Bartonella spp. are bacteria responsible for neglected diseases worldwide. Bartonella henselae is the species most associated with human infections. It is associated with a large spectrum of clinical manifestations and is potentially fatal. The identification of Bartonella spp. is considered a challenge in clinical routine. These bacteria are fastidious, and the time required to isolate them varies from one to six weeks. MALDI-TOF mass spectrometry has emerged as an application for research on Bartonella spp. , and has still been little explored. We investigated whether three different B. henselae strains with different growth times-14 and 28 days-could be correctly identified by MALDI-TOF mass spectra fingerprint comparison and matching. We found that the spectra from strains with different growth times do not match each other, leading to misidentification. We suggest creating database entries with multiple spectra from strains with different growth times to increase the chances of accurate identification of Bartonella spp. by MALD-TOF MS.

In this study, rapid diagnostic of multidrug-resistant (MDR) sepsis pathogens, directly from positive blood culture (BC) bottles, was evaluated by combining MALDI-TOF and the EUCAST Rapid Antimicrobial Susceptibility Testing (RAST). Carbapenemase production in Escherichia coli and Klebsiella pneumoniae isolates was also evaluated by RAST. From 171 positive BC bottles analyzed, 79 (46%) MDR species, including E. coli (4/34, 12%), K. pneumoniae (33/48, 69%), Pseudomonas aeruginosa (12/12, 100%), Acinetobacter baumannii (15/15, 100%), and Staphylococcus aureus (14/37, 38%) displaying resistance to beta-lactams, fluoroquinolones, aminoglycosides, and/or trimethoprim/sulphamethoxazole, were identified. In this regard, turnaround time of direct MALDI-TOF identification and RAST was < 7 h, which was significantly (p< 0.05) lower than our routine method. Carbapenemase detection by RAST displayed 100% sensitivity and 88.7% specificity at 8 h. This protocol could offer advantages for the treatment and clinical outcomes of septic patients, improving the rapid diagnostic of sepsis by MDR pathogens.

Sewage sludge (SS) presents a high agronomic potential due to high concentrations of organic matter and nutrients, encouraging its recycling as a soil conditioner. However, the presence of toxic substances can preclude this use. To enable the safe disposal of this waste in agriculture, SS requires additional detoxification to decrease the environmental risks of this practice. Although some alternatives have been proposed in this sense, little attention is provided to eliminating endocrine-disrupting chemicals (EDCs). To fill this gap, this study aimed to develop effective and low-cost technology to eliminate EDCs from SS. For this, a detoxification process combining microorganisms and biostimulating agents (soil, sugarcane bagasse, and coffee grounds) was performed for 2, 4, and 6 months with aerobic and anaerobic SSs. The (anti-)estrogenic, (anti-)androgenic, retinoic-like, and dioxin-like activities of SSs samples were verified using yeast-based reporter-gene assays to prove the effectiveness of the treatments. A fractionation procedure of samples, dividing the target sample extract into several fractions according to their polarity, was conducted to decrease the matrix complexity and facilitate the identification of EDCs. A decrease in the abundance and microbial diversity of the SS samples was noted along the biostimulation with the predominance of filamentous fungal species over yeasts and gram-positive bacteria and non-fermenting rods over enterobacteria. Among the 9 EDCs quantified by LC-ESI-MS/MS, triclosan and alkylphenols presented the highest concentrations in both SS. Before detoxification, the studied SSs induced significant agonistic activity, especially at the human estrogen receptor α (hERα) and the human aryl hydrocarbon receptor (AhR). The raw anaerobic sludge also activated the androgen (hAR), retinoic acid (RARα), and retinoid X (RXRα) receptors. However, no significant endocrine-disrupting activities were observed after the SS detoxification, showing that the technology applied here efficiently eliminates receptor-mediated toxicity.

