Projektet bestod i att ge underlag för lämpliga och användbara lösningar för effektivisering av Kockums Maskins skärvätskehantering och kostnadsrelaterade faktorer gällande företagets skärvätska. En grundlig undersökning av lämpligt material inom projektets huvudsakliga område gjordes till en början för att därefter resultera i en vidare analys av företagets nuvarande system och hantering av skärvätskor. Utifrån denna analys påbörjades en vidare undersökning om möjliga förslag om förbättring gällande de berörda problemen. Genom direktkontakt med flertalet företag och personer inom hantering/inköp samt befintlig dokumentation gällande skärvätska, erhölls ett flertal lösningar och förbättringsförslag som senare utvärderades och användes vid framtagning av det sökta projektresultatet. Problematiken gällande valet av lösningar har att göra med det faktum att det krävs omfattande kompetens inom varje typ av lösningsgrupp för att uppfylla de krav och förhållanden som finns hos Kockums Maskin. Därmed har avgränsningar gjorts gällande direkta val av lösningar för att istället ge förslag på lämpliga lösningar gällande förbättring av kvalitet, som indirekt bör sänka den årliga kostnaden av skärvätska, utifrån Kockums Maskins egentliga behov. / The project was to provide the basis for appropriate and useful solutions for any eventually streamlining of Kockums Maskin's cutting fluid management. Cost-related factors regarding the company's cutting fluid are also included in this part. A detailed examination of suitable material within the project's main area were initially and subsequently result in a further analysis of the company's existing systems and management, regarding cutting fluids. Based on this analysis a further investigation on possible proposals was started for improvement regarding the issues involved with the solutions and the cutting fluids. Through direct contact with many companies and individuals in management/purchasing and existing documentation regarding cutting fluid, obtained a several solutions and suggestions for improvement regarding the searched area. This result was then later evaluated and used in the development of the project results. The problem regarding the choice of solutions has to do with the fact that it requires extensive expertise in every type of area to meet the requirements and conditions that Kockums Maskin demands. Thus, the boundaries claimed the election of solutions to instead provide suggestions for suitable solutions regarding the improvement of quality, which indirectly should lower the annual cost of cutting fluid, based on Kockums Maskin's actual needs.

The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication. The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication.

Le suivi de la viabilitié, la croissance et le métabolisme cellulaire des bactéries peut contribuer de manière significative au diagnostic précoce de la maladie, mais peut aussi aider à améliorer le rendement des produits bactériens dans des expériences industrielle ou à petite echelle. Les méthodes conventionnelles utilisées pour l'étude de la sensibilité des bactéries aux antibiotiques sont basées principalement sur la culture, une technique qui prend au moins 12 heures pour rendre un résultat. Ce retard conduit au surtraitement d'un large éventail d'infections par des antibiotiques à large spectre, ce qui est coûteux et peut conduire à l'apparition de résistance à ces antibiotiques précieux, tandis que la détection rapide d'une infection virale ou l'absence de bactéries pourrait prévenir de tels traitements et, dans le cas d'une infection bactérienne, l'identification de la sensibilité aux antibiotiques pourrait permettre l'utilisation d'antibiotiques à spectre étroit. Le projet décrit dans le présent document vise à surveiller les activités biologiques des bactéries vivantes immobilisées sur les surfaces biofonctionnalisées de microstructures composées de semi-conducteurs quantiques (QS). Le procédé dépend de la sensibilité de la photoluminescence (PL) émise par des semi-conducteurs à la perturbation du champ électrique induit par la charge électrique des bactéries immobilisées sur la surface de ces structures. Dans la première phase du projet, nous avons étudié une méthode innovante impliquant la surveillance par PL de l'effet de photocorrosion dans des hétérostructures GaAs/AlGaAs. Le maintien d'un équilibre entre la sensibilité et la stabilité du biocapteur dans l'environnement aqueux nous a permis de détecter Escherichia coli K12 dans des solutions salines tamponnées au phosphate (PBS) avec une limite de détection attrayante de 103 UFC/ml en moins de 2 heures. Suite à cette recherche, nous avons émis l'hypothèse que ces hétérostructures pourraient être utilisés pour développer une méthode à faible coût et quasiment en temps reel de la croissance et de la sensibilité des bactéries aux antibiotiques. L'un des éléments clés dans le développement de cette plate-forme de biocapteurs était de démontrer que le GaAs (001), normalement utilisé pour recouvrir les hétérostructures de GaAs/AlGaAs, ne nuira pas à la croissance des bactéries. Dans la deuxième