Este trabalho apresenta um estudo empregando duas técnicas óticas para monitorar o crescimento de culturas de cepas de Escherichia coli em dois meios de cultura líquidos: Espectroscopia de absorção UV-Vis (turbidimetria) e espectroscopia Raman. Na primeira técnica, a turbidez permite avaliar as diferentes fases naturais de crescimento de uma cultura bacteriana (lag, exponencial, estacionária e decaimento) por meio da densidade ótica, medida com um espectrômetro UV-VIS. Na segunda, o espalhamento Raman (medido com um espectrômetro dispersivo), a partir de amostras de água contaminada, fornece não apenas informações sobre as fases de crescimento, mas também abre a possibilidade de identificação bacteriana através da sua impressão digital característica. Mediu-se a dinâmica de duas cepas de E. coli – (nomeadas como H2/11 e H3C2/12) em um caldo líquido nutriente e quatro cepas de E. coli (nomeadas como H2/11, H3C2/12, 109 e 110) em caldo líquido EC, mantidas a 37,0°C ao longo de 24 horas. Alíquotas das amostras foram removidas da cultura em intervalos de tempo regulares para medições espectrais. A análise da turbidez permitiu medir o tempo de geração (isto é, o tempo de duplicação de uma população), que foi maior para cepas crescidas em caldo EC. Os espectros Raman forneceram informações sobre a evolução temporal das bandas a 942 cm-1, 977 cm-1, 1036 cm-1, 1086 cm-1, 1140 cm-1, 1188 cm-1, 1182 cm-1, 1207 cm-1 e 1251 cm-1, associadas com impressões digitais de componentes biológicos específicos. Os dados espectrais foram analisados por Análise de Componentes Principais (PCA). Os resultados obtidos em ambas as técnicas permitiram identificar as fases lag, exponencial e estacionária das cepas estudadas. / This work is a study using two optical techniques to monitor the growth of cultures of Escherichia coli strains in two liquid culture medium: Raman spectroscopy and UV-Vis absorption spectroscopy (turbidimetry). In one hand, turbidity allows evaluating the different phases of growth of a bacterial culture (lag, exponential, stationary and decay) by optical density, measured with an UV-VIS spectrometer. On the other hand, Raman scattering (measured with a dispersive spectrometer) from contaminated water samples not only provides information about the grow phases, but also opens a possible identification of bacterial by its characteristic fingerprint. Two strains of E. coli (named as H2 / 11 and H3C2 / 12) were measured in liquid nutrient broth and four E. coli strains (named H2 / 11, H3C2 / 12, 109 and 110) in EC liquid broth, kept at 37.0 ° C over 24 hours. Aliquots of the samples were removed from the culture at regular time intervals for spectral measurements. The turbidity analysis allowed to measure the generation time, which was higher for strains grown in EC broth. Raman spectra provided information about the time evolution of the bands at 942 cm-1, 977 cm-1, 1036 cm-1, 1086 cm-1, 1140 cm-1, 1188 cm-1, 1182 cm-1, 1207 cm-1 e 1251 cm-1, associated with fingerprints of biological components. Data were analyzed by Principal Component Analysis (PCA). These results of both techniques allowed identifying the phases lag, exponential and stationary of the studied strains.

