Antibiotic resistance is a serious global public health problem. ESBLs are enzymes that destroy expanded-spectrum beta-lactam antibiotics rendering these drugs ineffective. Infection with ESBL-producing K.pneumoniae are hard to treat and result in longer hospital stay and higher mortality rates. The Clinical Laboratory Standard Institute (CLSI) have standard methods for detection of ESBL producing strains of bacteria in infected patients to guide antibiotic therapy, reduce the risk of mortality and risk of transmission. The presence of K.pneumoniae and E.coli which produce ESBLs have been confirmed in natural environments such as soil and water but no standard methods exist to identify directly and quantify these bacteria to understand the risk of human exposure in these settings. The purpose of this research is to assess the ability of an agar dilution method, using a differential agar Bio-Rad Rapid E.coli 2 agar utilized in environmental water quality studies, to identify correctly ESBL-producing K.pneumoniae. The minimum inhibitory concentration (MIC) of ceftriaxone antibiotic for wild-type ESBL producing K.pneumoniae isolates were compared on Mueller-Hinton broth (MHB) and Bio-Rad Rapid E.coli 2 agar. Using the MIC values, the isolates were classified as susceptible, intermediate or resistant. The MIC of wild-type strains of K.pneumoniae were above 4μg/mL for both methods on all susceptibility tests performed. The results of this research suggest that Bio-Rad Agar dilution method performed well, correctly identifying these strains as resistant to ceftriaxone, an indication of ESBL production. The Bio-Rad agar dilution method can be considered as a viable standard method for direct identification of ESBL-producing K.pneumoniae in natural environments.

Klebsiella spp. are opportunistic pathogens that cause hospital and community acquired infections such as pneumonia, urinary tract infection, septicaemia, soft tissue infections, liver abscess, and meningitis. Multidrug-resistant strains possessing extended-spectrum β-lactamases (ESBLs) has become an increasing problem worldwide. The over use and, in some cases, misuse of antibiotics in humans and in animal husbandry has been cited as a responsible factor in the development of drug resistance in all bacterial species. The advancing age; female gender, hospital crossinfection, the food chain trade and human migrations have contributed to increase the risk for community-acquired ESBL. A total of 223 isolates collected in 2006 and 2007 at Royal Infirmary of Edinburgh, Scotland, 219 K. pneumoniae, 2 K. oxytoca, 1 Enterobacter cloacae, and one isolate Salmonella enterica were identified by API 20E and confirmed genotypically with gyrA PCR-RFLP method. The antimicrobial susceptibility results showed that 34 (15.2%), 36 (16.1%), 35 (15.7%), 45 (20.2%), 30 (13.5%) and 55 (24.7%) of these strains were found to be resistant to cefotaxime, ceftazidime, ceftriaxone, naladixic acid, ciprofloxacin and cefoxitin. None of the isolates were found resistant to meropenem keeping carbapenems the drug of choice for the treatment of multiresistant isolates. The overall frequency of ESBL producers observed in this study was 35 (15.7%) most of them 32/35 (91.4%) were from K. pneumoniae. The genetic analysis showed that SHV β-lactamases were detected in 32, whereas TEM and CTX-M were detected in 24 and 16 respectively. From the ESBL-producing isolates, molecular methods identified nine strains possessing ESBL-SHV genes (1 strain blaSHV-5, 1 strain blaSHV-80 and 8 strains blaSHV-12), whereas the remaining were from the “non-ESBL” producing strains. Conjugation methods demonstrated that 29/32 isolates harboured transferable blaSHV genes. The large SHV transposon-borne promoters were amplified from only one non-transferable blaSHV-11, 15 isolates produced the small SHV transposon-borne promoters. Furthermore, the IS26 was found 73bp upstream of the blaSHV gene in all small SHV transposon-borne promoters. A new blaLEN gene was identified from K. pneumoniae (KpII) phylogenetic group but remained susceptible to all cephalosporins. Sixteen (7.3%) of K. pneumoniae isolates were found to be producers of the CTX-M- 15 ESBL, of which two isolates (12.5%) were reported to be from communityacquired