Antimicrobial resistance characterization of Enterococcus faecium, Enterococcus faecalis and Enterococcus hirae isolated from marine coastal recreational waters in the State of São Paulo, Brazil.

Coastal water quality is facing increasing threats due to human activities. Their contamination by sewage discharges poses significant risks to the environment and public health. We aimed to investigate the presence of antibiotic-resistant Enterococcus in beach waters. Over a 10-month period, samples were collected from four beaches in the State of São Paulo (Brazil). Enterococcus isolates underwent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and molecular analysis for accurate genus and species identification. The antimicrobial susceptibility for 14 antibiotics was evaluated using the disc diffusion method followed by a multidrug-resistance (MDR) classification. PCR amplification method was used to detect antimicrobial resistance genes (ARGs). Our findings revealed the prevalence of Enterococcus faecalis, E. faecium and E. hirae. Out of 130 isolates, 118 were resistant to multiple antibiotics. The detection of resistance genes provided evidence of the potential transfer of antibiotic resistance within the environment. Our findings underscore the necessity for continuous research and surveillance to enhance understanding of the pathogenicity and antimicrobial resistance mechanisms of Enterococcus, which is crucial to implement effective measures to preserve the integrity of coastal ecosystems.

One-year surveillance of SARS-CoV-2 in wastewater from vulnerable urban communities in metropolitan São Paulo, Brazil.

The current COVID-19 pandemic has emphasized the vulnerability of communities living in the urban outskirts and informal settlements. The lack of reliable COVID-19 case data highlights the importance and application of wastewater-based epidemiology. This study aimed to monitor the COVID-19 trends in four vulnerable urban communities (slums and low-income neighborhoods) in metropolitan São Paulo by assessing the SARS-CoV-2 RNA viral load in wastewater. We analyzed 160 samples from May 2020 to June 2021 with weekly or fortnightly samplings. The samples were ultracentrifuged with glycine elution and quantified by N1/N2 SARS-CoV-2 RT-qPCR. The results of positivity were 100% (Paraisópolis, Heliópolis and Cidade Tiradentes) and 76.9% (Vila Brasilândia). The new case numbers of COVID-19, counted from the onset of symptoms, positively correlated with SARS-CoV-2 N1 viral loads from the two largest communities (p<0.001). SARS-CoV-2 infectivity was tested in Vero E6 cells after concentration with the two techniques, ultrafiltration (Centricon® Plus-70 10 kDa) and sucrose cushion ultracentrifugation, but none of the evaluated samples presented positive results. Next-generation sequencing (NGS) analysis from samples collected in March and August 2021 revealed the presence of the clade 20 J (lineage P.1) belonging to the most prevalent circulating variant in the country. Our results showed that wastewater surveillance data can be used as complementary indicators to monitor the dynamics and temporal trends of COVID-19. The infectivity test results strengthened the evidence of low risk of infection associated with SARS-CoV-2 in wastewater.

Real-time PCR detection of Toxoplasma gondii in surface water samples in São Paulo, Brazil.

Water is considered an important vehicle for the spread of human toxoplasmosis in several countries. Toxoplasma gondii oocysts can persist in the environment for long periods, being highly resistant to the various chemical inactivation processes commonly used by water supply systems, distinctly from simple filtration and flocculation that are efficient in removing oocysts from drinking water. The existing methodologies for identification and quantification of this parasite in water samples are not standardized and have limitations. This study aimed to evaluate the presence of T. gondii oocysts in surface water samples used as a source for the production of drinking water in the State of São Paulo, through the implementation of a specific methodology using real-time PCR technique (qPCR). Volumes of 20 L of the sample were concentrated by filtration in Envirocheck® HV capsules. For DNA extraction, the PowerSoil DNA isolation® kit (currently DNeasy PowerSoil®) was used. The target sequence selected for qPCR was a 62-base-pair fragment of the B1 gene. In the initial recovery evaluation of the method in four replicates of reverse osmosis water, the mean recovery was 48.5% (SD ± 11.5), while the mean recovery for method performance in matrices was 3.2% (SD ± 3.2) (rainy season) and 62.0% (SD ± 6.2) (dry period), suggesting that the characteristics of the samples and the climatic conditions interfere in the recovery efficiency. Of the 39 samples analyzed (May to December 2015), 7.7% (3/39) were positive for T. gondii, and among the ten sources studied; the occurrence of the oocysts was detected in 30% (3/10).

