Fecal matter is considered as one of the worst pollutants in waterbodies due to the potential spread of waterborne diseases. This study aimed to determine the host-specific fecal contamination in two Brazilian watersheds and to predict the possible impacts on human health. Fecal sources were enumerated using host-specific genetic markers to swine (16S rRNA), human and bovine (archaeal nifH), and equine (archaeal mcrA). A single cycling condition was established for four markers aiming to decrease the analysis time. Fifteen samples from São João watershed (75%) and 25 from Guandu (62.5%) presenting Escherichia coli enumeration in compliance with Brazilian guidelines (<1,000 MPN/100 mL) showed the human marker. Furthermore, the bovine, swine, and equine markers were present in 92% (59/64), 89% (57/64), and 81% (52/64) of the water samples, respectively. The molecular markers proposed for qPCR in our study were sensitivity and specific enough to detect host-specific fecal pollution in all samples regardless of E. coli levels reaffirming the low correlation among them and supporting their use in water quality monitoring programs. To our knowledge, this is the first study using this approach for quantification of nifH, mcrA, and rrs gene-associated human and animal fecal pollution in waters intended for drinking water supply in Brazil.

To investigate the occurrence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and their clonal relationships from hospital and municipal wastewater treatment plants (WWTPs). Eighteen K. pneumoniae strains recovered from three WWTPs were identified by MALDI-TOF. The antimicrobial susceptibility were evaluated by disk-diffusion and the carbapenemases production by Carbapenembac®. The carbapenemases genes were investigated by real-time PCR and the clonal relationship through multi-locus sequence typing (MLST). Thirty nine percent (7/18) of isolates were classified as multidrug-resistant (MDR), 61.1% (11/18) extensively drug-resistant (XDR) and 83.3% (15/18) showed carbapenemase activity. Three carbapenemase-encoding genes were found, bla_KPC (55%), bla_NDM (27.8%) and bla_OXA-370 (11.1%) as well five sequencing types ST11, ST37, ST147, ST244 and ST281. ST11 and ST244, sharing four alleles were grouped into clonal complex 11 (CC11). Our results show the importance of monitoring antimicrobial resistance in WWTPs effluents to minimize the risk of spreading bacterial load and ARGs in aquatic ecosystems, using advanced treatment technologies to reduce these emerging pollutants at WWTPs.

Antibiotic resistance genes (ARGs) are widespread in the environment due to the overuse of antibiotics and other pollutants, posing a threat to human and animal health. In this study, we evaluated antimicrobial residues, bacterial diversity and ARGs in two important watersheds, Guandu and São João, that supply drinking water to Rio de Janeiro city, Brazil. In addition, tap water samples were collected from three different cities in Rio de Janeiro State, including the metropolitan area of Rio de Janeiro city. Clarithromycin, sulfamethoxazole and azithromycin were found in untreated water and drinking water in all samples. A greater abundance of Proteobacteria was observed in Guandu and São João watersheds, with most of the sequences belonging to the Gammaproteobacteria class. A plasmidome-focused metagenomics approach revealed 4881 (Guandu), 3705 (São João) and 3385 (drinking water) ARGs mainly associated with efflux systems. The genes encoding metallo-β-lactamase enzymes (bla_AIM, bla_GIM, bla_IMP, and bla_VIM) were detected in the two watersheds and in drinking water samples. Moreover, we demonstrated the presence of the colistin resistance genes mcr-3 and mcr-4 (both watersheds) and mcr-9 (drinking water and Guandu) for the first time in Brazil. Our data emphasize the importance of introducing measures to reduce the disposal of antibiotics and other pollutants capable of promoting the occurrence and spread of the microbial resistome on aquatic environments and predicting possible negative impacts on human health.

The emergence of vancomycin-resistant Enterococcus faecium (Efm) harboring vanA gene and multidrug-resistant determinants is a relevant public health concern. It is an opportunistic pathogen responsible for nosocomial infections widely distributed in the environment, including wastewater treatment plants (WWTPs). Our study addresses a genomic investigation of vanA -carrying Efm from WWTPs in Brazil. Samples from five WWTPs supplied with sewage from different sources were evaluated. Here we present whole-genome sequencing of eight vanA -Efm isolates performed on Illumina MiSeq platform. All these isolates presented multidrug-resistant profile, and five strains were from treated wastewater. Multiple antimicrobial resistance genes (ARGs) were found, such as aph(3′)-IIIa , ant(6′)-Ia , erm(B) , and msrC , some of them being allocated in plasmids. The virulence profile was predominantly constituted by efaAfm and acm genes and all isolates, except for one, were predicted as human pathogens. Multilocus sequence typing analysis revealed a new allele and five different STs, three previously described (ST32, ST168, and ST253) and two novel ones (ST1893 and ST1894). Six strains belonged to CC17, often associated with hospital outbreaks. As far as our knowledge, no genomic studies of vanA -Efm recovered from WWTPs revealed isolates belonging to CC17 in Brazil. Therefore, our findings point to the environmental spread of Efm carrying multiple ARGs.

Wastewater-based epidemiology can be a complementary approach for monitoring SARS-CoV-2 prevalence, diversity, and geographic distribution. It is a complementary approach regarding its prevalence and diversity, and geographic distribution. The study aimed to evaluate the genetic diversity of SARS-CoV-2 in two wastewater treatment plants (WWTPs) in Rio de Janeiro, Brazil. Samples were collected over a period of January to December 2021 and were concentrated with PEG8000 and the presence of SARS-CoV-2 was detected using E and N1 genes. Partial sequencing of the SARS-CoV-2 genomes resulted in the identification of variants of concern and variants of interest throughout the collection period. It was possible to identify the Mu, Delta, Gamma and Omicron variants in WWTP1; on the contrary, no variants were observed in WWTP2. To the best of our knowledge, we detected the variant Mu (B.1.621) containing characteristic mutations (S:E484K, S:N501Y) from WWTP, for the first time, in Brazil. Another Mu variant detected from clinical surveillance was announced one month after our finding. The detection of SARS-CoV-2 in wastewater can serve as a tool to monitor the prevalence and epidemiology in each community, helping to understand the spread of the virus among the population.

Bivalve mollusks face a crisis due to infectious diseases, resulting in high mortality and economic losses. The need for continuous monitoring to prevent contamination from sewage and rainwater in aquaculture is evident. The recent mass mortality of scallops in Ilha Grande Bay (IGB), Rio de Janeiro’s largest scallop producer, due to environmental contaminants underscores the need for further research. This study aims to investigate the recent collapse of the scallop population and assess the human impact by analyzing the circulation of pathogens. Materials and Methods: Mollusks were collected from three sites in Ilha Grande Bay (IGB), a region known for its significant scallop production, and from scallop farms in Angra dos Reis, RJ. A total of 216 gill and adductor tissue samples from lion’s foot scallops were analyzed. Bacterial contamination was identified using MALDI-TOF, while antimicrobial susceptibility and carbapenem production were assessed via disk diffusion tests. Results: Mollusks were contaminated with V. alginolyticus, V. fluvialis, V. harveyi, Pseudomonas putida, and Pseudomonas monteilii. All isolates were sensitive to meropenem, but P. putida showed higher resistance to ciprofloxacin. Conclusions: The presence of these pathogenic and resistant bacteria in scallop adductor tissues is a concern for the aquaculture industry and a significant public health risk. The potential for these bacteria to enter the human food chain through consuming contaminated seafood or recreational activities such as bathing is a serious issue that needs to be addressed.