phase du projet, nous avons exploré la capture et la croissance de E. coli K12 sur des surfaces nues et biofonctionnalisées de GaAs (001). Il a été déterminé que la couverture initiale et les taux de croissance de bactéries dépendent de l'architecture de biofonctionnalisation utilisée pour capturer les bactéries: les surfaces biofonctionnalisées avec d'anticorps présentaient une efficacité de capture significativement plus élevée. En outre, on a trouvé que pour des suspensions contenant des bactéries à moins de 105 UFC/ml, la surface des plaquettes de GaAs ne supportait pas la croissance des bactéries, quel que soit le type d'architecture de biofonctionnalisation. Dans la troisième phase du projet, nous avons suivi la croissance et la sensibilité aux antibiotiques de E. coli K12 et E. coli HB101. Tandis que la présence de bactéries retardaient d’apparition du maximum de PL, la croissance des bactéries retardaient encore plus ce maximum. Par contre, en presence d’antibiotiques efficaces, la croissance des bactéries était arrêtée et le maximum de PL est arrivé plus tôt. Ainsi, nous avons pu distinguer entre des E. coli sensibles ou résistantes à la pénicilline ou à la ciprofloxacine en moins de 3h. En raison de la petite taille, du faible coût et de la réponse rapide du biocapteur, l'approche proposée a le potentiel d'être appliquée dans les laboratoires de diagnostic clinique pour le suivi rapide de la sensibilité des bactéries aux antibiotiques. / Abstract : Monitoring the viability, growth and cellular metabolism of bacteria can contribute significantly to the early diagnosis of disease, but can also help improve yield of bacterial products in industrial- or small-scale experiments. Conventional methods applied for investigation of antibiotic sensitivity of bacteria are mostly culture-based techniques that are time-consuming and take at least 12 h to reveal results. This delay leads to overtreatment of a wide range of infections with broad spectrum antibiotics which is costly and may lead to the development of resistance to these precious antibiotics, whereas rapid detection of a viral infection or absence of bacteria could prevent such treatments and, in the case of bacterial infection, identification of antibiotic susceptibility could allow use of narrow spectrum antibiotics. The project outlined in this document aims at monitoring biological activities of live bacteria immobilized on biofunctionalized surfaces of quantum semiconductor (QS) microstructures. The method takes advantage of the sensitivity of photoluminescence (PL) emitting semiconductors to the perturbation of the electric field induced by the electric charge of bacteria immobilized on the surface of these structures. Our hypothesis was that bacteria growing on the surface of biofunctionalized QS biochips would modify their PL in a different, and measurable way in comparison with inactivated bacteria. In the first phase of the project, we investigated an innovative method involving PL monitoring of the photocorrosion effect in GaAs/AlGaAs heterostructures. Maintaining the balance between device sensitivity and stability in the biosensing (aqueous) environment allowed us to detect Escherichia coli K12 in phosphate buffered saline solutions (PBS) at an attractive limit of detection of 103 CFU/mL in less than 2 hours. Following this research, we hypothesised that these heterostructures could be employed to develop a method for inexpensive and quasi-real time monitoring of the growth and antibiotic susceptibility of bacteria. One of the key elements in the development of this biosensing platform was to demonstrate that GaAs (001), normally used for capping PL emitting GaAs/AlGaAs heterostructures, would not inhibit the growth of bacteria. In the second phase of the project, we explored the capture and growth of E. coli K12 on bare and biofunctionalized surfaces of GaAs (001). It has been determined that the initial coverage, and the subsequent bacterial growth rates are dependent on the biofunctionalization architecture used to capture bacteria, with antibody biofunctionalized surfaces exhibiting significantly higher capture efficiencies. Moreover, for suspensions containing bacteria at less than 105 CFU/mL, it has been found that the surface of GaAs wafers could not support the growth of bacteria, regardless of the type of biofunctionalization architecture. In the third phase of the project, we used PL to monitor the growth and antibiotic susceptibility of E. coli K12 and E. coli HB101 bacteria. While immobilization of bacteria on the surface of GaAs/AlGaAs heterostructures retards the PL monitored photocorrosion, growth of these bacteria further amplifies this effect. By comparing the photocorrosion rate of QS wafers exposed to bacterial solutions with and without antibiotics, the sensitivity of bacteria to the specific antibiotic could be determined in less than 3 hours. Due to the small size, low cost and rapid response of the biosensor, the proposed approach has the potential of being applied in clinical diagnostic laboratories for quick monitoring of antibiotic susceptibility of different bacteria.

