Yeasts of genus Cryptococcus belong to the Basidiomycetes, a wide group with different geographical sharing in nature. Many of them rank among human pathogens that endanger immunocompromised patients. Thanks to the big diversity at the level of subspecies, various species of the genus Cryptococcus show different molecular characteristics. Their identification is difficult because of the presence of polysaccharide capsule that surrounds the cell of some species. This work deals with the identification of species occurring in meadows and gardens. The 79 yeasts samples are identified by MALDI-TOF MS. The influence of different culture media on the identification results is monitored simultaneously with the identification. Since a capsule is present in many species, another parameter to be monitored is the influence of the sample preparation method and the matrix on the identification. 51 samples of yeasts of the genus Cryptococcus are identified at the species level in the experimental part. Selected samples are further subjected to the determination of characteristics by conventional microbiological methods. The determined parameters are the presence of urease, radial growth constant, susceptibility to antimycotics, fermentation and assimilation of sugars, growth in the presence of alcohols and growth in the absence of vitamins. The yeast samples are classified into yeast species based on microbiological results. Biotyping resulted in identification of selected samples in the species Filobasidium magnum, Filobasidium oeirense and Filobasidium wieringae. Other samples showed ambiguous identification. As these species have the same properties, they could not be distinguished by the selected microbiological tests.

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“

Les moustiques sont les principaux vecteurs incriminés dans la transmission d’agents pathogènes à l’homme. L’identification précise des espèces de moustiques est importante pour distinguer les espèces vectrices des non vectrices. La détermination de l’origine du repas sanguin des moustiques vecteurs est indispensable dans la compréhension du comportement des espèces vectrices. Nous avons mise à jour la littérature actuelle sur la faune Culicidienne du Mali. Ainsi, nous avons listé 106 espèces de moustiques actuellement enregistrée au Mali dont 28 Anophelinae et 78 Culicinae. Nous avons ensuite évalué l’efficacité du MALDI-TOF MS à identifier des moustiques collectés au Mali et déterminer leur source de repas sanguin. Nous avons confirmé la robustesse du MALDI-TOF MS à identifier un grand nombre de sang d’animaux. Nous avons artificiellement gorgé des femelles de An. gambiae et An. coluzzii sur différents types de sang d’animaux. Nous avons obtenu 100% d'identification correcte du repas de sang pour les spécimens collectés 1h à 24h après le gorgement. Ensuite nous avons expérimentalement gorgés An. gambiae, An. coluzzii et Ae. albopictus sur des repas de sang successif et mixte par MALDI-TOF MS. Nos résultats révèlent que le MALDI-TOF MS est tout à fait capable d’identifier le repas mixte. Mais en ce qui concerne le repas successif seul le dernier repas de sang est identifié. Enfin nous avons utilisé la culturomique et le MALDI-TOF pour l’étude du microbiote digestif de moustiques collectés sur le terrain au Mali et à Marseille. Cette approche a révélé une grande diversité du microbiote digestif des moustiques An. gambiae, Ae. albopictus et Cx. quinquefasciatus. / Mosquitoes are the main vectors involved in the transmission of pathogens to humans. Accurate identification of mosquito species is crucial to distinguish between vector and non-vector species. The mosquito blood meal determination is fundamental in understanding the behavior of vector species. Thus, we have listed 106 mosquito species currently recorded in Mali, including 28 Anophelinae and 78 Culicinae. Then, we evaluated the effectiveness of MALDI-TOF MS for identified mosquitoes collected in Mali and to determine their blood meal source. The results obtained show the ability of MALDI-TOF MS to identify mosquitoes collected in Mali and their source of blood meal. Subsequently, we were able to confirm the robustness of MALDI-TOF MS to identify other animal blood samples. We artificially engorged Anopheles gambiae and Anopheles coluzzii on eight animal bloods samples. We obtained 100% correct identification of the blood source for samples taken 1 to 24 hours after feeding. Then, we experimentally engorged An. gambiae, An. coluzzii and Ae. albopictus on successive and mixed blood meals using MALDI-TOF MS. The results revealed that MALDI-TOF MS is able to identify mixed blood meals. In addition we used MALDI-TOF and culturomics for the microbiota study of the mosquito collected in the field, notably in Marseille and Mali. The culturomics approach revealed a great diversity of the digestive microbiota of the An. gambiae, Ae. albopictus and Cx. quinquefasciatus mosquitoes.