The application of biodegradable and biocompatible polymer poly(lactic acid) (PLA) in the medical field has been widely studied. In this study, films of neat PLA and PLA containing 1 wt% ZnO nanofibers obtained by electrospinning (PLA/ZnO) were successfully molded by solvent-cast three-dimensional (3D) printing. Mechanical behavior was assessed by conventional dynamic mechanical analysis (DMA) and by adapting the equipment conditions to simulate a mechanical fatigue test at human body temperature-cyclic stress in isotherm at 36.5 degrees C. DMA results showed that for temperatures above 30 degrees C, the storage module of PLA/ZnO was higher when compared to neat PLA, and in the fatigue test, PLA/ZnO withstood more than 3600 cycles while neat PLA failed after an average of 1768 cycles. Differential scanning calorimetry (DSC) tests revealed that cyclic stress did not cause changes in the degree of crystallinity of the PLA. The microdilution and plating methods were applied to bacteria (Staphylococcus aureus, Salmonella, and Escherichia coli) and to yeast (Candida albicans) and revealed the antimicrobial effect of ZnO nanofibers and the PLA/ZnO composite. The antimicrobial activity and fatigue resistance of PLA/ZnO nanocomposites indicate that the material has potential for application in bone implants.

Aeromonas spp. are opportunistic and ubiquitous bacteria considered emerging pathogens that can cause infections in animals, especially fish, as well as humans. In humans, these bacteria are associated with gastroenteritis but can also be related to extraintestinal diseases. Its main infection route is through water, but it has been increasingly associated with foods. Their association with ready-to-eat foods may be a concern, especially because these products are for immediate consumption. This study aimed to investigate the prevalence of Aeromonas spp. in ready-to-eat foods (temakis, cheeses and minimally processed fruits) and to characterize the virulence profile and antimicrobial resistance of the isolates. The species A. hydrophila, A. caviae and A. veronii were identified using polymerase chain reaction (PCR), which was later compared with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The performance of two isolation selective agars (starch-ampicillin agar-SAA and Aeromonas agar-AA) was also evaluated. Aeromonas spp. was isolated in 66.67 % (20/30) of temaki, 3.23 % (1/31) of fruits and none (0/30) of cheeses, observing high microorganism counts from <102 to 2.6 × 105 CFU/g. A. caviae (26.39 %) was the most prevalent species, followed by A. hydrophila (20.83 %) and A. veronii (8.34 %), and 44.44 % were classified as Aeromonas sp. The performance analysis between PCR and MALDI-TOF/MS for Aeromonas identification was not statistically significant, and the Kappa index showed moderate agreement (p < 0.01 and Kappa = 0.718). The SAA selective medium performed better than AA. We identified seventeen virulence profiles, and 59.72 % of the isolates had some of the genes studied. The aerA gene (47.2 %) was the most abundant, followed by act (41.7 %), hlyA and alt (38.9 %), and ast (18.1 %). A. hydrophila was the species most associated with these genes. The antimicrobial susceptibility test showed that 90 % of the isolates were resistant to amoxicillin-clavulanic acid, 17 % to tetracycline, 10 % to imipenem and 3 % to aztreonam. The results showed that temakis are carriers of potentially pathogenic Aeromonas spp. and therefore should be avoided by children, elderly individuals, pregnant women, and immunocompromised people. We also found strains resistant to antimicrobials, meaning that these microorganisms need constant monitoring.

Convergence of resistance and virulence in Klebsiella pneumoniae is a critical public health issue worldwide. A multidrug-resistant CTX-M-15-producing K. pneumoniae (TIES-4900 strain) was isolated from a highly impacted urban river, in Brazil. The genome was sequenced by MiSeq Illumina platform and de novo assembled using Unicycler. In silico prediction was accomplished by bioinformatics tools. The size of the genome is 5.4 Mb with 5145 protein-coding genes. TIES-4900 strain belonged to the sequence type ST15, yersiniabactin sequence type YbST10, ICEKp4, KL24 (wzi-24) and O1v1 locus. Phylogenomics confirmed genomic relatedness with ST15 clones from human and animal hosts. Convergence of broad resistome (antibiotics, heavy-metals and biocides) and virulome, including the Kpi pilus system involved in host-pathogen interaction and persistence of ST15 clone to hospital environments, were predicted. Virulent behavior was confirmed in the Galleria mellonella infection model. This study may give genomic insights on the spread of critical-priority WHO pathogens beyond hospital settings.

