Mycobacterium avium and Mycobacterium intracellulare are environmental opportunistic pathogens whose source for human infection is water and soil. M. avium and M. intracellulare cause pulmonary infections (tuberculosis) in immunocompetent individuals and bacteremia in immunodeficient individuals (e.g. AIDS). One factor likely influencing the lack of success of antibiotic therapy in patients would be their ability to form biofilms. Growth in biofilms might result in antimicrobial resistance because (1) cells are protected by layers of other cells and extracellular material (2) and differences in physiologic state of cells as a consequence of growing on surfaces. The objectives of the work were to (1) establish methods for reproducible growth of mycobacterial biofilms (2) measure the formation of biofilms on surfaces by cells of M. avium and M. intracellulare (3) measure the antibiotic- and chlorine- susceptibility of M. avium and M. intracellulare strain TMC1406T in cell grown in suspension, cells grown in biofilms and suspended and of cells grown in biofilms (4) measure the hydrophobicity of M. avium and M. intracellulare grown in suspension and in biofilms. Methods were developed for growing mycobacteria in biofilms in polystyrene flasks and on glass beads. Although both strains formed biofilms, M. intracellulare strain TMC 1406T more readily formed biofilms than M. avium strain A5 in polystyrene flasks. The majority of M. intracellulare strain TMC 1406T cells grew on the walls of the flasks rather than in suspension like M. avium strain A5. The susceptibility of M7H9 medium-grown cells of M. avium strain A5 and M. intracellulare strain TMC 1406T cells grown in suspension, cells grown in biofilms and suspended and cells grown in biofilms was measured against clarithromycin, ethambutol, kanamycin, rifampicin and streptomycin. Cells grown in biofilms and exposed to antibiotics in biofilms were five-fold resistant to antibiotics than were cells grown in biofilms and exposed in suspension. Cells grown and exposed in suspension were ten-fold more sensitive to antibiotics than were cells grown in biofilms and exposed in suspension. The chlorine susceptibility of cells grown in medium and water was also measured. Cells grown in biofilms were more resistant to chlorine than cells grown in biofilms and suspended. Cells grown in suspension were more sensitive to chlorine than cells grown in biofilms and suspended. The hydrophobicity data (i.e., hexadecane adherence and contact angle measurements) showed that cells grown in biofilms are more hydrophobic than cells grown in biofilms and suspended and cells grown in suspension. It is clear that there are physiological changes between cells grown in suspension, cells grown in biofilms and suspended and cells in biofilms. / Master of Science

This thesis describes a series of naphthoquinone reactions employing pyridine carboxylic acid derivatives (nicotinic acid derivatives). The products of these reactions will be tested by Parke, Davis and Company for their activity against the tubercle bacillus and other pathogenic microorganisms.

Bacterial evolution has been shaped by the acquisition of clusters of genes called genomic islands through means other than vertical inheritance. These gene clusters provide beneficial traits to the recipient bacteria such as virulence, resistance and the ability to utilize different metabolites, thereby facilitating bacterial adaptation to diverse environments and leading to the emergence of multi-drug resistant pathogens. As identification of genomic islands are of immense biomedical importance, we have developed a novel genomic island detection method, DICEP, to robustly identify genomic islands in bacterial genomes. Once genomic islands were identified, we focused on functional characterization of genes harbored by these islands as an essential step towards understanding their role in providing fitness to the recipient bacterium. We have used a gene co-expression network-based approach to gain insights into the functional association of genes within an island. The network analysis revealed novel pathogenicity associated genes and helped in functional characterization of island genes.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第25270号 / 工博第5229号 / 新制||工||1997(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 西村 文武, 教授 松田 知成, 准教授 浅田 安廣, 准教授 井原 賢(高知大学) / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The global spread of antibiotic resistance warrants concerted surveillance in the clinic and in the environment. The widespread use of metagenomics for various studies has led to the generation of a large amount of sequencing data. Next-generation sequencing of microbial communities provides an opportunity for proactive detection of emerging antibiotic resistance genes (ARGs) from such data, but there are a limited number of pipelines that enable the identification of novel ARGs belonging to diverse antibiotic classes at present. Therefore, there is a need for the development of computational pipelines that can identify these putative novel ARGs. Such pipelines should be scalable, accessible and have good performance. To address this problem we develop a new method for predicting novel ARGs from genomic or metagenomic sequences, leveraging known ARGs of different resistance categories. Our method takes into account