Development of a standarised protocol for analyses somatic coliphages in sludge, soil and treated biowastes [Final Report]

Projecte: Final report. Deliverable 3_4.2. EU project (HORIZONTAL-HYG) Horizontal Standards on Hygienic Microbiological parameters for Implementation of EU Directives on Sludges, Soils, Soil Improvers, growing Media and Biowastes. Project/Contract no. SSPI-CT-2004-513660.Informe final HORIZONTAL – HYG. Bacteriòfags.An initial desk study indicated that many biosolids contain human virus, but also that even in the more contaminated ones, they are not very abundant and that they are difficult to recover and quantify. Moreover, feasible methods for detecting infectious viruses are only available for enteroviruses, and it is well known that infectious viruses are needed for risk assessment. The need of indicators seemed obvious, as it was clear that the traditional bacterial indicators are not a good option for predicting presence and behaviour of human viruses in biosolids, biowastes and soils. Bacteriophages appeared as more suitable indicators, and among the proposed groups of phages, somatic coliphages aroused as those potentially more useful attending to their numbers in biosolids, feasibility of the detection methods and for sharing behaviour with viruses in biosolid and biowastes processing. Consequently, at present, somatic coliphages appear as a very useful indicator. Moreover, feasible standardised methods (ISO (adopted by CEN) and USEPA) for detection and quantification of somatic coliphages suspended in water, and consequently in aqueous solutions, are available. As well, literature available indicated that as human viruses, bacteriophages present in biosolids tend to be either included in or adsorbed to particles. Consequently, an extraction steep is necessary. There is a relatively abundant literature regarding extraction of human viruses and bacteriophages from solids. The methods for extracting bacteriophages (and also human viruses) from solids require the following steeps: homogenization, elution, clarification and decontamination. Described methods mostly vary in the elution steep. In a pair of papers comparing several elution methods showed elution with beef extract as the more efficient one. However the described methods required optimization in the various steeps. The optimization of the various steeps was the main aim of this work. Unfortunately, inoculation of biosolids with known concentrations of viruses (as it is feasible with water samples) are not mimicking what happens in the real world because of inclusion and adsorption of phages in/to solid particles. Therefore, it was decided to perform the experiments of optimization of the extraction method with matrixes containing concentrations as high as possible of naturally occurring somatic coliphages. The studied matrixes were raw sludge, digested and dewatered sludge, selected (for its content of somatic coliphages) compost and soil contaminated with raw sewage. Some of these matrixes contained very homogeneous and steady numbers of phages and consequently it was possible to compare modifications of each one of the steeps of the extraction method and how they affected the efficiency of recovery. The method was optimised, published in scientific literature (Guzman et al. 2007. J. Virol. Methods 144: 41-48) and a draft of a “standard method” for the extraction of somatic coliphages from biosolids, biowastes and soils has been presented for approval to the CEN/TC308/WG1/TG5. The non-convenience of inoculating these sort of matrices, since as said previously, the inoculated material does not mimic what happens in nature, complicated the performance of validation studies. Reference materials are needed for validation studies. To get round this problem, a few natural biosolids were tested as potential reference materials. For it, the matrices were distributed in a great number of containers, and placed at 4ºC. Then, somatic coliphages were enumerated after different days of storage from two containers and also from two subsamples of each one of the containers. This allowed testing the intra and inter-container homogeneity and the time elapsed without significant descent in the number of phages detected. Digested-dewatered sludge probed to be an excellent reference material lasting in perfect condition for at least 2 months. This will allow to make validation multilaboratory studies and have a reference material for “in lab” quality control. In fact, a small validation study of the extraction method with three laboratories was performed with satisfactory results. A few experiments done with other phages, as for example F-specific RNA bacteriophages, indicate that the method will also be applicable to other phages. As well, it may be useful for extracting human and animal viruses, though this should be further verified. In conclusion, a feasible, fast and low cost method for determining somatic coliphages from biosolids, biowastes and soils is available. Besides the feasibility of the methods for extraction, detection and enumeration, somatic coliphages have, in our opinion, several advantages to follow the higienization processes of sludges as well as to have an indication of the viral contamination of these solid matrixes

Assessing the usefulness of clostridia spores for evaluating sewage sludge hygienization

