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1.
Nat Commun ; 11(1): 378, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953385

RESUMO

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.


Assuntos
Colífagos/fisiologia , Escherichia coli/virologia , Fezes/virologia , Proteínas de Transporte , Colífagos/classificação , Colífagos/genética , Colífagos/isolamento & purificação , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Microbioma Gastrointestinal , Genoma Viral , Especificidade de Hospedeiro , Humanos , Lactente , Lisogenia , Especificidade da Espécie
2.
Proc Natl Acad Sci U S A ; 116(48): 24206-24213, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31719208

RESUMO

We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage øX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available-23 Bullavirinae genomes related to øX174. Using bioinformatic tools, we first identified 315 potential open reading frames (ORFs) within the genome, including the 11 established essential genes and 82 highly conserved ORFs that have no known gene products or assigned functions. Using genome-scale design and synthesis, we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved but cryptic ORFs. The resulting genome is not viable. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ∼50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation that modulates expression of gene H, a known essential gene, and fully suppresses the fitness defect. Taken together, we conclude that the annotation of currently functional ORFs for the øX174 genome is formally complete. More broadly, we show that sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.


Assuntos
Colífagos/genética , Genoma Viral , Anotação de Sequência Molecular/métodos , Fases de Leitura Aberta , Sequência de Bases , Códon , Sequência Conservada , Evolução Molecular Direcionada , Regulação Viral da Expressão Gênica , Genes Essenciais , Genômica/métodos , Microrganismos Geneticamente Modificados , Mutação , Proteínas Virais/genética
3.
Elife ; 82019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31650957

RESUMO

Temperate bacteriophages are viruses that can incorporate their genomes into their bacterial hosts, existing there as prophages that refrain from killing the host cell until induced. Prophages are largely quiescent, but they can alter host phenotype through factors encoded in their genomes (often virulence factors) or by disrupting host genes as a result of integration. Here we describe another mechanism by which a prophage can modulate host phenotype. We show that a temperate phage that integrates in Escherichia coli reprograms host regulation of an anaerobic respiratory system, thereby inhibiting a bet hedging strategy. The phage exerts this effect by upregulating a host-encoded signal transduction protein through transcription initiated from a phage-encoded promoter. We further show that this phenomenon occurs not only in a laboratory strain of E. coli, but also in a natural isolate that contains a prophage at this site.


Assuntos
Colífagos/genética , Metabolismo Energético , Escherichia coli/metabolismo , Escherichia coli/virologia , Regulação Bacteriana da Expressão Gênica , Prófagos/genética , Integração Viral , Anaerobiose , Transdução de Sinais
4.
Environ Sci Process Impacts ; 21(12): 2031-2041, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31612888

RESUMO

Coliphages, viruses that infect Escherichia coli, have been used for decades to assess surface water quality yet there is no guideline for interpreting their concentrations. The present study uses a quantitative microbial risk assessment (QMRA) framework to derive risk-based surface water quality thresholds for somatic and F+ or male-specific coliphages. The risk-based threshold is the concentration at which the risk of gastro-intestinal illness is simulated to be 32/1000. The framework specifically investigates a simplified hazard scenario where recreational swimmers come into contact with water contaminated with untreated sewage containing coliphages and enteric pathogens. The framework considers exposure to sewage of diverse ages and thus accounts for the decay of coliphages and pathogens over time. As decay rate constants depend on temperature, the model considers the effect of temperature on the risk-based threshold. When exposure to fresh, unaged sewage contamination occurs, the risk-based water quality threshold for somatic and F+ coliphages is 60 PFU per 100 mL and 30 PFU per 100 mL, respectively, and temperature independent. The risk-based threshold decreases as the contamination ages because, on average, coliphages decay more quickly than norovirus, the pathogen that contributes the most to risk. The decrease in the risk-based threshold with contaminant age is equal to the difference in the first order decay rate constants of coliphages and norovirus. Since coliphage decay rate constants are larger at 25 °C than at 15 °C, and norovirus decay rate constants are a weak function of temperature, risk-based thresholds decrease more quickly with age at 25 °C than at 15 °C. For the common case where the age of contamination is unknown, the risk-based threshold for both coliphages is between ∼1 PFU per 100 mL and ∼10 PFU per 100 mL, depending on model assumptions. Future work can apply this QMRA framework for identifying risk-based thresholds for coliphages from different hazards (treated wastewater or animal feces) or from mixtures of contamination of different ages and sources.