infections. The insertion sequence ISEcp1 was detected by sequencing 48 nucleotides upstream of blaCTX-M-15 in all isolates but one. Five different clones of CTX-M-15-producing isolates were identified by PFGE. The findings indicated a higher prevalence of qnr genes than in previous studies but still low in general. By PCR, 18 (8%) (11 qnrB1, 2 qnrB6 and 5 qnrA1) genes were identified from K. pneumoniae isolates. Also, the findings indicated the frequent coexpression of fluoroquinolones and ESBLs resistance in the same isolate. Two K. oxytoca strains were isolated from urine and blood specimens of hospitalized patients. Both strains were positive for the blaOXY-2 gene. One strain showed resistance to pencillins, monbactams, cephalosporins including cefotaxime and ceftazidime but was not inhibited by clavulanic acid. It differed by an amino acid substitution Ala237→Thr, which enhances the binding of cefotaxime. S1-nuclease plasmid profiles were obtained for some isolates. A total of one to two plasmids, ranging in size from approximately 40 to 210 kb, were observed per strain. The plasmids from 24 ESBL K. pneumoniae strains were assigned to be IncN or IncFII replicons. Analysis of phylogenetic groups showed that the majority of K. pneumoniae isolates were belonged to KpI-type. Both K. oxytoca strains were assigned to be KoII phylogenetic group based on rpoB and gyrA sequencing. Integrons are capable of capturing and mobilizing genes called gene cassettes which play an important role in the dissemination of antimicrobial resistance through horizontal transmission. In fact, the present study indicated a high frequency of occurrence of class 1 integrons among ESBL-positive K pneumoniae. Three isolates positive for class 1 integrons were found positive for class 2 integrons as well. Class 1 integrons including dfr, aadA and ereA2 gene cassettes have been identified by sequencing, which confer resistance to trimethoprim, streptomycin/spectinomycin and erythromycin respectively. In conclusion, the results from this thesis report the emergence of hospital and community-acquired highly resistant CTX-15 β-lactamase in the Edinburgh, Scotland. The prevalence of ESBL-producing isolates in Scotland is still much lower than in many other European countries. The dissemination of SHV- and TEM- β- lactamase types in this study is more predominate than CTX-M-15.

OBJETIVO: Determinar los factores asociados a producción de betalactamasas de espectro extendido por enterobacterias en pacientes hospitalizados del Hospital Nacional Daniel Alcides Carrión durante Septiembre 2008-Diciembre 2009. MATERIALES Y MÉTODOS: Diseño analítico, observacional, controlado, de casos y control. La población estuvo formada por todo paciente hospitalizado que tuvo registro de cultivo de Escherichia coli, Klebsiella pneumoniae y Klebsiella oxytoca durante el período septiembre 2008 - diciembre 2009. Se estudiaron 92 pacientes (40 casos y 52 controles); se definió como caso a todo paciente con registro de un cultivo de enterobacteria positivo a betalactamasas de espectro extendido y como control a todo paciente con registro de un cultivo de enterobacteria no positivo a betalactamasa de espectro extendido. Se calculó la fuerza de asociación del uso previo de antibiótico, presencia de comorbilidad grave subyacente, y exposición a método invasivo con riesgo de producción de betalactamasas de espectro extendido por enterobacterias y por E. coli. Se mostró la sensibilidad y resistencia antibiótica de acuerdo a los resultados del antibiograma. Los resultados se analizaron y graficaron mediante el programa estadístico EPI INFO versión 3.5.1 y hoja de cálculo de Microsoft Excel. RESULTADOS: Se encontró que quienes tuvieron uso previo de antibiótico tuvieron 3,0 veces más riesgo de producción de BLEE comparado con quienes no tuvieron dicha exposición; y quienes usaron ceftriaxona tuvieron 3,4 veces más riesgo de producción de BLEE. La exposición a catéter endovenoso tuvo 3,1 mayor riesgo de producción de BLEE. El uso de sonda nasogástrica tuvo 4,7 más riesgo de producción de BLEE. La afección de tejido blando presentó 5,3 veces más riesgo de producción de BLEE. El uso de sonda urinaria no se halló asociada estadísticamente con producción BLEE por enterobacterias ni por E. coli.