No controle da qualidade da água, parâmetros microbiológicos são avaliados e devem estar de acordo com os limites estipulados em portarias e resoluções. Todas as metodologias de monitoramento microbiano demandam o cultivo dos organismos-alvo. Em muitos casos, as cepas isoladas devem ser analisadas por teses fenotípicos para determinação da espécie. Por meio de inóculos de E. coli, demonstrou-se que a variação na técnica de plaqueamento se deve especialmente à falta de consistência entre as diluições. Além disso, para a análise de réplicas, comarando-se as médias de dois métodos de diluição, foi demonstrado que ser utilizado apenas uma série de diluição derivada de um único inóculo. Utilizando-se culturas puras de E. coli, E. faecalis e P. aeruginosa, a recuperação foi similar entre os meios seletivos respectivos (m-Endo, m-EI, m-PA-C) e meio não seletivo (Tripticase Soy Agar). A utilização da técnica de membrana filtrante resultou em contagens significativamente maiores para E.coli e E. faecalis em comparação ao método de spread plate. A microbiota natural presente na água potável (torneira) não influenciou significativamente as contagens de E. coli, E. faecalis e P. aeruginosa em meios seletivos. Entretanto, na água mineral engarrafada inoculada artificialmente com P. aeruginosa, a contagem foi significativamente maior na réplica estéril. Na análise de água marinha, e na réplica não estéril inoculada com E. faecalis, a contagem foi signifivativamente menor e as células de P. aeruginosa produziram colônias atípicas. Analisando-se amostras do meio ambiente (biofilmes de estação de tratamento de água, lodo de esgoto e água de córrego contaminado), 45 colônias típicas isoladas em meios de cultura seletivos foram identificadas por meio do sequenciamento do gene 16S rRNA, testes bioquímicos e MALDI-TOF. Os resultados de sequenciamento do gene 16SrRNA tiveram baixa correlação com a identificação dos organismos por testes bioquímicos (46,6% em nível de gênero e 20% em nível de espécie) e MALDI-TOF (60% em nível de gênero e 48,8% em nível de espécie). Para as cepas identificadas como E. coli e Enterococcus spp., a correlação entre o sequenciamento e MALDI-TOF foi de 75% e 62%, respectivamente. Uma vez que a quantificação de micro-organismo por membrana filtrante depende do micro-organismo em análise e do tipo de água, é necessário que os laboratórios realizem testes de padronização antes de implementar essa metodologia. Os resultados demonstram que os métodos convencionais utilizados não são adequados e suficientes para a análise da qualidade da água e, portanto, novas metodologias devem ser empregadas. Idealmente devem ser utilizadas técnicas baseadas em características fenotípicas, genômica e proteômica cujos resultados são complementares e fornecem uma identificação mais acurada. / All over the world regulatory agencies specify microbiological water quality parameters to guarantee water safety. Conventional microbiological water quality analysis is based on the cultivation of the target organisms. In many analysis protocols, phenotypic assays of isolated strains are mandated for species determination. Reference samples are required for quality assessment and quality control of analytical protocols. Using E. coli as a model organism, it was demonstrated here that the variability of plate counts of reference cultures is caused mainly by the spread of counts in individual serial dilutions. Besides, comparing the means obtained by two dilution approaches, it was demonstrated that in the analysis of replicates it is possible to use only a set of dilutions derived for a unique inoculum. Recovery of pure cultures of E. coli, E. faecalis and P. aeruginosa was similar on selective culture media (m-Endo, m-EI, m-PA-C) and with the non-selective medium (Tripticase Soy Agar). The membrane filter technique yielded significantly higher counts for E.coli and E. faecalis in comparison to spread plate method. The autochthonous microbiota of potable water (tap water) did not influence the counts of E. coli, E. faecalis and P. aeruginosa on selective culture media. However, the sterile replica of mineral water spiked with P. aeruginosa showed higher counts for these bacteria. For marine water analysis, the non-sterile replica spiked with E. faecalis yielded low counts and P. aeruginosa produced an atypical colony. Both, sample-induced variation in target strain recovery and colony appearance on culture plates indicates the requirement for method evaluation tests on particular sample matrices before implementing routine microbiological analysis by culture-based methods in the laboratory. 45 typical colonies obtained from environmental samples in selective culture media (biofilms from activated sludge, drinking water treatment plants and water from creek contaminated with raw sewage) were analyzed by 16S rRNA gene sequencing, biochemical phenotypic assays and MALDI-TOF. Sequencing showed low correlation with phenotypic assays (46,6% at the genus level and 20% at the species level) and MALDI-TOF (60% at the genus level and 48,8% at the species level). On the other hand, the strains identified as E. coli an Enterococcus spp. by 16S rDNA gene sequencing demonstrated a high correlation with MALDI-TOF (75% and 62%, respectively). Since the results showed that conventional methods aren´t suitable and sufficient to assess water quality, new technologies should be employed. Ideally, techniques should be based on phenotypic, genomic and proteomic features since the results are complementary and provide a more accurate identification.