Distribution of human fecal marker GB-124 bacteriophages in urban sewage and reclaimed water of São Paulo city, Brazil.

Bacteriophages infecting Bacteroides fragilis GB-124 have been described as potential markers of human fecal contamination in water sources. The aim of this study was to evaluate the occurrence of GB-124 phages in raw sewage, secondary effluents and reclaimed water of the São Paulo city using a low-cost microbial source tracking method. Samples were collected monthly from April 2015 to March 2016 in four municipal wastewater treatment plants that operate with activated sludge processes followed by different tertiary treatments (sand-anthracite filtration, membrane bioreactor/reverse osmosis) and final chlorination. GB-124 phages were detected in 100% of the raw sewage samples, with viral loads varying from 7.5 × 103 to 1.32 × 106 PFU/L. Virus removal efficiency in activated sludge processes ranged from 1.89 to 2.31 log10. Frequencies of phage detection were lower in reclaimed water samples (0-22.2%). The results indicated that GB-124 phage could be a complementary low-cost viral marker for the detection of human fecal pollution in waters impacted with urban sewage in this region. However, the datasets of tertiary effluents resulted in several samples with concentrations below the detection limit (DL ≤1 PFU/mL) suggesting the need to obtain analytical methods with lower DL for greater accuracy of negative results.

Novel members of the order Picornavirales identified in freshwater from Guarapiranga reservoir in São Paulo.

The global challenge of water resource availability is exacerbated by anthropogenic influences that promote the emergence of pollutants. Among these pollutants are microbiological agents, including viruses, which are ubiquitous in the biosphere and play a pivotal role in both ecological balance and the occurrence of diseases in animals and plants. Consequently, monitoring viruses in water sources becomes indispensable for the establishment of effective prevention, promotion, and control strategies. Within this context, the study focuses on the identification of novel viruses belonging to the Picornavirales order in freshwater from the Guarapiranga Reservoir in the state of São Paulo, Brazil. The samples were subjected to viral metagenomics. Our analysis led to the characterization of four distinct sequences (GinkV-05, AquaV_10, MarV_14, and MarV_64), which exhibited significant divergence compared to other members of the Picornavirales order. This remarkable diversity prompted the identification of a potential new genus within the Marnaviridae family, tentatively named Ginkgonavirus. Additionally, we characterized four sequences in a very distinct clade and propose the recognition of a novel family (named Aquaviridae) within the Picornavirales order. Our findings contribute valuable insights into the previously uncharted diversity of Picornavirales present in water sources, shedding light on an important facet of viral ecology and evolution in aquatic environments.

Environmental surveillance of Cryptosporidium and Giardia in surface supply water and treated sewage intended for reuse from an urban area in Brazil.

Environmental monitoring of protozoa, with the potential to trigger diseases, is essential for decision-making by managing authorities and for the control of water surveillance. This study aimed to detect and quantify Cryptosporidium oocysts and Giardia cysts in surface water for drinking water supply and treated sewage for reuse in the city of São Paulo. Samples collected bimonthly for one year were concentrated using the USEPA 1623.1 and 1693 methods for surface water and treated effluents, respectively. Immunofluorescence and nucleic acid amplification techniques were used to detect and quantify (oo)cysts. The cloning technique followed by sequencing and phylogenetic analyses were performed to characterize species and genotypes. The immunofluorescence detected Cryptosporidium spp. and Giardia spp. in 69.2% (9/13) and 100% (13/13) of the surface water samples (0.1-41 oocysts/L and 7.2-354 cysts/L, respectively). In the reuse samples, 85.7% (12/14) were positive for both protozoa and the concentrations varied from 0.4 to 100.6 oocysts/L and 1.2 and 93.5 cysts/L. qPCR assays showed that 100% of surface water (0.1-14.6 oocysts/L and 0.3-639.8 cysts/L) and reused samples (0.1-26.6 oocysts/L and 0.3-92.5 cysts/L) were positive for both protozoa. Species C. parvum, C. hominis, and C. muris were identified using the 18S rRNA gene, demonstrating anthroponotic and zoonotic species in the samples. Multilocus SSU rRNAanalyses of the SSU rRNA, tpi, and gdh genes from Giardia intestinalis identified the AII, BII, and BIV assemblages, revealing that contamination in the different matrices comes from human isolates. The study showed the circulation of these protozoa in the São Paulo city area and the impairment of surface water supply in metropolitan regions impacted by the discharge of untreated or inadequately treated sewage regarding the removal of protozoa, emphasizing the need to implement policies for water safety, to prevent the spread of these protozoa in the population.