Assuntos
Colífagos/efeitos dos fármacos , Monitoramento Ambiental/métodos , Água Doce , Microbiologia da Água/normas , Poluição da Água/análise , Qualidade da Água , Animais , Escherichia coli/virologia , Fezes/virologia , Água Doce/química , Água Doce/virologia , Humanos , Modelos Teóricos , Medição de Risco , Esgotos/virologia , Temperatura
5.
Mar Pollut Bull ; 149: 110570, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31542593

RESUMO

Microbial safety of recreational waters is a significant public health issue. In this study we assessed the occurrence and quantity of enteric viruses in bathing and non-bathing waters in Italy, in parallel with microbial faecal indicators, somatic coliphages and Vibrio spp. Enteric viruses (aichivirus, norovirus and enterovirus) were detected in 55% of bathing water samples, including samples with bacterial indicator concentrations compliant with the European bathing water Directive. Aichivirus was the most frequent and abundant virus. Adenovirus was detected only in non-bathing waters. Somatic coliphages were identified in 50% bathing water samples, 80% of which showed simultaneous presence of viruses. Vibrio species were ubiquitous, with 9 species identified, including potential pathogens (V. cholerae, V. parahaemoylticus and V. vulnificus). This is the first study showing the occurrence and high concentration of Aichivirus in bathing waters and provides original information, useful in view of a future revision of the European Directive.


Assuntos
Praias , Água do Mar/microbiologia , Água do Mar/virologia , Colífagos , Enterovirus , Monitoramento Ambiental , Fezes/microbiologia , Fezes/virologia , Humanos , Itália , Mar Mediterrâneo , Norovirus/genética , Norovirus/isolamento & purificação , Vibrio/genética , Vibrio/isolamento & purificação , Vibrio/patogenicidade , Microbiologia da Água
6.
PLoS One ; 14(9): e0222719, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553761

RESUMO

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.


Assuntos
Carvão Vegetal/química , Colífagos/isolamento & purificação , Escherichia coli/isolamento & purificação , Águas Residuárias/microbiologia , Purificação da Água/instrumentação , Cidades , Fezes/microbiologia , Filtração/instrumentação , Chuva , Estações do Ano
7.
Water Res ; 164: 114898, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31404902

RESUMO

Surface waters are essential natural resources. They are also receiving waters for a variety of anthropogenic waste streams that carry a myriad of pollutants including pathogens. Watershed and fate and transport models can help inform the spatial and temporal extent of microbial pollution from point and non-point sources and thus provide useful information for managing surface waters. Viruses are particularly important water-related pathogens because they often have a low infectious dose, which means that ingestion of even a small volume of water containing a low concentration of virions has the potential to cause disease. We conducted a systematic review of the literature, following best practices, to gather decay rate constants (k) of mammalian waterborne viruses (enteroviruses, adenoviruses, noroviruses, astroviruses, rotaviruses, and hepatitis A viruses) and coliphages in raw surface waters to aid in the parameterization of virus fate and transport models. We identified 562 k values from the literature, with the largest number identified for enteroviruses and coliphages and the smallest for astrovirus, hepatitis A virus, and norovirus. Average k values for each virus varied from 0.07 to 0.9 per day, in order from smallest to largest: Norwalk virus (i.e., noroviruses) < Human astrovirus < Mastadenovirus (i.e., adenoviruses) < Hepatovirus A (i.e., hepatitis A viruses) < Rotavirus A < coliphages < Enterovirus. A meta-analysis investigated how k varied among viruses for experiments conducted with different virus serotypes or species at different temperatures, salinities, and sunlight exposures, and for experiments that enumerated viruses using different methodologies. Virus species or serotype did not affect k among decay experiments. k values were generally larger for experiments conducted at higher temperatures, in sunlight, and in estuarine waters, and enumerated using culture methods. k values were statistically different between virus types with Norwalk virus, Hepatovirus A, and Mastadenovirus having smaller k values than other viruses, controlling for experimental condition and enumeration method. While F+ coliphage k values were similar to those of Enterovirus, Human astrovirus, and Rotavirus A, they were different from those of the other mammalian viruses. This compilation of coliphage and mammalian virus k values provides essential information for researchers and risk assessors who model virus fate and transport in surface waters and identifies avenues for future research to fill knowledge gaps.


