Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan / Caracterização de um colifago AS1 isolado de efluente de esgoto no Paquistão

The emergence of multi-drug resistant (MDR) bacterial strains, which are posing a global health threat has developed the interest of scientists to use bacteriophages instead of conventional antibiotics therapy. In light of an increased interest in the use of phage as a bacterial control agent, the study aimed to isolate and characterize lytic phages from sewage effluent. During the current study, bacteriophage AS1 was isolated from sewage effluent against E.coli S2. The lytic activity of phageAS1 was limited to E.coli S2 strain showing monovalent behavior. The calculated phage titer was 3.5×109 pfu/ml. PhageAS1 was stable at a wide range of pH and temperature. The maximum stability was recorded at 37ºC and pH 7.0, while showing its normal lytic activity at temperature 60ºC and from pH 5.0 to 11.0 respectively. At temperature 70ºC, phage activity was somewhat reduced whereas, further increase in temperature and decrease or increase in pH completely inactivated the phage. From the current study, it was concluded that waste water is a best source for finding bacteriophages against multi-drug resistant bacterial strains and can be used as bacterial control agent.

O surgimento de cepas bacterianas multirresistentes (MDR), que representam uma ameaça global à saúde, desenvolveu o interesse dos cientistas em usar bacteriófagos em vez da terapia convencional com antibióticos. Diante do crescente interesse no uso de fago como agente de controle bacteriano, o estudo visou isolar e caracterizar fagos líticos de efluente de esgoto. Durante o estudo atual, o bacteriófago AS1 foi isolado de efluente de esgoto contra E. coli S2. A atividade lítica de phageAS1 foi limitada à cepa E. coli S2, apresentando comportamento monovalente. O título de fago calculado foi de 3,5 x 109 ufp/ml. PhageAS1 foi estável em uma ampla faixa de pH e temperatura. A estabilidade máxima foi registrada a 37ºC e pH 7,0, enquanto mostrou atividade lítica normal em temperatura de 60ºC e pH 5,0 a 11,0, respectivamente. Na temperatura de 70ºC, a atividade do fago foi um pouco reduzida, enquanto o aumento adicional da temperatura e a diminuição ou aumento do pH inativaram completamente o fago. Com base no estudo atual, concluiu-se que a água residual é a melhor fonte para encontrar bacteriófagos contra cepas bacterianas multirresistentes e pode ser usada como agente de controle bacteriano.

Applicability Evaluation of Male-Specific Coliphage-Based Detection Methods for Microbial Contamination Tracking.

Outbreaks of food poisoning due to the consumption of norovirus-contaminated shellfish continue to occur. Male-specific (F+) coliphage has been suggested as an indicator of viral species due to the association with animal and human wastes. Here, we compared two methods, the double agar overlay and the quantitative real-time PCR (RT-PCR)-based method, for evaluating the applicability of F+ coliphage-based detection technique in microbial contamination tracking of shellfish samples. The RT-PCR-based method showed 1.6-39 times higher coliphage PFU values from spiked shellfish samples, in relation to the double agar overlay method. These differences indicated that the RT-PCR-based technique can detect both intact viruses and non-particle-protected viral DNA/RNA, suggesting that the RT-PCR based method could be a more efficient tool for tracking microbial contamination in shellfish. However, the virome information on F+ coliphage-contaminated oyster samples revealed that the high specificity of the RT-PCR- based method has a limitation in microbial contamination tracking due to the genomic diversity of F+ coliphages. Further research on the development of appropriate primer sets for microbial contamination tracking is therefore necessary. This study provides preliminary insight that should be examined in the search for suitable microbial contamination tracking methods to control the sanitation of shellfish and related seawater.

Pathogenic Escherichia coli-Specific Bacteriophages and Polyvalent Bacteriophages in Piglet Guts with Increasing Coliphage Numbers after Weaning.