Wastewater reuse is an important strategy for water resource management. For this reason, the disinfection process must be appropriated, eliminating pathogenic microorganisms. Ozonation (O-3) and UV/H2O2 treatments can be used for effluent disinfection, but few studies just address the Escherichia coli quantification. In this study, secondary effluents from two wastewater treatment plants with different characteristics were exposed to O-3 (5 and 10 mg L-1) or UV/H2O2 (H2O2: 90 mg L-1) treatments and evaluated by BD Phoenix (TM) 100 (Becton Dickinson, USA) and MALDI-TOF for the characterization of the indigenous microorganisms in the effluents, before and after treatments. Additionally, all the samples were tested for phytotoxicity by Lactuca sativa bioassay. The results showed that the highest ozone dose and the UV/H2O2 treatment were effective in removing E. coli. UV/H2O2 was more efficient as it eliminated most of the microorganisms. Acinetobacter sp., Aeromonas and Pseudomonas were still found after O-3 treatment. Bacillus sp. was found after O-3 and UV/H2O2 treatments. The results with L. sativa showed inhibition of mot growth for all dry period (low rainfall) samples of one of the WWTP, due to the high concentration of the phytotoxicity compounds. For environmental and human health safety, treated effluents should be evaluated for their toxic and pathogenic potential before being released into the environment. Pathogens evaluation on treated effluents should cover a wider range of pathogenic micro-organisms than those routinely required by legislation.

Among the treatment-related acute toxic effects, risks for bloodstream infections (BSIs) are associated with several variables. The authors carried out a retrospective cohort study with 259 children and adolescents with ALL, treated with the GBTLI–LLA 2009 protocol, in order to assess the incidence of BSIs in the induction phase; to determine the risk factors for these BSIs; and to identify the related microorganisms and sensitivity profile of the microorganisms related to these infections. BSIs were documented in 19.3% of patients. The isolated microorganisms were 39 Gram-negative bacteria, 21 Gram-positive bacteria, and four fungi. There was a statistically significant risk of BSI between the variables: protocol for T-line-derived leukemia (Derived T Protocol) (p = 0.020), oral manifestations (p = 0.015), central venous catheter (p = 0.008), and bladder catheter (p = 0.004). BSI is a frequent event in ALL patients during the induction phase. The identification of these factors can allow the elaboration and improvement of strategies for the intensification of supportive care, prevention, and rapid treatment of infections.

Visceral leishmaniasis remains a serious public health issue, and Brazil was among the seven countries with the highest prevalence of this disease worldwide. The measures to control this disease are not easily developed, and the improvement of its diagnosis, surveillance, and control is still needed. This study aimed to carry out the polymerase chain reaction (PCR) diagnosis of Leishmania infantum in vector samples in some municipalities of the State of São Paulo, which included two municipalities with human disease transmission and two with dog transmission only. Vectors were collected in traps with luminous bait. Next, they were killed at −4 °C and kept in 70% alcohol. Groups of ten female insects (pools) were mashed on cation exchange paper (fine cellulose phosphate with 18 µEq/cm² ionic exchange capacity) for DNA extraction. The PCR was carried out to identify the natural infection of the Leishmania genus in female Lutzomyia longipalpis (Lu. Longipalpis). Out of the 3,880 Lu. longipalpis phlebotomines, 1060 were female and 2820 were male (3:1). The method used to extract the DNA in pools of ten phlebotomines and the PCR resulted in sensitivity, specificity, practicality, and faster analyses when compared to the individual analysis method. The procedure described can be used on a large scale in the leishmaniasis epidemiological surveillance, enabling a higher number of analyses and the optimization of human resources because the traditional diagnostic method is carried out via desiccation of the insect digestive system and microscopic examination, which is time-demanding and there is the need of manual skills.

Multidrug-resistant tuberculosis (MDR-TB) represents a significant impact in transmission, outcome, and health costs. The World Health Organization recommends implementation of rapid diagnostic methods for multidrug-resistance detection. This study was performed to evaluate the frequency of pre- and extensively drug resistant tuberculosis (pre-XDR-TB and XDR-TB) among MDR-TB patients, the pattern of resistance mutations for fluoroquinolones and the clinical outcome. Adult patients followed at a Brazilian regional reference center for TB, from January 2013 to June 2019 were included. Stored Mycobacterium tuberculosis (Mtb) cultures were recovered, the DNA was extracted, and the susceptibility test was performed using the line probe assay for second line antimycobacterial drugs, Genotype MTBDRsl version 2.0 (Hain Lifescience, CmbH, Germany). Among 33 MDR-TB included patients, we diagnosed XDR-TB or pre-XDR in five (15%) cases. Of these, mutations related to fluoroquinolones resistance were observed in four Mtb isolates, including one who had no phenotypic resistance profile. In two other patients with phenotypic resistance to ofloxacin, genotypic resistance was not found. Case fatality rate was 60% in pre/XDR-TB group, compared to 3.6% in the remaining of patients. This study observed few cases of pre-XDR and XDR-TB among a MDR-TB cohort. Phenotypic and genotypic assays presented good agreement. Clinical outcome was more favorable for patients with susceptibility to fluoroquinolones and injectable drugs.