Assuntos
Enterovirus , Rotavirus , Vírus , Animais , Colífagos , Humanos , Microbiologia da Água
8.
Vet Microbiol ; 234: 61-71, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213273

RESUMO

Anti-phage activity of serum is of importance in repeated phage therapy. Higher serum anti-phage activity has been associated with greater susceptibility of phages to neutralisation and phage therapy failure. In this study, in vivo and in vitro survivability and immunogenicity of four coliphages (TM1, TM2, TM3 and TM4) were investigated in naive chickens and chickens pre-immunised with phage TM1. Furthermore, two phages that displayed different survivability and immunogenicity (TM1 and TM3) were compared with respect to their efficacy in treating naive or pre-immunised (TM1) chickens suffering from colibacillosis. The efficacy of the treatments was evaluated based on body weight, relative organ weights, mortality, E. coli counts in the lungs as well as severity and frequency of internal organ lesions. At the end of the experiment, both naive and pre-immunised chickens treated with TM3 showed significantly lower mortality and higher body weights than untreated chickens and those treated with TM1. The same trend was observed in incidence and severity of organ lesions as well as relative spleen weight. However, naive chickens treated with TM1 also showed a shortened inflammation period as indicated by spleen weights. E. coli counts in the lungs of chicken treated with TM3 were lower than those of chickens treated with TM1 on days 3 and 10 post challenge. These data indicate that the outcome of phage therapy and the impact of serum anti-phage activity are highly phage-type dependent in broilers.


Assuntos
Anticorpos Antivirais/sangue , Colífagos/imunologia , Infecções por Escherichia coli/veterinária , Terapia por Fagos/efeitos adversos , Doenças das Aves Domésticas/terapia , Animais , Galinhas , Escherichia coli , Infecções por Escherichia coli/terapia , Imunidade Humoral , Imunização , Imunoglobulina M/sangue , Imunoglobulinas/sangue , Inflamação , Pulmão/microbiologia , Pulmão/patologia , Terapia por Fagos/métodos , Doenças das Aves Domésticas/microbiologia , Soro , Baço/microbiologia , Baço/patologia
9.
Nucleic Acids Res ; 47(13): 7118-7129, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31180482

RESUMO

The gene cro promotes lytic growth of phages through binding of Cro protein dimers to regulatory DNA sites. Most Cro proteins are one-to-one orthologs, yet their sequence, structure and binding site sequences are quite divergent across lambdoid phages. We report the cocrystal structure of bacteriophage N15 Cro with a symmetric consensus site. We contrast this complex with an orthologous structure from phage λ, which has a dissimilar binding site sequence and a Cro protein that is highly divergent in sequence, dimerization interface and protein fold. The N15 Cro complex has less DNA bending and smaller DNA-induced changes in protein structure. N15 Cro makes fewer direct contacts and hydrogen bonds to bases, relying mostly on water-mediated and Van der Waals contacts to recognize the sequence. The recognition helices of N15 Cro and λ Cro make mostly nonhomologous and nonanalogous contacts. Interface alignment scores show that half-site binding geometries of N15 Cro and λ Cro are less similar to each other than to distantly related CI repressors. Despite this divergence, the Cro family shows several code-like protein-DNA sequence covariations. In some cases, orthologous genes can achieve a similar biological function using very different specific molecular interactions.


