Detection of Multiple Human Viruses, including Mpox, Using a Wastewater Surveillance Approach in Brazil.

Sewage surveillance can be used as an effective complementary tool for detecting pathogens in local communities, providing insights into emerging threats and aiding in the monitoring of outbreaks. In this study using qPCR and whole genomic sewage surveillance, we detected the Mpox virus along with other viruses, in municipal and hospital wastewaters in Belo Horizonte, Brazil over a 9-month period (from July 2022 until March 2023). MPXV DNA detection rates varied in our study, with 19.6% (11 out of 56 samples) detected through the hybrid capture method of whole-genome sequencing and 20% (12 out of 60 samples) through qPCR. In hospital wastewaters, the detection rate was higher, at 40% (12 out of 30 samples) compared to 13.3% (4 out of 30 samples) in municipal wastewaters. This variation could be attributed to the relatively low number of MPXV cases reported in the city, which ranged from 106 to 341 cases during the study period, and the dilution effects, given that each of the two wastewater treatment plants (WWTP) investigated serves approximately 1.1 million inhabitants. Additionally, nine other virus families were identified in both hospitals and municipal wastewaters, including Adenoviridade, Astroviridae, Caliciviridae, Picornaviridade, Polyomaviridae, Coronaviridae (which includes SARS-CoV-2), Herspesviridae, Papillomaviridae and Flaviviridae (notably including Dengue). These findings underscore the potential of genomic sewage surveillance as a robust public health tool for monitoring a wide range of viruses circulating in both community and hospitals environments, including MPXV.

Partial nitritation/anammox applied to real anaerobically pretreated domestic sewage under subtropical climate: aeration strategies and nitrogen cycle bacteria.

The combination of sewage anaerobic treatment and partial nitritation/anammox process (PN/A) can make wastewater treatment plants energetically self-sufficient. However, PN/A application has been a challenge in low-nitrogen wastewaters and it is little explored in anaerobically pretreated domestic sewage, as well as aeration strategies and the PN/A feasibility at ambient temperature. This study investigated PN/A in a sequential batch reactor (SBR) treating real anaerobically pretreated domestic sewage. After the startup, SBR was fed with real wastewater and operated at 35°C and at ambient temperature (20-31°C) without total nitrogen (TN) removal decrease (71 ± 8 and 75 ± 6%, respectively). The median ammonium and TN removals were 68 ± 21 and 59 ± 9%, respectively with 7 min on/14 min off strategy, which represents 12.3 ± 4.2 mg L-1 N-NH4+ effluent, which is lower than Brazilian discharge limits. The qPCR results showed anammox abundance in the range of 108-109 n° copies gVSS-1. Thus, results were very promising and showed the feasibility of the PN/A process for treating real anaerobically pretreated domestic sewage at ambient temperature.

Antibiotic resistance profile of wastewater treatment plants in Brazil reveals different patterns of resistance and multi resistant bacteria in final effluents.

Wastewater treatment plants (WWTPs) are recognized as important sources of Antibiotic Resistant Bacteria (ARBs) and Antibiotic Resistant Genes (ARGs), and might play a role in the removal and dissemination of antimicrobial resistance (AMR) in the environment. Detailed information about AMR removal by the different treatment technologies commonly applied in urban WWTPs is needed. This study investigated the occurrence, removal and characterization of ARBs in WWTPs employing different technologies: WWTP-A (conventional activated sludge-CAS), WWTP-B (UASB reactor followed by biological trickling filter) and WWTP-C (modified activated sludge followed by UV disinfection-MAS/UV). Samples of raw sewage (RI) and treated effluent (TE) were collected and, through the cultivation-based method using 11 antibiotics, the antibiotic resistance profiles were characterized in a one-year period. MAS was effective in reducing ARB counts (2 to 3 log units), compared to CAS (1 log unit) and UASB/BTF (0.5 log unit). The composition of cultivable ARB differed between RI and TE samples. Escherichia was predominant in RI (56/118); whilst in TE Escherichia (31/118) was followed by Bacillus (22/118), Shigella (14/118) and Enterococcus (14/118). Most of the isolates identified (370/394) harboured at least two ARGs and in over 80 % of the isolates, 4 or more ARG (int1, blaTEM, TetA, sul1 and qnrB) were detected. A reduction in the resistance prevalence was observed in effluents after CAS and MAS processes; whilst a slight increase was observed in treated effluents from UASB/BTF and after UV disinfection stage. The multi-drug resistance (MDR) phenotype was attributed to 84.3 % of the isolates from RI (27/32) and 63.6 % from TE (21/33) samples and 52.3 % of the isolates (34/65) were resistant to carbapenems (imipenem, meropenem, ertapenem). The results indicate that treated effluents are still a source for MDR bacteria and ARGs dissemination to aquatic environments. The importance of biological sewage treatment was reinforced by the significant reductions in ARB counts observed. However, implementation of additional treatments is needed to mitigate MDR bacteria release into the environment.

