Elucidation of fecal inputs into the River Tagus catchment (Portugal) using source-specific mitochondrial DNA, HAdV, and phage markers.

Determining the source of fecal contamination in a water body is important for the application of appropriate remediation measures. However, it has been suggested in the extant literature that this can best be achieved using a 'toolbox' of molecular- and culture-based methods. In response, this study deployed three indicators (Escherichia coli (EC), intestinal enterococci (IE) and somatic coliphages (SC)), one culture-dependent human marker (Bacteroides (GB-124) bacteriophage) and five culture-independent markers (human adenovirus (HAdV), human (HMMit), cattle (CWMit), pig (PGMit) and poultry (PLMit) mitochondrial DNA markers (mtDNA)) within the River Tagus catchment (n = 105). Water samples were collected monthly over a 13-month sampling campaign at four sites (impacted by significant specific human and non-human inputs and influenced by differing degrees of marine and freshwater mixing) to determine the dominant fecal inputs and assess geographical, temporal, and meteorological (precipitation, UV, temperature) fluctuations. Our results revealed that all sampling sites were not only highly impacted by fecal contamination but that this contamination originated from human and from a range of agricultural animal sources. HMMit was present in a higher percentage (83%) and concentration (4.20 log GC/100 mL) than HAdV (32%, 2.23 log GC/100 mL) and GB-124 bacteriophage with the latter being detected once. Animal mtDNA markers were detected, with CWMit found in 73% of samples with mean concentration of 3.74 log GC/100 mL. Correlation was found between concentrations of fecal indicators (EC, IE and SC), CWMit and season. Levels of CWMit were found to be related to physico-chemical parameters, such as temperature and UV radiation, possibly as a result of the increasing presence of livestock outside in warmer months. This study provides the first evaluation of such a source-associated 'toolbox' for monitoring surface water in Portugal, and the conclusions may inform future implementation of surveillance and remediation strategies for improving water quality.

Human-specific phages infecting Enterococcus host strain MW47: are they reliable microbial source tracking markers?

AIM: The aim of this study was to determine the morphological diversity and environmental survival of human-specific phages infecting Enterococcus faecium host strain MW47, to support their use as microbial source tracking (MST) markers. METHODS AND RESULTS: Twenty phages capable of infecting strain MW47 were propagated and their morphologies were determined using transmission electron microscopy (TEM), which revealed that a heterogeneous group of phages was able to infect strain MW47. Three distinct morphologies from two different families (Myoviridae and Siphoviridae) were observed. In situ inactivation experiments were subsequently conducted to determine their environmental persistence. CONCLUSION: The findings revealed a statistically significant link between morphology and the rate of inactivation, with phages belonging to the Myoviridae family demonstrating more rapid inactivation in comparison to those belonging to the Siphoviridae family. SIGNIFICANCE AND IMPACT OF STUDY: The results suggest that while Enterococcus MW47 phages appear to be a potentially valuable MST tools, significant variations in the persistence of the different phages mean that the approach should be used with caution, as this may adversely affect the reliability of the approach, especially when comparing MW47 phage levels or the presence across different matrices (e.g. levels in sediments or shellfish). This highlights the importance of elucidating the ecological characteristics of newly proposed MST markers before they are used in full-scale MST investigations.

The application of phage-based faecal pollution markers to predict the concentration of adenoviruses in mussels (Mytilus edulis) and their overlying waters.

AIM: This study set out to determine whether phage-based indicators may provide a 'low-tech' alternative to existing approaches that might help maintain the microbial safety of shellfish and their overlying waters. METHODS AND RESULTS: Mussels and their overlying waters were collected biweekly from an estuary in southeast England over a 2-year period (May 2013-April 2015) (n = 48). Levels of bacterial indicators were determined using membrane filtration and most probable number methods and those of bacteriophages were determined by direct plaque assay. The detection of adenovirus was determined using real-time polymerase chain reaction. The results revealed that somatic coliphages demonstrated the most significant correlations with AdV F and G in mussels (ρ = 0·55) and overlying waters (ρ = 0·66), followed by GB124 phages (ρ = 0·43) while Escherichia coli showed no correlation with AdV F and G in mussels. CONCLUSION: This study demonstrates that the use of somatic coliphages and GB124 phages may provide a better indication of the risk of adenovirus contamination of mussels and their overlying waters than existing bacterial indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Phage-based detection may be particularly advantageous in low-resource settings where viral infectious disease presents a significant burden to human health.

Bioaccumulation and persistence of faecal bacterial and viral indicators in Mytilus edulis and Crassostrea gigas.

This study investigated the response of two shellfish species - mussels (Mytilus edulis) and oysters (Crassostrea gigas) to microbial contamination in order to gain a better understanding of the bioaccumulation and persistence of microorganisms under controlled conditions. M. edulis and C. gigas were placed in sterile laboratory-prepared artificial seawater and initial tests were carried out to ensure both faecal indicator bacteria (FIB) and bacteriophages were below detection limits. FIB and phages were isolated, purified and dosed into experimental tanks containing the shellfish species. The GB124 phages were bioaccumulated to the highest concentration in M. edulis (1.88Log10) and C. gigas (1.27Log10) after 24h. Somatic coliphages were bioaccumulated to the highest concentration in both M. edulis (4.84log10) and C. gigas (1.73log10) after 48h. F-RNA phages were bioaccumulated to the highest concentration in M. edulis (3.51log10) after 6h but were below detection limit in C. gigas throughout the exposure period. Escherichia coli, faecal coliforms and intestinal enterococci were bioaccumulated to the highest concentrations in M. edulis (5.05log10, 5.06log10 and 3.98log10, respectively) after 48h. In C. gigas, E. coli reached a maximum concentration (5.47log10) after 6h, faecal coliforms (5.19log10) after 12h and intestinal enterococci (3.23log10) after 24h.M. edulis bioaccumulated phages to a greater extent than the faecal bacteria, and in both shellfish species, faecal bacteria persisted for longer periods over 48h than the phages. This study highlights significant variation in the levels and rate of accumulation and persistence with respect to both shellfish species and the indicators used to assess risk. The results suggest that phage-based indicators could help elucidate risks to human health associated with pathogenic organisms.