Cross-continental comparative experiences of wastewater surveillance and a vision for the 21st century.

The COVID-19 pandemic has brought the epidemiological value of monitoring wastewater into sharp focus. The challenges of implementing and optimising wastewater monitoring vary significantly from one region to another, often due to the array of different wastewater systems around the globe, as well as the availability of resources to undertake the required analyses (e.g. laboratory infrastructure and expertise). Here we reflect on the local and shared challenges of implementing a SARS-CoV-2 monitoring programme in two geographically and socio-economically distinct regions, São Paulo state (Brazil) and Wales (UK), focusing on design, laboratory methods and data analysis, and identifying potential guiding principles for wastewater surveillance fit for the 21st century. Our results highlight the historical nature of region-specific challenges to the implementation of wastewater surveillance, including previous experience of using wastewater surveillance, stakeholders involved, and nature of wastewater infrastructure. Building on those challenges, we then highlight what an ideal programme would look like if restrictions such as resource were not a constraint. Finally, we demonstrate the value of bringing multidisciplinary skills and international networks together for effective wastewater surveillance.

Wastewater-based epidemiology for preventing outbreaks and epidemics in Latin America - Lessons from the past and a look to the future.

Wastewater-based epidemiology (WBE) is an approach with the potential to complement clinical surveillance systems. Using WBE, it is possible to carry out an early warning of a possible outbreak, monitor spatial and temporal trends of infectious diseases, produce real-time results and generate representative epidemiological information in a territory, especially in areas of social vulnerability. Despite the historical uses of this approach, particularly in the Global Polio Eradication Initiative, and for other pathogens, it was during the COVID-19 pandemic that occurred an exponential increase in environmental surveillance programs for SARS-CoV-2 in wastewater, with many experiences and developments in the field of public health using data for decision making and prioritizing actions to control the pandemic. In Latin America, WBE was applied in heterogeneous contexts and with emphasis on populations that present many socio-environmental inequalities, a condition shared by all Latin American countries. This manuscript addresses the concepts and applications of WBE in public health actions, as well as different experiences in Latin American countries, and discusses a model to implement this surveillance system at the local or national level. We emphasize the need to implement this sentinel surveillance system in countries that want to detect the early entry and spread of new pathogens and monitor outbreaks or epidemics of infectious agents in their territories as a complement of public health surveillance systems.

Serotyping and Antimicrobial Susceptibility Profiling of Glaesserella parasuis Isolated from Diseased Swine in Brazil.

Glaesserella parasuis is one of the major pathogens in swine intensive production systems. To date, 15 serovars have been described, and the prevalence of these serotypes in different geographical regions has been identified by several methods. G. parasuis outbreaks could be controlled with vaccination if it were not for serovar diversity and limited cross-serovar protection; consequently, antibiotic therapy continues to be necessary for infection control. Here, we present the isolation, identification, serotyping, and antibiotic susceptibility profiling of G. parasuis from diseased swine in Brazil. A total of 105 G. parasuis strains, originating from nine different Brazilian states, were evaluated, and serotypes 4 and 5 were found to be the most prevalent (27.6% and 24.8% respectively). Aminoglycosides, florfenicol, tiamulin, and ß-lactams were tested, and they presented lower resistant rates against G. parasuis strains. The highest resistance rates were observed against tylosin (97.1%), sulfadimethoxine (89.5%), danofloxacin (80%), trimethoprim/sulfamethoxazole (62.5%), enrofloxacin (54.3%), and clindamycin (50.5%). Multidrug resistance was detected in 89.5% of tested strains, and a total of sixty resistance profiles were identified. The cluster analysis of resistance patterns showed no correlation with the isolation year or G. parasuis serotype.