Quantification of SARS-CoV-2 in wastewater samples from hospitals treating COVID-19 patients during the first wave of the pandemic in Brazil.

The COVID-19 pandemic has caused a global health crisis, and wastewater-based epidemiology (WBE) has emerged as an important tool to assist public health decision-making. Recent studies have shown that the SARS-CoV-2 RNA concentration in wastewater samples is a reliable indicator of the severity of the pandemic for large populations. However, few studies have established a strong correlation between the number of infected people and the viral concentration in wastewater due to variations in viral shedding over time, viral decay, infiltration, and inflow. Herein we present the relationship between the number of COVID-19-positive patients and the viral concentration in wastewater samples from three different hospitals (A, B, and C) in the city of Belo Horizonte, Minas Gerais, Brazil. A positive and strong correlation between wastewater SARS-CoV-2 concentration and the number of confirmed cases was observed for Hospital B for both regions of the N gene (R = 0.89 and 0.77 for N1 and N2, respectively), while samples from Hospitals A and C showed low and moderate correlations, respectively. Even though the effects of viral decay and infiltration were minimized in our study, the variability of viral shedding throughout the infection period and feces dilution due to water usage for different activities in the hospitals could have affected the viral concentrations. These effects were prominent in Hospital A, which had the smallest sewershed population size, and where no correlation between the number of defecations from COVID-19 patients and viral concentration in wastewater was observed. Although we could not determine trends in the number of infected patients through SARS-CoV-2 concentrations in hospitals' wastewater samples, our results suggest that wastewater monitoring can be efficient for the detection of infected individuals at a local level, complementing clinical data.

Virtual screening of molecular databases for potential inhibitors of the NSP16/NSP10 methyltransferase from SARS-CoV-2.

COVID-19 is a disease caused by the SARS-CoV-2 virus and represents one of the greatest health problems that humanity faces at the moment. Therefore, efforts have been made with the objective of seeking therapies that could be effective in combating this problematic. In the search for ligands, computational chemistry plays an essential role, since it allows the screening of thousands of molecules on a given target, in order to save time and money for the in vitro or in vivo pharmacological stage. In this paper, we perform a virtual screening by docking looking for potential inhibitors of the NSP16-NSP10 protein dimer (methyltransferase) from SARS-CoV-2, by evaluating a homemade databank of molecules found in plants of the Caatinga Brazilian biome, compounds from ZINC online molecular database, as well as structural analogues of the enzymatic cofactor s-adenosylmethionine (SAM) and a known inhibitor in the literature, sinefungin (SFG), provided at PubChem database. All the evaluated sets presented molecules that deserve attention, highlighting four compounds from ZINC as the most promising ligands. These results contribute to the discovery of new molecular hits, in the search of potential agents against SARS-CoV-2 virus, still unveiling a pathway that can be used in combined therapies.

Effects of activated sludge and UV disinfection processes on the bacterial community and antibiotic resistance profile in a municipal wastewater treatment plant.

Wastewater tertiary treatment has been pointed out as an effective alternative for reducing the concentration of antibiotic resistant bacteria and genes (ARB and ARGs) in wastewaters. The present work aimed to build on the current knowledge about the effects of activated sludge and UV irradiation on antibiotic resistance determinants in biologically treated wastewaters. For that, the microbial community and ARGs' composition of samples collected after preliminary (APT), secondary (AST), and tertiary (ATT) treatments in a full-scale wastewater treatment plant using a modified activated sludge (MAS) system followed by an UV stage (16 mJ/cm2) were investigated through culture-dependent and independent approaches (including metagenomics). A total of 24 phyla and 460 genera were identified, with predominance of Gammaproteobacteria in all samples. Pathogenic genera corresponded to 8.6% of all sequences on average, mainly Acinetobacter and Streptococcus. Significant differences (p < 0.05) in the proportion of pathogens were observed between APT and the other samples, suggesting that the secondary treatment reduced its abundance. The MAS achieved 64.0-99.7% average removal efficiency for total (THB) and resistant heterotrophic bacteria, although the proportions of ARB/THB have increased for sulfamethoxazole, cephalexin, ciprofloxacin, and tetracycline. A total of 107 copies/mL of intI1 gene remained in the final effluent, suggesting that the treatment did not significantly remove this gene and possibly other ARGs. In accordance, metagenomic results suggested that number of reads recruited to plasmid-associated ARGs became more abundant in the pool throughout the treatment, suggesting that it affected more the bacteria without these ARGs than those with it. In conclusion, disinfected effluents are still a potential source for ARB and ARGs, which highlights the importance to investigate ways to mitigate their release into the environment.