Les maladies transmises par les arthropodes vecteurs sont responsables de centaines de milliers de cas d’infections humaines et de décès chaque année à travers le monde. Ces maladies, causées par des bactéries, virus ou parasites, parfois émergents ou réémergents, sont parfois peu connues ou sous-estimées. Les arthropodes peuvent être utilisés comme outil de suivi épidémiologique des micro-organismes qui leur sont associés et dont certains pourront être transmis à des hôtes vertébrés. L’identification des arthropodes reste cruciale dans les enquêtes entomologiques.Nous avons pu ainsi détecter de potentielles nouvelles bactéries dans les tiques de Tahiti et les triatomes de Guyane.Nous avons exploré le microbiote salivaire de près de mille moustiques de 3 pays différents par métagénomique 16S. Nous avons ainsi détecté un large nombre de bactéries pathogènes opportunistes mais aussi un très grand nombre de génotypes correspondant probablement à des espèces et genres bactériens nouveaux. Enfin notre axe majeur a été le développement de l’utilisation de la spectrométrie de masse MALDI-TOF en entomologie médicale. Pour pallier les limites des méthodes de référence d’identification des arthropodes existantes, nous avons validé l’utilisation de cet outil pour l’identification des moustiques (collectés sur terrain en Australie) et de puces (Espagne, Corse, Algérie). Nous avons également mis au point son utilisation pour l’identification de nouvelles familles d’arthropodes, comme les punaises de lits et les triatomes. Nous avons pu mettre en évidence, la capacité de la spectrométrie de masse MALDI-TOF pour différencier les anophèles infectés ou non par des plasmodies. / Vector-borne diseases are responsible for hundreds of thousands of cases of human infections and deaths each year worldwide. Generally, little is known about these diseases, caused by bacteria, viruses or parasites, sometimes emerging or re-emerging. Arthropods can be used as a tool for epidemiological monitoring of their associated microorganisms, some of which being able to be transmitted to vertebrate hosts. The identification of arthropods remains crucial in entomological investigations.We were able to detect potential new bacteria in ticks from Tahiti and triatomines in French Guiana.We explored the salivary microbiota of nearly a thousand mosquitoes from 3 different countries by 16S rRNA metagenomics. We have thus detected a large number of opportunistic pathogenic bacteria but also a very large number of genotypes probably corresponding to new bacterial species and genera. Finally, our major focus has been the development of the use of MALDI-TOF mass spectrometry in medical entomology. To overcome the limitations of existing arthropod identification reference methods, we validated the use of this tool for the identification of mosquitoes (collected in the field in Australia) and fleas (Spain, Corsica, Algeria). We have also developed its use for the identification of new families of arthropods, such as bed bugs (Cimicidae) and triatomines (Reduviidae). We were able to highlight the capacity of MALDI-TOF mass spectrometry to differentiate between anopheles infected or not by malaria parasites.

Ce travail est composé de 3 parties. La première est une étude épidémiologique avec la détection moléculaire des spécimens appartenant à six espèces d’Argasidés collectées en Algérie et identifiées morphologiquement et par biologie moléculaire. Nous avons pu détecter Borrelia hispanica dans des Ornithodoros occidentalis et Borrelia cf turicatae dans des Carios Carpensis. Dans des Argas persicus nous avons pu identifier un nouveau génotype de Bartonella spp ainsi qu’un génotype appartenant à une nouvelle espèce dans la famille des Anaplasmataceae. Dans la 2e partie, nous avons évalué la capacité vectorielle des punaises de lit à transmettre Borrelia recurrentis, l’agent de la fièvre récurrente. Pour ce fait, nous avons utilisé un modèle expérimental d’infection artificielle de Cimex lectularius par B. recurrentis pour ensuite détecter la présence de la bactérie dans les fèces. Nous avons utilisé quatre approches : la détection par qPCR, la culture à partir des fèces, la FDA (Fluorescein Diacetate) et l’inoculation des fèces aux souris. Nous avons également utilisé l’Immunofluorescence pour localiser la bactérie dans le corps de la punaise. Nous avons constaté que les punaises de lit acquièrent la bactérie et excrètent des microorganismes vivants dans les fèces. Elles peuvent être considérées comme vecteur potentiel de Borrelia recurrentis. La troisième partie s’intéresse à l’évaluation de la capacité du MALDI-TOF MS à identifier les puces, les punaises et les