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The reliance of the global population on urban aquifers is steadily increasing, and urban aquifers are susceptible to pathogenic contamination through sources such as sewer leakage or urban runoff. However, there is insufficient monitoring of groundwater quality in urban areas. In this study, quantitative polymerase chain reaction (qPCR) was employed to evaluate the presence of human fecal viral indicators and viral pathogens in urban wastewater (n = 13) and groundwater (n = 12) samples from four locations in Barcelona with different degrees of urbanization, as well as in runoff samples (n = 2). Additionally, a target enrichment sequencing (TES) approach was utilized to explore the viral diversity within groundwater and runoff samples, offering insights into viral contamination and potential virus transmission routes in urban areas. Human adenovirus (HAdV) was identified in all wastewater samples, 67 % (8/12) of groundwater samples, and one runoff sample by qPCR indicating human viral fecal contamination. The viral pathogen Norovirus genogroup GI (NoV GI) was detected in wastewater and two winter groundwater samples from highly and medium urbanized areas. NoV genogroup GII (NoV GII), Enterovirus (EV) and SARS-CoV-2 were exclusively detected in wastewater. Human and other vertebrate viruses were detected in groundwater and runoff samples using TES. This study gives insights about the virome present in urban water sources, emphasizing the need for thorough monitoring and deeper understanding to address emerging public health concerns.
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Monitoramento Ambiental , Água Subterrânea , Água Subterrânea/virologia , Humanos , Águas Residuárias/virologia , Microbiologia da Água , Cidades , Norovirus/isolamento & purificação , Norovirus/genética , Fezes/virologia , Espanha , SARS-CoV-2 , Vírus/isolamento & purificaçãoRESUMO
The COVID-19 pandemic has underscored the importance of understanding the role of animals in the transmission of coronaviruses (CoVs) and their impact on human health. A One Health approach, integrating human, animal, and environmental health, is essential for effective CoVs control. Next-generation sequencing has played a pivotal role in identifying and monitoring the evolution of novel CoVs strains, like SARS-CoV-2. However, viral occurrence and diversity studies in environmental and animal samples are challenging because of the complexity of viral communities and low abundance of viruses in these samples. Target enrichment sequencing (TES) has emerged as a valuable tool for investigating viral families in challenging samples. This approach involves the specific capture and enrichment of viral genomes using sequence-specific probes, thereby enhancing the efficiency of detection and characterization. In this study, we aimed to develop and validate a TES panel to study CoVs in various complex environmental and animal derived samples. The results demonstrated the panel's effectiveness in capturing and sequencing a wide diversity of CoVs providing valuable insights into their abundance and host diversity in urban wastewater, farm animal corpses lixiviates and bat guano samples. In sewage samples, CoVs were detected solely when TES was employed while in guano samples, sequencing of CoVs species was achieved in 2 out of 4 samples showing an almost three-logarithmic increase in the number of reads obtained in comparison with the untargeted approach. For animal lixiviates, only the TES application enabled the acquisition of CoVs reads. The information obtained can significantly contribute to early detection, surveillance, and control measures for CoVs, including viral discovery and potential spillover events. Additionally, this sequencing panel shows potential for studying other significant viral families and monitoring viral diversity in different animal populations.