Review

Philosophiae Doctor - PhD / The aim of this thesis is first, to evaluate various culture conditions to improve embryo development, and secondly, to analyze spent culture media for any biomarkers that may be predictive of embryo health. Single-step and sequential culture media were compared in both Planer and EmbryoScope™ incubators. Single-step media resulted in better blastocyst development compared to sequential media and the EmbryoScope™ incubation system showed slight improvements in embryo development than the Planer system. The benefits of supplementing the culture medium with either insulin or insulin-like growth factor 1 (IGF-1) or culturing in a 2% O2 environment, using two different strains of mice (hybrid and C57), as well as the suitability of these strains for quality control were compared. In insulin, hybrid embryos were slower to blastulate and had a lower blastocyst rate, whereas C57 embryos were slower to the morula and faster to blastocyst stages, and lower blastocyst rate than the controls. IGF-1 showed no difference in time-lapse morphokinetics (TLM) or blastocyst rates compared to controls in both hybrid and C57 embryos. Under 2% O2, hybrid embryos showed no significant difference in TLM up to the 8-cell stage, but slowed down afterwards, resulting in blastocysts with significantly lower cell counts than the 6% O2 group. The C57 embryos were slower to reach morula and expanded blastocyst, and had lower blastocyst rates in 2%O2 vs 6%O2. The C57 strain had significant slower overall embryo development for all time points than hybrid embryos in insulin, IGF-1 and ultra-low O2, as well as lower blastocyst rates. Measurement of growth differentiation factor 9 (GDF-9) and oxidation-reduction potential (ORP) in spent media as markers for embryo health were evaluated. Day 5 human blastocysts yielded higher pregnancy rates and GDF-9 levels in spent media compared to Day 6 blastocysts, but TLM parameters showed no impact on pregnancy outcome. In Day 6 blastocysts, the non-pregnant group showed significantly faster embryo development compared to the clinically pregnant group up to the 8-cell stage and start of blastulation. GDF-9 did not show any significant differences between non-pregnant and pregnant groups of Day 5 or Day 6 embryo transfers. ORP in spent media from good quality Day 3 embryos that developed into blastocysts were significantly higher than from those that did not, with no difference in control medium ORP. Spent media from arrested embryos showed lower ORP than their corresponding controls. Arrested embryos had slower development at syngamy, morula, blastulation and blastocyst stages. The single step medium in the EmbryoScope™ is the preferred choice for embryo culture. Insulin or IGF-1 media supplementation or 2% O2 culture did not provide any benefit to embryo development. The C57 mouse strain is more sensitive and may be better to detect changes in culture conditions, and therefore better model for quality control assays. GDF-9 values decrease from Day 5 to Day 6 which gives new insight to understanding the role of GDF-9 during embryogenesis. ORP in spent media indicate that embryos that developed into blastocysts did not contribute to ROS, but maintained ORP balance.

