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

Determination of crAssphage in water samples and applicability for tracking human fecal pollution

In recent decades, considerable effort has been devoted to finding microbial source-tracking (MST) markers that are suitable to assess the health risks of faecally polluted waters, with no universal marker reported so far. In this study, the abundance and prevalence of a crAssphage-derived DNA marker in wastewaters of human and animal origins were studied by a new qPCR assay with the ultimate aim of assessing its potential as an MST marker. crAssphage showed up to 106 GC/ml in the sewage samples of human origin, in both the total DNA and the viral DNA fraction. In wastewaters containing animal faecal remains, 39% of the samples were negative for the presence of the crAssphage sequence, while those showing positive results (41% of the samples) were at least 1 log10 unit lower than the samples of human origin. Noteworthy, the log10 values of the ratio (R) crAssphage (GC/ml)/Escherichia coli (CFU/ml) varied significantly depending on the human or animal origin (R > 1.5 for human samples and R < −1.5 for animal wastewater samples. This study opens the way for further research to explore if different specific animal variants of crAssphage exist and whether other zones of the crAssphage genome are better suited to source discrimination

Is Genetic Mobilization Considered When Using Bacteriophages in Antimicrobial Therapy?

The emergence of multi-drug resistant bacteria has undermined our capacity to control bacterial infectious diseases. Measures needed to tackle this problem include controlling the spread of antibiotic resistance, designing new antibiotics, and encouraging the use of alternative therapies. Phage therapy seems to be a feasible alternative to antibiotics, although there are still some concerns and legal issues to overcome before it can be implemented on a large scale. Here we highlight some of those concerns, especially those related to the ability of bacteriophages to transport bacterial DNA and, in particular, antibiotic resistance genes

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

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

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

Analysis of a phase variable restriction modification system of the human gut symbiont Bacteroides fragilis

The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum

Detection of bacteriophage particles containing antibiotic resistance genes in the sputum of cystic fibrosis patients

Cystic fibrosis (CF) is a chronic disease in which the bacterial colonization of the lung is linked to an excessive inflammatory response that leads to respiratory failure. The microbiology of CF is complex. Staphylococcus aureus is the first bacterium to colonize the lungs in 30% of pediatric CF patients, and 80% of adult patients develop a chronic Pseudomonas aeruginosa infection, but other microorganisms can also be found. The use of antibiotics is essential to treat the disease, but antibiotic performance is compromised by resistance mechanisms. Among various mechanisms of transfer of antibiotic resistance genes (ARGs), the recently been reported bacteriophages are the least explored in clinical settings. To determine the role of phages in CF as mobile genetic elements (MGEs) carrying ARGs, we evaluated their presence in 71 CF patients. 71 sputum samples taken from these patients were screened for eight ARGs (blaTEM, blaCTX-M-1-group, blaCTX-M-9-group, blaOXA-48, blaVIM, mecA, qnrA, and qnrS) in the bacteriophage DNA fraction. The phages found were also purified and observed by electron microscopy. 32.4% of CF patients harbored ARGs in phage DNA. β-lactamase genes, particularly blaVIM and blaTEM, were the most prevalent and abundant, whereas mecA, qnrA, and qnrS were very rare. Siphoviridae phage particles capable of infecting P. aeruginosa and Klebsiella pneumoniae were detected in CF sputum. Phage particles harboring ARGs were found to be abundant in the lungs of both CF patients and healthy individuals and could contribute to the colonization of multiresistant strains