The rapid and reliable detection of pathogens is of utmost importance in healthcare settings to ensure the appropriate treatment thereby reducing morbidity and mortality for the patient. Current culturing, PCR based and NGS species detection methods are time consuming (Opota et al., 2015), limited in their detection (Buckley et al., 2015), or require specialist skills and are expensive (Basho and Eterovic., 2015). Oxford Technologies Nanopore devices could provide detailed genomic sequencing at a fraction of the cost and without the need for technical bioinformatic skills. This study evaluates the MinION device and analysis tools to suggest best practice. Classification and genotyping of 12 Klebsiella isolates were performed using EPI2ME automated workflows and manual de novo assembly.  Automated workflows using raw MinION reads provided clinically relevant information identified in ~6hrs. Manual de novo assembly and analysis used hybrid, and single source data took >24hrs. The inclusion of MinION long reads overcome problems assembling short reads. Hybrid genomes provided the most contiguous and highly detailed contigs. MinION only read assemblies contained more errors but still identified similar genotypic findings. Automated workflows are rapid and require minimal bioinformatic know-how. There should be a dialogue between clinicians and bioinformaticians to develop bespoke analysis tools.  Although challenges remain around compatible kits and vulnerable flowcells long read sequencing can be an effective tool for species detection and pathogen typing. Furthermore, hybrid assemblies have the potential to advance our genome detailing and discovery.

Submitted by admin tede (tede@pucgoias.edu.br) on 2016-08-17T14:55:11Z No. of bitstreams: 1 DANIELLA FABIOLA DOS SANTOS.pdf: 702349 bytes, checksum: b479d0d411d5a404d1e3f2dba2f21c37 (MD5) / Made available in DSpace on 2016-08-17T14:55:11Z (GMT). No. of bitstreams: 1 DANIELLA FABIOLA DOS SANTOS.pdf: 702349 bytes, checksum: b479d0d411d5a404d1e3f2dba2f21c37 (MD5) Previous issue date: 2007-08-01 / Klebsiella pneumoniae is an important ethiological agent of infections in the nosocomial environment and due to indiscriminate use of broad spectrum antimicrobials, especially of third generation cephalosporins; a selective pressure is produced, what favors the growth of strains producing extended spectrum β-lactamases (ESBLS). These enzymes hydrolyse broad spectrum cephalosporins and monobactams, but are inhibited by β-lactamases inhibitors. ESBLs have been identified in other pathogens but are more often found in Klebsiella pneumoniae and Escherichia coli. The fails in the detection of ESBL increase the mortality and morbity rates, favors the occurance of multirresistant drug outbreaks and increase the costs to control these microorganisms. The purpose of this study was to evaluate the prevalence, susceptibility to antimicrobial agents and genomic variability of ESBL Klebsiella pneumoniae in the hospital with the highest prevalence of ESBL producing isolates in Goiânia. A total of 61 strains of Klebsiella pneumoniae (89,9%) and 7 of Klebsiella oxytoca (10,1%) were isolated from January 2005 to May 2006 from hospitalized patients in three hospitals in Goiânia. All the isolates were identified and the susceptibility to antimicrobials tested by the authomatized semiquantitative method (MicroScan WalkAway® - Dade Behring, USA). The double-disk diffusion was used to detect the ESBL producing strains; a disk containing amoxicillin/clavulanate was placed as the inhibitor of β-lactamase 20mm from the oxymino-β-lactam. Enhancement of the zone inhibition of the oxymino β-lactam caused by the clavulanate disk was considered as evidence of ESBL production. The chomossomal DNA analysis was performed by Pulsed-field gel electrophoresis (PFGE). The prevalence of ESBL producing Klebsiella pneumoniae was high (25%, 28.5% and 66.7% respectively) and the antimicrobial resistance rates were higher among ESBL producing isolates. Only the imipenem showed excellent activity in vitro. The chomossomal DNA analysis showed a great variability among 5 strains (multiclonal) and similarity among 6 strains (clonal) producing ESBL. / Klebsiella pneumoniae é importante agente etiológico de infecções no meio ambiente hospitalar e o uso indiscriminado