the physio-chemical properties that are intrinsic to different ARG families. Traditionally, new ARGs are predicted by making sequence alignment and calculating sequence similarity to existing ARG reference databases, which can be very time consuming. Here we introduce an alignment free and deep learning prediction method that incorporates both the primary protein sequences of ARGs and their physio-chemical properties. We compare our method with existing pipelines including hidden Markov model based Resfams and fARGene, sequence alignment and machine learning-based DeepARG-LS, and homology modelling based Pairwise Comparative Modelling. We also use our model to detect novel ARGs from various environments including human-gut, soil, activated sludge and the influent samples collected from a waste water treatment plant. Results show that our method achieves greater accuracy compared to existing models for the prediction of ARGs and enables the detection of putative novel ARGs, providing promising targets for experimental characterization to the scientific community. / Master of Science / Various bacteria contain genes that allow them to survive and grow even after the application of antibiotics. Such genes are called antibiotic resistance genes (ARGs). Each ARG has properties that make it resistant to a particular class of antibiotics. This class is called the resistance class/category of the gene. Antimicrobial resistance (AMR) is one of the biggest challenges to public health in recent times. It has been projected that a large number of deaths might occur due to AMR in the future. Therefore, there is a need for monitoring AMR in various environments. Currently, developed methods use the sequence's similarity with the existing database as a feature for ARG prediction. Some tools also use the 3D structure of proteins as a feature for ARG prediction. In this thesis, we develop a tool that incorporates both the sequence similarity and the structural information of proteins for ARG prediction. The structural information is encoded with physio-chemical properties (such as hydrophobicity, molecular weight etc.) of the amino acids. Our results show the efficacy of the pipeline in various environments. Results also show that our method achieves accuracy greater than existing models for the prediction of ARGs from metagenomic data. It also enables the detection of putative novel ARGs, providing promising targets for experimental characterization to the scientific community.

Thesis (M. Tech. ) - Central University of Technology, Free State, 2006 / Antibiotics have been used on animals since the Second World War for therapeutic purposes. The risks associated with the use of antibiotics in food animals are enormous, due to the possibility of resistance of organisms caused by the usage of low levels of antibiotics. The goals of this study were to determine the prevalence of antibiotic residues in C grade milk cows, and to determine the practices and knowledge of producers regarding the use of antibiotics as well as the knowledge and habits of consumers. In the study the presence of antibiotic residues was tested by means of microbiological inhibition testing. Fifteen percent of all tested samples tested positive for the presence of antibiotic residues. Eleven samples that tested positive on the microbiological inhibition test were sent to the Onderstepoort Veterinary Institute for confirmation. The knowledge of the consumer as well as that of the producer was tested by means of a questionnaire and then processed statistically. Producers were, in general, fairly ignorant regarding the withdrawal period of antibiotic residues. Age and the combination of age and reproduction were the common reasons for the selling of milk cows. Both the sampling and the questionnaires proved that the presence of antibiotic residues is higher than that stated in literature. The selling of the animals was mostly by auction or directly to the abattoirs. Consumer practices regarding the antibiotic residues were mostly in order, although consumers did not have knowledge regarding antibiotic residues. High temperatures, for example in the frying or grilling of meat, will reduce the presence of antibiotic residues because these effects have an influence on the presence of antibiotic residues (Rose et al., 1995). Almost 75% of consumers describe antibiotics in a positive manner. Milk and meat are regarded as posing the greatest risk for the origin of antibiotic residues. A lack of general knowledge regarding antibiotic residues was noteworthy, especially from the consumers of Thaba ’Nchu and Heidedal. The wrong usage of antibiotics by medical practitioners and consumers will certainly have consequences. Most of the consumers (112/227 respondents) indicated that they always completed a course of antibiotics. Forty-nine respondents indicated that they had already used antibiotics and that the therapy was unsuccessful. Antibiotic residues are still present so the risk to the consumer is still viable. Improved sampling and monitoring at abattoirs both for local and export abattoirs is necessary. The “farm to fork” concept and financial incentives by way of subsidies for residue-free products is a positive step. South African consumers are not quality conscious and further studies are necessary to improve the quality and safety of meat.