The capability of clostridia spores to act as pathogen indicators in sewage sludge treatment was investigated. Sulfite-reducing clostridia and E. coli levels were monitored during waste activated sludge pre-treatments (alkali and ultrasound) and its subsequent mesophilic anaerobic digestion. E. coli was maintained or reduced depending on treatment type and intensity. However, alkali pre-treatment (35.3 gNaOH/kg TS) by itself and alkali (157 gNaOH/kg TS) and ultrasound (27,000 kJ/kg TS) pre-treatments followed by anaerobic digestion provoked reproducible clostridia increases. Specifically, up to 2.7 log10 after 35.3 gNaOH/kg TS pre-treatment and up to 1.9 and 1.1 log10 after digesting the 157 gNaOH/kg TS and 27,000 kJ/kg TS pre-treated sludge, respectively. Having rejected the hypotheses of sporulation and floc dissipation, the most plausible explanation for these clostridia increases is re-growth. These results question the suitability of clostridia spores as indicators of sludge treatment and other biological treatments where clostridia may have a role

Unravelling the composition of tap and mineral water microbiota: Divergences between next-generation sequencing techniques and culture-based methods

The complex and highly diverse microbial environment of drinking water, consisting mainly of bacteria at different metabolic states, is still underexplored. The aim of this work was to characterize the bacterial communities in tap water and bottled mineral water, the two predominant sources of drinking water in modern societies. A total of 11 tap water samples from a range of locations and distribution networks and 10 brands of bottled natural mineral water were analysed using two approaches: a) heterotrophic plate counts by matrixassisted laser desorption/ionization time of flight mass-spectrometry (MALDI-TOF MS) for the culturable heterotrophic communities, and b) Illumina amplicon sequencing for total bacteria including non-culturable bacteria. Culturable heterotrophic bacteria were isolated in WPCA (ISO) agar at 22 ± 2 °C for 72 h and 2046 isolates were identified using MALDI-TOF MS. The Bruker Daltonics Library and a previously customized library (Drinking Water Library) were used as reference databases. For the total bacteria fraction, DNA was extracted from 6 L of water and submitted to Illumina 16S rRNA sequencing of the v4 region. Significant differences were observed between mineral and tap water, with a general dominance of Alphaproteobacteria (mainly the genus Blastomonas) in tap water and Gammaproteobacteria in mineral water with Acidovorax being the dominant genus in 3 out of 7 mineral water brands. The bacterial communities in the different brands of mineral water were highly diverse and characteristic of each one. Moreover, the season in which the water was bottled also affected the species distribution, with some of them identified in only one season. Among the culturable bacteria, the most abundant phylum was Proteobacteria (around 85% of the isolates), followed by Actinobacteria, Firmicutes and Bacteroidetes. Proteobacteria was also the most abundant phylum detected with Illumina sequencing (> 99% of the reads). The two methods gave distinct results at the different taxonomic levels and could therefore have a complimentary application in the study of microbiota in mineral water environments. MALDI-TOF MS is a promising method for the rapid identification of heterotrophic bacteria in routine water analysis in the bottling industry. Significance and impact of the study: The complementarity of MALDI-TOF MS and NGS in the assessment of bacterial community diversity has been demonstrated in water intended for human consumption. The two methods are suitable for routine use in the water industry for water quality management

Virus entèrics a aigües urbano-industrials

Increasing amounts of wastewater are reeled for human use. Neither the natural mechanism of virus inactivation, which vary from water to water, nor the methods used by man for water purification can guarantee the complete elimination of infectious viruses. Although the importance of the presence of human viruses in waters has not been fully stablished as the cause of viral infectious diseases, it becomes evident that the evaluation of viruses in waters is of major relevance. Such evaluation presents a great deal of difficulties, the most important one being the lack of indicator cells for the majority of human enteric viruses. So far, only Enteroviruses are well evaluated. The usual need of virus concentration prior to the evaluation of the viral load is another difficulty. There are a good deal of concentration methods which represent the main difference among the procedures used to evaluate the amount of virus in waters. Using the technique of adsorption-elution on glass powder and BGM cells as indicator system we have been evaluating in the last years the presence of Enterovirus in superficial waters in Barcelona and Surroundings. Poliovirus, Coxsackievirus and Echovirus have been detected either in the rivers (Llobregat and Besos) and in seawater

Effect of ultrasound, low-temperature thermal and alkali pre-treatments on waste activated sludge rheology, hygienization and methane potential

