Negative impacts of cleaning agent DEPTAL MCL® on activated sludge wastewater treatment system.

DEPTAL MCL® is a professional cleaning agent approved by the Portuguese Food Regulatory Authority and is used in agro-food industries, namely in fish canning industries in the north of Portugal. Its extensive use during cleaning procedures results in potential significant negative impacts on the performance of the downstream municipal wastewater treatment plant (WWTP). A lab-scale extended areation activated sludge wastewater treatment system, continuously fed by influent collected at a municipal WWTP, was used to assess the impact of a range of DEPTAL MCL® concentrations during 72 h. Despite distinct activated sludge community composition (due to its dynamic nature) and variations in real influent characteristics, a relevant impact was observed. DEPTAL MCL® effect was underscored through the use of a multivariate analysis using seventeen physicochemical operational factors and nineteen quantitative image analysis (QIA) parameters. DEPTAL MCL® exerted a severe negative impact on phosphorous (P-PO4) removal, total nitrogen (TN) removal and sludge volume index (SVI). With increasing DEPTAL MCL® concentrations, both P-PO4 and TN removal were affected and diminished proportionally. Moreover, several QIA parameters indicate defloculation when DEPTAL MCL® was present, in particular for intermediate size aggregates with significant impacts. Optical density of the effluent (Ode), displayed an increase of effluent turbidity. Percentage of area covered by small aggregates (%Areasml) was also significantly higher for the intermediate and higher DEPTAL MCL® concentrations tested. Principal component analysis exhibited 3 distinct ordenations: (i) control without addition of DEPTAL MCL®; (ii) addition of 0.03% and 0.06% and of (iii) 0.13 and 0.26% (v DEPTAL MCL®/v aeration tank). Canonical correspondence analysis (CCA) was used to correlate the physicochemical data, QIA and the filamentous bacteria species prevalence to DEPTAL MCL® concentration and incubation time. A time persistent DEPTAL MCL® effect was observed, underscoring the need of a pretreatment of wastewater containing this cleaning agent.

Flow-FISH Using Nucleic Acid Mimic Probes for the Detection of Bacteria.

Flow-Fluorescence in situ hybridization (Flow-FISH) enables multiparametric high-throughput detection of target nucleic acid sequences at the single cell-level, allowing an accurate quantification of different cell populations by using a combination of flow cytometry and fluorescent in situ hybridization (FISH). In this chapter, a flow-FISH protocol is described with labeled nucleic acid mimics (NAMs) (e.g. LNA/2'OMe and PNA) acting as the reporter molecules. This protocol allows for the specific detection of bacterial cells. Hence, this protocol can be carried out with minor adjustments, in order to simultaneously detect different species of bacteria in different types of clinical, food, or environmental samples.

The first sequenced Sphaerotilus natans bacteriophage- characterization and potential to control its filamentous bacterium host.

Bacteriophages (phages) are ubiquitous entities present in every conceivable habitat as a result of their bacterial parasitism. Their prevalence and impact in the ecology of bacterial communities and their ability to control pathogens make their characterization essential, particularly of new phages, improving knowledge and potential application. The isolation and characterization of a new lytic phage against Sphaerotilus natans strain DSM 6575, named vB_SnaP-R1 (SnaR1), is here described. Besides being the first sequenced genome of a Sphaerotilus natans infecting phage, 99% of its 41507 bp genome lacks homology with any other sequenced phage, revealing its uniqueness and previous lack of knowledge. Moreover, SnaR1 is the first Podoviridae phage described infecting this bacterium. Sphaerotilus natans is an important filamentous bacterium due to its deleterious effect on wastewater treatment plants (WWTP) and thus, phages may play a role as novel biotechnological tools against filamentous overgrowth in WWTP. The lytic spectrum of SnaR1 was restricted to its host strain, infecting only one out of three S. natans strains and infection assays revealed its ability to reduce bacterial loads. Results suggest SnaR1 as the prototype of a new phage genus and demonstrates its potential as a non-chemical alternative to reduce S. natans DSM 6575 cells.

Antibacterial Effects of Bimetallic Clusters Incorporated in Amorphous Carbon for Stent Application.