Pour enrayer la progression des résistances bactériennes aux antibiotiques (AB), liée, en partie, à un mésusage de ces médicaments, l’Union européenne (UE) s’est mobilisée en adoptant, en 2001, une recommandation prônant un usage prudent des AB. En 2008, sous l’égide de la Commission européenne, nous avons évalué la mise en œuvre de cette recommandation et son impact dans les Etats membres, et nous avons réalisé une analyse du cadre juridique développé dans 12 de ces Etats. De plus, une étude a été conduite entre 2005 et 2009 sur 74 établissements de santé (ES) volontaires du Sud-Ouest de la France pour identifier l’impact du cadre juridique sur l’évolution des consommations d’AB. Les Etats de l’UE ont engagé un grand nombre d’actions pour rationaliser l’utilisation des AB, mais des progrès dans le champ de l’évaluation demeurent nécessaires. Il ressort de l’analyse sur 12 Etats qu’un cadre juridique plus important semble associé à une plus grande sensibilisation de la population générale et à une meilleure maîtrise de la consommation de fluoroquinolones. Dans les ES du Sud-Ouest, les politiques locales de bon usage des AB ont progressé. Une combinaison de mesures telles que la présence d’un référent « AB », l’organisation de formations et le recours à des prescriptions à durée limitée, était associée à une réduction significative de la consommation des fluoroquinolones. L’amélioration de l’utilisation des AB implique de préciser les conditions d’efficacité optimale des actions et de renforcer l’encadrement juridique dans les Etats de l’UE, notamment en matière de surveillance, d’évaluation, de moyens d’incitation, en l’adaptant aux caractéristiques nationales. / Antimicrobial resistance (AMR) is a public health problem worldwide. As antibiotic (AB) use is one of the drivers of AMR, the Council of the European Union adopted in 2001 a recommendation on the prudent use of antimicrobial agents. To analyze the way Member States (MS) had implemented this recommendation and to approach its efficacy, we performed a survey under the auspices of the European Commission in 2008, completed by a thorough analysis of the legal framework in twelve Member States. In addition, relationships between AB stewardship programmes (ABS) and trends in AB consumption were studied from 2005 to 2009 in 74 voluntary hospitals in Southwestern France. MS had implemented a broad range of activities to improve AB use, but differences were seen namely in evaluation systems. Further analysis in 12 MS highlighted discrepancies regarding the scope of the legal framework, incentives for its enforcement, and means of evaluation. A legal framework regarding surveillance and national organisation seemed in favor of higher citizens’ knowledge and awareness and appeared to be associated with lower increase in fluoroquinolone (FQ) use. In French hospitals, ABS had sharply improved and AB consumption remained stable when adjusted on activity. The presence of an antibiotic advisor combined with provision of training and use of prescriptions with stop-orders was associated with a significant decrease in FQ use. Progress in the use of AB could be achieved by sharing experience on best practices and by enforcing legal framework, tailored to MS organisation and epidemiology, targeting activities such as surveillance and evaluation

Introduction: Nosocomial infections remain a major cause of mortality and morbidity. The problem is most apparent in intensive care units (ICUs). Most ICU patients are compromised and vulnerable as a result of disease or severe trauma. One in ten people admitted to hospital is given an antibiotic for infection. The risk of acquiring a nosocomial infection in a European ICU is approximately 20%. It is vitally important that ways are found to prevent transmission between patients and personnel, and that local hygiene routines and antibiotic policies are developed. This thesis is a holistic work focused particularly on antimicrobial antibiotic resistance, antibiotic consumption and to some extent on hygiene in Swedish ICUs. Aims: The general aim of this thesis was to investigate bacterial resistance and antibiotic consumption in Swedish ICUs and to try to correlate ICU demographic data with antibiotic consumption and antibiotic resistance. Additional aims were to investigate on which clinical indications antibacterial drugs are prescribed in the ICU, and to investigate the emergence of resistance and transmission of Pseudomonas aeruginosa in the ICU using cluster analysis based on antibiograms and genotype data