Reduction and liquid-solid partitioning of SARS-CoV-2 and adenovirus throughout the different stages of a pilot-scale wastewater treatment plant.

Investigating waterborne viruses is of great importance to minimizing risks to public health. Viruses tend to adsorb to sludge particles from wastewater processes by electrostatic and hydrophobic interactions between virus, aquatic matrix, and particle surface. Sludge is often re-used in agriculture; therefore, its evaluation is also of great interest to public health. In the present study, a pilot scale system treating real domestic wastewater from a large city in Brazil was used to evaluate the removal, the overall reduction, and liquid-solid partitioning of human adenovirus (HAdV), the novel coronavirus (SARS-CoV-2) and fecal indicators (F-specific coliphages and E. coli). The system consists of a high-rate algal pond (HRAP) post-treating the effluent of an upflow anaerobic sludge blanket (UASB) reactor. Samples were collected from the influent and effluent of each unit, as well as from the sludge of the UASB and from the microalgae biomass in the HRAP. Pathogens and indicators were quantified by quantitative polymerase chain reaction (qPCR) (for HAdV), qPCR with reverse transcription (RTqPCR) (for SARS-CoV-2), the double agar plaque assay (for coliphages), and the most probable number (MPN) method (for E. coli). The removal and overall reduction of HAdV and SARS-CoV-2 was greater than 1-log10. Almost 60% of remaining SARS-CoV-2 RNA and more than 70% of remaining HAdV DNA left the system in the sludge, demonstrating that both viruses may have affinity for solids. Coliphages showed a much lower affinity to solids, with only 3.7% leaving the system in the sludge. The system performed well in terms of the removal of organic matter and ammoniacal nitrogen, however tertiary treatment would be necessary to provide further pathogen reduction, if the effluent is to be reused in agriculture. To our knowledge, this is the first study that evaluated the reduction and partitioning of SARS-CoV-2 and HAdV through the complete cycle of a wastewater treatment system consisting of a UASB reactor followed by HRAPs.

Aeration strategies and temperature effects on the partial nitritation/anammox process for nitrogen removal: performance and bacterial community assessment.

The partial nitritation/anammox process (PN/A) could be a promising alternative for nitrogen removal from high-strength wastewater. There is, however, a lack of information about suitable aeration and temperature for PN/A in single-stage reactors for high-strength wastewater, such as food waste (FW) digestate treatment. To this end, a laboratory-scale (10 L) partial nitritation/anammox sequencing batch reactor was operated for more than 230 days under four different intermittent aeration strategies and temperature variations (35°C and ambient temperature - 26-29°C) to investigate the feasibility of nitrogen removal from real FW digestate. High ammonium (NH4+-N) and total nitrogen (TN) removal median efficiencies of 81 and 63%, respectively (corresponding to median NH4+-N and TN loads removed of 76 and 67 g.m-3.d-1), were achieved when the aeration strategy comprised by 7 min/14 min off and an airflow rate of 0.050 L.min-1.Lreactor-1 was applied. Nitrogen removal efficiencies were not affected by temperature variations in southeastern Brazil. COD, chloride and organic nitrogen (520, 239 and 102.8 mg.L-1, respectively) did not prevent PN/A. Changes of the bacterial community in response to aeration strategies were observed. Candidatus Brocadia dominated most of the time being more resistant to aeration and temperature changes than Candidatus Jettenia. This study demonstrated that optimizations of anoxic periods and airflow rate support PN/A with high nitrogen removal from FW digestate.