The purpose of this work is the development of Ag/a:C and Ag-Au/a:C coatings for ureteral stents, to provide them with antimicrobial characteristics. Silver was selected because of its well-known antibacterial properties, while gold was included to assess its capacity to accelerate the silver ion release forming a galvanic couple between Au and Ag. Thus, the metallic (Ag) and bimetallic clusters (Ag-Au) were produced by three different configurations: (i) unbalanced magnetron sputtering (conventional sputtering), (ii) plasma gas condensation process, and by (iii) a combination between both previous approaches. Coatings with Ag-Au bimetallic clusters were characterized by transmission electron microscopy (TEM) in order to study the arrangement (alloy, core-shell, and galvanic couple) of these particles in the carbon matrix. Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to quantify the Ag ions released through artificial urine from the different coatings deposited on thermoplastic polyurethane tape (one of the materials used in the manufacture of the ureteral stent ). Then, the antibacterial and cytotoxicity properties of Ag and Ag-Au/a:C coatings were evaluated. TEM shows that a biphasic structure was not detected, thus not allowing to anticipate the establishment of a galvanic couple. The ICP-OES results demonstrate that the silver ionization is mainly a function of the amount of silver incorporated in the amorphous carbon (a:C) matrix, and the formation of a bimetallic alloy has a detrimental effect on release of the silver ions. The antibacterial activity was regulated by the silver ionization mechanisms because the coatings with higher Ag release had a higher antibacterial activity.

A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood.

Bloodstream infections (BSIs) are considered a major cause of death worldwide. Staphylococcus spp. are one of the most BSIs prevalent bacteria, classified as high priority due to the increasing multidrug resistant strains. Thus, a fast, specific and sensitive method for detection of these pathogens is of extreme importance. In this study, we have designed a novel assay for detection of Staphylococcus in blood culture samples, which combines the advantages of a phage endolysin cell wall binding domain (CBD) as a specific probe with the accuracy and high-throughput of flow cytometry techniques. In order to select the biorecognition molecule, three different truncations of the C-terminus of Staphylococcus phage endolysin E-LM12, namely the amidase (AMI), SH3 and amidase+SH3 (AMI_SH3) were cloned fused with a green fluorescent protein. From these, a higher binding efficiency to Staphylococcus cells was observed for AMI_SH3, indicating that the amidase domain possibly contributes to a more efficient binding of the SH3 domain. The novel phage endolysin-based flow cytometry assay provided highly reliable and specific detection of 1-5 CFU of Staphylococcus in 10 mL of spiked blood, after 16 hours of enrichment culture. Overall, the method developed herein presents advantages over the standard BSIs diagnostic methods, potentially contributing to an early and effective treatment of BSIs.

Evaluation by Flow Cytometry of Escherichia coli Viability in Lettuce after Disinfection.

Foodborne outbreaks due to the consumption of ready-to-eat vegetables have increased worldwide, with Escherichia coli (E. coli) being one of the main sources responsible. Viable but nonculturable bacteria (VBNC) retain virulence even after some disinfection procedures and constitute a huge problem to public health due to their non-detectability through conventional microbiological techniques. Flow cytometry (FCM) is a promising tool in food microbiology as it enables the distinction of the different physiological states of bacteria after disinfection procedures within a short time. In this study, samples of lettuce inoculated with E. coli were subject to disinfection with sodium hypochlorite at free chlorine concentrations of 5, 10, 25, 50, and 100 mg·L-1 or with 35% peracetic acid at concentrations of 5, 10, 25, and 50 mg·L-1. The efficiency of these disinfectants on the viability of E. coli in lettuce was evaluated by flow cytometry with LIVE/DEAD stains. Results from this study suggest that FCM can effectively monitor cell viability. However, peracetic acid is more effective than sodium hypochlorite as, at half the concentration, it is enough to kill 100% of bacteria and always induces a lower percentage of VBNC. Finally, we can conclude that the recommended levels of chemical disinfectants for fresh fruit and vegetables are adequate when applied in lettuce. More importantly, it is possible to ensure that all cells of E. coli are dead and that there are no VBNC cells even with lower concentrations of those chemicals. These results can serve as guidance for lettuce disinfection, improving quality and the safety of consumption.