Causes of Sow Mortality and Risks to Post-Mortem Findings in a Brazilian Intensive Swine Production System.

The present study was conducted to investigate the risk factors for post-mortem findings and causes of sow mortality. A post-mortem examination and microbiological investigation were conducted on 123 sows from a breeding herd with 15,000 dams. The mortality of spontaneous death in sows occurred mostly in the peripartum period (53%; p < 0.05). The spontaneous deaths were associated with heart failures, hemorrhagic and perforating gastric ulcers, and liver torsion, while in the euthanized sows, the post-mortem findings were associated with locomotor disorders. A higher body condition score (BCS ≥ 3.5) increased (p < 0.05) heart failure on the post-mortem examination. The excessive use of manual obstetric interventions increased sow deaths resulting from cervix/uterus ruptures and increased the odds of death (p < 0.05) due to metritis. Sow mortality had a multifactorial etiology. Infections were polymicrobial. The main microbial agents identified from a septic lesion in locomotor, genitourinary, and respiratory systems were Trueperella pyogenes, Escherichia coli, and Actinobacillus pleuropneumoniae, respectively. In conclusion, sow mortality involved multiple risk factors and several bacterial agents. These results indicate that better management practices can reduce sow mortality in swine production and increase sow welfare.

Hepatitis A Outbreaks and Environmental Circulation of Genotype IA Strains in the São Paulo City, 2017-2018.

Hepatitis A virus (HAV) is the major cause of enterically transmitted infectious hepatitis. Between 2016 and 2017, the number of confirmed cases of hepatitis A virus (HAV) increased from 64 to 786 in São Paulo affecting mainly adults aged between 18 and 39 years (80%) and males (88%). To support epidemiological surveillance, the present study monitored the presence of HAV in urban sewage samples collected bimonthly for 1 year (November 2017-November 2018) in the central region of the city, where most of cases were detected. Sewage samples were concentrated by polyethylene glycol precipitation and HAV RNA was quantified by RT-qPCR. Nucleotide sequencing targeting the VP1/2A junction region was carried out to genotype the HAV strains. HAV was detected in 76.9% (40/52) of the samples, with a geometric mean viral load of 5.09 × 104 (± SD 4.51 × 105) genome copies (GC/L) (Mauá Street) and 5.27 × 104 (± SD 1.26 × 106) GC/L (Prestes Maia Avenue). Of the 40 positive samples, 8 were typed as HAV subgenotype IA [100% nucleotide (nt) identity with HAV strain VRD_521_2016]. Highest homology was obtained with sequences from European countries (Italy, Spain) and Israel, all of which had reported recent HAV outbreaks associated with men who have sex with men. Our results highlight that wastewater surveillance is a useful tool to support investigating HAV outbreaks in the community, including circulating genotypes.

Vagococcus sp. a porcine pathogen: molecular and phenotypic characterization of strains isolated from diseased pigs in Brazil.

INTRODUCTION: Vagococcus spp. is known for its importance as a systemic and zoonotic bacterial pathogen even though it is not often reported in pigs. This is related to the pathogen misidentification due to the lack of usage of more discriminatory diagnostic techniques. Here we present the first report of Vagococcus lutrae in swine and the characterization of Vagococcus fluvialis and Vagococcus lutrae isolated from diseased animals. METHODOLOGY: Between 2012 and 2017, 11 strains with morphological characteristics similar to Streptococcus spp. were isolated from pigs presenting different clinical signs. Bacterial identification was performed by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry and confirmed by 16S rRNA sequencing and biochemical profile. Strains were further genotyped by single-enzyme amplified fragment length polymorphism (SE-AFLP). Broth microdilution was used to determine the minimal inhibitory concentration of the antimicrobials of veterinary interest. RESULTS: Ten strains were identified as V. fluvialis and one was identified as V. lutrae. The SE-AFLP analysis enabled the species differentiation with specific clustering of all V. fluvialis separately from the V. lutrae strain. Most strains presented growth in the maximum antibiotic concentration values tested for eight of the 10 analyzed antimicrobial classes. CONCLUSIONS: The observed resistance pattern can represent a problem for veterinary and producers in the treatment of diseases associated Vagococcus spp. in swine production. Vagococcus species may also be a risk for pig industry workers. The data described here will be of great value in further understanding the behavior of this pathogen in animal production.

