70+/-10) degrees C and using them directly to inoculate fresh broth allows the obtention of cultures ready for phage enumeration in about 2.5 h. All these developments have been incorporated into a procedure that makes the method for detecting phages infecting B. fragilis as workable as the standardised methods available for the detection of coliphages.

• Viral contamination of shellfish: evaluation of methods and analysis of bacteriophages and human viruses.

  Authors: I Muniain-Mujika, R Girones, F Lucena

  Journal of virological methods. 10/2000; 89(1-2):109-18.

  Viral outbreaks attributed to the consumption of contaminated shellfish have been clearly demonstrated. Thirty-five samples of mussels collected from areas with two different levels of faecalViral outbreaks attributed to the consumption of contaminated shellfish have been clearly demonstrated. Thirty-five samples of mussels collected from areas with two different levels of faecal pollution were analysed for somatic coliphages, F-RNA phages and bacteriophages infecting Bacteroides fragilis HSP40 and RYC2056 following standardised protocols, and for enterovirus, human adenovirus and hepatitis A virus by nucleic acid amplification (Nested-PCR and RT-PCR). Four methods for viral recovery from shellfish have been compared. The first method is based on borate buffer at pH 9.5 as eluent, the second is based on glycine buffer at pH 10 as eluent, a third method is based on glycine buffer at pH 7.5 and changes in conductivity and the fourth method on nutritive broth with Tween 80 as eluent. The results obtained were analysed statistically and the method based in glycine buffer at pH 10 seems to be the most efficient and useful for the recovery of phages and human viruses. The results also show a different pattern in the proportions between the viral parameters when the source of the faecal pollution is close to or distant from the shellfish growing area.

• Description of a DNA amplification procedure for the detection of bacteriophages of Bacteroides fragilis HSP40 in environmental samples.

  Authors: M Puig, S Pina, F Lucena, J Jofre, R Girones

  Journal of virological methods. 10/2000; 89(1-2):159-66.

  A molecular test based on DNA amplification by PCR was developed for the detection of bacteriophages of Bacteroides fragilis strain HSP40 in the environment. These specific phages are associated withA molecular test based on DNA amplification by PCR was developed for the detection of bacteriophages of Bacteroides fragilis strain HSP40 in the environment. These specific phages are associated with faecal contamination of human origin. A homologous DNA region of 1.5 kb, identified previously by hybridisation, was used to design primers for the detection of B. fragilis HSP40 phages. A nested-PCR procedure for the DNA amplification of those phages was developed. The sensitivity of the nested-PCR was between 10(-1) and 10(-2) PFU for purified HSP40 phage solutions, sewage and seawater samples, and between 1 and 10 PFU for river water samples. Specific amplification of HSP40 phages was observed when viral suspensions of 10(3) PFU/ml or lower were used. Common levels of B. fragilis phages found in sewage are 10(1)-10(2) PFU/ml. A total of 24 water samples (sewage, river water and seawater) were tested both by PCR and by plaque assay, to evaluate the efficiency of the molecular method in field samples. The data obtained by PCR in environmental samples showed good concordance with the PFU counts and a higher sensitivity.