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Occupational exposure to pathogens can pose health risks. This study investigates the viral exposure of workers in a wastewater treatment plant (WWTP) and a swine farm by analyzing aerosol and surfaces samples. Viral contamination was evaluated using quantitative polymerase chain reaction (qPCR) assays, and target enrichment sequencing (TES) was performed to identify the vertebrate viruses to which workers might be exposed. Additionally, Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate the occupational risk associated with viral exposure for WWTP workers, choosing Human Adenovirus (HAdV) as the reference pathogen. In the swine farm, QMRA was performed as an extrapolation, considering a hypothetical zoonotic virus with characteristics similar to Porcine Adenovirus (PAdV). The modelled exposure routes included aerosol inhalation and oral ingestion through contaminated surfaces and hand-to-mouth contact. HAdV and PAdV were widespread viruses in the WWTP and the swine farm, respectively, by qPCR assays. TES identified human and other vertebrate viruses WWTP samples, including viruses from families such as Adenoviridae, Circoviridae, Orthoherpesviridae, Papillomaviridae, and Parvoviridae. In the swine farm, most of the identified vertebrate viruses were porcine viruses belonging to Adenoviridae, Astroviridae, Circoviridae, Herpesviridae, Papillomaviridae, Parvoviridae, Picornaviridae, and Retroviridae. QMRA analysis revealed noteworthy risks of viral infections for WWTP workers if safety measures are not taken. The probability of illness due to HAdV inhalation was higher in summer compared to winter, while the greatest risk from oral ingestion was observed in workspaces during winter. Swine farm QMRA simulation suggested a potential occupational risk in the case of exposure to a hypothetical zoonotic virus. This study provides valuable insights into WWTP and swine farm worker's occupational exposure to human and other vertebrate viruses. QMRA and NGS analyses conducted in this study will assist managers in making evidence-based decisions, facilitating the implementation of protection measures, and risk mitigation practices for workers.
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Fazendas , Sequenciamento de Nucleotídeos em Larga Escala , Exposição Ocupacional , Águas Residuárias , Animais , Suínos , Águas Residuárias/virologia , Humanos , Medição de Risco , Vírus/isolamento & purificação , Vírus/genética , Monitoramento Ambiental/métodosRESUMO
BACKGROUND: Human viruses released into the environment can be detected and characterized in wastewater. The study of wastewater virome offers a consolidated perspective on the circulation of viruses within a population. Because the occurrence and severity of viral infections can vary across a person's lifetime, studying the virome in wastewater samples contributed by various demographic segments can provide valuable insights into the prevalence of viral infections within these segments. In our study, targeted enrichment sequencing was employed to characterize the human virome in wastewater at a building-level scale. This was accomplished through passive sampling of wastewater in schools, university settings, and nursing homes in two cities in Catalonia. Additionally, sewage from a large urban wastewater treatment plant was analysed to serve as a reference for examining the collective excreted human virome. RESULTS: The virome obtained from influent wastewater treatment plant samples showcased the combined viral presence from individuals of varying ages, with astroviruses and human bocaviruses being the most prevalent, followed by human adenoviruses, polyomaviruses, and papillomaviruses. Significant variations in the viral profiles were observed among the different types of buildings studied. Mamastrovirus 1 was predominant in school samples, salivirus and human polyomaviruses JC and BK in the university settings while nursing homes showed a more balanced distribution of viral families presenting papillomavirus and picornaviruses and, interestingly, some viruses linked to immunosuppression. CONCLUSIONS: This study shows the utility of building-level wastewater-based epidemiology as an effective tool for monitoring the presence of viruses circulating within specific age groups. It provides valuable insights for public health monitoring and epidemiological studies.
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Viroses , Vírus , Humanos , Águas Residuárias , Vigilância Epidemiológica Baseada em Águas Residuárias , Viroma/genética , Vírus/genéticaRESUMO
The recent monkeypox virus (MPXV) outbreak was of global concern and has mainly affected gay, bisexual and other men who have sex with men (GBMSM). Here we assess prevalence of MPXV in high-risk populations of GBMSM, trans women (TW) and non-binary people without symptoms or with unrecognized monkeypox (Mpox) symptoms, using a self-sampling strategy. Anal and pharyngeal swabs are tested by MPXV real-time PCR and positive samples are tested for cytopathic effect (CPE) in cell culture. 113 individuals participated in the study, 89 (78.76%) were cis men, 17 (15.04%) were TW. The median age was 35.0 years (IQR: 30.0-43.0), 96 (85.02%) individuals were gay or bisexual and 72 (63.72%) were migrants. Seven participants were MPXV positive (6.19% (95% CI: 1.75%-10.64%)). Five tested positive in pharyngeal swabs, one in anal swab and one in both. Six did not present symptoms recognized as MPXV infection. Three samples were positive for CPE, and showed anti-vaccinia pAb staining by FACS and confocal microscopy. This suggests that unrecognized Mpox cases can shed infectious virus. Restricting testing to individuals reporting Mpox symptoms may not be sufficient to contain outbreaks.