pathogènes associés. / This work focuses on three main parts, a first part presents an epidemiological study of bacteria associated with soft ticks in Algeria, or we identified morphologically and confirmed by molecular biology six species of Argasidae. In addition, looking further we could detect Borrelia hispanica in Ornithodoros occidentalis and Borrelia cf turicatae in Carios Carpensis. On the other hand, in Argas persicus a new genotype of Bartonella spp has been identified as well as a new species of Anaplasmatacea bacteria.A second part evaluates the vectorial capacity of bed bugs to transmit Borrelia recurrentis, the agent of the relapsing fever. For this reason an experimental model of artificial infection of Cimex lectularius by Borrelia recurrentis has been developed, to study the presence of bacteria in feces. In this model, four approaches were used: qPCR, fece’s culture, FDA (Fluorescein Diacetate) and fece’s inoculation to mice. Immunofluorescence was also used to detect the location of the bacteria in the body of the bed bug. We confirmed that bed bugs acquire the bacteria and excrete live microorganisms in the feces. They can be considered as potential vector of Borrelia recurrentis.The third part is an assessment of the capacity of MALDI-TOF MS to identified fleas, bed bugs and associated pathogens. This innovative tool, which has revolutionized medical entomology and has shown its efficiency to identify several species of arthropods, has also been able to distinguish between infected and uninfected fleas and bugs, and even distinguish between fleas and bugs infected by the same species of bacteria.

Métodos microbiológicos tradicionais como isolamento, coloração de Gram e testes bioquímicos auxiliam na identificação do gênero Streptococcus, no entanto, as espécies apresentam ampla variação fenotípica, tornando difícil a identificação ou diferenciação das mesmas apenas por estes métodos. Uma das espécies mais importantes em suínos, Streptococcus suis, tem provocado grandes prejuízos em todo o mundo e tem sido descrito como uma importante zoonose em alguns países. S. suis está presente nas vias respiratórias superiores, colonizando principalmente tonsilas, cavidades oral e nasal facilitando a alta disseminação por contato direto, principalmente em leitões entre 4 e 12 semanas de vida. Os quadros clínicos mais frequentes em suínos infectados pelo S. suis são meningite, artrite e pneumonia. O objetivo do presente estudo foi identificar estirpes do gênero Streptococcus mediante as técnicas de reação em cadeia pela polimerase (PCR), sequenciamento parcial do gene 16S rRNA e espectrometria de massa MALDI-TOF (MALDI-TOF MS). As análises por PCR e por MALDI-TOF MS resultaram na identificação de 215 estirpes como S. suis e 35 como diferentes espécies pertencentes ao gênero Streptococcus. Os resultados da identificação das 35 estirpes pertencentes a outras espécies do gênero Streptococcus pelo MALDI-TOF MS foram confirmados pelo sequenciamento parcial do gene 16S rRNA, sendo que as duas técnicas apresentaram 100% de concordância. Os resultados obtidos indicam grande eficácia na utilização das técnicas avaliadas para a identificação de S suis e de outras espécies do gênero Streptococcus. A técnica de MALDI-TOF MS, apesar do custo elevado do equipamento, apresentou a vantagem de ser rápida, apresentar baixo custo por análise e reduzida utilização de material / Traditional microbiological methods such as isolation, Gram staining and biochemical tests help to identify the Streptococcus genus, however, the species present broad phenotypic variation, making it difficult for their identification or even differentiation just by these methods. One of the most important species in swine, Streptococcus suis, has led to great losses worldwide and has been described as an important zoonosis in some countries. S. suis is present in the upper airways, especially colonizing tonsils, oral and nasal cavities facilitating the high dissemination by direct contact, especially among piglets between 4 to 12 weeks of age. The most common clinical manifestations in pigs infected by S. suis are meningitis, arthritis and pneumonia. The aim of this study was to identify Streptococcus strains by polymerase chain reaction (PCR), 16S rRNA gene partial sequencing and MALDI-TOF mass spectrometry (MALDI-TOF MS). PCR and MALDI-TOF MS analysis resulted in the identification of 215 strains as S. suis and 35 as different species of the Streptococcus genus. The identification of the 35 strains belonging to other species of the genus by MALDI-TOF MS was confirmed by 16S rRNA gene partial sequencing, and both techniques presented 100% concordance. These results demonstrate the high efficiency in the use of the evaluated techniques for the identification of S. suis and the other species of the Streptococcus genus. The MALDI-TOF MS technique, despite the equipment high cost, presented the advantage of being fast, have low cost per analysis and reduced material usage