Strongyloidiasis, a parasitosis caused by Strongyloides stercoralis in humans, is a very prevalent infection in tropical or subtropical areas. Gaps on public health strategies corroborates to the high global incidence of strongyloidiasis especially due to challenges involved on its diagnosis. Based on the lack of a gold-standard diagnostic tool, we aimed to present a metabolomic study for the assessment of stool metabolic alterations. Stool samples were collected from 25 patients segregated into positive for strongyloidiasis (n = 10) and negative control (n = 15) and prepared for direct injection high-resolution mass spectrometry analysis. Using metabolomics workflow, 18 metabolites were annotated increased or decreased in strongyloidiasis condition, from which a group of 5 biomarkers comprising caprylic acid, mannitol, glucose, lysophosphatidylinositol and hydroxy-dodecanoic acid demonstrated accuracy over 89% to be explored as potential markers. The observed metabolic alteration in stool samples indicates involvement of microbiota remodeling, parasite constitution, and host response during S. stercoralis infection.

ABSTRACT The aim of this study was to describe the frequency of nontuberculous mycobacteria (NTM) isolation and their related outcomes among pediatric patients of a Brazilian university hospital from 2012 to 2019. NTM were identified in different clinical samples by microbiological culture and molecular-based methods. NTM were isolated from 14 patients, out of whom four (27%) were infected and were treated accordingly. Two were infected with Mycobacterium avium complex (MAC), two with M. abscessus complex (MABSC) and one with M. intracellulare. Two patients had cystic fibrosis-related lung disease and improved after successful NTM eradication. One patient was HIV-positive and died. One patient had severe combined immunodeficiency (SCID)-related pneumonia and is currently being followed-up. We conclude that NTM frequency in our center was low among pediatric patients. Whether this is inherent to Brazilian patients, due to the broad coverage of the Bacille Calmette-Guérin (BCG) vaccine in Brazil, or a result of underdiagnosis remains to be elucidated.

Klebsiella quasipneumoniae is an emerging pathogen in human medicine. We report draft genome sequences of NDM-1- and KPC-2-producing K. quasipneumoniae strains from inpatients in Brazil. K. quasipneumoniae subsp. quasipneumoniae and K. quasipneumoniae subsp. similipneumoniae harbored broad resistomes. These data could contribute to a better understanding of acquired resistance in K. quasipneumoniae.

Serratia fonticola is a human pathogen widely found in the environment, with birds being reported as possible natural hosts. During an epidemiological and genomic surveillance study conducted to monitor the occurrence of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales in South American wild birds, we identified an ESBL-positive S. fonticola in a fecal sample collected from a Hudsonian Whimbrel, during its non-breeding range on the Pacific Coast of Chile. Whole genome sequencing analysis and "in silico" modeling revealed a novel variant of the class A ESBLs FONA family, designated FONA-7, which shows 96.28% amino acid identity with FONA-6; with amino acid substitutions occurring in the signal peptide sequence (Thr22→Ser), and in the mature protein (Ser39→Asn and Thr227→Ile). This finding denotes that migratory birds can be potential vectors for the transboundary spread of ESBL-producing bacteria, creating a further theoretical risk for the origin of novel plasmid-encoded β-lactamases.