The effect of UV-C radiation (254 nm) on candidate microbial source tracking phages infecting a human-specific strain of Bacteroides fragilis (GB-124).

The enumeration of phages infecting host-specific strains of Bacteroides has been widely recognised as an effective and low-cost method of microbial source tracking (MST). A recently described human-specific Bacteroides host strain (GB-124) has been shown to detect bacteriophages exclusively in human-impacted waters and is emerging as a useful MST tool. However, a better understanding of the morphology and ecological behaviour of the phages, especially in wastewater disinfection processes, is now required in order to validate their role as MST markers. Bacteriophages infecting Bacteroides fragilis GB-124 (n = 21) were isolated from wastewater effluent and irradiated using laboratory-based UV-C (254 nm) collimated beam experiments. Bacteriophages were found to be both a morphologically and ecologically homogeneous group, with all specimens showing highly similar first order log-linear inactivation profiles (mean fluence required to inactivate phages by 4-log(10) was 36 mJ/cm(2)). These findings present the first evidence that phages infecting GB-124 are inactivated by the levels of UV-C radiation routinely delivered during tertiary wastewater treatment processes. More importantly, comparison with previously published inactivation data suggests that their response to UV-C radiation makes GB-124 phages more suitable surrogates for selected enteric viruses in UV disinfection processes than traditional faecal indicator bacteria or human-specific molecular markers.

Geographical stability of enterococcal antibiotic resistance profiles in Europe and its implications for the identification of fecal sources.

This manuscript describes the use of a recently developed microbial source tracking (MST) technique to determine sources of fecal bacteria (enterococci) from four separate European countries. The investigation aimed to evaluate whether the origin of bacterial populations from France, Spain, and Sweden (n = 456) could be predicted using a library of antibiotic resistance patterns of enterococci of known origin assembled in the United Kingdom (n = 2739). Bacterial isolates were exposed to a battery of 31 antibiotic tests and classified into source groups using discriminant function analysis (DFA). Results showed that while on average 72% of the U.K. isolates could be correctly classified as originating from either municipal wastewater (MW), livestock, or wild birds, only 43% of non-U.K. isolates could be successfully classified into the same source categories. The results suggested that patterns of resistance amongst isolates contained in the U.K. library were not representative of those found in the other locations and that it may not be possible to share libraries over large distances, such as those in this study. Future MST studies using antibiotic resistance analysis (ARA) in Europe may therefore require the assembly of watershed specific libraries, increasing the cost of such studies.

Synthesis and properties of amphiphilic networks 3: preparation and characterization of block conetworks of poly(butyl methacrylate-block-(2,3 propandiol-1-methacrylate-stat-ethandiol dimethacrylate)).

Amphiphilic conetwork polymers were prepared and studied as substrates in the culture of dermal fibroblasts. Both block and random conetworks polymers were produced by radical polymerization of either low-molecular weight monomers or oligomeric macromonomers. The oligomeric macromonomers were prepared by methacrylation of biscarboxy oligo(butyl methacrylates) (OBMA). The latter were synthesized by ozononolysis of poly(butyl methacrylate-co-butadiene) materials. The hydrophilic component was derived from copolymerization with 2,3 propandiol-1-methacrylate and cross-linking was provided by inclusion of ethandiol dimethacrylate (EDMA). None of the synthesized materials showed indications of cytotoxicity to human dermal fibroblasts. All of the block conetworks were highly phase separated and possessed pores on the micron length scale. The equilibrium water contents of the latter could be controlled by addition of EDMA. Block conetworks that did not contain EDMA were highly swollen and had smoother surfaces than those that contained EDMA. The former were poor substrates for cell proliferation (as measured by monitoring DNA content) whilst the latter class gave increasing levels of DNA during culture; an indicator proliferation. The performance of these materials in cell culture was also dependent on the fraction of OBMA in the formulation. Increasing the fractions of BMA, either in the random terpolymer or block networks, system had the effect of increasing both cell proliferation and viability (as measured by the Alamar Blue assay).

A simplified low-cost approach to antibiotic resistance profiling for faecal source tracking.

Antibiotic resistance profiling (ARP) is a potentially useful method for distinguishing faecal bacteria according to host source. This phenotypic approach has cost benefits over genotypic methods, but existing protocols are time-consuming and manual data handling is open to human error. A simplified, low-cost approach to the ARP technique was developed that used automated data recording techniques combined with simple statistical analyses to compare isolates of the genus Enterococcus from various faecal sources. An initial battery of 21 antibiotics (at up to four concentrations) was chosen for source discrimination. Images of growth or non-growth in microplate wells were stored as bitmaps and converted to binary data to form a database of known antibiotic resistance profiles. Discriminant function analysis (DFA) showed that the average rate of isolates correctly classified by the database (wastewater vs non-wastewater) was 86%. Once the more discriminating antibiotics and their concentrations had been identified, it was possible to reduce the number of tests from 80 to 18 whilst increasing the number of correctly classified human isolates. ARP could offer a low-cost and rapid means of identifying sources of faecal pollution. As such, the technique may be of particular benefit to developing countries, where water quality may have a significant impact on health and where cost is a major factor when choosing environmental management technology.