O objetivo deste estudo foi avaliar a técnica de espectrometria de massas por ionização/dessorção a laser assistida por matriz tempo-de-vôo (MALDI-TOF) para a identificação direta em amostras de leite (sem cultivo microbiológico) de bactérias causadoras de mastite. Para tanto foram realizados dois experimentos (1 e 2). No experimento 1, o objetivo foi determinar a sensibilidade diagnóstica da técnica MALDI-TOF MS para a identificação direta em amostras de leite de Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae e Escherichia coli. Foram realizadas contaminações experimentais de S. aureus, S. uberis, S. agalactiae, S. dysgalactiae e E. coli em amostras de leite, para a obtenção de contagens de 103 a 109 ufc/mL. As amostras de leite contaminadas foram processadas com o uso do kit Maldi Sepsityper® (Bruker Daltonics) e submetidas ao protocolo de lise bacteriana para posterior análise por MALDI-TOF MS. Espectros de massas foram coletados na faixa de massas de 2.000-20.000 m/z e foram analisados pelo programa MALDI Biotyper 3.0 (Bruker Daltonics) com as configurações padrão para obtenção da identificação bacteriana. A identificação direta de patógenos causadores de mastite a partir de amostras de leite foi possível em contagem ≥106 ufc/mL para S. aureus, ≥107 ufc/mL para E. coli, e ≥108 ufc/mL para S. agalactiae, S. dysgalactiae e S. uberis. No experimento 2, o objetivo foi avaliar o efeito da pré-incubação de amostras de leite de quartos mamários com mastite subclínica sobre a eficácia da identificação sem cultivo de patógenos causadores de mastite por MALDI-TOF MS. Foram selecionados 2 rebanhos leiteiros para coletas de leite de quartos mamários de todas as vacas em lactação. As amostras de leite foram submetidas às análises de: a) cultura microbiológica; b) pré-incubação seguida de identificação por espectrometria de massas diretamente do leite; c) contagem bacteriana total (CBT). Para a realização da CBT, as amostras de leite foram submetidas à citometria de fluxo; e para a identificação direta de patógenos causadores de mastite a partir do leite, as amostras foram submetidas ao desnate por centrifugação (10.000 Xg por 10 minutos), e pré-incubação a 37ºC por 12 horas. Posteriormente, as amostras foram processadas com o uso do kit Maldi Sepsityper® (Bruker Daltonics) e submetidas ao protocolo de lise bacteriana para posterior análise por MALDI-TOF MS. Do total de 810 amostras de leite analisadas por cultura microbiológica (método referência), 347 apresentaram crescimento bacteriano, sendo 305 identificadas como agentes de interesse na identificação direta pelo método MALDI-TOF MS: Staphylococcus coagulase negativa (n=191), S. aureus (n=31), S. agalactiae (n=42), S. uberis (n=37), S. dysgalactiae (n= 4). Sendo assim, 305 amostras foram analisadas pelo método de idenfificação direta MALDI-TOF MS, a qual apresentou baixa sensibilidade quando comparado com a cultura microbiológica (método referência): Staphylococcus coagulase negativa (14,08%), S. agalactiae (15,25%), S. uberis (1,69%) S. aureus (6,12%) e S. dysgalactiae (0%). A pré-incubação de amostras de leite não aumentou a sensibilidade de identificação direta de microrganismos causadores de mastite pelo método MALDI-TOF MS. / The purpose of the present study was to evaluate the technique of mass spectrometry by desorption / ionization assisted laser array time-of-flight (MALDI-TOF MS) for the direct identification in milk samples (no microbiological culture) of mastitis causing bacteria. Therefore, we carried out two experiments (1 and 2). In experiment 1, we determined the diagnostic sensitivity of MALDI-TOF MS technique for the direct identification in milk samples of Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae and Escherichia coli. Experimental contamination of S. aureus, S. uberis, S. agalactiae, S. dysgalactiae and E. coli in samples of milk to a concentration of 103-109 cfu/mL were performed. The contaminated milk samples were processed using kit Maldi Sepsityper® (Bruker Daltonics) and subjected to bacterial lysis protocol for analysis by MALDI-TOF MS. Mass spectra were collected in the mass range of 2000-20000 m/z and were analyzed by MALDI Biotyper 3.0 software (Bruker Daltonics) with default settings to obtain bacterial identification. Direct identification of mastitis causing pathogens from milk samples was possible