Assuntos
Colífagos/metabolismo , Regiões Operadoras Genéticas , Proteínas Repressoras/química , Proteínas Virais Reguladoras e Acessórias/química , Bacteriófago P22/metabolismo , Bacteriófago lambda/metabolismo , Sequência Consenso , Cristalografia por Raios X , DNA Bacteriano/metabolismo , Evolução Molecular , Ligação de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
10.
Res Vet Sci ; 125: 153-161, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31228739

RESUMO

An Escherichia coli (E. coli) O2:K1 bacterial ghost was produced by controlled expression of bacteriophage PhiX 174 lysis gene E. Temperature controlled expression of this gene caused tunnels and holes in the cell wall of E. coli O2:K1 bacterium, leading to loss of cytoplasmic contents. Formation of E. coli O2:K1 ghost was confirmed by scanning electron microscopy and determination of colony forming units. To evaluate the efficiency of this bacterial ghost vaccine to elicit cellular and humoral immune responses, 85 one day old chickens from Ross 308 breed were divided into the following 5 groups; group 1 (non-immunized control), group 2 (vaccine administered by injection of E. coli O2:K1 killed vaccine), group 3 (vaccine administered by injection of E. coli O2:K1 ghost), group 4 (vaccine administered by inhalation of E. coli O2:K1 ghost), and group 5 (neither immunized, nor challenged as negative control). The groups of 2, 3, and 4 were received vaccines at days 7, 14, and 22. Groups 1 to 4 were challenged with the wild type at day 33. Evaluation of post-mortem lesions and immune responses in all groups showed that chicken injected with the killed vaccine and the bacterial ghost had the best protection. These findings suggest that this bacterial ghost has the potential to be used as a poultry colibacillosis vaccine.


Assuntos
Galinhas , Infecções por Escherichia coli/veterinária , Vacinas contra Escherichia coli/imunologia , Escherichia coli/imunologia , Imunidade Celular , Imunidade Humoral , Doenças das Aves Domésticas/prevenção & controle , Adenosina/análogos & derivados , Animais , Colífagos/fisiologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Fenetilaminas , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Sorogrupo , Vacinas de Produtos Inativados/imunologia
11.
Food Environ Virol ; 11(2): 120-125, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30919239

RESUMO

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.


Assuntos
Colífagos/isolamento & purificação , Crassostrea/virologia , DNA Viral/genética , Frutos do Mar/virologia , Animais , Colífagos/classificação , Colífagos/genética , Masculino , Água do Mar/virologia , Esgotos/virologia , Especificidade da Espécie , Temperatura , Poluição da Água
12.
Nutrients ; 11(3)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897686

RESUMO

The gut microbiota is increasingly recognized as an important modulator of human health. As such, there is a growing need to identify effective means of selectively modifying gut microbial communities. Bacteriophages, which were briefly utilized as clinical antimicrobials in the early 20th century, present an opportunity to selectively reduce populations of undesirable microorganisms. However, whether intentional consumption of specific bacteriophages affects overall gut ecology is not yet known. Using a commercial cocktail of Escherichia coli-targeting bacteriophages, we examined their effects on gut microbiota and markers of intestinal and systemic inflammation in a healthy human population. In a double-blinded, placebo-controlled crossover trial, normal to overweight adults consumed bacteriophages for 28 days. Stool and blood samples were collected and used to examine inflammatory markers, lipid metabolism, and gut microbiota. Reductions in fecal E. coli loads were observed with phage consumption. However, there were no significant changes to alpha and beta diversity parameters, suggesting that consumed phages did not globally disrupt the microbiota. However, specific populations were altered in response to treatment, including increases in members of the butyrate-producing genera Eubacterium and a decreased proportion of taxa most closely related to Clostridium perfringens. Short-chain fatty acid production, inflammatory markers, and lipid metabolism were largely unaltered, but there was a small but significant decrease in circulating interleukin-4 (Il-4). Together, these data demonstrate the potential of bacteriophages to selectively reduce target organisms without global disruption of the gut community.


Assuntos
Colífagos , Gastroenteropatias/microbiologia , Inflamação/microbiologia , Inflamação/terapia , Adolescente , Adulto , Idoso , Suplementos Nutricionais , Método Duplo-Cego , Escherichia coli/virologia , Feminino , Microbioma Gastrointestinal , Humanos , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Adulto Jovem
13.
Environ Microbiol ; 21(6): 2112-2128, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30884081

RESUMO

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.