Epidemiology of dermatophytoses in 31 municipalities of the province of Buenos Aires, Argentina: A 6-year study / Epidemiología de las dermatofitosis en 31 municipios de la provincia de Buenos Aires, Argentina: estudio de 6 años

Background: No reliable data are available in the province of Buenos Aires regarding the frequency of dermatophytoses and other fungal diseases. The distribution of the clinical forms and the species involved are also unknown. Aims: To present the data collected by the laboratories participating in the Mycology Network of the province of Buenos Aires (MNPBA) from a retrospective epidemiological survey on dermatophytoses. Methods: A descriptive and exploratory analysis was performed on the cases of dermatophytoses gathered between 2002 and 2007 by the Mycology Network of the province of Buenos Aires. Results: Of the 3966 dermatophytosis cases reported by 41 laboratories in 31 municipalities, more than a half occurred in three highly populated urban municipalities. The male:female ratio was 1:1.5. The most frequent clinical form was tinea unguium, diagnosed in 904 cases (51.83%), followed by tinea capitis (19.32%), tinea corporis (15.19%), tinea pedis (6.77%), tinea cruris (3.73%), and tinea manuum (2.18%). The species involved was identified in 1368 (33.49%) cases. Trichophyton rubrum was the most common species, with a frequency of 42.03%. An association was found between urban municipalities and T. rubrum or the Trichophyton mentagrophytes complex. Conclusions: Results from the MNPBA survey provide valuable information that should enable further interventions to be designed in order to prevent and control the disease

Antecedentes: No existen datos fiables acerca de la frecuencia de las dermatofitosis y otras enfermedades fúngicas en la provincia de Buenos Aires. Por otra parte, la distribución en la provincia de las formas clínicas y las especies involucradas no es conocida. Objetivos: El objetivo de este estudio fue reportar los datos recogidos por los laboratorios que participan en la Red de Micología de la Provincia de Buenos Aires (MNPBA) a través del análisis de encuestas epidemiológicas retrospectivas sobre casos notificados de dermatofitosis. Métodos: Se realizó un análisis descriptivo y exploratorio de los casos de dermatofitosis recogidos por los laboratorios de la Red de Micología de la provincia de Buenos Aires durante un período de 6 años (2002-2007). Resultados: Se registraron 3.966 casos procedentes de 41 laboratorios distribuidos en 31 municipios. Más de la mitad de los casos tuvieron lugar en tres municipios urbanos muy poblados. La proporción varón:mujer fue de 1:1,5. La forma clínica más frecuente fue tinea unguium, diagnosticada en 904 casos (51,83%), seguida de tinea capitis (19,32%), tinea corporis (15,19%), tinea pedis (6,77%), tinea cruris (3,73%) y tinea manuum (2,18%). La identificación de las especies de dermatofitos se realizó en 1.368 casos (33,49%). La especie predominante fue Trichophyton rubrum (42,03%). Se observó asociación entre los municipios de alta densidad poblacional y la presencia de T. rubrum y del complejo de especies Trichophyton mentagrophytes. Conclusiones: Los resultados de las encuestas de MNPBA generan información de calidad que permite el diseño de nuevas intervenciones para la prevención y control de estas micosis

Epidemiology of dermatophytoses in 31 municipalities of the province of Buenos Aires, Argentina: A 6-year study.

BACKGROUND: No reliable data are available in the province of Buenos Aires regarding the frequency of dermatophytoses and other fungal diseases. The distribution of the clinical forms and the species involved are also unknown. AIMS: To present the data collected by the laboratories participating in the Mycology Network of the province of Buenos Aires (MNPBA) from a retrospective epidemiological survey on dermatophytoses. METHODS: A descriptive and exploratory analysis was performed on the cases of dermatophytoses gathered between 2002 and 2007 by the Mycology Network of the province of Buenos Aires. RESULTS: Of the 3966 dermatophytosis cases reported by 41 laboratories in 31 municipalities, more than a half occurred in three highly populated urban municipalities. The male:female ratio was 1:1.5. The most frequent clinical form was tinea unguium, diagnosed in 904 cases (51.83%), followed by tinea capitis (19.32%), tinea corporis (15.19%), tinea pedis (6.77%), tinea cruris (3.73%), and tinea manuum (2.18%). The species involved was identified in 1368 (33.49%) cases. Trichophyton rubrum was the most common species, with a frequency of 42.03%. An association was found between urban municipalities and T. rubrum or the Trichophyton mentagrophytes complex. CONCLUSIONS: Results from the MNPBA survey provide valuable information that should enable further interventions to be designed in order to prevent and control the disease.