L’imagerie médicale a longtemps été limitée à cause des performances médiocres des fluorophores organiques. Récemment la recherche sur les nanocristaux semi-conducteurs a grandement contribué à l’élargissement de la gamme d’applications de la luminescence dans les domaines de l’imagerie et du diagnostic. Les points quantiques (QDs) sont des nanocristaux de taille similaire aux protéines (2-10 nm) dont la longueur d’onde d’émission dépend de leur taille et de leur composition. Le fait que leur surface peut être fonctionnalisée facilement avec des biomolécules rend leur application particulièrement attrayante dans le milieu biologique. Des QDs de structure « coeur-coquille » ont été synthétisés selon nos besoins en longueur d’onde d’émission. Dans un premier article nous avons modifié la surface des QDs avec des petites molécules bi-fonctionnelles portant des groupes amines, carboxyles ou zwitterions. L’effet de la charge a été analysé sur le mode d’entrée des QDs dans deux types cellulaires. À l’aide d’inhibiteurs pharmacologiques spécifiques à certains modes d’internalisation, nous avons déterminé le mode d’internalisation prédominant. L’endocytose par les radeaux lipidiques représente le mode d’entrée le plus employé pour ces QDs de tailles similaires. D’autres modes participent également, mais à des degrés moindres. Des disparités dans les modes d’entrée ont été observées selon le ligand de surface. Nous avons ensuite analysé l’effet de l’agglomération de différents QDs sur leur internalisation dans des cellules microgliales. La caractérisation des agglomérats dans le milieu de culture cellulaire a été faite par la technique de fractionnement par couplage flux-force (AF4) associé à un détecteur de diffusion de la lumière. En fonction du ligand de surface et de la présence ou non de protéines du sérum, chacun des types de QDs se sont agglomérés de façon différente. À l'aide d’inhibiteur des modes d’internalisation, nous avons corrélé les données de tailles d’agglomérats avec leur mode d’entrée cellulaire. Les cellules microgliales sont les cellules immunitaires du système nerveux central (CNS). Elles répondent aux blessures ou à la présence d’inflammagènes en relâchant des cytokines pro-inflammatoires. Une inflammation non contrôlée du CNS peut conduire à la neurodégénérescence neuronale et est souvent observée dans les cas de maladies chroniques. Nous nous sommes intéressés au développement d’un nanosenseur pour mesurer des biomarqueurs du début de l’inflammation. Les méthodes classiques pour étudier l’inflammation consistent à mesurer le niveau de protéines ou molécules relâchées par les cellules stressées (par exemple monoxyde d’azote, IL-1β). Bien que précises, ces méthodes ne mesurent qu’indirectement l’activité de la caspase-1, responsable de la libération du l’IL-1β. De plus ces méthode ne peuvent pas être utilisées avec des cellules vivantes. Nous avons construit un nanosenseur basé sur le FRET entre un QD et un fluorophore organique reliés entre eux par un peptide qui est spécifiquement clivé par la caspase-1. Pour induire l’inflammation, nous avons utilisé des molécules de lipopolysaccharides (LPS). La molécule de LPS est amphiphile. Dans l’eau le LPS forme des nanoparticules, avec des régions hydrophobes à l’intérieure. Nous avons incorporé des QDs dans ces régions ce qui nous a permis de suivre le cheminement du LPS dans les cellules microgliales. Les LPS-QDs sont internalisés spécifiquement par les récepteurs TLR-4 à la surface des microglies. Le nanosenseur s’est montré fonctionnel dans la détermination de l’activité de la caspase-1 dans cellules microgliales activées par le LPS. Éventuellement, le senseur permettrait d’observer en temps réel l’effet de thérapies ciblant l’inflammation, sur l’activité de la caspase-1. / Medical imaging based on fluorescence has suffered from the poor photostability and mediocre performance of organic fluorophores. The discovery and subsequent improvements in nanocrystal synthesis and functionalization has greatly benefited the applications in medical imaging and the development of nanocrystal-based sensors for diagnostics. QDs are semi-conductor nanocrystals which have similar sizes as proteins (2-10 nm). They are highly luminescent, and can be made to emit at any desired wavelength by varying their size and composition. The surface of QDs can