de antimicrobianos de amplo espectro, principalmente cefalosporinas de terceira geração, produz pressão seletiva que favorece a proliferação de isolados produtores de β-lactamases de espectro ampliado (ESBLS). Estas enzimas hidrolisam cefalosporinas de amplo espectro e monobactâmicos, mas são sensíveis aos inibidores de β- lactamases. As ESBLs já foram identificadas em outros patógenos, mas são encontradas principalmente em Klebsiella pneumoniae e Escherichia coli. As falhas na detecção de ESBL aumentam as taxas de morbidade, de mortalidade, favorece a ocorrência de surtos por organismos multirresistentes aos antibióticos e aumentam os custos hospitalares. Os objetivos do estudo foram avaliar a prevalência, o perfil de sensibilidade e a variedade genética de amostras de Klebsiella pneumoniae produtoras de ESBL no hospital com prevalência elevada de infecções causadas por este microrganismo em Goiânia. Foram avaliadas 61 amostras de Klebsiella pneumoniae (89,9%) e 7 de Klebsiella oxytoca (10,1%) isoladas de janeiro de 2005 a maio de 2006 de pacientes hospitalizados em três hospitais de Goiânia. Todos os isolados foram identificados e testados quanto à suscetibilidade aos antimicrobianos por meio do método automatizado semi-quantitativo (MicroScan WalkAway® - Dade Behring, USA). O teste de difusão dupla em disco foi utilizado para detectar as amostras produtoras de ESBL. Um disco contendo amoxicilina/clavulanato foi usado como inibidor de β-lactamase e posto a 20 mm do oximino-β-lactâmico. O aumento da zona de inibição do oximino-β-lactâmico causado pelo clavulanato evidenciou a produção ESBL. A tipagem das amostras produtoras de ESBL do Hospital C foi realizada pelo método Pulsed-field gel electrophoresis (PFGE). A prevalência de amostras de Klebsiella pneumoniae produtoras de ESBL foi elevada (25%, 28,5% e 66,7% respectivamente) e as taxas de resistência aos antimicrobianos β-lactâmicos foram maiores entre isolados produtores de ESBL. Somente o imipenem teve atividade ótima in vitro. A análise do DNA cromossomal das cepas mostrou uma variedade genética em 5 amostras (natureza multiclonal) e similaridade entre 6 (natureza clonal) produtoras de ESBL.

by Yuen Pui-yee, Joyce. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 213-221). / Acknowledgments --- p.i / Abstract --- p.ii / List of Tables --- p.ix / List of Figures --- p.xi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Problems of Pollution From Textile Industries --- p.1 / Chapter 1.2 --- Current Treatment Methods of Wastewater from Textile Industries --- p.5 / Chapter 1.3 --- Adverse Effects of Dyes on the Environment --- p.11 / Chapter 1.4 --- Classification of Dyes --- p.16 / Chapter 1.5 --- Azo Dyes --- p.17 / Chapter 1.6 --- Metabolisms of Azo Dyes in Microbial and Animal Systems --- p.21 / Chapter 1.7 --- "Toxicity, Mutagenicity and Carcinogenicity of Azo Dyes" --- p.31 / Chapter 1.8 --- Removal of Azo Dyes --- p.35 / Chapter 1.8.1 --- Biological Methods --- p.35 / Chapter 1.8.2 --- Physico-chemical Methods --- p.49 / Chapter 1.9 --- Purposes of Study --- p.50 / Chapter 2. --- Objectives --- p.53 / Chapter 3. --- Materials and Methods --- p.54 / Chapter 3.1 --- "Isolation, Selection and Characterization of Methyl Red-degrading and N,N-Dimethyl-p-phenylene diamine-degrading Microbial Isolates" --- p.54 / Chapter 3.1.1 --- "Isolation of Methyl Red-degrading Microbial Isolates from Dye- containing Wastewater, Activated Sludge and Soil" --- p.54 / Chapter 3.1.2 --- Selection of Methyl Red-degrading Microbial Isolates --- p.56 / Chapter 3.1.3 --- "Enrichment of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria from Dye-containing wastewater, Activated Sludge and Soil" --- p.59 / Chapter 3.1.4 --- "Isolation of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.60 / Chapter 3.1.5 --- Selection of N，N-Dimethyl-p-phenylene diamine-degrading Bacteria --- p.60 / Chapter 3.1.6 --- "Identification of the Selected Methyl Red-degrading and N,N- Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.61 / Chapter 3.1.7 --- Correlationship of Dry Weight and Absorbance of Cells of Selected Methyl Red-degrading Bacterial Isolates --- p.63 / Chapter 3.2 --- "Characterization of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.64 / Chapter 3.2.1 --- "Chemical Stability