Postweaning diarrhea in pigs is mainly caused by pathogenic Escherichia coli and is a major source of revenue loss to the livestock industry. Bacteriophages dominate the gut virome and have the potential to regulate bacterial communities and thus influence the intestinal physiology. To determine the biological characterization of intestinal coliphages, we isolated and identified the fecal coliphages of healthy preweaned and postweaned piglets from the Nanjing and Chuzhou pig farms. First, ahead of coliphage isolation, 87 E. coli strains were isolated from healthy or diarrheal fecal samples from three pig farms, of which 8 were pathogenic strains, including enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC). Of the E. coli strains, 87.3% possessed drug resistance to three antibiotics. Using these 87 E. coli strains as indicator hosts, we isolated 45 coliphages and found a higher abundance in the postweaning stage than in the preweaning stage (24 versus 17 in the Nanjing and 13 versus 4 in the Chuzhou farm). Furthermore, each farm had a single most-prevalent coliphage strain. Pathogenic E. coli-specific bacteriophages were commonly detected (9/10 samples in the Nanjing farm and 7/10 in the Chuzhou farm) in guts of sampled piglets, and most had significant bacteriostatic effects (P < 0.05) on pathogenic E. coli strains. Three polyvalent bacteriophages (N24, N30, and C5) were identified. The N30 and C5 strains showed a genetic identity of 89.67%, with mild differences in infection characteristics. Our findings suggest that pathogenic E. coli-specific bacteriophages as well as polyvalent bacteriophages are commonly present in piglet guts and that weaning is an important event that affects coliphage numbers. IMPORTANCE Previous studies based on metagenomic sequencing reported that gut bacteriophages profoundly influence gut physiology but did not provide information regarding the host range and biological significance. Here, we screened coliphages from the guts of preweaned and postweaned piglets against indicator hosts, which allowed us to identify the pathogenic E. coli-specific bacteriophages and polyvalent bacteriophages in pig farms and quantify their abundance. Our approach complements sequencing methods and provides new insights into the biological characterizations of bacteriophage in the gut along with the ecological effects of intestinal bacteriophages.

Characteristics of Bacteriophage Isolates and Expression of Shiga Toxin Genes Transferred to Non Shiga Toxin-Producing E. coli by Transduction.

A risk analysis of Shiga toxin (Stx)-encoding bacteriophage was carried out by confirming the transduction phage to non-Stx-producing Escherichia coli (STEC) and subsequent expression of the Shiga toxin genes. The virulence factor stx1 was identified in five phages, and both stx1 and stx2 were found in four phages from a total of 19 phage isolates with seven non-O157 STEC strains. The four phages, designated as φNOEC41, φNOEC46, φNOEC47, and φNOEC49, belonged morphologically to the Myoviridae family. The stabilities of these phages to temperature, pH, ethanol, and NaClO were high with some variabilities among the phages. The infection of five non-STEC strains by nine Stx-encoding phages occurred at a rate of approximately 40%. Non-STEC strains were transduced by Stx-encoding phage to become lysogenic strains, and seven convertant strains had stx1 and/or stx2 genes. Only the stx1 gene was transferred to the receptor strains without any deletion. Gene expression of a convertant having both stx1 and stx2 genes was confirmed to be up to 32 times higher for Stx1 in 6% NaCl osmotic media and twice for Stx2 in 4% NaCl media, compared with expression in low-salt environments. Therefore, a new risk might arise from the transfer of pathogenic genes from Stx-encoding phages to otherwise harmless hosts. Without adequate sterilization of food exposed to various environments, there is a possibility that the toxicity of the phages might increase.

A rigorous assessment and comparison of enumeration methods for environmental viruses.