obtained by AFLP. Material and methods: In paper 1-3, antibiotic consumption data together with bacterial antibiotic resistance data and specific ICU-demographic data were collected from an increasing number of ICUs over the years 1997-2001. Data from ICUs covering up to six million out of Sweden’s nine million inhabitants were included. In paper 4, the indications for antibiotic prescribing were studied during two weeks in 2000. Paper 5 investigated Pseudomonas aeruginosa isolates in order to detect cross-transmission with genotype obtained by AFLP, and antibiogram-based cluster analysis was also performed in order to see if this could be a quicker and easier substitute for AFLP. Results: This thesis has produced three important findings. Firstly, antibiotic consumption in participating ICUs was relatively high during the study period, and every patient received on average more than one antimicrobial drug per day (I-IV). Secondly, levels of antimicrobial drug resistance seen in S. aureus, E. coli and Klebsiella spp remained low when data were pooled from all ICUs throughout the study period, despite relatively high antibiotic consumption (I-V). Thirdly, the prevalence of antibiotic resistance in CoNS and E. faecium, cefotaxime resistance in Enterobacter, and ciprofloxacin and imipenem resistance in P. aeruginosa was high enough to cause concern. Conclusion: For the period studied, multidrug resistance in Swedish ICUs was not a major problem. Signs of cross-transmission with non-multiresistant bacteria were observed, indicating a hygiene problem and identifying simple improvements that could be made in patient care guidelines and barrier precautions. A need for better follow up of prescribed antibiotics was evident. With further surveillance studies and monitoring of antibiotics and bacterial resistance patterns in the local setting as well as on a national and international level, some of the strategic goals in the prevention and control of the emergence of antimicrobial-resistant microbes may be achievable.

The primary objectives of this research were to demonstrate that: 1.) antibiotic resistant bacteria can promote the survival of antibiotic sensitive organisms when grown simultaneously as biofilms in antibiotics, 2.) community-level multiple antibiotic resistance of polymicrobial consortia can lead to biofilm formation despite the presence of multiple antibiotics, and 3.) biofilms may benefit plasmid retention and heterologous protein production in the absence of selective pressure. Quantitative analyses of confocal data showed that ampicillin resistant organisms supported populations of ampicillin sensitive organisms in steady state ampicillin concentrations 13 times greater than that which would inhibit sensitive cells inoculated alone. The rate of reaction of the resistance mechanism influenced the degree of protection. Spectinomycin resistant organisms did not support their sensitive counterparts, although flow cytometry indicated that GFP production by the sensitive strain was improved. When both organisms were grown in both antibiotics, larger numbers of substratum-attached pairs at 2 hours resulted in greater biofilm formation at 48 hours. For biofilms grown in both antibiotics, a benefit to spectinomycin resistant organism’s population size was detectable, but the only benefit to ampicillin resistant organisms was in terms of GFP production. Additionally, an initial attachment ratio of 5 spectinomycin resistant organisms to 1 ampicillin resistant organism resulted in optimal biofilm formation at 48 hours. Biofilms also enhanced the stability of high-copy number plasmids and heterologous protein production. In the absence of antibiotic selective pressure, plasmid DNA was not detected after 48 hours in chemostats, where the faster growth rate of plasmid-free cells contributed to the washout of plasmid retaining cells. The plasmid copy number per cell in biofilms grown without antibiotic selective pressure steadily increased over a six day period. Flow cytometric monitoring of bacteria grown in biofilms indicated that 95 percent of the population was producing GFP at 48 hours. This research supports the idea that ecological interactions between bacteria contribute to biofilm development in the presence of antibiotics, and demonstrates that community-level multiple antibiotic resistance is a factor in biofilm recalcitrance against antibiotics. Additionally, biofilms may provide an additional tool for stabilizing high copy number plasmids used for heterologous protein production.