at ≥106 cfu/mL for S. aureus, ≥107 cfu/mL for E. coli and ≥108 cfu/mL for S. agalactiae, S. dysgalactiae and S. uberis. In experiment 2, the objective was to evaluate the effect of pre-incubation of milk samples from mammary quarters with subclinical mastitis on the effectiveness of identification without cultivation, of mastitis-causing pathogens by MALDI-TOF MS. We selected mammary quarter milk samples from all lactating cows on two dairy herds. The milk samples were subjected to analyzes of: a) microbiological culture; b) pre-incubation followed by identification by mass spectrometry directly from milk; c) total bacterial count (TBC). Flow cytometry was used to determine TBC and; to directly identify the mastitis-causing pathogens from milk, fat was separated by centrifugation (10,000 Xg for 10 minutes) and; samples were pre-incubated at 37°C for 12 hours. Subsequently, the skim milk samples were submitted to kit Maldi Sepsityper® (Bruker Daltonics) and to the bacterial lysis protocol for analysis by MALDI-TOF MS. A total of 810 milk samples were analyzed by microbiological culture (reference method), of which 347 showed bacterial growth. Considering all culture positive samples 305 were identified as agents of interest in the direct identification by MALDI-TOF MS method: coagulase negative staphylococci (n = 191), S. aureus (n = 31), S. agalactiae (n = 42), S. uberis (n = 37) and S. dysgalactiae (n = 4). Therefore, 305 samples were directly identified by MALDI-TOF MS, which presented low sensitivity when compared to microbiological culture (Reference method): coagulase-negative staphylococci (14.08%), S. agalactiae (15.25 %), S. uberis (1.69%), S. aureus (6.12%) and S. dysgalactiae (0%). Pre-incubation of milk samples did not increase the sensitivity of the MALDI-TOF MS method directly identify mastitis-causing microorganisms.

Escherichia fergusonii é um patógeno emergente na medicina humana e veterinária, nos relatos de casos de infecção em aves são escassos. O objetivo desse trabalho foi identificar e caracterizar estirpes de E. fergusonii isoladas de anatídeos de vida livre, aves cativas silvestres e aves comerciais criadas em sistemas convencionais e orgânico. Dentre as 431 amostras, foram isoladas 29 estirpes de Escherichia fergusonii, sendo a maior prevalência em galinhas no sistema de criação convencional. O perfil de sensibilidade das amostras foi avaliado, revelando diferenças entre os grupos, com a presença de estirpes de frangos no sistema de criação convencional apresentando maiores índices de resistência aos antimicrobianos testados, enquanto estirpes de irerês apresentaram sensibilidade a todos os antibióticos. Foram identificadas duas estirpes multirresistentes com resistência a mais de três classes de antimicrobianos. A caracterização molecular dessas amostras revelou estirpes de irerês (Dendrocygna viduata) com maior presença de genes de virulência (iroN, cvi, cva, astA e ompT). O ensaio de produção de hemolisinas em ágar sangue revelou amostras negativas, sem formação de hemólise ao redor da colônia. A técnica de AFLP classificou as estirpes em clados distintos sugerindo padrões de heterogeneidade entre as amostras. Esse é o primeiro relato de achados de Escherichia fergusonii em irerês de vida livre e gavião-pombo pequeno. / Escherichia fergusonii is an emerging pathogen in human and veterinary medicine, in cases reports associated with birds are scarce. The aims of this work were to identify and to characterize strains of E. fergusonii isolated from free-living ants, wild captive birds and poultry from conventional and organic farms. Among the 431 samples, 29 Escherichia fergusonii were isolated, being the highest prevalence in chickens from conventional breeding systems. The sensitivity profile of the samples was evaluated, revealing differences between the groups, with the higher antimicrobial resistance indexes in isolates from chicken in the conventional farms, while strains of white-faced whistling-duck showed sensitivity to all antibiotics. Two multiresistant strains with resistance to more than three classes of antimicrobial agents were identified. The molecular characterization of these samples revealed strains of white-faced whistlingduck (Dendrocygna viduata) with higher presence of virulence genes (iroN, cvi, cva, astA and ompT). The assay of hemolysin production on blood agar revealed negative strains, with no hemolysis forming around the colony. The AFLP technique classified the strains into distinct clades suggesting patterns of heterogeneity between the strains. This is the first report of Escherichia fergusonii in free-living white-faced whistling duck and white-necked hawk.