Determining exact viral titers in a given sample is essential for many environmental and clinical applications, e.g., for studying viral ecology or application of bacteriophages for food safety. However, virus quantification is not a simple task, especially for complex environmental samples. While clonal viral isolates can be quantified with relative high accuracy using virus-specific methods, i.e., plaque assay or quantitative real-time PCR, these methods are not valid for complex and diverse environmental samples. Moreover, it is not yet known how precisely laser-based methods, i.e., epifluorescence microscopy, flow cytometry, and nanoparticle tracking analysis, quantify environmental viruses. In the present study, we compared five state-of-the-art viral quantification methods by enumerating four model viral isolates of different genome and size characteristics as well as four different environmental water samples. Although Nanoparticle tracking analysis combined with gentle staining at 30 °C could be confirmed by this study to be a reliable quantification technique for tested environmental samples, environmental samples still lack an universally applicable and accurate quantification method. Special attention has to be put on optimal sample concentrations as well as optimized sample preparations, which are specific for each method. As our results show the inefficiency when enumerating small, or single-stranded DNA or RNA viruses, the global population of viruses is presumably higher than expected.

Exploring the Remarkable Diversity of Culturable Escherichia coli Phages in the Danish Wastewater Environment.

Phages drive bacterial diversity, profoundly influencing microbial communities, from microbiomes to the drivers of global biogeochemical cycling. Aiming to broaden our understanding of Escherichiacoli (MG1655, K-12) phages, we screened 188 Danish wastewater samples and isolated 136 phages. Ninety-two of these have genomic sequences with less than 95% similarity to known phages, while most map to existing genera several represent novel lineages. The isolated phages are highly diverse, estimated to represent roughly one-third of the true diversity of culturable virulent dsDNA Escherichia phages in Danish wastewater, yet almost half (40%) are not represented in metagenomic databases, emphasising the importance of isolating phages to uncover diversity. Seven viral families, Myoviridae, Siphoviridae, Podoviridae,Drexlerviridae,Chaseviridae,Autographviridae, and Microviridae, are represented in the dataset. Their genomes vary drastically in length from 5.3 kb to 170.8 kb, with a guanine and cytosine (GC) content ranging from 35.3% to 60.0%. Hence, even for a model host bacterium, substantial diversity remains to be uncovered. These results expand and underline the range of coliphage diversity and demonstrate how far we are from fully disclosing phage diversity and ecology.

Male-Specific and Somatic Coliphage Profiles from Major Aquaculture Areas in Republic of Korea.

Human and animal feces are important sources of various types of microbial contamination in water. Especially, enteric viruses, the major agents of waterborne infection, can attain long-term survival in water environments due to their strong resistance to various environmental factors including pH, salinity, and temperature. Coliphages are promising viral indicators for fecal contamination in water environments. Here, we investigated the seasonal and spatial distribution of male-specific and somatic coliphages in surface water and seawater at three major aquaculture areas, including Goseong Bay, Aphae Island, and Gomso Bay, in Republic of Korea over a period of 1 year. We selected 6 surface water and 14 seawater sampling sites for each study area and collected a total of 480 water samples from March 2014 to February 2015. Overall, surface water samples contained higher occurrences of coliphages than seawater samples. The high coliphage concentrations were detected in spring (March to May 2014). The differences in geographical features and patterns in land usage of the three aquaculture areas may have affected the coliphage concentration and occurrence. Moreover, environmental factors such as cumulative precipitation were strongly correlated with coliphage concentrations. Therefore, we suggest that further longitudinal studies on coliphage concentrations and distributions should be performed to support the application of coliphages in tracking fecal contamination in water.

A Somatic Coliphage Threshold Approach To Improve the Management of Activated Sludge Wastewater Treatment Plant Effluents in Resource-Limited Regions.