Assuntos
Colífagos/genética , Água do Mar/virologia , Colífagos/classificação , Colífagos/isolamento & purificação , Colífagos/fisiologia , Escherichia coli/genética , Escherichia coli/virologia , Genoma Viral , Genômica , Especificidade de Hospedeiro , Myoviridae/classificação , Myoviridae/genética , Myoviridae/isolamento & purificação , Myoviridae/fisiologia , Filogenia , Polônia , Proteômica , Siphoviridae/classificação , Siphoviridae/genética , Siphoviridae/isolamento & purificação , Siphoviridae/fisiologia , Reino Unido
14.
Anal Bioanal Chem ; 411(12): 2487-2492, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30903224

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Colífagos/enzimologia , Colífagos/isolamento & purificação , Endopeptidases/isolamento & purificação , Escherichia coli/virologia , Técnicas de Transferência de Genes , Grafite/química , Nanopartículas , Peptídeos/química , Sequência de Aminoácidos , Colífagos/genética , Contagem de Colônia Microbiana , Endopeptidases/genética , Fluorescência , Corantes Fluorescentes/química , Genes Virais , Limite de Detecção , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Estudo de Prova de Conceito
15.
Water Environ Res ; 91(9): 830-842, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30848516

RESUMO

A year-long sampling campaign at nine water resource recovery facilities (WRRFs) was conducted to assess the treatability and fate of bacterial indicators, viral indicators, and viruses. Influent concentrations of viral indicators (male-specific and somatic coliphages) and bacterial indicators (Escherichia coli and enterococci) remained relatively constant, typically varying by one order of magnitude over the course of the year. Annual average bacterial indicator reduction ranged from 4.0 to 6.7 logs, and annual average viral indicator reduction ranged from 1.6 to 5.4 logs. Bacterial and viral indicator reduction depended on the WRRF's treatment processes, and bacterial indicator reduction was greater than viral indicator reduction for many processes. Viral reduction (adenovirus 41, norovirus GI, and norovirus GII) was more similar to viral indicator reduction than bacterial indicator reduction. Overall, this work suggests that viral indicator reduction in WRRFs is variable and depends on specific unit processes. Moreover, for the same unit treatment process, viral indicator reduction and bacterial indicator reduction can vary. PRACTITIONER POINTS: A year-long sampling campaign was conducted at nine water resource recovery facilities (WRRFs). The treatability and fate of bacterial indicators, viral indicators, and viruses were assessed. Viral indicator reduction in WRRFs is variable and depends on specific unit processes. For the same unit treatment process, viral indicator reduction and bacterial indicator reduction can vary.


Assuntos
Vírus , Recursos Hídricos , Bactérias , Colífagos , Microbiologia da Água
16.
Water Res ; 155: 233-244, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30851594

RESUMO

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.


Assuntos
Escherichia coli , Microbiologia da Água , Bacteroides , Colífagos , Fezes , Humanos , Esgotos
17.
Molecules ; 24(4)2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795631

RESUMO

CRISPR/Cas is an adaptive bacterial immune system, whose CRISPR array can actively change in response to viral infections. However, Type I-E CRISPR/Cas in E. coli (an established model system), appears not to exhibit such active adaptation, which suggests that it might have functions other than immune response. Through computational analysis, we address the involvement of the system in non-canonical functions. To assess targets of CRISPR spacers, we align them against both E. coli genome and an exhaustive (~230) set of E. coli viruses. We systematically investigate the obtained alignments, such as hit distribution with respect to genome annotation, propensity to target mRNA, the target functional enrichment, conservation of CRISPR spacers and putative targets in related bacterial genomes. We find that CRISPR spacers have a statistically highly significant tendency to target i) host compared to phage genomes, ii) one of the two DNA strands, iii) genomic dsDNA rather than mRNA, iv) transcriptionally active regions, and v) sequences (cis-regulatory elements) with slower turn-over rate compared to CRISPR spacers (trans-factors). The results suggest that the Type I-E CRISPR/Cas system has a major role in transcription regulation of endogenous genes, with a potential to rapidly rewire these regulatory interactions, with targets being selected through naïve adaptation.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Colífagos/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Genoma Viral , Sequência de Bases , Biologia Computacional , DNA/genética , DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virologia , Loci Gênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Transcrição Genética
18.
Water Res ; 154: 62-71, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30771708