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Mpox , Minorias Sexuais e de Gênero , Masculino , Humanos , Feminino , Adulto , Espanha/epidemiologia , Homossexualidade Masculina , Mpox/diagnóstico , Mpox/epidemiologia , Monkeypox virus/genéticaRESUMO
Water treatment and reuse is gaining acceptance as a strategy to fight against water contamination and scarcity, but it usually requires complex treatments to ensure safety. Consequently, the electrochemical advanced processes have emerged as an effective alternative for water remediation. The main objective here is to perform a systematic study that quantifies the efficiency of a laboratory-scale electrochemical system to inactivate bacteria, bacterial spores, protozoa, bacteriophages and viruses in synthetic water, as well as in urban wastewater once treated in a wetland for reuse in irrigation. A Ti|RuO2-based plate and Si|BDD thin-film were comparatively employed as the anode, which was combined with a stainless-steel cathode in an undivided cell operating at 12 V. Despite the low resulting current density (<15 mA/cm2), both anodes demonstrated the production of oxidants in wetland effluent water. The disinfection efficiency was high for the bacteriophage MS2 (T99 in less than 7.1 min) and bacteria (T99 in about 30 min as maximum), but limited for CBV5 and TuV, spores and amoebas (T99 in more than 300 min). MS2 presented a rapid exponential inactivation regardless of the anode and bacteria showed similar sigmoidal curves, whereas human viruses, spores and amoebas resulted in linear profiles. Due the different sensitivity of microorganisms, different models must be considered to predict their inactivation kinetics. On this basis, it can be concluded that evaluating the viral inactivation from inactivation profiles determined for bacteria or some bacteriophages may be misleading. Therefore, neither bacteria nor bacteriophages are suitable models for the disinfection of water containing enteric viruses. The electrochemical treatment added as a final disinfection step enhances the inactivation of microorganisms, which could contribute to safe water reuse for irrigation. Considering the calculated low energy consumption, decentralized water treatment units powered by photovoltaic modules might be a near reality.
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Desinfecção , Purificação da Água , Humanos , Desinfecção/métodos , Bactérias , Oxirredução , Purificação da Água/métodos , OxidantesRESUMO
Viruses linked to animals inhabiting Antarctic latitudes remain poorly studied. Remote environments hosting large pinniped populations may be prone to exposure of immunologically naïve animals to new infectious agents due to increasing human presence or introduction of new animal species. Antarctic fur seals (Arctocephalus gazella) inhabiting the Western Antarctic Peninsula and the South Shetland Islands are challenged because of climate change and increased anthropogenic activity. In the present study, the fecal and serum virome of A. gazella was characterized by applying target enrichment next generation sequencing. The resulting viromes were dominated by CRESS-DNA sequences. Viruses known to infect vertebrate and invertebrate hosts were also observed in fecal samples. Fur seal picornavirus was present in all the fecal pools studied suggesting it is a prevalent virus in these species. Six different viruses presenting similarities with previously described A. gazella viruses or other otariids and mammal viruses were identified as potential new A. gazella viruses. Also, diet-derived viruses such as crustacean viruses were present in fecal content. Penguin viruses, but not fish viruses, were also detected. Obtained results contribute to a better understanding of the viral community present in these species, which is relevant for its conservation.
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Otárias , Vírus , Animais , Humanos , Vírus/genética , Metagenômica , Mudança Climática , Dieta , Regiões AntárticasRESUMO
Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.