be easily functionalized with biomolecules. Hence, it is interesting to study how QDs interact in the biological world. Highly luminescent core-shell QDs emitting at different wavelengths were prepared according to our needs. In a first study, the surface of the QDs was modified with various small bi-functional thiolated ligands (carboxylated, aminated and zwitterionic). The modified-QDs of nearly identical sizes were administered in vitro to study the impact of surface charge and cell type on the mode and extent of cell uptake and elimination. Using specific inhibitors of cell uptake we determined which modes contributed to the internalization of the QDs. Endocytosis mediated by lipid rafts represented the predominant pathway for the internalization of QDs. However, other modes contributed to a lesser degree, depending on the surface ligand. We then analyzed the effect of QD agglomeration in cell culture media on its cellular uptake by microglia. Thorough characterization of QD agglomerate size distribution was conducted by asymmetrical flow field-flow fractionation (AF4) with a dynamic light scattering detector. Depending on the type of surface ligand and if serum proteins were present, the agglomeration pattern of the QDs was significantly different. With inhibitors of specific modes of cell uptake, we showed that the size distribution data, obtained by AF4, correlated with the modes of cell uptake. Microglia cells are immune cells of the central nervous system (CNS). They respond to injury or the presence of inflammagens by producing pro-inflammatory cytokine. Inflammation in the CNS may lead to loss of neurons, and can found in many chronic diseases. We were interested in building nanosensors to measure the onset of inflammation. Current methods to study inflammation consist in measuring levels of certain proteins or chemicals released by stressed cell (e.g. Western blot or ELISA assay for IL-1β). Although precise, these methods measure indirectly the activity of the enzyme responsible for releasing IL-1β, i.e. caspase-1. Moreover, these methods cannot be applied to live cells. We designed a sensor based on FRET between a QD and a dye linked by a peptide specifically cleaved by the caspase-1. To induce inflammation, we applied lipopolysaccharides (LPS), which are endotoxins present in Gram negative bacteria responsible for sceptic shock. The LPS form nanoparticles due to their amphiphilicity. The interior hydrophobic regions were used to load hydrophobic QDs, making the LPS luminescent. The microglia internalized LPS-QD predominantly through TLR-4 membrane receptors. We describe how the LPS induce inflammation and demonstrated the functionality of the QD-based sensor. Eventually, the sensor could be used to monitor in real time the action of therapeutics against inflammation.

INTRODUÇÃO: As infecções por bactérias anaeróbias são geralmente de origem endógena, polimicrobianas e mistas. Devido a sua natureza fastidiosa, essas bactérias necessitam de uma prévia incubação em meios líquidos enriquecidos, como o caldo Thioglicolato (CT) para serem recuperadas, o isolamento desses microrganismos é trabalhoso e o tempo de resposta - TAT (turn around time) estendido desse exame pode estar associado a falhas terapêuticas e ao aumento da resistência bacteriana. A cultura de anaeróbios (CANA) ainda é um desafio para os laboratórios clínicos de rotina e novas estratégias para diminuir o TAT são fundamentais para que esse exame forneça um impacto clínico significativo. OBJETIVO: Otimizar o processo de triagem da CANA pela modificação do CT; comparar a identificação dos anaeróbios pelas metodologias fenotípicas ANC (Vitek 2- bioMérieux, France) e MALDI-TOF (Vitek MS - bioMérieux, France) e verificar o impacto econômico das ações propostas MÉTODOS: O caldo de triagem CT foi modificado eluindo individualmente discos comerciais de antibióticos (em concentrações fixas) selecionados por apresentarem baixa ou nenhuma ação contra microrganismos anaeróbios e com um bom espectro de ação para os principais aeróbios associados em culturas mistas e foram escolhidos aqueles que após uma bateria de testes frente a 15 cepas dos principais anaeróbios envolvidos em infecções humanas mantiveram a viabilidade inicial. O caldo Thioglicolato modificado (CTM) foi composto pela adição dos antibióticos que apresentaram a melhor \"performance\" acima descrita. A sensibilidade e especificidade do CTM foram avaliadas paralelamente com CT na rotina de CANA do HCFMUSP. Para a avaliar a identificação fenotípica, 421 anaeróbios isolados no período de seis meses foram submetidos a identificação pelo ANC (Vitek 2) e MALDI-TOF (Vitek MS). Os resultados discordantes ou com baixa discriminação da espécie foram avaliados pelo sequenciamento 16S rRNA. O impacto econômico da introdução do CTM bem como os custos diretos da identificação pelo MALDI-TOF foram avaliados. RESULTADOS: O CTM foi composto por amicacina, gentamicina e aztreonam. Das 159 amostras clínicas triadas pelo CT e CTM, 11 (7%) foram positivas para CANA com as mesmas espécies isoladas em ambos os meios. Utilizando o CTM, foi obtida uma redução dos falsos positivos de 97 (61%) para 69 (43%) quando comparado ao CT (p < 0,05). O TAT do resultado negativo da CANA com o CTM foi reduzido de 14 para sete dias em 28 (18%) amostras; o CTM permitiu a liberação do resultado positivo da CANA 48 horas à frente do CT. A sensibilidade do CTM foi igual ao CT, porém a especificidade foi superior em 19%. Das 421 cepas avaliadas, 35 foram identificadas somente pelo MALDI-TOF (Vitek MS) sendo que uma (Clostridium innocum) foi identificada somente pelo sequenciamento 16S rRNA. Das 386 avaliadas por ambas as metodologias, houve uma concordância de 97% e os resultados das 13 (3%) cepas submetidas ao sequenciamento foram concordantes em 92% com o MALDI-TOF (Vitek MS) que promoveu a redução do TAT do resultado positivo em cinco dias. A implementação do CTM possibilitou uma redução de custos nessa amostragem, de R$ 2.240,00 e a identificação pelo MALDI-TOF proporcionou uma economia de R$ 7.786,00. Considerando os valores econômicos encontrados nesse estudo e projetando-os nas estatísticas de CANA do HCFMUSP em 2017, o CTM poderia proporcionar uma economia de R$ 132.560,00 /ano e o MALDI-TOF uma redução nos gastos de R$ 13.579,00/ ano CONCLUSÕES: A padronização e implementação do CTM permitiu uma um aumento significativo de especificidade da cultura anaeróbia com redução do TAT e dos custos. A utilização do MALDI-TOF diminuiu o TAT das identificações aliado a uma melhor performance de forma custo efetiva / INTRODUCTION: Anaerobic bacterial infections are usually of endogenous origin, polymicrobial and mixed. Because of their fastidious nature, these bacteria require prior incubation in enriched liquid media, such as Thioglycolate broth (TB) to be recovered, the isolation of these microorganisms is laborious, and the TAT (turn around time) extended time of this examination may be associated with therapeutic failures and increased bacterial resistance. Anaerobic culture (AC) is still a challenge for routine clinical laboratories, and new strategies for lowering TAT are critical to provide a significant clinical impact. OBJECTIVE: To optimize the AC screening process by modifying the TB; Compare anaerobical identification between (Vitek 2- bioMérieux, France) and MALDI-TOF (Vitek MS - bioMérieux, France) and to verify the economic impact of the proposed actions. METHODS: TB broth was modified by eluting individually antibiotic commercial discs (at fixed concentrations) selected for low or no action against anaerobic microorganisms and with a good action spectrum for the main associated aerobes in mixed cultures. Those who maintained the initial viability after a battery of tests against 15 strains of the major anaerobes involved in human infections were selected. Modified Thioglycolate Broth (MTB) was composed of the antibiotics that presented the best performance described above. The sensitivity and specificity of MTB were evaluated in parallel with TB in the HCFMUSP AC routine. To evaluate the phenotypic identification, 421 anaerobes isolated in the six-month period were submitted to identification by ANC (Vitek 2) and MALDI-TOF (Vitek MS). Discordant results or those with low discrimination of the species were submitted to 16S rRNA sequencing. The economic impact of the introduction of MTB as well as the direct costs of MALDI-TOF identification were assessed. RESULTS: MTB was composed of amikacin, gentamicin and aztreonam. Of the 159 clinical samples screened by TB and MTB, 11 (7%) were positive for AC with the same species isolated in both media. Using MTB, a reduction of false positives