Waste activated sludge is slower to biodegrade under anaerobic conditions than is primary sludge due to the glycan strands present in microbial cell walls. The use of pre-treatments may help to disrupt cell membranes and improve waste activated sludge biodegradability. In the present study, the effect of ultrasound, low-temperature thermal and alkali pre-treatments on the rheology, hygienization and biodegradability of waste activated sludge was evaluated. The optimum condition of each pre-treatment was selected based on rheological criteria (reduction of steady state viscosity) and hygienization levels (reduction of Escherichia coli, somatic coliphages and spores of sulfite-reducing clostridia). The three pre-treatments were able to reduce the viscosity of the sludge, and this reduction was greater with increasing treatment intensity. However, only the alkali and thermal conditioning allowed the hygienization of the sludge, whereas the ultrasonication did not exhibit any notorious effect on microbial indicators populations. The selected optimum conditions were as follows: 27,000 kJ/kg TS for the ultrasound, 80 °C during 15 min for the thermal and 157 g NaOH/kg TS for the alkali. Afterward, the specific methane production was evaluated through biomethane potential tests at the specified optimum conditions. The alkali pre-treatment exhibited the greatest methane production increase (34%) followed by the ultrasonication (13%), whereas the thermal pre-treatment presented a methane potential similar to the untreated sludge. Finally, an assessment of the different treatment scenarios was conducted considering the results together with an energy balance, which revealed that the ultrasound and alkali treatments entailed higher costs

Coliphages as model organisms in the characterization and management of water resources

Two groups of bacteriophages that infect Escherichia coli, somatic and F-specific coliphages, have been used in academia as both fecal and viral indicators for many years. Regulatory authorities in different parts of the world are beginning to consider coliphages as indicators of water quality in a range of settings. However, issues such as their potential replication in natural water environments, the cumbersome detection and enumeration methods, a lack of definition concerning which of the two groups should be included in future regulations, and the lack of a clear correlation between coliphages and human viruses and health risks in different water settings remain controversial. This review attempts to shed some light on these contentious issues. The conclusions are that: 1) supposing that they can replicate in some natural water settings, the contribution of coliphages replicated outside the gut will not affect the numbers contributed by fecal pollution and detected by strains recommended for standardized methods; 2) there are easy, fast, and cost-effective methods that can be used in routine laboratories after a little training; 3) perhaps the best option is to determine both groups in a single step; and 4) the low correlation of coliphages with human viruses and health risks is no worse than the correlation between different human viruses

Bacteriophages in bathing wàters: A feasibility study on the development of a method based on bacteriophages for the determination of microbiological quality of bathing waters

Projecte: Project report. BCR Information. EU project KINA19506ENC_001. EUROPEAN COMMISSION. Community Research. DG XII/C - Competitive and Sustainable Growth Programme. Published by EU Directorate General ΧΠ - Science, Research and Development ISBN 92-828-9145-3Informe final projecte europeu aigües de bany i bacteriòfagsMethods for the detection and enumeration of somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis had been standardised and validated. Conditions for the preparation, transport and distribution of bacteriophage reference materials and preservation of samples had been defined. A method based on flocculation with Mg(OH2) with concentration efficiencies from about 40% was settled to concentrate phages from bathing waters. All methods were successfully implemented in routine laboratories all around the EU. Data on the occurrence of bacteriophages as compared to E. coli and Enterococci are available from diverse situations encountered in the EU. Results allow to conclude that the potential of phages for the determination of the microbiological quality of bathing waters merits to be considered since their determination is feasible and their behaviour in natural water differs from the behaviour of bacterial indicators and consequently they add valuable information

Impact of treated sewage effluent on the bacterial community composition in an intermittent mediterranean stream