Effective wastewater management is crucial to ensure the safety of water reuse projects and effluent discharge into surface waters. Multiple studies have demonstrated that municipal wastewater treatment with conventional activated sludge processes is inefficient for the removal of a wide spectrum of viruses in sewage. In this study, a well-accepted statistical approach was used to investigate the relationship between viral indicators and human enteric viruses during wastewater treatment in a resource-limited region. Influent and effluent samples from five urban wastewater treatment plants (WWTPs) in Costa Rica were analyzed for somatic coliphage and human enterovirus, hepatitis A virus, norovirus genotypes I and II, and rotavirus. All WWTPs provide primary treatment followed by conventional activated sludge treatment prior to discharge into surface waters that are indirectly used for agricultural irrigation. The results revealed a statistically significant relationship between the detection of at least one of the five human enteric viruses and somatic coliphage. Multiple logistic regression and receiver operating characteristic curve analysis identified a threshold of 3.0 × 103 (3.5 log10) somatic coliphage PFU per 100 ml, which corresponded to an increased likelihood of encountering enteric viruses above the limit of detection (>1.83 × 102 virus targets/100 ml). Additionally, quantitative microbial risk assessment was executed for farmers indirectly reusing WWTP effluent that met the proposed threshold. The resulting estimated median cumulative annual disease burden complied with World Health Organization recommendations. Future studies are needed to validate the proposed threshold for use in Costa Rica and other regions.IMPORTANCE Effective wastewater management is crucial to ensure safe direct and indirect water reuse; nevertheless, few countries have adopted the virus log reduction value management approach established by the World Health Organization. In this study, we investigated an alternative and/or complementary approach to the virus log reduction value framework for the indirect reuse of activated sludge-treated wastewater effluent. Specifically, we employed a well-accepted statistical approach to identify a statistically sound somatic coliphage threshold value which corresponded to an increased likelihood of human enteric virus detection. This study demonstrates an alternative approach to the virus log reduction value framework which can be applied to improve wastewater reuse practices and effluent management.

Fast and easy methods for the detection of coliphages.

Somatic and F-specific coliphages are gaining ground as indicators of fecal/viral pollution. Guidelines and regulations worldwide for monitoring water, biosolids and food are including them as parameters to assess quality and treatment efficiency. Robust methods to detect and quantify both groups of phages in water samples have been launched by agencies such as the International Standardization Organization (ISO) and the USA Environmental Protection Agency (USEPA). Although these methods have proved readily implementable in routine microbiology laboratories, faster and more user-friendly protocols will be highly welcome if coliphage detection becomes routine in water quality analysis. We here provide an overview of new approaches seeking to facilitate the detection of infectious coliphages included in guidelines and regulations. The improvements achieved suggest that streamlined kits able to provide results in a few hours at very reasonable costs will become available in the near future. The potential of molecular procedures and methods based on microelectronic sensors is also briefly discussed.

Evaluation of New Components in Modified Scholten's Medium for the Detection of Somatic Coliphages.

Enteric bacteriophages (somatic coliphages, F-specific coliphages or both together) are now recognized as useful viral indicators in water, shellfish, and biosolids and are being progressively included in national and international sanitary regulations. Among them, somatic coliphages have an advantage in that they usually outnumber F-RNA coliphages in water environments. Their enumeration using Modified Scholten's (MS) media, following the ISO 10705-2 standard for the growth of Escherichia coli host strain WG5, is highly efficient and a common practice worldwide. These media contain a high concentration of nutrients, which may be modified to save costs without loss of bacterial growth host efficiency. This study explored reducing the concentration of nutrients in the current formulation and/or incorporating new components to improve the host bacterial growth and/or the enumeration of somatic coliphages at an affordable analytical cost. A twofold dilution of the original MS media was found not to affect the bacterial growth rate. The addition of combinations of assayed compounds to twofold diluted MS media slightly enhanced its analytical performance without altering bacterial growth. By generating savings in both cost and time while maintaining optimal results, media dilution could be applied to design new simple applications for coliphage enumeration.

Virulent coliphages in 1-year-old children fecal samples are fewer, but more infectious than temperate coliphages.