was obtained from 97 (61%) to 69 (43%) when compared to TB (p < 0.05). The TAT of the negative result of the AC with the MTB was reduced from 14 to 7 days in 28 (18%) samples; the MTB allowed the release of the AC positive result 48 hours ahead of the TB. The sensitivity of MTB was equal to TB, but the specificity was higher in 19%. Of the 421 strains evaluated, 35 were identified only by MALDI-TOF (Vitek MS) and one (Clostridium innocum) was identified only by 16S rRNA sequencing. Of the 386 evaluated by both methodologies, there was a concordance of 97% and the results of the 13 (3%) strains submitted to the sequencing were concordant in 92% with the MALDI-TOF (Vitek MS) that promoted TAT of the positive result reduction in five days. The implementation of the MTB made possible a reduction of costs in this sampling, of US $ 677,00 and the identification by MALDI-TOF provided a saving of US $ 2354,00. Considering the economic values found in this study and projecting them in the HCFMUSP AC statistics in 2017, the MTB could provide savings of US $40,070.00 / year and MALDI-TOF a reduction in expenses of US $ 4,100.00 / year. CONCLUSIONS: Standardization and implementation of MTB allowed a significant increase of anaerobic culture specificity with TAT and costs reduction. The use of MALDI-TOF reduced the TAT of the identifications and also resulted in a better performance in a cost effective way

INTRODUÇÃO: As infecções por bactérias anaeróbias são geralmente de origem endógena, polimicrobianas e mistas. Devido a sua natureza fastidiosa, essas bactérias necessitam de uma prévia incubação em meios líquidos enriquecidos, como o caldo Thioglicolato (CT) para serem recuperadas, o isolamento desses microrganismos é trabalhoso e o tempo de resposta - TAT (turn around time) estendido desse exame pode estar associado a falhas terapêuticas e ao aumento da resistência bacteriana. A cultura de anaeróbios (CANA) ainda é um desafio para os laboratórios clínicos de rotina e novas estratégias para diminuir o TAT são fundamentais para que esse exame forneça um impacto clínico significativo. OBJETIVO: Otimizar o processo de triagem da CANA pela modificação do CT; comparar a identificação dos anaeróbios pelas metodologias fenotípicas ANC (Vitek 2- bioMérieux, France) e MALDI-TOF (Vitek MS - bioMérieux, France) e verificar o impacto econômico das ações propostas MÉTODOS: O caldo de triagem CT foi modificado eluindo individualmente discos comerciais de antibióticos (em concentrações fixas) selecionados por apresentarem baixa ou nenhuma ação contra microrganismos anaeróbios e com um bom espectro de ação para os principais aeróbios associados em culturas mistas e foram escolhidos aqueles que após uma bateria de testes frente a 15 cepas dos principais anaeróbios envolvidos em infecções humanas mantiveram a viabilidade inicial. O caldo Thioglicolato modificado (CTM) foi composto pela adição dos antibióticos que apresentaram a melhor \"performance\" acima descrita. A sensibilidade e especificidade do CTM foram avaliadas paralelamente com CT na rotina de CANA do HCFMUSP. Para a avaliar a identificação fenotípica, 421 anaeróbios isolados no período de seis meses foram submetidos a identificação pelo ANC (Vitek 2) e MALDI-TOF (Vitek MS). Os resultados discordantes ou com baixa discriminação da espécie foram avaliados pelo sequenciamento 16S rRNA. O impacto econômico da introdução do CTM bem como os custos diretos da identificação pelo MALDI-TOF foram avaliados. RESULTADOS: O CTM foi composto por amicacina, gentamicina e aztreonam. Das 159 amostras clínicas triadas pelo CT e CTM, 11 (7%) foram positivas para CANA com as mesmas espécies isoladas em ambos os meios. Utilizando o CTM, foi obtida uma redução dos falsos positivos de 97 (61%) para 69 (43%) quando comparado ao CT (p < 0,05). O TAT do resultado negativo da CANA com o CTM foi reduzido de 14 para sete dias em 28 (18%) amostras; o CTM permitiu a liberação do resultado positivo da CANA 48 horas à frente do CT. A sensibilidade do CTM foi igual ao CT, porém a especificidade foi superior em 19%. Das 421 cepas avaliadas, 35 foram identificadas somente pelo MALDI-TOF (Vitek MS) sendo que uma (Clostridium innocum) foi identificada somente pelo sequenciamento 16S rRNA. Das 386 avaliadas por ambas as metodologias, houve uma concordância de 97% e os resultados das 13 (3%) cepas