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COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , Humanos , Pandemias , RNA Viral , Esgotos , Águas Residuárias , Vigilância Epidemiológica Baseada em Águas ResiduáriasRESUMO
Assessing the presence of viruses in large-volume samples involves cumbersome methods that require specialized training and laboratory equipment. In this study, a large volume concentration (LVC) method, based on dead-end ultrafiltration (DEUF) and Wet Foam Elution™ technology, was evaluated in different type of waters and different microorganisms. Its recovery efficiency was evaluated through different techniques (infectivity assays and molecular detection) by spiking different viral surrogates (bacteriophages PhiX174 and MS2 and Coxsackie virus B5 (CVB5) and Escherichia coli (E. coli). Furthermore, the application of a secondary concentration step was evaluated and compared with skimmed milk flocculation. Viruses present in river water, seawater and groundwater samples were concentrated by applying LVC method and a centrifugal ultrafiltration device (CeUF), as a secondary concentration step and quantified with specific qPCR Human adenoviruses (HAdV) and noroviruses (NoVs). MS2 was used as process control, obtaining a mean viral recovery of 22.0 ± 12.47%. The presence of other viruses was also characterized by applying two different next-generation sequencing approaches. LVC coupled to a secondary concentration step based on CeUF allowed to detect naturally occurring viruses such as HAdV and NoVs in different water matrices. Using HAdV as a human fecal indicator, the highest viral pollution was found in river water samples (100% of positive samples), followed by seawater (83.33%) and groundwater samples (66.67%). The LVC method has also proven to be useful as a virus concentration method in the filed since HAdV and NoVs were detected in the river water and groundwater samples concentrated in the field. All in all, LVC method presents high concentration factor and a low limit of detection and provides viral concentrates useful for subsequent molecular analysis such as PCR and massive sequencing.
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Adenovírus Humanos , Norovirus , Escherichia coli , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Ultrafiltração , Água , Microbiologia da ÁguaRESUMO
SARS-CoV-2 variants are emerging worldwide, and monitoring them is key in providing early warnings. Here, we summarize the different analytical approaches currently used to study the dissemination of SARS-CoV-2 variants in wastewater and discuss their advantages and disadvantages. We also provide preliminary results of two sensitive and cost-effective approaches: variant-specific reverse transcription-nested PCR assays and a nonvariant-specific amplicon deep sequencing strategy that targets three key regions of the viral spike protein. Next-generation sequencing approaches enable the simultaneous detection of signature mutations of different variants of concern in a single assay and may be the best option to explore the real picture at a particular time. Targeted PCR approaches focused on specific signature mutations will need continuous updating but are sensitive and cost-effective.
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Fresh fruits and vegetables are susceptible to microbial contamination at every stage of the food production chain, and as a potential source of pathogens, irrigation water quality is a critical factor. Next-generation sequencing (NGS) techniques have been flourishing and expanding to a wide variety of fields. However, their application in food safety remains insufficiently explored, and their sensitivity requires improvement. In this study, quantitative polymerase chain reaction (qPCR) assays showed low but frequent contamination of common circulating viral pathogens, which were found in 46.9% of samples of fresh produce: 6/12 lettuce samples, 4/12 strawberries samples, and 5/8 parsley samples. Furthermore, the application of two different NGS approaches, target enrichment sequencing (TES) for detecting viruses that infect vertebrates and amplicon deep sequencing (ADS), revealed a high diversity of viral pathogens, especially Norovirus (NoV) and Human Papillomavirus (HPV), in fresh produce and irrigation water. All NoV and HPV types found in fresh fruit and vegetable samples were also detected in irrigation water sources, indicating that these viruses are common circulating pathogens in the population and that irrigation water may be the most probable source of viral pathogens in food samples.