The main aim of this study was to assess the “top-down” and “bottom-up” control of bacterial production in the small temporarily open/closed East Kleinemonde Estuary, situated on the south-eastern coastline of southern Africa. Spatial and temporal patterns in bacterial abundance, biomass and production and the importance of abiotic and biotic factors were investigated over the period May 2006 to April 2007. The trophic interactions between bacteria, phytoplankton, nanoflagellates (< 20 μm), microzooplankton (< 200 μm) and mesozooplankton (< 2 000 μm) were investigated during winter and summer. Bacterial abundance, biomass and production ranged between 1.00 × 10⁹ and 4.93 × 10⁹ cells 1⁻¹, 32.4 and 109 μg C 1⁻¹ and 0.01 and 1.99 μg C 1⁻¹ h⁻¹, respectively. With a few exceptions there were no spatial patterns in the values. Bacterial abundance, biomass and production, however, demonstrated a distinct temporal pattern with the lowest values consistently recorded during the winter months. Nanoflagellate and bacterial abundances were significantly correlated to one another (lower reaches: r = 0.818, p < 0.001; middle reaches: r = 0.628, p < 0.001; upper reaches: r = 0.484, p < 0.05) suggesting a strong predator-prey relationship. The frequency of visibly infected bacterial cells and the mean number of virus particles within each bacterial cell during this study demonstrated no temporal or spatial patterns and ranged from 0.5 to 6.1 % and 12.0 to 37.5 virus particles per bacterium, respectively. Viral infection and lysis was thus a constant source of bacterial mortality throughout the year. The estimated percentage of bacterial production removed by viral lysis ranged between 7.8 and 88.9% of the total which suggests that viral lysis represented a very important source of bacterial mortality during this study. The biological interactions between the selected components of the plankton community demonstrated that among the heterotrophic components of the plankton, the nanoflagellates were identified as the most important consumers of bacteria and small phytoplankton cells (< 20 μm). In the presence of microzooplankton the impact of the nanoflagellates on both the bacteria and phytoplankton was reduced, indicating that larger heterotrophs were preying upon the nanoflagellates. Mesozooplankton, however, appeared to exert the greatest impact on nanoflagellates. In the cascading experiments, the data suggest that mesozooplankton consume nanoflagellates, which resulted in a decrease in the predation impact of these organisms on the bacteria. This result is consistent with predator-prey cascades. The presence of the larger heterotrophs therefore, mediates the interactions between the primary bacterivores, the nanoflagellates, and the bacteria within the temporarily open/closed East Kleinemonde Estuary.

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

La litière de fumier recyclé (LFR) est utilisée dans les fermes laitières québécoises depuis quelques années, et ce, malgré le manque de connaissances scientifiques quant aux risques reliés à l’utilisation de ce produit pour la santé des animaux. Le premier objectif de cette thèse était de décrire les méthodes de production de LFR dans un contexte québécois et les pratiques de régie associées. Le deuxième objectif du projet consistait à déterminer le potentiel de recroissance de certaines espèces bactériennes dans la LFR lorsqu’elle est contaminée. Par la suite, le niveau de propreté et la prévalence de lésions aux jarrets chez les vaches laitières exposées ou non à ce produit ont été estimés. Finalement, l’association entre l’utilisation de LFR et l’incidence de mammite sous-clinique et clinique a été évaluée. Des études observationnelles cohorte ou transversale (selon l’objectif de recherche) ont été réalisées en 2018-2019 sur 27 fermes utilisant de la LFR et 61 fermes utilisant de la litière de paille, à titre comparatif. Les visites de fermes ont permis de constater que les méthodes utilisées pour produire de la LFR n’étaient pas standardisées et qu’elles ne permettaient pas un réel compostage de la fraction solide du fumier. Les essais de recroissance ont permis de démontrer que les différentes LFR (traitée en tas, contenant fermé ou cuve rotative) ne réagissaient pas de la même façon à une inoculation par des coliformes. La LFR de cuve rotative contenait une plus faible concentration initiale de Klebsiella spp. et a montré une croissance bactérienne significative dans les premières 24 h post-inoculation. Les LFR conditionnées dans des tas ou contenants avaient quant à elles une concentration initiale importante de Klebsiella spp. et n’ont pas démontré de croissance bactérienne significative suivant leur inoculation, ce qui suggère un effet de saturation de croissance bactérienne. Au cours de l’étude transversale, 30 vaches par troupeau, en moyenne, ont été notées à l’aide d’un score de propreté sur trois zones du corps ainsi que d’un score sur l’état des jarrets, afin d’estimer leur propreté et la présence de lésions aux jarrets. Le score de propreté attribué à chaque zone du corps des vaches allait de 1 à 4, 1 étant très propre et 4 très sale. Nous avons observé que les vaches logées sur LFR avaient, en général, une meilleure propreté du pis et du bas des pattes que celles logées sur paille. La LFR avait un effet protecteur pour le risque d’avoir un score de propreté du pis ≥ 3 (rapport de cotes (RC) : 0,43) ou d’avoir un score de 4 (RC : 0,29). Les vaches logées