BACKGROUND:The upper airways (UAW) are a niche and a reservoir of Pseudomonas aeruginosa strains that cause chronic infection of the lower airways (LAW) in cystic fibrosis (CF). Here, we assessed the role of anti-P. aeruginosa immunoglobulin A (IgA) and IgG antibodies in upper and lower airway infections in cystic fibrosis patients.METHODS:Nasal lavage fluid and induced sputum samples of 40 CF patients were microbiologically cultured. We searched for correlations between anti-P. aeruginosa IgA and IgG levels, measured by enzyme-linked immunosorbent assay (optical density), and unspecific immune mediators in both specimens.RESULTS:Anti-P. aeruginosa IgA (median optical density: 0.953 vs 0.298) and IgG (0.120 vs 0.059) were significantly higher in nasal lavage than in sputum, but not significantly different between patients with and without chronic P. aeruginosa infection in UAW. Matrix metallopeptidase-9 (MMP-9) in nasal lavage and neutrophil elastase (NE) in sputum were predictors of IgA in nasal lavage and IgA in sputum, respectively. IgA was a predictor of myeloperoxidase (MPO) in nasal lavage. Tissue inhibitor of metalloproteinases-1 (TIMP-1) was a predictor of IgG in sputum. IgG, TIMP-1, and NE in sputum were predictors of IgG in nasal lavage.CONCLUSION:The anti-P. aeruginosa IgA response was more prominent in CF patients' UAW, indicating a lower degree of inflammatory responses. Proteases may play a role in the anti-P. aeruginosa humoral response in the upper and LAW, and anti-P. aeruginosa IgG may be involved in the crosstalk between upper and lower airways in cystic fibrosis patients.

Cystic fibrosis (CF) presents with progressive and chronic deterioration of lung function due to inflammation and colonization/infection of the lungs. This study evaluated spirometry and colonization/infection with Staphylococcus aureus and/or Pseudomonas aeruginosa over a 24-month follow-up period. A total of 52 CF patients were studied with spirometry: forced vital capacity (FVC), forced expiratory volume in one second of FVC (FEV1), FEV1/FVC and forced expiratory flow between 25% and 75% of FVC (FEF25–75%). Colonization/infection was evaluated as predominantly S. aureus, predominantly P. aeruginosa or concomitance of these microorganisms. In CF, there was a higher prevalence of p.Phe508del/p.Phe508del genotype (16/52; 30.8%) and female gender (33/52; 63.5%). Spirometry (% predicted) markers worsened for the following groups over the 24-month period: (i) male: FVC, FEV1, FEV1/FVC, FEF25–75%; (ii) female: FVC%, FEV1, (iii) predominantly S. aureus: FVC, FEV1, FEV1/FVC, FEF25–75%; (iv) predominantly P aeruginosa: FEV1/FVC; (v) concomitant S. aureus and P. aeruginosa: FVC, FEV1. Age correlated with reduction of FVC(Liter) (Rho = −0.50) and FEV1(Liter) (Rho = −0.46). Pancreatic insufficiency and severe cystic fibrosis transmembrane regultador (CFTR) mutations were associated with deteriorating lung function. In CF, deterioration of lung function as evaluated by spirometry was continuous and varied according to sex, pancreatic insufficiency, and severe CFTR mutations. No differences were observed between groups in terms of predominant type of bacteria, but the reduction of spirometry parameters was significant in the predominantly S. aureus and concomitant infection groups.

Cronobacter spp. are opportunistic pathogens that cause serious infections, especially in infants, elderly, and immunocompromised people. Dehydrated infant foods are the main vehicle associated with infections caused by these bacteria. Thus, this study aims to investigate the occurrence of Cronobacter spp. in 152 commercial samples of dehydrated infant formulas (77 samples) and dehydrated infant cereals (75 samples), as well as characterize the isolates. Polymerase Chain Reaction (PCR) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) methods for isolate identification were used, and their results compared. Furthermore, the susceptibility to 11 antibiotics was tested, and DNA sequencing of one isolate with multi-drug resistance was analyzed. No contamination in the infant formula samples was found, whereas 17.33% (13/75) of the infant cereal samples presented contamination with Cronobacter sakazakii. The identification results by PCR and MALDI-TOF/MS were divergent for some isolates. The antimicrobial resistance results showed a high incidence of resistance to cefazolin (94.4%) besides resistance to amoxicillin (9.45%), cefpodoxime (5.55%), streptomycin (1.35%), and trimethoprim/sulfamethoxazole (1.35%). Whole genome sequencing of one multidrug resistant isolate showed six genes associated with antimicrobial resistance and an 82% possibility of being a human pathogen based on the presence of virulence factors. The presence of Cronobacter spp. in infant foods represents a risk for the infant's health. Moreover, the presence of a pathogenic multi-drug resistant isolate in infant's food reinforces the necessity of improving food safety policies to protect young children.