Bacteriophages constitute an important part of the human gut microbiota, but their impact on this community is largely unknown. Here, we cultivate temperate phages produced by 900 E. coli strains isolated from 648 fecal samples from 1-year-old children and obtain coliphages directly from the viral fraction of the same fecal samples. We find that 63% of strains hosted phages, while 24% of the viromes contain phages targeting E. coli. 150 of these phages, half recovered from strain supernatants, half from virome (73% temperate and 27% virulent) were tested for their host range on 75 E. coli strains isolated from the same cohort. Temperate phages barely infected the gut strains, whereas virulent phages killed up to 68% of them. We conclude that in fecal samples from children, temperate coliphages dominate, while virulent ones have greater infectivity and broader host range, likely playing a role in gut microbiota dynamics.

A Quest of Great Importance-Developing a Broad Spectrum Escherichiacoli Phage Collection.

Shigella ssp. and enterotoxigenic Escherichiacoli are the most common etiological agents of diarrheal diseases in malnourished children under five years of age in developing countries. The ever-growing issue of antibiotic resistance and the potential negative impact of antibiotic use on infant commensal microbiota are significant challenges to current therapeutic approaches. Bacteriophages (or phages) represent an alternative treatment that can be used to treat specific bacterial infections. In the present study, we screened water samples from both environmental and industrial sources for phages capable of infecting E. coli laboratory strains within our collection. Nineteen phages were isolatedand tested for their ability to infect strains within the ECOR collection and E. coli O157:H7 Δstx. Furthermore, since coliphages have been reported to cross-infect certain Shigella spp., we also evaluated the ability of the nineteen phages to infect a representative Shigella sonnei strain from our collection. Based on having distinct (although overlapping in some cases) host ranges, ten phage isolates were selected for genome sequence and morphological characterization. Together, these ten selected phages were shown to infect most of the ECOR library, with 61 of the 72 strains infected by at least one phage from our collection. Genome analysis of the ten phages allowed classification into five previously described genetic subgroups plus one previously underrepresented subgroup.

Fecal indicator bacteria and virus removal in stormwater biofilters: Effects of biochar, media saturation, and field conditioning.

Stormwater biofilters are used to attenuate the flow and volume of runoff and reduce pollutant loading to aquatic systems. However, the capacity of biofilters to remove microbial contaminants remains inadequate. While biochar has demonstrated promise as an amendment to improve microbial removal in laboratory-scale biofilters, it is uncertain if the results are generalizable to the field. To assess biochar performance in a simulated field setting, sand and biochar-amended sand biofilters were periodically dosed with natural stormwater over a 61-week conditioning phase. Impact of media saturation was assessed by maintaining biofilters with and without a saturated zone. Biochar-amended biofilters demonstrated improved Escherichia coli removal over sand biofilters during the first 31 weeks of conditioning though media type did not impact E. coli removal during the last 30 weeks of conditioning. Presence of a saturated zone was not a significant factor influencing E. coli removal across the entire conditioning phase. Following conditioning, biofilters underwent challenge tests using stormwater spiked with wastewater to assess their capacity to remove wastewater-derived E. coli, enterococci, and male-specific (F+) coliphage. In challenge tests, biochar-amended biofilters demonstrated enhanced removal of all fecal indicators relative to sand biofilters. Additionally, saturated biofilters demonstrated greater removal of fecal indicators than unsaturated biofilters for both media types. Discrepant conclusions from the conditioning phase and challenge tests may be due to variable influent chemistry, dissimilar transport of E. coli indigenous to stormwater and those indigenous to wastewater, and differences in E. coli removal mechanisms between tests. Mobilization tests conducted following challenge tests showed minimal (<2.5%) observable mobilization of fecal indicators, regardless of media type and presence of a saturated zone. While our results emphasize the challenge of translating biochar's performance from the laboratory to the field, findings of this study inform biofilter design to remove microbial contaminants from urban stormwater.

Aptness of Escherichia coli host strain CB390 to detect total coliphages in Colombia.