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In the wake of the COVID-19 pandemic, the use of next generation sequencing (NGS) has proved to be an important tool for the genetic characterization of SARS-CoV-2 from clinical samples. The use of different available NGS tools applied to wastewater samples could be the key for an in-depth study of the excreted virome, not only focusing on SARS-CoV-2 circulation and typing, but also to detect other potentially pandemic viruses within the same family. With this aim, 24-hours composite wastewater samples from March and July 2020 were sequenced by applying specific viral NGS as well as target enrichment NGS. The full virome of the analyzed samples was obtained, with human Coronaviridae members (CoV) present in one of those samples after applying the enrichment. One contig was identified as HCoV-OC43 and 8 contigs as SARS-CoV-2. CoVs from other animal hosts were also detected when applying this technique. These contigs were compared with those obtained from contemporary clinical specimens by applying the same target enrichment approach. The results showed that there is a co-circulation in urban areas of human and animal coronaviruses infecting domestic animals and rodents. NGS enrichment-based protocols might be crucial to describe the occurrence and genetic characteristics of SARS-CoV-2 and other Coronaviridae family members within the excreted virome present in wastewater.
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COVID-19 , Pandemias , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , SARS-CoV-2 , EsgotosRESUMO
The objective of the present study was to characterize Salmonella enterica serovar Infantis isolated from chicken meat determining their clonal relationships with S. Infantis isolated from children with diarrhea. Fifteen meat-recovered S. Infantis were analyzed. Susceptibility levels to 14 antibacterial agents, the presence of ESBL and that of inducible plasmid-mediated AmpC (i-pAmpC) were determined by phenotypical methods. The presence of ESBL and pAmpC was confirmed by PCR, and detected ESBL-encoding genes were sequenced and their transferability tested by conjugation. The presence of gyrA mutations as well as Class 1 integrons was determined by PCR. Clonal relationships were established by REP-PCR and RAPD. In addition, 25 clinical isolates of S. Infantis were included in clonality studies. All meat-recovered S. Infantis were MDR, showing resistance to ampicillin, nitrofurans and quinolones, while none was resistant to azithromycin, ceftazidime or imipenem. ESBL (blaCTX-M-65) and i-pAmpC (blaDHA) were detected in 2 and 5 isolates respectively (in one case concomitantly), with blaCTX-M-65 being transferable through conjugation. In addition, 1 isolate presented a blaSHV gene. All isolates presented D87Y at GyrA, nalidixic acid active efflux pump and a Class 1 integron of ~1000 bp (aadA1). Clonal analysis showed that all isolates were related. Further they were identical to MDR blaCTX-M-65-producing S. Infantis isolates causing children diarrhea in Lima. The dissemination of MDR blaCTX-M-65-producing S. Infantis between marketed meat and children highlights a public health problem which needs be controlled at livestock level.
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Proteínas de Bactérias/genética , Carne/microbiologia , Intoxicação Alimentar por Salmonella/transmissão , Salmonelose Animal/transmissão , Salmonella enterica/genética , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Galinhas/microbiologia , Criança , Humanos , Integrons/genética , Plasmídeos/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Intoxicação Alimentar por Salmonella/microbiologia , Salmonelose Animal/microbiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/isolamento & purificação , Sorogrupo , beta-Lactamases/metabolismoRESUMO
Acute infectious gastroenteritis is an important illness worldwide, especially on children, with viruses accounting for approximately 70% of the acute cases. A high number of these cases have an unknown etiological agent and the rise of next generation sequencing technologies has opened new opportunities for viral pathogen detection and discovery. Viral metagenomics in routine clinical settings has the potential to identify unexpected or novel variants of viral pathogens that cause gastroenteritis. In this study, 124 samples from acute gastroenteritis patients from 2012-2014 previously tested negative for common gastroenteritis pathogens were pooled by age and analyzed by next generation sequencing (NGS) to elucidate unidentified viral infections. The most abundant sequences detected potentially associated to acute gastroenteritis were from Astroviridae and Caliciviridae families, with the detection of norovirus GIV and sapoviruses. Lower number of contigs associated to rotaviruses were detected. As expected, other viruses that may be associated to gastroenteritis but also produce persistent infections in the gut were identified including several Picornaviridae members (EV, parechoviruses, cardioviruses) and adenoviruses. According to the sequencing data, astroviruses, sapoviruses and NoV GIV should be added to the list of viral pathogens screened in routine clinical analysis.
