RESUMO
Background: Urinary tract infection is a worldwide health problem. According to the Clinical Laboratory Improvement Amendments and the European Urinalysis Guideline, urine samples should be tested within 2 h of collection. Thus, using chemical preservatives that guarantee the pre-analytical conditions is a practical tool. However, the effects of temperature and storage time as uropathogenic bacteria stressors are unclear. Methods: Gram-negative and -positive ATTC strains, E. coli, P. mirabilis, E. faecalis, and S. aureus, were used in this study. Strains in liquid media were stored at 4, 25, and 37 °C for 0, 2, 12, 24, and 48 h in tubes with and without preservatives. Then, reactive oxygen species (ROS) levels, viable but non-culturable bacteria (VBNC), and bacteria growth were analyzed. Results: A high ROS level was associated with the presence of VBNC and dead bacteria with low CFU counts, but a low ROS level increased the CFU number, depending on temperature and storage time in tubes without preservatives (boric acid, sodium borate, and formate). The BD Vacutainer™ Urine Culture & Sensitivity Preservative PLUS Plastic Tubes (C&S-PP) prevent this ROS increase, maintaining the CFU number for longer. Conclusions: C&S-PP tubes minimize the stressor effects (temperature and time storage) on uropathogenic bacteria when stored, improving the pre-analytical conditions of cultures realized by the clinical laboratory.
RESUMO
Primary sludge (PS) is associated with public health and environmental risks, so regulations focus on reducing the pathogenic and heavy metal contents of the treated material (biosolids), intended for soil amendments and land reclamation. The regulations set limits for Escherichia coli (or fecal coliforms), Salmonella spp., helminth eggs and enterovirus. However, the potential risk due to antibiotic resistant bacteria (ARB) and other human potential pathogenic bacteria (HPB) are not considered. In this work, three sludge treatment processes, having in common an anaerobic digestion step, were applied to assess the removal of regulated bacteria (fecal coliforms, Salmonella spp), ARB and HPB. The treatment arrangements, fed with PS from a full-scale wastewater treatment plant were: 1) Mesophilic anaerobic digestion followed by alkaline stabilization post-treatment (MAD-CaO); 2) Thermophilic anaerobic digestion (TAD) and, 3) Pre-treatment (mild thermo-hydrolysis) followed by TAD (PT-TAD). The results address the identification, quantification (colony forming units) and taxonomic characterization of ARB resistant to ß-lactams and vancomycin, as well as the taxonomic characterization of HPB by sequencing with PacBio. In addition, quantification based on culture media of fecal coliforms and Salmonella spp. is presented. The capabilities and limitations of microbiological and metataxonomomic analyses based on PacBio sequencing are discussed, emphasizing that they complement each other. Genus Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Ochrobactrum, Pseudomonas and Raoultella, among others, were found in the PS, which are of clinical or environmental importance, being either HPB, HPB-ARB, or non-pathogenic ARB with the potentiality of horizontal gene transfer. Based on the analysis of fecal coliforms and Salmonella spp., the three processes produced class A (highest) biosolids, suitable for unrestricted agriculture applications. Mild thermo-hydrolisis was effective in decreasing ARB cultivability, but it reappeared after the following TAD. O. intermedium (HPB-ARB) was enriched in MAD and TAD while Laribacter hongkongensis (HPB) did persist after the applied treatments.