Fecal bacteria have been used for more than a century as indicators of fecal contamination in water. In recent years, the monitoring of somatic and F-specific coliphages has been gradually included in guidelines and regulations as an additional parameter to reinforce water safety. The Escherichia coli host strain CB390 was tailored to detect both somatic and F-specific coliphages in a single test. The efficacy of this strain for bacteriophage detection, previously evaluated in Western Europe and North America, was assessed here for the first time in South America. The detection of somatic and F-specific coliphages by the strain CB390, as well as by standardized methods, was performed in drinking and river water and municipal and abattoir wastewaters. No statistical difference was found in the numbers of total coliphages detected by strain CB390 and the sum of somatic and F-specific coliphages determined separately by the standardized ISO methods. The data presented here provide further validation of the effectiveness of the host strain E. coli CB390 for the detection of total coliphages in waters in a single test and demonstrate its suitability for application in upper-middle income countries of the Americas (World Bank category).

Still Something to Discover: Novel Insights intoEscherichia coli Phage Diversity and Taxonomy.

The aim of this study was to gain further insight into the diversity of Escherichia coli phagesfollowed by enhanced work on taxonomic issues in that field. Therefore, we present the genomiccharacterization and taxonomic classification of 50 bacteriophages against E. coli isolated fromvarious sources, such as manure or sewage. All phages were examined for their host range on a setof different E. coli strains, originating, e.g., from human diagnostic laboratories or poultry farms.Transmission electron microscopy revealed a diversity of morphotypes (70% Myo-, 22% Sipho-, and8% Podoviruses), and genome sequencing resulted in genomes sizes from ~44 to ~370 kb.Annotation and comparison with databases showed similarities in particular to T4- and T5-likephages, but also to less-known groups. Though various phages against E. coli are already describedin literature and databases, we still isolated phages that showed no or only few similarities to otherphages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny andclassification of the newly isolated phages using VICTOR resulted in the proposal of new generaand led to an enhanced taxonomic classification of E. coli phages.

Evaluation of a Male-Specific DNA Coliphage Persistence Within Eastern Oysters (Crassostrea virginica).

Male-specific coliphages (MSCs) are currently used to assess the virologic quality of shellfish-growing waters and to assess the impact of sewage release or adverse weather events on bivalve shellfish. Since MSC can have either DNA or RNA genomes, and most research has been performed exclusively on RNA MSCs, persistence of M13, a DNA MSC, was evaluated for its persistence as a function of time and temperature within Eastern oysters (Crassostrea virginica). Oysters were individually exposed to seawater containing a total of 1010 to 1012 pfu of M13 for 24 h at 15 °C followed by maintenance in tanks with as many as 21 oysters in continuously UV-sterilized water for up to 6 weeks at either 7, 15, or 22 °C. Two trials for each temperature were performed combining three shucked oysters per time point which were assayed by tenfold serial dilution in triplicate. Initial contamination levels averaged 106.9 and ranged from 106.0 to 107.0 of M13. For oysters held for 3 weeks, log10 reductions were 1.7, 3.8, and 4.2 log10 at 7, 15, and 22 °C, respectively. Oysters held at 7 and 15 °C for 6 weeks showed average reductions of 3.6 and 5.1 log10, respectively, but still retained infectious M13. In total, this work shows that DNA MSC may decline within shellfish in a manner analogous to RNA MSCs.

Riding the wave of genomics to investigate aquatic coliphage diversity and activity.

Bacteriophages infecting Escherichia coli (coliphages) have been used as a proxy for faecal matter and water quality from a variety of environments. However, the diversity of coliphages that is present in seawater remains largely unknown, with previous studies largely focusing on morphological diversity. Here, we isolated and characterized coliphages from three coastal locations in the United Kingdom and Poland. Comparative genomics and phylogenetic analysis of phage isolates facilitated the identification of putative new species within the genera Rb69virus and T5virus and a putative new genus within the subfamily Tunavirinae. Furthermore, genomic and proteomic analysis combined with host range analysis allowed the identification of a putative tail fibre that is likely responsible for the observed differences in host range of phages vB_Eco_mar003J3 and vB_Eco_mar004NP2.