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Gastroenterite/virologia , Metagenoma , Metagenômica , Viroses/virologia , Fatores Etários , Criança , Pré-Escolar , Biologia Computacional/métodos , Fezes/virologia , Feminino , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Metagenômica/métodos , Filogenia , Carga ViralRESUMO
Much of the knowledge on viruses is focused on those that can be propagated using cell-cultures or that can cause disease in humans or in economically important animals and plants. However, this only reflects a small portion of the virosphere. Therefore, in this study, we explore by targeted next-generation sequencing, how the virome varies between Atlantic horse mackerels and gilthead seabreams from fisheries and aquaculture from the center and south regions of Portugal. Viral genomes potentially pathogenic to fish and crustaceans, as well as to humans, were identified namelyese included Astroviridae, Nodaviridae, Hepadnaviridae, Birnaviridae, Caliciviridae, and Picornaviridae families. Also bacteriophages sequences were identified corresponding to the majority of sequencese detected, with Myoviridae, Podoviridae, and Siphoviridae, the most widespread families in both fish species. However, these findings can also be due to the presence of bacteria in fish tissues, or even to contamination. Overall, seabreams harbored viruses from a smaller number of families in comparison with mackerels. Therefore, the obtained data show that fish sold for consumption can harbor a high diversity of viruses, many of which are unknown, reflecting the overall uncharacterized virome of fish. While cross-species transmission of bonafide fish viruses to humans is unlikely, the finding of human pathogenic viruses in fish suggest that fish virome can be a potential threat regarding food safety.
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As the novel SARS-CoV-2 was detected in faeces, environmental researchers have been using centrifugal ultrafiltration, polyethylene glycol precipitation and aluminium hydroxide flocculation to describe its presence in wastewater samples. High recoveries (up to 65%) are described with electronegative filtration when using surrogate viruses, but few literature reports recovery efficiencies using accurate quantification of enveloped viruses. Considering that every single virus will have a different behaviour during viral concentration, it is recommended to use an enveloped virus, and if possible, a betacoronaviruses as murine hepatitis virus, as a surrogate. In this review, we show new data from a newly available technology that provides a quick ultrafiltration protocol for SARS-CoV-2. Wastewater surveillance is an efficient system for the evaluation of the relative prevalence of SARS-CoV-2 infections in a community, and there is the need of using reliable concentration methods for an accurate and sensitive quantification of the virus in water.
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The mechanisms of resistance to nitrofurans from 18 meat samples with Salmonella enterica (chicken: 15; beef: 2; pork: 1) collected in Lima (Peru) were analyzed. The isolates were serotyped and the susceptibility levels to furazolidone and nitrofurantoin [with and without the efflux pump inhibitor Phenyl-Arginine- ß-naphthylamide (PAßN)], the presence of mutations in the snrA and cnr genes and the transferability of resistance by conjugation were established. Fifteen samples with S. infantis (13 from chicken samples), 2 with S. enteritidis and 1 with S. anatum were identified. All isolates except the S. anatum were resistant to both nitrofurans showing MICs (minimum inhibitory concentration) of furazolidone and nitrofurantoin of 32-64 µg/mL and 128-256 µg/mL, respectively. The addition of PAßN had no effect on the MIC levels. All nitrofuran-resistant isolates showed amino acid codon alterations at both snrA and cnr (S. infantis: snrA STOP-151; cnr STOP-137; S. enteritidis: snrA STOP-180; cnr STOP-179). No transferable mecha nisms of nitrofuran resistance were detected.