Terapia empírica com antimicrobiano de amplo espectro faz parte do manejo inicial padrão de pacientes com neutropenia febril (NF). Evidências suficientes de quais esquemas antibióticos devem ser inicialmente prescritos já existem; embora, nenhum estudo randomizado tenha avaliado se a aderência a programas de controle de antimicrobianos (PCAs) resulta em diminuição das taxas de mortalidade por esta síndrome. No presente estudo de coorte prospectivo, realizado em um hospital terciário no período de outubro de 2009 a agosto de 2011, avaliou-se o impacto da aderência ao PCA, aferida através da prescrição antimicrobiana inicial, na mortalidade em 295 episódios de NF (em 145 indivíduos adultos) que necessitaram de tratamento endovenoso hospitalar. Após análise multivariada através de regressão de Cox, incluindo outros preditores de mortalidade, a aderência ao PCA mostrou-se fator de proteção independente para morte 28 dias após início do episódio de NF (razão de hazard ajustada[HR], 0.29; intervalo de confiança de 95% [IC 95%], 0.11 a 0.72). Os fatores de risco encontrados para a não-aderência ao PCA foram presença de hipotensão (risco relativo ajustado[RR], 1.90; IC 95%, 1.37 a 2.63), diarreia (RR, 2.13; IC 95%, 1.66 a 2.73), dor perianal (RR, 2.08; IC 95%, 1.54 a 2.82), suspeita de foco infeccioso em cavidade oral (RR, 2.45; IC 95%, 1.75 a 3.43) e manifestações cutâneas de infecção (RR, 2.34; IC 95%, 1.81 a 3.04). A escolha antimicrobiana inicial é particularmente importante no manejo inicial do paciente com febre em vigência de neutropenia; a aderência ao PCA, que preconiza o uso racional de antibióticos, mostrou ser efetiva na redução de mortalidade durante o curso da doença. A presença de fatores modificadores da terapia inicial representa risco para não-adesão ao programa de controle de antimicrobianos. / Empirical therapy with broad-spectrum antimicrobial is part of the initial management of patients with febrile neutropenia (FN). Enough evidence on which antibiotics schemes should be initially prescribed already exists; however, no randomized study has evaluated whether adherence to antimicrobial stewardship programs (ASPs) results in lower rates of mortality from this syndrome. In the present prospective cohort study performed in a tertiary hospital, from October 2009 to August 2011, we evaluated the impact of adherence to ASP, measured by initial antimicrobial prescribing, in mortality of 295 episodes of FN (in 145 adults) that required intravenous inpatient treatment. After multivariate analysis through Cox regression, including other predictors of mortality, adherence to ASP proved to be an independent protective factor for death 28 days after the beginning of the episode of FN (adjusted hazard ratio [HR], 0.29; 95% confidence interval [95% CI], 0.11 to 0.72). The risk factors found to noncompliance to ASP were presence of hypotension (adjusted relative risk [RR], 1.90; 95% CI, 1.37 to 2.63), diarrhea (RR, 2.13; 95% CI, 1.66 to 2.73), perianal pain (RR, 2.08; 95% CI, 1.54 to 2.82), suspected source of infection in oral cavity (RR, 2.45; 95% CI 1.75 to 3.43) and cutaneous manifestations of infection (RR, 2.34; 95% CI, 1.81 to 3.04). The choice of antimicrobial is particularly important in the initial management of patients with fever in the presence of neutropenia; the adherence to ASP, which calls for rational use of antibiotics, was effective in reducing mortality during the course of the disease. The presence of signs or symptoms that demand changes in the initial therapy poses risks to nonadherence to the antimicrobial management program.