Water quality monitoring is essential to safeguard human and environmental health. The advent of next-generation sequencing techniques in recent years, which allow a more in-depth study of environmental microbial communities in the environment, could broaden the perspective of water quality monitoring to include impact of faecal pollution bacteria on ecosystem. In this study, 16 S rRNA amplicon sequencing was used to evaluate the impact of wastewater treatment plant (WWTP) effluent on autochthonous microbial communities of a temporary Mediterranean stream characterized by high flow seasonality (from 0.02 m(3)/s in winter to 0.006 m(3)/s in summer). Seven sampling campaigns were performed under different temperatures and streamflow conditions (winter and summer). Water samples were collected upstream (Upper) of the WWTP, the secondary effluent (EF) discharge and 75 m (P75) and 1000 m (P1000) downstream of the WWTP. A total of 5,593,724 sequences were obtained, giving rise to 20,650 amplicon sequence variants (ASV), which were further analysed and classified into phylum, class, family and genus. Each sample presented different distribution and abundance of taxa. Although taxon distribution and abundance differed in each sample, the microbial community structure of P75 resembled that of EF samples, and Upper and P1000 samples mostly clustered together. Alpha diversity showed the highest values for Upper and P1000 samples and presented seasonal differences, being higher in winter conditions of high streamflow and low temperature. Our results suggest the microbial ecology re-establishment, since autochthonous bacterial communities were able to recover from the impact of the WWTP effluent in 1 km. Alpha diversity results indicates a possible influence of environmental factors on the bacterial community structure. This study shows the potential of next-generation sequencing techniques as useful tools in water quality monitoring and management within the climate change scenari

Assessment of dead-end ultrafiltration for the detection and quantification of microbial indicators and pathogens in the drinking water treatment processes

A safe water supply requires distinct treatments and monitoring to guarantee the absence of pathogens and substances potentially hazardous for human health. In this study we assessed the efficiency of the dead-end ultrafiltration (DEUF) method to concentrate faecal indicator organisms (FIO) and pathogens in water samples with different physicochemical characteristics. Water samples were collected at the treatment stages of two drinking water treatment plants to analyse the concentration of a variety of 7 FIO and 4 reference microbes which have some species that are pathogenic to humans: Campylobacter spp., enteroviruses, Cryptosporidium spp. and Giardia spp. The samples were analysed before and after concentration by DEUF, detecting FIO concentrations about 1 log10 higher in non-concentrated samples from both catchments. Percent recoveries were highly variable with a mean of 43.8 ± 17.5%, depending on the FIO and inherent sample characteristics. However, DEUF enabled FIO concentration in high volumes of water (100-500 l), allowing a reduction in the detection limit compared to the non-concentrated samples due to the high volume processing capabilities of the method. As a consequence, the detection of FIO removal from water in the drinking water treatment process was 1.0-1.5 logarithms greater in DEUF-treated water compared to unfiltered samples. The DEUF method improved the detection of target indicators and allowed for the detection of pathogens in low concentrations in water after the treatment stages, confirming the suitability of DEUF to concentrate high volumes of different types of water. This method could be useful for microbial analysis in water treatment monitoring and risk assessment, allowing the identification of critical points during the water treatment process and potential hazards in water destined for several uses

Dynamics of crAssphage as a human source tracking marker in potentially faecally polluted environments

Recent studies have shown that crAssphage is abundant in human faecal samples worldwide. It has thus been postulated as a potential microbial source tracking (MST) marker to detect human faecal pollution in water. However, an effective implementation of crAssphage in water management strategies will depend on an understanding of its environmental dynamics. In this work, the abundance and temporal distribution of crAssphage was analysed in the effluent of wastewater treatment plants using different sewage treatments, and in two rivers (water and sediments) that differ in pollution impact and flow regime. Additionally, the influence of environmental conditions (temperature and rainfall) on the removal of the marker was studied along a river section, and natural inactivation was assessed by a mesocosms approach. Molecular and culture-based tools were used to compare crAssphage abundance and dynamics with those of bacteria and bacteriophages currently applied as global indicators (E. coli, somatic coliphages, Bacteroides GA17 bacteriophages, and the human-associated MST markers HF183 and HMBif). CrAssphage concentrations in sewage effluent and river samples were similar to those of HF183 and HMBif and higher than other general and/or culture-based indicators (by 2-3 orders of magnitude). Measurement of crAssphage abundance revealed no temporal variability in the effluent, although rainfall events affected the dynamics, possibly through the mobilisation of sediments, where the marker was detected in high concentrations, and an increase in diffuse and point pollution. Another factor affecting crAssphage inactivation was temperature. Its persistence was longer compared with other bacterial markers analysed by qPCR but lower than culturable markers. The results of this study support the use of crAssphage as a human source tracking marker of faecal pollution in water, since it has similar abundances to other molecular human MST markers, yet with a longer persistence in the environment. Nevertheless, its use in combination with infectious bacteriophages is probably advisable