Monitoramento do esgoto como ferramenta de vigilância epidemiológica para controle da COVID-19: estudo de caso na cidade de Belo Horizonte / Sewage monitoring as a surveillance epidemiological tool for COVID-19 control: a case study in the city of Belo Horizonte

RESUMO A recente detecção de material genético (RNA) do novo coronavírus em amostras de fezes e no esgoto aponta para a possibilidade de se identificar a circulação do vírus e até mesmo estimar o número de pessoas infectadas em determinada região pelo monitoramento sistemático do esgoto, configurando-se em importante ferramenta epidemiológica de testagem massiva indireta, incluindo portadores sintomáticos e assintomáticos. Nesse sentido, concebeu-se um projeto para a detecção e a quantificação do novo coronavírus em amostras de esgoto coletadas em 15 sub-bacias de esgotamento sanitário dos ribeirões Arrudas e Onça, visando entender a dinâmica de circulação e a prevalência do vírus nas regiões investigadas. Tais sub-bacias esgotam os efluentes gerados por uma população da ordem de 1,5 milhão de pessoas no município de Belo Horizonte e parte de Contagem. O plano de amostragem contemplou 17 pontos (15 sub-bacias + afluente às 2 estações de tratamento de esgoto) de monitoramento semanal, com coletas compostas durante todo o período da manhã. A detecção e a quantificação do RNA viral efetuaram-se em laboratório por meio de ensaios de RT-qPCR. Os resultados obtidos em quatro semanas de monitoramento (semanas epidemiológicas 21 a 24) mostraram um incremento da ocorrência do vírus, atingindo 100% das regiões investigadas na semana epidemiológica 24. A estimativa da população infectada pelo novo coronavírus pelo monitoramento do esgoto em Belo Horizonte apresentou tendência de crescimento exponencial, sendo até 20 vezes maior do que o número de casos confirmados acumulados. Quanto à circulação do vírus, as concentrações do RNA viral têm se mostrado bastante variáveis nas regiões monitoradas, com maiores porcentagens de população infectada estimada ao norte e nordeste da capital mineira.

ABSTRACT The recent detection of SARS-CoV-2 RNA in stool and sewage samples highlights the possibility of mapping the circulation of the virus and even estimating the number of infected people through the systematic monitoring of sewage in a specific region. Therefore, this is an important epidemiological tool for large-scale indirect testing, including symptomatic and asymptomatic carriers. This project was conceived for the detection and quantification of the SARS-CoV-2 in sewage samples collected in 15 watersheds of the Arrudas and Onça streams, aiming to understand the dynamics of spread and the prevalence of the virus in these regions/watersheds. These sub-basins exhaust the effluents generated by a population of approximately 1.5 million people in the municipality of Belo Horizonte and part of Contagem. Weekly composite samples were collected during the morning periods in seventeen monitoring points (15 water sheds + influent to 2 sewage treatment plants). RNA detection and quantification were performed in the laboratory using RT-qPCR. The results obtained in four weeks of monitoring (epidemiological weeks 21 to 24) showed an increase in the occurrence of the virus, reaching 100% of the monitored regions investigated in epidemiological week 24. The infected population, estimated by sewage monitoring in Belo Horizonte, showed exponential growth, being up to 20 times higher than those of accumulated confirmed cases. As for the dynamics of virus spread, RNA concentrations have shown to be quite variable in the monitored regions with higher percentages of the estimated infected population in the northern and north-eastern portions of Belo Horizonte.

Assessing spatial distribution of COVID-19 prevalence in Brazil using decentralised sewage monitoring.

Brazil has become one of the epicentres of the COVID-19 pandemic, with cases heavily concentrated in large cities. Testing data is extremely limited and unreliable, which restricts health authorities' ability to deal with the pandemic. Given the stark demographic, social and economic heterogeneities within Brazilian cities, it is important to identify hotspots so that the limited resources available can have the greatest impact. This study shows that decentralised monitoring of SARS-CoV-2 RNA in sewage can be used to assess the distribution of COVID-19 prevalence in the city. The methodology developed in this study allowed the identification of hotspots by comprehensively monitoring sewers distributed through Belo Horizonte, Brazil's third largest city. Our results show that the most vulnerable neighbourhoods in the city were the hardest hit by the pandemic, indicating that, for many Brazilians, the situation is much worse than reported by official figures.

Nitratation in pilot-scale bioreactors fed with effluent from a submerged biological aerated filter used in the treatment of dog wastewater.