En el presente estudio, se analizaron los mecanismos de resistencia a nitrofuranos en 18 muestras cár nicas con Salmonella enterica (15 de pollo, 2 de ternera y 1 de cerdo) de mercados de Lima (Perú). Determinaron los serotipos de los aislamientos y la sensibilidad a furazolidona y nitrofurantoina (con y sin el inhibidor de bombas de expulsión Phenyl-Arginine-ß-Naphthylamide [PAßN]), las mutaciones en los genes snrA y cnr por PCR y la transferabilidad de la resistencia por conjugación. Se identificaron 15 muestras con S. infantis (13 muestras de pollo), 2 con S. enteritidis y 1 con S. anatum. Todos los aisla mientos, excepto S. anatum, fueron resistentes a ambos nitrofuranos (concentración mínima inhibidora [CMI] a furazolidona: 32-64 µg/mL, CMI a nitrofurantoina: 128-256 µg/mL), sin diferencias al adicio narse PAßN. Todos los aislamientos resistentes a nitrofuranos presentaron sustituciones en snrA y cnr (S. infantis: snrA STOP-151; cnr STOP-137; S. enteritidis: snrA STOP-180; cnr STOP-179). No se detectaron mecanismos transferibles de resistencia a nitrofuranos.
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Farmacorresistência Bacteriana , Microbiologia de Alimentos , Carne , Nitrofurantoína , Salmonella enterica , Animais , Bovinos , Galinhas , Humanos , Carne/microbiologia , Testes de Sensibilidade Microbiana , Nitrofurantoína/farmacologia , Peru , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/isolamento & purificação , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/isolamento & purificação , SuínosRESUMO
Rotavirus A (RVA) is the most common virus associated with infantile gastroenteritisworldwide, being a public health threat, as it is excreted in large amounts in stool and can persist inthe environment for extended periods. In this study, we performed the detection of RVA and humanadenovirus (HAdV) by TaqMan qPCR and assessed the circulation of RVA genotypes in threewastewater treatment plants (WWTPs) between 2015 and 2016 in Catalonia, Spain. RVA wasdetected in 90% and HAdV in 100% of the WWTP samples, with viral loads ranging between 3.96 ×104 and 3.30 × 108 RT-PCR Units/L and 9.51 × 104 and 1.16 × 106 genomic copies/L, respectively. RVAVP7 and VP4 gene analysis revealed the circulation of G2, G3, G9, G12, P[4], P[8], P[9] and P[10].Nucleotide sequencing (VP6 fragment) showed the circulation of I1 and I2 genotypes, commonlyassociated with human, bovine and porcine strains. It is important to mention that the RVA strainsisolated from the WWTPs were different from those recovered from piglets and calves living in thesame area of single sampling in 2016. These data highlight the importance of monitoring watermatrices for RVA epidemiology and may be a useful tool to evaluate and predict possibleemergence/reemergence of uncommon strains in a region.
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Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/virologia , Rotavirus/classificação , Rotavirus/isolamento & purificação , Esgotos/virologia , Animais , Proteínas do Capsídeo/genética , Bovinos , Fezes/virologia , Variação Genética , Genótipo , Humanos , Filogenia , Prevalência , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Rotavirus/genética , Análise de Sequência , Espanha , Suínos , Carga ViralRESUMO
RESUMEN En el presente estudio, se analizaron los mecanismos de resistencia a nitrofuranos en 18 muestras cár nicas con Salmonella enterica (15 de pollo, 2 de ternera y 1 de cerdo) de mercados de Lima (Perú). Determinaron los serotipos de los aislamientos y la sensibilidad a furazolidona y nitrofurantoina (con y sin el inhibidor de bombas de expulsión Phenyl-Arginine-β-Naphthylamide [PAβN]), las mutaciones en los genes snrA y cnr por PCR y la transferabilidad de la resistencia por conjugación. Se identificaron 15 muestras con S. infantis (13 muestras de pollo), 2 con S. enteritidis y 1 con S. anatum. Todos los aisla mientos, excepto S. anatum, fueron resistentes a ambos nitrofuranos (concentración mínima inhibidora [CMI] a furazolidona: 32-64 µg/mL, CMI a nitrofurantoina: 128-256 µg/mL), sin diferencias al adicio narse PAβN. Todos los aislamientos resistentes a nitrofuranos presentaron sustituciones en snrA y cnr (S. infantis: snrA STOP-151; cnr STOP-137; S. enteritidis: snrA STOP-180; cnr STOP-179). No se detectaron mecanismos transferibles de resistencia a nitrofuranos.