RESUMO

The purpose of this study was to evaluate the efficacy of a waste stabilization pond (WSP) system to reduce pathogen contaminants in sludge. This included examining the factors that influence the fate and concentration of human pathogens and their indicators in two sludge layers. The decay rates of five study microorganisms were determined under in-situ conditions at a WSP. The background levels of fecal origin microorganisms were consistently detected (ranging: Escherichia coli 104 to 106, enterococci 101 to 103, F-specific bacteriophage (MS2) 101 to 103 and somatic coliphage 101 to 104 colony-forming units (CFU) mL-1, as well as 101 to 102 human adenovirus gene copies mL-1) in the primary facultative pond. Among microorganisms tested, the bacteria generally decayed faster than adenovirus and bacteriophage, particularly in the upper sludge layer. Due to the observed regrowth of E. coli, it may have a limited value as an indicator for pathogen removal in the wastewater stabilization ponds. The abundance of E. coli numbers within the pond biome followed changes in pond temperature over time. The results of the study suggest that viruses could survive for a long time, particularly in deeper layers (>1 metre) in the sludge, during winter months (T90 = 156 d). The presence of human pathogens in WSP sludge, in particular viruses, may be a barrier to its beneficial reuse in agriculture. The results indicate that additional treatment of sludge may be required to mitigate potential public health risks from reuse of sludge for agricultural purposes.


Assuntos
Adenovírus Humanos , Esgotos , Colífagos , Enterococcus , Escherichia coli , Humanos , Levivirus , Tanques
19.
Water Res ; 153: 263-273, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30735956

RESUMO

Coliphage have been proposed as indicators of fecal contamination in recreational waters because they better reflect the persistence of pathogenic viruses in the environment and through wastewater treatment than traditional fecal indicator bacteria. Herein, we conducted a systematic literature search of peer-reviewed publications to identify coliphage density data (somatic and male-specific, or MSC) in raw wastewater and ambient waters. The literature review inclusion criteria included scope, study quality, and data availability. A non-parametric two-stage bootstrap analysis was used to estimate the coliphage distributions in raw wastewater and account for geographic region and season. Additionally, two statistical methodologies were explored for developing coliphage density distributions in ambient waters, to account for the nondetects in the datasets. In raw wastewater, the analysis resulted in seasonal density distributions of somatic coliphage (SC) (mean 6.5 log10 plaque forming units (PFU)/L; 95% confidence interval (CI): 6.2-6.8) and MSC (mean 5.9 log10 PFU/L; 95% CI: 5.5-6.1). In ambient waters, 49% of MSC samples were nondetects, compared with less than 5% for SC. Overall distributional estimates of ambient densities of coliphage were statistically higher for SC than for MSC (mean 3.4 and 1.0 log10 PFU/L, respectively). Distributions of coliphage in raw wastewater and ambient water will be useful for future microbial risk assessments.


Assuntos
Vírus , Águas Residuárias , Colífagos , Fezes , Humanos , Masculino , Microbiologia da Água
20.
Water Res ; 151: 439-446, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30639729

RESUMO

Sunlight inactivation is important for disinfection of viruses in sunlit waters. As such, attempts have been made to predict the endogenous photoinactivation rate of bacteriophage MS2 using biological weighting functions, which describe microorganism sensitivity to sunlight inactivation as a function of wavelength. In this study, four biological weighting function models were compared to assess their ability to predict endogenous inactivation rates (kendo) of MS2. Previously-published and newly-collected datasets consisting of an incident irradiance spectrum (used as an input to the model) and a measured inactivation rate (kobs) were used for model validation and comparison. kendo values predicted by each model were compared with measured kobs to evaluate the ability of each biological weighting function to predict endogenous sunlight inactivation rates. A model previously developed by Mattle et al. (Env. Sci. Technol. 49, 334-342) over-predicted inactivation rates, whereas the other three models - a model from Fisher et al. (Env. Sci. Technol. 45, 9249-9255), a new model developed in this study, and a modification of the model by Mattle et al. (developed as part of this study) - were better able to estimate inactivation rates. The biological relevance of the spectral shape of each biological weighting function is discussed.


Assuntos
Luz Solar , Microbiologia da Água , Colífagos , Desinfecção , Levivirus , Inativação de Vírus
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