Nitrification is a biochemical process that allows oxidation of ammonium ion to nitrite, and nitrite to nitrate in a system. Aerobic processes, such as use of submerged biological aerated filter (SBAF), enable nitrification. However, some variables that are entirely unavailable or not available at the required concentration range may hamper the process. In this study, nitratation under high dissolved oxygen (DO) concentrations was evaluated in laboratory-scale bioreactors containing 10% inoculum (0.5 kg kg-1) fed with affluent from a SBAF that receive the sewage generated from washing the bays of a dog kennel. The following variables were monitored over time: ammoniacal nitrogen (12.44-29.62 mg L-1), nitrite (0.28-0.54 mg L-1), nitrate (1.75-3.55 mg L-1), pH (8.11 ± 0.62), temperature (21.61 ± 1.24°C) and DO (9.69 ± 0.36 mg L-1). Quantification of nitrifying bacteria by the multiple tube technique showed the value of 1.4 × 1012 MPN mL-1for ammonia-oxidizing bacteria (AOB) and 9.2 × 1014 MPN mL-1 for nitrite-oxidizing bacteria. These values were higher than those found in a synthetic medium, which can be explained by the greater availability of ammonium and nitrite in the effluent. By the extraction of genomic DNA, and PCR, with specific primers, the presence of the AmoA (Ammonia monooxygenase) gene for AOB and of the Nitrobacter was detected in the bioreactor samples. By PCR-DGGE, the sequenced bands showed high similarity with denitrifying bacteria, such as Pseudomonas, Limnobacter, Thauera, Rhodococcus, and Thiobacillus. Thus, the saturation of dissolved oxygen in the system resulted in improvement in the nitratation step and allowed detection of bacterial genera involved in the process.

Detecção e quantificação de bactérias resistentes aos antibióticos ampicilina e cloranfenicol em estações de tratamento de esgoto doméstico / Detection and quantification of bacteria resistant to ampicillin and chloranphenicol in domestic sewage treatment plants

RESUMO Estações de tratamento de esgotos (ETEs) estão entre as principais fontes de disseminação de bactérias resistentes a antibióticos (BRAs) e genes de resistência (GRAs) no ambiente. Este trabalho quantificou a ocorrência de bactérias resistentes aos antibióticos ampicilina e cloranfenicol no esgoto bruto (EB), no efluente tratado (ET) e no lodo de duas ETEs em escala plena por um período de nove meses. As unidades investigadas utilizavam os seguintes sistemas de tratamento: ETE-A, sistema de lodos ativados convencional; e a ETE-B, reatores anaeróbios (UASB) seguidos de filtros biológicos percoladores (FBP). Os resultados evidenciaram que a ETE-A foi mais eficiente na redução das concentrações de bactérias resistentes à ampicilina e ao cloranfenicol (cerca de 1,1 e 0,7 log10UFC.mL−1 de remoção, respectivamente), quando comparada com a ETE-B (0,5log10 UFC.mL−1 de remoção para as bactérias resistentes ao cloranfenicol e nenhuma remoção para as resistentes à ampicilina). As amostras de lodo, de ambas ETEs, apresentaram elevadas concentrações de bactérias heterotróficas totais — BHTs (4,8-7,6 log10UFC.mL−1) e de BRAs (3,0-6,3 log10UFC.mL−1). A maioria das cepas resistentes à ampicilina e ao cloranfenicol isoladas foi identificada como sendo da família Enterobacteriaceae. Algumas das espécies identificadas são bactérias potencialmente patogênicas, tais como: Klebsiella pneumoniae, Aeromonas hydrophila, Escherichia coli, Enterococcus faecium, Salmonella spp. Os resultados chamam a atenção para a disseminação de BRAs, potencialmente patogênicas, no meio ambiente a partir do efluente final (proveniente do tratamento secundário) das ETEs, independentemente do tipo de sistema adotado. Fica evidente que para reduzir significativamente a concentração das BRAs no ET, este deveria passar por tratamento adicional e desinfecção.

ABSTRACT Sewage treatment plants (STP) are among the main sources of dissemination of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARG) into the environment. This work quantified the occurrence of cultivable ampicilin-resistant and chloramphenicol-resistant bacteria in raw sewage, treated effluent and sludge samples from two full-scale STP over nine months. The STP investigated used the following treatment systems: STP-A used conventional activated sludge system; and STP-B, anaerobic reactors (UASB) followed by percolating biological filters (PBF). Results showed that was more efficient in reducing the concentrations of ampicilin- and chloramphenicol-resistant bacteria (around 1.1 and 0.7 log10UFC.mL−1, respectively) when compared to STP-B (0.5 log10 UFC.mL−1 removal of cloramphenicol-resistant bacteria and no-removal of ampicilin-resistant bacteria). Sludge samples, from both STP, showed high concentrations of total heterotrophic bacteria (THB; 4.8-7.6 log10UFC.mL−1) and ARB (3.0-6.3 log10UFC.mL−1). Most of the isolated ampicilin- and chloramphenicol-resistant strains were identified as members of the Enterobacteriaceae family. Some of the identified species are potential pathogenic bacteria, such as Klebsiella pneumoniae, Aeromonas hydrophila, Escherichia coli, Enterococcus faecium, Salmonella spp. These results call attention to the spread of ARB, potentially pathogenic, in the environment from the final effluent (from secondary effluent) on the STP, regardless of the type of system adopted. It is evident that in order to significantly reduce the concentration of ARB in the treated effluent, it should undergo additional treatment and disinfection.