Noroviruses in raw sewage, secondary effluents and reclaimed water produced by sand-anthracite filters and membrane bioreactor/reverse osmosis system.

The importance of noroviruses (NoVs) in the epidemiology of waterborne diseases has increased globally in the last decades. The present study aimed to monitor genogroup I and II noroviruses in different treatment stages of four wastewater treatment plants (WWTPs) in the metropolitan São Paulo. WWTPs consist of secondary (activated sludge) and tertiary treatments (coagulation, sand-anthracite filters, membrane bioreactor (MBR)/reverse osmosis (RO) and chlorination). Raw sewage (500mL) and treated effluents (1L) were concentrated by celite and reclaimed water (40L) by hollow-fiber ultrafiltration system. Quantitative (qPCR) and nested PCR with nucleotide sequencing were used for quantification and molecular characterization. NoVs were widely distributed in raw wastewater samples (83.3%-100% NoV GI and 91.6%-100% NoV GII) and viral loads varied from 3.8 to 6.66log10gcL-1 for NoV GI and 3.8 to 7.3log10gcL-1 for NoV GII. Mean virus removal efficiencies obtained for activated sludge processes ranged from 0.3 to 0.8 log10 for NoV GI and 0.4 to 1.4 log10 for NoV GII. NoVs were not detected in the reuse water produced by MBR/RO system, while sand-anthracite filters resulted in a NoV GI and GII decay of 1.1-1.6 log10 and 0.7-1.6 log10, respectively. A variety of genotypes (GI.2, GI.3a, GI.3b, GI.5, GII.1, GII.4 Sydney 2012, GII.5, GII.6, GII.17) was observed, with a predominance of GI.2 and GII.17 in the different genogroups. These results corroborate with recent data about the entry and dissemination of the emerging genotype GII.P17-GII.17 Kawasaki 2014 in the country, and may indicate a change in the epidemiological patterns of norovirus strains circulation in this region. This is the first large-scale study to evaluate burden and genotypes of noroviruses in WWTPs in Brazil, providing a rapid diagnosis of viruses circulating in the population.

Performance of wastewater reclamation systems in enteric virus removal.

Analysis of virus removal by tertiary or advanced sewage treatment processes is an emerging topic due to importance of reusing water on a global level. This study aimed to monitor important human viral pathogens: the human adenovirus (HAdV), JC polyomavirus (JCV) and Species A rotaviruses (RVA) in urban sewage, secondary effluents and reclaimed water from metropolitan São Paulo (MSP), Brazil. Four large wastewater treatment plants (WWTPs) in MSP were sampled monthly during a one-year period (April 2015 to March 2016). The viruses were quantified by quantitative PCR (qPCR), and HAdV viability was tested by the integrated cell culture (ICC)-qPCR assay. WWTPs are composed of activated sludge processes and different tertiary treatments (coagulation/sedimentation, sand-anthracite filters, membrane bioreactors (MBRs)/reverse osmosis (RO) and disinfection by chlorination). Physicochemical parameters were also evaluated to verify association with density of viruses detected in different treatment stages. HAdV, JCV and RVA were consistently detected (100%) in the sewage influent samples (range: 106-108 genome copies GC/L). In the secondary effluent, HAdV was detected in 100% (48/48) of the analysed samples, JCV in 85.4% and RVA in 97.9% (range: 104-107 GC/L for all viruses tested). HAdV was the most frequently detected virus in the tertiary effluent (62.2%) (28/45), exhibiting a viability between 0 and 44% of the tested samples in the wastewater reclamation systems. The MBR/RO systems demonstrated better virus removal efficiencies (range: 2.3-2.9 log10). Temperature, pH, turbidity and total organic carbon presented association with the viral density in the reclaimed water samples. Presence of viruses in treated effluents can indicates health risks depending on uses of recovery water. Further risk assessment studies should be conducted to better assess health risks under different exposure scenarios for water recovery in urban settings.