Establishment of a Quality Management System Based on ISO 9001 Standard in a Public Service Fungal Culture Collection.

Collaborations between different Microbiological Resource Centres (mBRCs) and ethical sourcing practices are mandatory to guarantee biodiversity conservation, successful and sustainable preservation and fair share of benefits that arise from the use of genetic resources. Since microbial Culture Collections (CCs) are now engaged in meeting high quality operational standards, they are facing the challenge of establishing quality control criteria to certify their biological materials. The authentication/certification of strains is nowadays a demand from the bioeconomy sector for the global operation of mBRCs. The achievement of consistent quality assurance and trust within the mBRCs and microbial CCs context is a dynamic and never-ending process. A good option to facilitate that process is to implement a Quality Management System (QMS) based on the ISO 9001 standard. Here, we report a detailed description of all the steps taken for the QMS implementation at the Portuguese CC of filamentous fungi: Micoteca da Universidade do Minho (MUM). Our aim is to provide guidelines for the certification of other CCs, so that they can also enhance the search and choice of the most consistent, reliable, and effective operating methods, with assured procedures and validation of preservation; and guarantee trustworthy relations with all stakeholders.

The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing

AIMS: In this study, the potential of matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) was investigated for the identification of clinical isolates. The isolates were analyzed at the species and strain level. Methods Spectral identification by MALDI-TOF ICMS was performed for all strains, and compared with the results of sequencing of the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA region. PCR fingerprinting analysis using primers M13, (GACA)4, and (AC)10 was performed in order to assess the intra-specific variability of Trichophyton rubrum strains. Results The identification of strains at species level by MALDI-TOF ICMS was in agreement with the previously performed morphological and biochemical analysis. Sequence data confirmed spectral mass identification at species level. Intra-specific variability was assessed. Within the T. rubrum cluster, strains were distributed into smaller highly related sub-groups with a similarity values above 85%.ConclusionsMALDI-TOF ICMS was shown to be a rapid, low-cost and accurate alternative tool for the identification and strain typing of T. rubruM

OBJETIVO: En este estudio se investigó el potencial de matrix-assisted laser desorption/ionization time-of-flight intact cell mass (MALDI-TOF ICMS) para la identificación de aislados clínicos. Los aislados fueron analizados al nivel de especie y de cepa. MÉTODOS: Se realizó una identificación espectral mediante MALDI-TOF ICMS de todas las cepas que se comparó con los resultados de secuenciación de la región espaciadores transcritos internos (ITS1 e ITS2) y el ADNr 5,8S. Los análisis PCR fingerprinting usando los primers M13, (GACA)4 y (AC)10 se realizaron con la intención de evaluar la variabilidad intraespecífica de las cepas de Trichophyton rubrum. RESULTADOS: La identificación de las cepas al nivel de especie mediante MALDI-TOF ICMS concordó con la ID realizada retrospectivamente en el análisis morfológico y bioquímico. La secuencia de datos confirmó la identificación por espectro de masas a nivel de especie. Se evaluó la variabilidad intraespecífica. Dentro del clúster de T. rubrum, las cepas se distribuyeron en subgrupos menores y muy relacionados con valores de similaridad superiores a 85%. CONCLUSIONES: Se demuestra que MALDI-TOF ICMS es una herramienta alternativa rápida, de bajo coste y precisa para la identificación y tipificación de cepas de T. rubrum

The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing.