To propose a new coating to silicone implants using Manganese dioxide. We present bacterial adhesion and proliferation when implants are challenged with Escherichia coli. Coated and control silicon implants were placed in two independent subcutaneous pouches in the dorsum of Wistar rats. After skin closure, 0.5 ml of E. coli solution was injected in each incision. The animals were euthanized at 7 and 28 days. Extracted material was cultured and analyzed by confocal microscopy. At 1 week, uncoated implants had a 17-fold higher infection rate (p < 0.001). Coated samples showed a mean bacterial count of 28,700 CFU/ml, while the control ones 503,000 CFU/ml, with a significant mean difference of 474,300 CFU/ml (95% CI 165,900–782,600). At 4 weeks, the mean bacterial growth in coated group was 7600; while in control one was 53,890. The mean difference between groups was 46,200 (95% CI 21,100–71,400). Confocal microscopy presented the percentage of implant’s surface with attached bacteria: at 7 days, coated implants had 6.85% and controls 10.9% and the difference was not significant (p =0.32). At 4 weeks, the coated group showed 0.98% of the surface with attached bacteria, while control group showed 7.64%, which resulted in a significant 11-fold difference (p = 0.004). Manganese dioxide coating inhibits bacterial proliferation and adhesion in subcutaneous silicon implants in an animal model. These findings can be useful to improve development of biomaterials.

BACKGROUND:The low rate of nontuberculous mycobacteria (NTM) among Brazilian patients with cystic fibrosis (CF) may be due to cross-reactive Bacille Calmette-Guérin (BCG) vaccination. In the present pilot study, we aimed to compare the lymphocyte responses against Mycobacterium tuberculosis(Mtb) and Mycobacterium bovis (BCG) in BCG-vaccinated CF patients and healthy controls.METHODS:The lymphocyte responses of CF patients (n = 10) and healthy controls (n = 10) were assessed in terms of lymphocyte proliferation index (LPI), using flow cytometry. Median rates of each cell subtype - CD4, CD8, γδ T cells and CD19 (B) cells - were also determined.RESULTS:Median LPIs (CF vs. controls) were 22.9% vs. 13.0% (p = 0.481) and 23.1% vs. 17.6% (p = 0.481), upon stimulation with Mtb and BCG, respectively. Both groups had a predominant CD4 T cell response to Mtb (median rate = 82.5% vs. 79.7%; p = 0.796) and BCG (LPI = 84.3% vs. 83.0%; p = 0.853), which were significantly higher than the CD8, CD19 and γδ responses within both groups. CF patients tended to have a higher CD8 T cell response upon stimulation with the phytohemagglutinin mitogen than healthy controls (median rate = 42.8% vs. 31.7%, p = 0.075).CONCLUSION:The responses of BCG-vaccinated CF patients to Mtb and BCG are at least similar to those of healthy individuals. These are probably memory responses elicited by the BCG vaccination, which can cross-react with NTM and may explain the low frequency of NTM lung infection in our CF center.

Sewage sludge (SS) exhibits a relevant agronomic potential due to the high content of organic matter and nutrients. However, the presence of several toxic substances can prevent its agricultural application. This study evaluated if the incorporation of stimulating agents (coffee grounds and sugarcane bagasse) could contribute to an effective increase of the SS biodegradability in order to decrease its toxicity. The samples were prepared mixing aerobic or anaerobic sludge with soil, soil and bagasse, and soil and coffee grounds. Respirometric tests showed that stimulating agents enhanced the CO2 production. However, in terms of biodegradation efficiency, more satisfactory results were verified for the anaerobic SS, especially when mixed with coffee grounds. The biodegradation also favored the SS sanitization, eliminating the Enterobacteria. For baseline toxicity (Microtox with Aliivibrio fischeri) and phytotoxicity (Lactuca sativa), all the initial samples showed higher effects. Nevertheless, after the biodegradation, this toxicity was significantly decreased and the best results were obtained for the mixtures containing only soil and sludge. For the AREc32 assay (NRF2 mediated oxidative stress response), although a very weak response was observed, this effect was attenuated for the aerobic SS or completely eliminated for the anaerobic SS after the biodegradation. Thus, even though the use of biostimulation agents during the biodegradation led to an enhancement of microbial respiration, their incorporation to the samples do not seem to interfere in the decrease of the toxic potential of the studied SSs. However, the SS biodegradation in aerobiosis was crucial for toxicity reduction and to accelerate its maturity.