Evaluation of nitrogen removal and the microbial community in a submerged aerated biological filter (SABF), secondary decanters (SD), and horizontal subsurface flow constructed wetlands (HSSF-CW) for the treatment of kennel effluent.

To ensure microbial activity and a reaction equilibrium with efficiency and energy saving, it is important to know the factors that influence microbiological nitrogen removal in wastewater. Thus, it was investigated the microorganisms and their products involved in the treatment of kennel effluents operated with different aeration times, phase 1 (7 h of continuous daily aeration), phase 2 (5 h of continuous daily aeration), and phase 3 (intermittent aeration every 2 h), monitoring chemical and physical parameters weekly, monthly microbiological, and qualitative and quantitative microbiological analyzes at the end of each applied aeration phase. The results showed a higher mean growth of nitrifying bacteria (NB) (106) and denitrifying bacteria (DB) (1022) in phase with intermittent aeration, in which better total nitrogen (TN) removal performance, with 33%, was achieved, against 21% in phase 1 and 17% in phase 2, due to the longer aeration time and lower carbon/nitrogen ratio (15.7), compared with the other phases. The presence of ammonia-oxidizing bacteria (AOB), the genus Nitrobacter nitrite-oxidizing bacteria (NOB), and DB were detected by PCR with specific primers at all phases. The analysis performed by 16S-rRNA DGGE revealed the genres Thauera at all phases; Betaproteobacteria and Acidovorax in phase 3; Azoarcus in phases 2 and 3; Clostridium, Bacillus, Lactobacillus, Turicibacter, Rhodopseudomonas, and Saccharibacteria in phase 1, which are related to the nitrogen removal, most of them by denitrifying. It is concluded that, with the characterization of the microbial community and the analysis of nitrogen compounds, it was determined, consistently, that the studied treatment system has microbiological capacity to remove TN, with the phase 3 aeration strategy, by simultaneous nitrification and denitrification (SND). Due to the high density of DB, most of the nitrification occurred by heterotrophic nitrification-aerobic. And denitrification occurred by heterotrophic and autotrophic forms, since the higher rate of oxygen application did not harm the DB. Therefore, the aeration and carbon conditions in phase 3 favored the activity of the microorganisms involved in these different routes. It is considered that, in order to increase autotrophic nitrification-aerobic, it is necessary to exhaust the volume of sludge in the secondary settlers (SD), further reducing the carbon/nitrogen ratio, through more frequent cleaning, whose periodicity should be the object of further studies. Graphical abstract.

Effect of alkaline treatment on pathogens, bacterial community and antibiotic resistance genes in different sewage sludges for potential agriculture use.

Alkaline treatment is widely used to reduce pathogens in sewage sludge in developing countries and guarantee that it is safe for use in agriculture. The aim of this study was to investigate the effect of alkaline treatment applied to waste-activated (WAS) and Upflow Anaerobic Sludge Blanket (UASB)-sludge on the bacterial community, pathogens (viable helminths eggs and Salmonella spp), and antibiotic resistance genes (ARG). The bacterial community structure was examined through denaturing gel gradient electrophoresis (DGGE), targeting 16S rRNA genes. Polymerase chain reaction (PCR) was applied to evaluate the presence of several ARGs. The conducted alkaline experiment consisted of adding hydrated lime (Ca(OH)2) to sewage sludges. Samples were taken before and after 2, 24, 48, and 72 hours of treatment. Alkaline treatment changed considerably the bacterial community structure and after 24 hours, shifts in bacterial profiles were more pronounced in the UASB sludge sample than in WAS. Some bacteria remained under extreme pH conditions (pH > 12), such as Azospira oryzae and Dechloromonas denitrificans in the WAS samples, and Geothrix and Geobacter in the UASB sludge samples. The values of pathogens and indicators in the sludge after 24 hours of alkaline treatment meet sanitary law regulations and thus the sludges could have the potential to agricultural distribution. It is important to highlight that ARG, which are not currently present in sanitary regulations, were detected in the sludge samples after the alkaline treatment, which could be a concern for human health.