AIMS: In this study, the potential of matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) was investigated for the identification of clinical isolates. The isolates were analyzed at the species and strain level. METHODS: Spectral identification by MALDI-TOF ICMS was performed for all strains, and compared with the results of sequencing of the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA region. PCR fingerprinting analysis using primers M13, (GACA)4, and (AC)10 was performed in order to assess the intra-specific variability of Trichophyton rubrum strains. RESULTS: The identification of strains at species level by MALDI-TOF ICMS was in agreement with the previously performed morphological and biochemical analysis. Sequence data confirmed spectral mass identification at species level. Intra-specific variability was assessed. Within the T. rubrum cluster, strains were distributed into smaller highly related sub-groups with a similarity values above 85%. CONCLUSIONS: MALDI-TOF ICMS was shown to be a rapid, low-cost and accurate alternative tool for the identification and strain typing of T. rubrum.

Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes.

Toxicity of two azo dyes (Reactive Orange 16 (RO16); Congo Red (CR)) and two anthraquinone dyes (Remazol Brilliant Blue R (RBBR); Disperse Blue 3 (DB3)) were compared using bacterium Vibrio fischeri, microalga Selenastrum capricornutum and ciliate Tetrahymena pyriformis. The following respective endpoints were involved: acute toxicity measured as bacterial luminescence inhibition, algal growth inhibition, and the effects on the protozoa including viability, growth inhibition, grazing effect and morphometric effects. In addition, mutagenicity of the dyes was determined using Ames test with bacterium Salmonella typhimurium His(-). DB3 dye was the most toxic of all dyes in the bacterial, algal and protozoan tests. In contrast to other dyes, DB3 exhibited mutagenic effects after metabolic activation in vitro in all S. typhimurium strains used. Of the methods applied, the algal test was the most sensitive to evaluate toxicity of the dyes tested.

Morphological and physiological changes in Tetrahymena pyriformis for the in vitro cytotoxicity assessment of Triton X-100.

Non-ionic surfactants such as Triton X-100 have been widely used in industrial processing and in cleaning products for almost 50 years, being effective and economic emulsifying, wetting agents, dispersants and solubilizers. Cleaning products containing these surfactants are disposed of mainly by discharge into wastewater, which receives biological treatment in wastewater treatment systems. However, surface-active agents interact with eukaryotic cell membranes leading to biological damage at high concentrations. Tetrahymena pyriformis was used here as model organism to assess the effects of Triton X-100 through a series of in vitro cytotoxicity tests. Growth rates and morphological changes were, by their simplicity and reproducibility, the simplest toxicological assays. Cytoskeleton analysis seemed to be related with phagocytosis rate. Viability was evaluated by two different tests. Calcein AM/EthD-1 was used to assess T. pyriformis membrane damage during the 48-h experiment. The colorimetric MTT assay proved to be highly sensitive even at very short periods of Triton X-100 exposure. Tests performed in this study included simple and fast bioassays that provide overall information on the morphological and physiological state of cells exposed to different non-lytic and lytic concentrations of Triton X-100.

A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis.

A novel cellular cytotoxicity assay using two fluorescent dyes was developed as an alternative method to the standard direct count of viable protozoa under light microscopy. The compound calcein AM is a non-fluorescent substance that diffuses passively across intact cell membranes and is converted by intracellular esterases to the green fluorescent calcein, which is retained in viable cells. The addition of EthD-1 that binds to DNA stained nuclei of dead cells red. The experiments were carried out in order to assess viability in the freshwater ciliate Tetrahymena pyriformis after exposure to eight surfactants, two of each representing one of four ionic class (non-ionic, anionic, cationic and amphoteric), and two heavy metals, copper and zinc, at several concentrations. In earlier time exposure, less than one hour of contact with surfactants at sublethal concentrations, the fluorescent method is more sensitive and provides more accurate results than direct counting under light microscopy. In contrast, with increasing time exposure, the results obtained by the two methods were similar. Calcein was shown to be a poor viability marker in the presence of zinc and copper since the fluorescence intensity was affected by the metal presence. However, the fluorescent method offers new opportunities to use advanced techniques, such as flow cytometry, to assess cytotoxicity in protozoa.