Métodos microbiológicos tradicionais como isolamento, coloração de Gram e testes bioquímicos auxiliam na identificação do gênero Streptococcus, no entanto, as espécies apresentam ampla variação fenotípica, tornando difícil a identificação ou diferenciação das mesmas apenas por estes métodos. Uma das espécies mais importantes em suínos, Streptococcus suis, tem provocado grandes prejuízos em todo o mundo e tem sido descrito como uma importante zoonose em alguns países. S. suis está presente nas vias respiratórias superiores, colonizando principalmente tonsilas, cavidades oral e nasal facilitando a alta disseminação por contato direto, principalmente em leitões entre 4 e 12 semanas de vida. Os quadros clínicos mais frequentes em suínos infectados pelo S. suis são meningite, artrite e pneumonia. O objetivo do presente estudo foi identificar estirpes do gênero Streptococcus mediante as técnicas de reação em cadeia pela polimerase (PCR), sequenciamento parcial do gene 16S rRNA e espectrometria de massa MALDI-TOF (MALDI-TOF MS). As análises por PCR e por MALDI-TOF MS resultaram na identificação de 215 estirpes como S. suis e 35 como diferentes espécies pertencentes ao gênero Streptococcus. Os resultados da identificação das 35 estirpes pertencentes a outras espécies do gênero Streptococcus pelo MALDI-TOF MS foram confirmados pelo sequenciamento parcial do gene 16S rRNA, sendo que as duas técnicas apresentaram 100% de concordância. Os resultados obtidos indicam grande eficácia na utilização das técnicas avaliadas para a identificação de S suis e de outras espécies do gênero Streptococcus. A técnica de MALDI-TOF MS, apesar do custo elevado do equipamento, apresentou a vantagem de ser rápida, apresentar baixo custo por análise e reduzida utilização de material / Traditional microbiological methods such as isolation, Gram staining and biochemical tests help to identify the Streptococcus genus, however, the species present broad phenotypic variation, making it difficult for their identification or even differentiation just by these methods. One of the most important species in swine, Streptococcus suis, has led to great losses worldwide and has been described as an important zoonosis in some countries. S. suis is present in the upper airways, especially colonizing tonsils, oral and nasal cavities facilitating the high dissemination by direct contact, especially among piglets between 4 to 12 weeks of age. The most common clinical manifestations in pigs infected by S. suis are meningitis, arthritis and pneumonia. The aim of this study was to identify Streptococcus strains by polymerase chain reaction (PCR), 16S rRNA gene partial sequencing and MALDI-TOF mass spectrometry (MALDI-TOF MS). PCR and MALDI-TOF MS analysis resulted in the identification of 215 strains as S. suis and 35 as different species of the Streptococcus genus. The identification of the 35 strains belonging to other species of the genus by MALDI-TOF MS was confirmed by 16S rRNA gene partial sequencing, and both techniques presented 100% concordance. These results demonstrate the high efficiency in the use of the evaluated techniques for the identification of S. suis and the other species of the Streptococcus genus. The MALDI-TOF MS technique, despite the equipment high cost, presented the advantage of being fast, have low cost per analysis and reduced material usage

Submitted by Leticia Schettini (leticia@ufrrj.br) on 2016-09-20T14:14:37Z No. of bitstreams: 1 2016 - NAIARA DE MIRANDA BENTO RODRIGUES.pdf: 1508318 bytes, checksum: bbf2aebb5d43033339bf559d73461919 (MD5) / Made available in DSpace on 2016-09-20T14:14:37Z (GMT). No. of bitstreams: 1 2016 - NAIARA DE MIRANDA BENTO RODRIGUES.pdf: 1508318 bytes, checksum: bbf2aebb5d43033339bf559d73461919 (MD5) Previous issue date: 2016-02-26 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPQ / Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. These microorganisms are preferentially found in the habitat of animals in places contaminated with feces, urine, clay and also organic beds. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out and they can delay the antibiotic therapy by clinic veterinary. On the other hand the MALDI-TOF MS technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n=47) and fecal samples (n=94) collected from cows; also water (n=23) and milk line samples (n=19) collected from a farm in Rio de Janeiro with the purpose to present the MALDI-TOF MS technique as efficient methodology and also as a ?gold standard? to better understand the possible current biochemical errors in enterobacteria identification considering isolates from bovine environments. This proteomic technique confirmed 92.9% (170/183) of the enterobacteria species identified by biochemical tests that showed high sensitivity (> 81%) and specificity (> 89%). The gyrB sequencing was made in eigth from thirteen misidentified enterobacteria and confirmed 100% the MALDI-TOF results, so the proteomic technique was used as a ?gold standard? for this study. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp was the largest misidentified genus (76.9%, 10/13). E.coli was prevalent (83%, 152/183) in all samples and the bovine milk presented the most enterobacteria diversity. The Salmonella sp wasn?t detected in feces bovine samples and all water samples from different points in the farm presented unacceptable microbiological standards. Was identified enterobacteria in milkers hands and nasal cavity also in the milking machines used on the property. These results aim to contribute significantly to the characterization of the Enterobacteriaceae as well in understanding of its spread in dairy production environment , assisting in need diagnostic of possible agents involved in bovine mastitis as well as to implement properly targeted prophylactic measures. / A mastite bovina afeta negativamente a produ??o de leite dificultando a recupera??o dos n?veis de produ??o total das propriedades leiteiras, levando a perdas econ?micas consider?veis. Esta redu??o no percentual da produ??o de leite pode estar associada ao agente patog?nico espec?fico que causou a infec??o, sendo as enterobact?rias frequentemente respons?veis pela mastite ambiental. Estes microrganismos s?o preferencialmente encontrados no habitat normal dos animais como locais que apresentam esterco, urina, barro e camas org?nicas. Os testes fenot?picos est?o entre os m?todos dispon?veis atualmente utilizados para identificar as enterobact?rias; no entanto, eles podem ocasionalmente identitificar erroneamente algumas esp?cies apesar dos m?ltiplos ensaios realizados. Al?m disso, a demora na sua execu??o pode tardar a antibioticoterapia realizada em campo. Por outro lado, a t?cnica de MALDI-TOF MS tem atra?do a aten??o pela sua identifica??o precisa dos v?rios microorganismos em n?vel de esp?cie. No presente estudo, um total de 183 enterobact?rias foram isoladas a partir de amostras de leite (n=47) e fezes colhidas de vacas em lacta??o (n=94); amostras de ?gua (n=23) e na linha de ordenha (n=19) em uma propriedade situada no Rio de Janeiro. A proposta foi utilizar a t?cnica de MALDI-TOF MS como um m?todo eficaz de identifica??o bacteriana de enterobact?rias e descrever a permanencia destes microrganismos no ambiente de produ??o leiteira. A t?cnica prote?mica confirmou 92,9% (170/183) das esp?cies de enterobact?rias identificadas pelos testes bioqu?micos convencionais. O sequenciamento do gene gyrB, realizado em oito das 13 enterobact?rias que apresentaram identifica??o discordante, confirmou em 100% o resultado da t?cnica prote?mica, que foi utilizada como metodologia de refer?ncia no presente estudo. O g?nero Enterobacter foi o mais discordante pelo m?todo bioqu?mico (76,9%, 9/13). A E.coli foi a esp?cie predominante (83%, 152/183) em todas as amostras avaliadas, sendo que o leite bovino apresentou maior diversidade de enterobact?rias. N?o foi detectada a presen?a de Salmonella spp. nas amostras de fezes bovinas e todas as amostras de ?gua dos diferentes pontos de coleta da propriedade apresentaram padr?es microbiol?gicos inaceit?veis. Foram isoladas enterobact?rias das m?os e cavidades nasal dos ordenhadores, bem como nas ordenhadeiras mec?nicas utilizadas na propriedade. Estes dados visam contribuir de forma significativa para a caracteriza??o das enterobacterias bem como para a compreens?o e sua descri??o no ambiente de produ??o leiteira, auxiliando no diagn?stico preciso dos poss?veis agentes envolvidos na mastite bovina bem como na implementa??o de medidas profil?ticas devidamente direcionadas.