Effect of photodynamic antimicrobial chemotherapy on Candida albicans in the presence of glucose.

BACKGROUND: Candida albicans is an opportunistic commensal microorganism, often associated with severe infections in immunosuppressed individuals. C. albicans has hexose transporters that may favor the intracellular accumulation of photosensitizer (PS). the aims of this study were to investigate the influence of glucose load on photodynamic antimicrobial chemotherapy (PACT); and the role that membrane transport system plays on this therapy in the presence of glucose. MATERIAL AND METHODS: Strains of C. albicans were selected: ATCC 10231, YEM 12, YEM 13, YEM 14 and YEM 15. All strains were grown aerobically on Sabouraud agar and incubated at 30 °C for 24 h. The strains were treated with and without glucose, and divided into Control (no treatment), LED light (660 nm, 166 mW/cm2), Photosensitizer (100 µM methylene blue) and PACT at 1, 3 and 6 min of irradiation groups. The colony forming units were counted and data submitted to statistical analysis (ANOVA) and Tukey's test. The concentration of methylene blue (MB) outside the yeast was measured by fluorescence spectroscopy. RESULTS: PACT inactivate C. albicans and the presence of glucose did not affect the killing effect for most strains. Only YEM12 was partially affected by its presence. Regarding efflux systems, ABC overexpressing strain showed a protective effect on the yeast cells. We observed that yeast with overexpression of major facilitator superfamily (MFS) membrane pore tended to accumulate more MB in its cytoplasm, whereas strains that overexpressed ABC pumps (ATP-binding cassette transporters) tended to decrease MB uptake and survive the photodynamic challenge. CONCLUSION: Presence of glucose showed a small effect on PACT . The accumulation of MB on yeast induces more photodynamic inactivation; however, the photodynamic efficacy depends on the type and characteristics of the microbial strain.

Enhanced biodiesel industry wastewater treatment via a hybrid MBBR combined with advanced oxidation processes: analysis of active microbiota and toxicity removal.

In the present study, a multistage route is proposed for the treatment of biodiesel industry wastewater (BWW) containing around 1000 mg L-1 of total organic carbon (TOC), 3500 mg L-1 of chemical oxygen demand (COD), and 1325 mg L-1 of oil and grease. Initially, BWW aerobic biodegradability was assessed via Zhan-Wellens biodegradability test to confirm the appropriate treatment route. Then, a hybrid moving bed bioreactor (MBBR) system was chosen as the first treatment stage. The hybrid MBBR achieved 69 and 68% removal of COD and TOC removals, respectively, and provided great conditions for biomass growth. The bacterial community present in the hybrid MBBR was investigated by PCR-DGGE and potential biodegraders were identified such as: members of Desulfuromonadales, Nocardioidaceae and Pseudomonadaceae. Since biodegradation in the hybrid MBBR alone was unable to meet quality requirements, advanced oxidation processes, such as Fenton and photo-Fenton, were optimized for application as additional treatment stages. Physicochemical properties and acute toxicity of BWW were analyzed after the multistage routes: hybrid MBBR + Fenton, hybrid MBBR + photo-Fenton and hybrid MBBR + UV-C254nm/H2O2. Hybrid MBBR + Fenton or photo-Fenton showed overall COD removal efficiencies greater than 95% and removed acute toxicity, thus being appropriate integrated routes for the treatment of real BWW. Graphical abstract ᅟ.

Effect of temperature on microbial diversity and nitrogen removal performance of an anammox reactor treating anaerobically pretreated municipal wastewater.

The effects of temperature reduction (from 35 °C to 20 °C) on nitrogen removal performance and microbial diversity of an anammox sequencing batch reactor were evaluated. The reactor was fed for 148 days with anaerobically pretreated municipal wastewater amended with nitrite. On average, removal efficiencies of ammonium and nitrite were high (96%) during the enrichment period and phases 1 (at 35 °C) and 2 (at 25 °C), and slightly decreased (to 90%) when the reactor was operated at 20 °C. Deep sequencing analysis revealed that microbial community structure changed with temperature decrease. Anammox bacteria (Ca. Brocadia and Ca. Anammoximicrobium) and denitrifiers (Burkholderiales, Myxococcales, Rhodocyclales, Xanthomonadales, and Pseudomonadales) were favoured when the temperature was lowered from 35 °C to 25 °C, while Anaerolineales and Clostridiales were negatively affected. The results support the feasibility of using the anammox process for mainstream nitrogen removal from anaerobically pretreated municipal wastewater at typical tropical temperatures.