Detection of protease and engineered phage-infected bacteria using peptide-graphene oxide nanosensors.

A peptide-graphene oxide nanosensor has been developed to detect tobacco etch virus (TEV) protease and bacteria infected with an engineered bacteriophage. In the detection strategy, a peptide (sequence: RKRFRENLYFQSCP) is tagged with fluorophores and graphene oxide (GO) is used to adsorb the peptides while quenching their fluorescence. In the presence of TEV protease, fluoropeptides are cleaved between glutamine (Q) and serine (S), resulting in the recovery of fluorescence signal. Based on the fluorescent intensity, the detection limit of TEV protease is 51 ng/µL. Additionally, we have utilized the sensing system to detect bacteria cells. Bacteriophages, which were engineered to carry TEV protease genes, were used to infect target bacteria (Escherichia coli) resulting in the translation and release of the protease. This allowed the estimation of bacteria at the concentration of 104 CFU/mL. This strategy has the potential to be developed as a multiplex detection platform of multiple bacterial species. Graphical abstract.

Constructed wetlands combined with disinfection systems for removal of urban wastewater contaminants.

The removal efficiency of an urban wastewater treatment plant (WWTP) to obtain an effluent suitable for agriculture reuse was evaluated in a one-year period, taking into account the Italian wastewater limits and the recent European proposal for the minimum requirements water quality for agricultural irrigation. The secondary effluent of WWTP was treated by three full-scale horizontal sub-surface flow (H-SSF) constructed wetlands (CWs), working in parallel, planted with different macrophytes species, and combined with a UV device and a lagooning system running in series. The H-SSF CW system effectively reduced physico-chemical pollutants and its efficiency was steady over the investigation period, while, Escherichia coli densities always exceed the Italian limits required for wastewater reuse in agriculture. The UV system significantly reduced the microbiological indicators, eliminating E. coli, in compliance with the Italian regulation, and somatic coliphages, although a variable efficacy against total coliforms and enterococci, especially in winter season, was achieved. Although the lagooning unit provides a high removal of the main microbial groups, it did not reduce physico-chemical parameters. Even if the overall performance target, for the whole treatment chain, met the recent log10 reduction (≥5.0), required by the European Commission, the persistence of enterococci, especially in winter season, poses a matter of concern for public health, for the potential risk to serve as a genetic reservoir of transferable antibiotic-resistance.

Escherichia coli bacteriophage Gostya9, representing a new species within the genus T5virus.

Escherichia coli bacteriophage Gostya9 (genus T5virus) was isolated from horse feces collected in Moscow, Russia, in 2013. This phage was associated in a single plaque with the previously reported phage 9g and was subsequently purified. Analysis of the complete genomic sequence of Gostya9 revealed that it is closely related to the T5-like bacteriophage DT57C, which had been isolated at the same location in 2007. These two viruses share 79.5% nucleotide sequence identity, which is below the 95% threshold applied currently to demarcate bacteriophage species. The most significant features distinguishing Gostya9 from DT57C include 1) the presence of one long tail fiber protein gene, 122c (ltf), instead of the two genes, ltfA and ltfB, that are present in DT57C; 2) the absence of the gene for the receptor-blocking lytic conversion lipoprotein precursor llp; and 3) the divergence of the receptor-recognition protein, pb5, which is only distantly related at the amino acid sequence level. The observed features of the Gostya9 adsorption apparatus are suggestive of a possible novel specificity for the final receptor and make this phage interesting for possible direct application in phage therapy of E. coli infections or as a source of receptor-recognition protein for engineering new phage specificities.