sur LFR avaient aussi le bas des membres plus propres que celles logées sur paille avec de plus faibles cotes d’avoir un score ≥ 2 (RC : 0,45), un score ≥ 3 (RC : 0,16) ou un score de 4 (RC : 0,07). Cependant, nous n’avons pas trouvé de différence entre les deux groupes d’animaux au niveau de la propreté du flanc et du haut des pattes. Lors de leur évaluation pour la propreté, les vaches ont aussi été notées à l’aide d’un score de lésions aux jarrets allant de 0 à 3. Un score de 0 représentait un jarret parfaitement sain alors qu’un score de 3 était attribué à un jarret présentant une enflure de plus de 2,5 cm. Les deux jarrets étaient évalués et le jarret ayant reçu le score le plus élevé était inclus dans les analyses. Nous n’avons pas identifié de différence quant aux scores de jarret entre les deux groupes d’animaux. Afin d’évaluer la santé de la glande mammaire, une étude cohorte d’une durée d’un an a été mise en place à partir de la visite de la ferme. Les dynamiques de comptages de cellules somatiques ont été suivies sur 11 031 vaches durant cette période afin d’analyser l’incidence de mammite sous-clinique. Nous n’avons pas été en mesure de détecter une différence d’incidence entre les deux groupes d’animaux. Au cours de l’étude cohorte, les producteurs laitiers ont identifié et fait parvenir au laboratoire 1 144 échantillons de lait provenant de vaches atteintes de mammites cliniques. L’incidence totale de mammite clinique n’était pas plus élevée dans les fermes LFR qu’au sein des fermes paille. Cependant, lorsque nous avons analysé l’incidence de mammite clinique par agent pathogène spécifique, nous avons pu constater que les vaches logées sur LFR étaient 7,0 fois plus à risque d’expérimenter une mammite clinique causée par Klebsiella pneumoniae que celles du groupe comparatif. / Recycled manure solids (RMS) bedding has been used on Quebec dairy farms for a number of years, despite the lack of scientific knowledge about the health risks associated with the use of this product for animals. The first objective of this thesis was to describe the RMS production methods in a Quebec context and the associated management practices. The second objective of the project was to determine the potential for regrowth of certain bacterial species in the RMS when it is contaminated. Subsequently, the level of cleanliness and prevalence of hock lesions in dairy cows exposed or not to this product were estimated. Finally, the association between RMS use and the incidence of subclinical and clinical mastitis was evaluated. Observational cohort or cross-sectional studies (depending on the research objective) were conducted in 2018-2019 on 27 farms using RMS and 61 farms using straw bedding for comparison. The farm visits highlighted that the methods used to produce RMS were not standardized and did not allow for true composting of the manure solid fraction. The regrowth trials showed that different RMS (treated in heap, closed container or rotating drum) did not react in the same way to coliform inoculation. The rotating drum RMS contained a lower initial concentration of Klebsiella spp. and experienced significant bacterial growth in the first 24 hours post-inoculation. The heap or closed container RMS had a high initial concentration of Klebsiella spp. and did not show significant bacterial growth following inoculation, suggesting a saturation effect on bacterial growth. In the cross-sectional study, an average of 30 cows were measured using a cleanliness score on three body areas as well as a hock lesion score to estimate their cleanliness and the presence of hock lesions. The cleanliness score assigned to each area of the cows' body ranged from 1 to 4, with 1 being very clean and 4 being very dirty. We found that cows housed on RMS generally had better udder and lower leg cleanliness than those housed on straw. Recycled manure solids bedding had a protective effect for the risk of having an udder cleanliness score ≥ 3 (odds ratio (OR): 0,43) or having a score of 4 (OR: 0,29). Cows housed on RMS also had cleaner lower legs than those housed on straw with lower odds of having a score ≥ 2 (OR: 0,45), a score ≥ 3 (OR: 0,16) or a score of 4 (OR: 0,07). However, we found no difference between the two groups of animals in flank and upper leg cleanliness. When they were evaluated for cleanliness, cows were also measured using a hock lesion score ranging from 0 to 3. A score of 0 represented a perfectly healthy hock, while a score of 3 was assigned to a hock with swelling greater than 2.5 cm. Both hocks were scored and the hock with the higher score was included in the analyses. We did not identify any difference regarding hock lesions between the two groups of animals. To assess mammary gland health, a one-year cohort study was set up from the farm visit. Somatic cell count dynamics were followed on 11 031 cows during this period to analyze the incidence of subclinical mastitis. We were not able to detect a difference in incidence between the two groups of animals. During the cohort study, dairy farmers identified and sent to the laboratory 1 144 milk samples from cows with clinical mastitis. The total incidence of clinical mastitis was not higher on RMS farms than on straw farms. However, when we analyzed the incidence of clinical mastitis by specific pathogen, we found that cows housed on RMS were 7.0 times more likely to experience clinical mastitis caused by Klebsiella pneumoniae than those in the comparison group.