Antimicrobial photodynamic therapy on Streptococcus mutans is altered by glucose in the presence of methylene blue and red LED.

BACKGROUND: Dental caries are a multifactorial disease that progressively produces tooth destruction as a result of bacterial colonization of enamel surface, especially Streptococcus mutans. The objective of this work was to investigate the role of glucose in antimicrobial photodynamic therapy (aPDT) on S. mutans. METHODS: S. mutans ATCC 25175 were cultured on microaerophilia at 37°C for 48h, and we tested aPDT in the presence of 50mM glucose. Bacterial suspension was used to investigate aPDT with 100µM methylene blue (MB) under LED emitting radiation at ʎ=660nm and parameters as following (P=473 mW; I=166.8 mW/cm2, and doses of 5, 10 and 20J/cm2). A seventy-two hours biofilm was grown on 96 flat buttoned well-plate and irradiation was performed from 10 to 80J/cm2 at similar conditions. RESULTS: There was no dark toxicity nor bacterial death regarding LED irradiation on suspension and on biofilm. Nevertheless, aPDT presented expressive bacterial inactivation following 1 and 2min of irradiation on cell suspension. On the other hand, there was no inactivation in the presence of glucose under the same conditions. Biofilm was completely inactivated by MB-mediated aPDT after 6min of irradiation. However, the presence of glucose delayed the complete inactivation of the biofilm. CONCLUSION: The presence of glucose in the suspension drastically delayed the effect of aPDT on S. mutans and this effect is more pronounced in bacterial suspension than on biofilm.

Antimicrobial photodynamic therapy combined to periodontal treatment: Experimental model.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) has been used as an adjuvant treatment for periodontitis. It combines a photosensitizer with a light source to induce reactive oxygen species and kill microbial cells. PpNetNI is a protoporphyrin derivative, and it has a chemical binding site at biofilm and great affinity to microbial cells. The aim of this study was to investigate the effects of aPDT as an adjuvant treatment for periodontitis. METHODS: Thirty healthy male rats Wistar (Rattus norvegicus) were used in this study (Approved by UNINOVE Ethical committee AN0029/2015). Periodontitis was induced by placing a cotton ligature around the first mandibular molar in a subgengival position. The contralateral mandibular first molar received neither a ligature nor any treatment, and was used as a control. After 7 days, the ligature was removed and all animals received scaling and root planing (SRP) and were divided according to the following treatments: SRP group (received SRP and irrigation with PpNetNI, 10µM) and aPDT group (PpNetNI 10µM followed by LED irradiation). aPDT was performed with a LED (630nm) with an output power of 400mW (fluence-rate 200mW/cm2; fluence 18J/cm2). Rats were euthanized at 24h, 48h and 7days postoperatively. The area of bone loss in vestibular region of the first molar was evaluated by Optical Coherence Tomography (OCT, THORLABS LTD., Ely, UK). Data were analyzed statistically (ANOVA and Tukey tests, p<0.05). RESULTS: The animals treated by aPDT showed bone gain of approximately 30% compared to the SRP group following 7days from the treatment. CONCLUSION: aPDT promoted bone recovery 7days after periodontal intervention.

Relationship between analysis of laser speckle image and Knoop hardness on softening enamel.

BACKGROUND: In this study is presented the correlation between laser speckle images and enamel hardness loss. METHODS: In order to shift the enamel hardness, a dental demineralization model was applied to 32 samples of vestibular bovine teeth. After they were cleaned, cut and polished, the samples were divided into 4 groups and immersed in 30ml of a cola-based soft drink for 10, 20, 30 and 40min twice a day for 7 consecutive days with half the surface protected by two layers of nail polish. Each sample was analyzed by Knoop hardness and laser speckle imaging. RESULTS: Pearson's correlation analysis demonstrated that the laser speckle image technique presents a strong correlation with the hardness loss of the enamel (r=0.7085, p<0.0001). This finding is corroborated by Blend & Altman analysis, in which the data presented a constant behavior throughout the whole interval. For both analyses, more than 95% of the data is within the confidence interval, as expected. CONCLUSION: This work demonstrates, for the first time to our knowledge, an empirical model for correlating laser speckle images with the loss of tooth enamel hardness.