submetidas ao sequenciamento foram concordantes em 92% com o MALDI-TOF (Vitek MS) que promoveu a redução do TAT do resultado positivo em cinco dias. A implementação do CTM possibilitou uma redução de custos nessa amostragem, de R$ 2.240,00 e a identificação pelo MALDI-TOF proporcionou uma economia de R$ 7.786,00. Considerando os valores econômicos encontrados nesse estudo e projetando-os nas estatísticas de CANA do HCFMUSP em 2017, o CTM poderia proporcionar uma economia de R$ 132.560,00 /ano e o MALDI-TOF uma redução nos gastos de R$ 13.579,00/ ano CONCLUSÕES: A padronização e implementação do CTM permitiu uma um aumento significativo de especificidade da cultura anaeróbia com redução do TAT e dos custos. A utilização do MALDI-TOF diminuiu o TAT das identificações aliado a uma melhor performance de forma custo efetiva / INTRODUCTION: Anaerobic bacterial infections are usually of endogenous origin, polymicrobial and mixed. Because of their fastidious nature, these bacteria require prior incubation in enriched liquid media, such as Thioglycolate broth (TB) to be recovered, the isolation of these microorganisms is laborious, and the TAT (turn around time) extended time of this examination may be associated with therapeutic failures and increased bacterial resistance. Anaerobic culture (AC) is still a challenge for routine clinical laboratories, and new strategies for lowering TAT are critical to provide a significant clinical impact. OBJECTIVE: To optimize the AC screening process by modifying the TB; Compare anaerobical identification between (Vitek 2- bioMérieux, France) and MALDI-TOF (Vitek MS - bioMérieux, France) and to verify the economic impact of the proposed actions. METHODS: TB broth was modified by eluting individually antibiotic commercial discs (at fixed concentrations) selected for low or no action against anaerobic microorganisms and with a good action spectrum for the main associated aerobes in mixed cultures. Those who maintained the initial viability after a battery of tests against 15 strains of the major anaerobes involved in human infections were selected. Modified Thioglycolate Broth (MTB) was composed of the antibiotics that presented the best performance described above. The sensitivity and specificity of MTB were evaluated in parallel with TB in the HCFMUSP AC routine. To evaluate the phenotypic identification, 421 anaerobes isolated in the six-month period were submitted to identification by ANC (Vitek 2) and MALDI-TOF (Vitek MS). Discordant results or those with low discrimination of the species were submitted to 16S rRNA sequencing. The economic impact of the introduction of MTB as well as the direct costs of MALDI-TOF identification were assessed. RESULTS: MTB was composed of amikacin, gentamicin and aztreonam. Of the 159 clinical samples screened by TB and MTB, 11 (7%) were positive for AC with the same species isolated in both media. Using MTB, a reduction of false positives was obtained from 97 (61%) to 69 (43%) when compared to TB (p < 0.05). The TAT of the negative result of the AC with the MTB was reduced from 14 to 7 days in 28 (18%) samples; the MTB allowed the release of the AC positive result 48 hours ahead of the TB. The sensitivity of MTB was equal to TB, but the specificity was higher in 19%. Of the 421 strains evaluated, 35 were identified only by MALDI-TOF (Vitek MS) and one (Clostridium innocum) was identified only by 16S rRNA sequencing. Of the 386 evaluated by both methodologies, there was a concordance of 97% and the results of the 13 (3%) strains submitted to the sequencing were concordant in 92% with the MALDI-TOF (Vitek MS) that promoted TAT of the positive result reduction in five days. The implementation of the MTB made possible a reduction of costs in this sampling, of US $ 677,00 and the identification by MALDI-TOF provided a saving of US $ 2354,00. Considering the economic values found in this study and projecting them in the HCFMUSP AC statistics in 2017, the MTB could provide savings of US $40,070.00 / year and MALDI-TOF a reduction in expenses of US $ 4,100.00 / year. CONCLUSIONS: Standardization and implementation of MTB allowed a significant increase of anaerobic culture specificity with TAT and costs reduction. The use of MALDI-TOF reduced the TAT of the identifications and also resulted in a better performance in a cost effective way