ABSTRACT The mechanisms of resistance to nitrofurans from 18 meat samples with Salmonella enterica (chicken: 15; beef: 2; pork: 1) collected in Lima (Peru) were analyzed. The isolates were serotyped and the susceptibility levels to furazolidone and nitrofurantoin [with and without the efflux pump inhibitor Phenyl-Arginine- β-naphthylamide (PAβN)], the presence of mutations in the snrA and cnr genes and the transferability of resistance by conjugation were established. Fifteen samples with S. infantis (13 from chicken samples), 2 with S. enteritidis and 1 with S. anatum were identified. All isolates except the S. anatum were resistant to both nitrofurans showing MICs (minimum inhibitory concentration) of furazolidone and nitrofurantoin of 32-64 μg/mL and 128-256 μg/mL, respectively. The addition of PAßN had no effect on the MIC levels. All nitrofuran-resistant isolates showed amino acid codon alterations at both snrA and cnr (S. infantis: snrA STOP-151; cnr STOP-137; S. enteritidis: snrA STOP-180; cnr STOP-179). No transferable mecha nisms of nitrofuran resistance were detected.
Assuntos
Animais , Bovinos , Humanos , Salmonella enterica , Farmacorresistência Bacteriana , Microbiologia de Alimentos , Carne , Nitrofurantoína , Peru , Salmonella enteritidis/isolamento & purificação , Salmonella enteritidis/efeitos dos fármacos , Suínos , Testes de Sensibilidade Microbiana , Galinhas , Salmonella enterica/isolamento & purificação , Salmonella enterica/efeitos dos fármacos , Carne/microbiologia , Nitrofurantoína/farmacologiaRESUMO
Viruses (e.g., noroviruses and hepatitis A and E virus), bacteria (e.g., Salmonella spp. and pathogenic Escherichia coli) and protozoa (e.g., Cryptosporidium parvum and Giardia intestinalis) are well-known contributors to food-borne illnesses linked to contaminated fresh produce. As agricultural irrigation increases the total amount of water used annually, reclaimed water is a good alternative to reduce dependency on conventional irrigation water sources. European guidelines have established acceptable concentrations of certain pathogens and/or indicators in irrigation water, depending on the irrigation system used and the irrigated crop. However, the incidences of food-borne infections are known to be underestimated and all the different pathogens contributing to these infections are not known. Next-generation sequencing (NGS) enables the determination of the viral, bacterial and protozoan populations present in a water sample, providing an opportunity to detect emerging pathogens and develop improved tools for monitoring the quality of irrigation water. This is a descriptive study of the virome, bacteriome and parasitome present in different irrigation water sources. We applied the same concentration method for all the studied samples and specific metagenomic approaches to characterize both DNA and RNA viruses, bacteria and protozoa. In general, most of the known viral species corresponded to plant viruses and bacteriophages. Viral diversity in river water varied over the year, with higher bacteriophage prevalences during the autumn and winter. Reservoir water contained Enterobacter cloacae, an opportunistic human pathogen and an indicator of fecal contamination, as well as Naegleria australiensis and Naegleria clarki. Hepatitis E virus and Naegleria fowleri, emerging human pathogens, were detected in groundwater. Reclaimed water produced in a constructed wetland system presented a virome and bacteriome that resembled those of freshwater samples (river and reservoir water). Viral, bacterial and protozoan pathogens were occasionally detected in the different irrigation water sources included in this study, justifying the use of improved NGS techniques to get a comprehensive evaluation of microbial species and potential environmental health hazards associated to irrigation water.