of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.64 / Chapter 3.2.2 --- Change of UV-Vis Spectra of Methyl Red and N，N-Dimethyl-p- phenylene diamine at Different pH and Matrixes --- p.64 / Chapter 3.2.3 --- "UV-Vis Spectra and Standard Curves of Methyl Red, N，N- Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.66 / Chapter 3.2.4 --- "HPLC separation of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.67 / Chapter 3.3 --- Methyl Red Degradation by Selected Methyl Red-degrading Microbial Isolates --- p.68 / Chapter 3.3.1 --- "Monitoring of Percentage of Methyl Red Cleaved, Degradation Value of N,N-Dimethyl-p-phenylene diamine and o- Aminobenzoic acid, and Growth of Selected Methyl Red- degrading Bacteria by Spectrophotometric Analysis " --- p.68 / Chapter 3.3.2 --- Study of Degrading Products of Methyl Red by Selected Methyl Red-degrading Isolates --- p.71 / Chapter 3.4 --- Degradation of Other Azo Dyes by Selected Methyl Red-degrading Isolates --- p.73 / Chapter 4. --- Results --- p.74 / Chapter 4.1 --- "Isolation, Selection and Characterization of Methyl Red-degrading and N,N-dimethyl-p-phenylene diamine-degrading Microbial Isolates " --- p.74 / Chapter 4.1.1 --- "Isolation of Methyl Red-degrading Microbial Isolates from Dye- containing Wastewater, Activated Sludge and Soil " --- p.74 / Chapter 4.1.2 --- Selection of Methyl Red-degrading Microbial Isolates --- p.79 / Chapter 4.1.3 --- "Enrichment of N,N-dimethyl-p-phenylene diamine-degrading Bacteria from Dye-containing Wastewater, Activated Sludge and Soil " --- p.85 / Chapter 4.1.4 --- "Isolation of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.85 / Chapter 4.1.5 --- "Selection of N,N-Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.90 / Chapter 4.1.6 --- "Identification of the Selected Methyl Red-degrading and N,N- Dimethyl-p-phenylene diamine-degrading Bacteria " --- p.90 / Chapter 4.1.7 --- Correlationship of Dry Weight and Absorbance of Cells of Selected Methyl Red-degrading Bacterial Isolates --- p.94 / Chapter 4.2 --- "Characterization of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.94 / Chapter 4.2.1 --- "Chemical Stability of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.94 / Chapter 4.2.2 --- "Change of UV-Vis Spectra of Methyl Red and N,N-Dimethyl-p- phenylene diamine at Different pH and Matrixes " --- p.108 / Chapter 4.2.3 --- "UV-Vis Spectra and Standard Curves of Methyl Red, N，N- Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.123 / Chapter 4.2.4 --- "HPLC Separation of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.129 / Chapter 4.3 --- Methyl Red Degradation by Selected Methyl Red-degrading Microbial Isolates --- p.138 / Chapter 4.3.1 --- "Monitoring of Percentage of Methyl Red Cleaved and Degradation Value of N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid and Growth of Selected Methyl Red- degrading Bacterial Isolates by Spectrophotometric Analysis " --- p.138 / Chapter 4.3.2 --- Study of Degradation Products of Methyl Red by Selected Methyl Red-degrading Isolates by HPLC --- p.175 / Chapter 4.4 --- Degradation of Other Azo Dyes by Selected Methyl Red-degrading Isolates --- p.175 / Chapter 5. --- Discussion --- p.181 / Chapter 5.1 --- "Isolation, Selection and Characterization of Methyl Red-degrading and N,N-dimethyl-p-phenylene diamine-degrading Microbial Isolates " --- p.181 / Chapter 5.1.1 --- "Isolation and Selection of Methyl Red-degrading Microbes from Dye-containing Wastewater, Activated Sludge and Soil " --- p.181 / Chapter 5.1.2 --- "Isolation and Selection of N,N-Dimethyl-p-phenylene diamine- degrading Microbial Isolates from Dye-containing Wastewater, Activated Sludge and Soil " --- p.183 / Chapter 5.1.3 --- Identification of the Selected Methyl Red-degrading and N，N- Dimethyl-p-phenylene diamine-degrading Bacteria --- p.185 / Chapter 5.1.4 --- Correlationship of Dry Weight and Absorbance of Cells of Selected Methyl Red-degrading Bacterial Isolates --- p.185 / Chapter 5.2 --- "Characterization of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.186 / Chapter 5.2.1 --- "Chemical Stability of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid in 0.05 M phosphate buffer and 0.2MHC1 " --- p.186 / Chapter 5.2.2 --- "Change of UV-Vis Spectra of Methyl Red and N,N-Dimethyl-p- phenylene diamine at Different pH and Matrixes " --- p.187 / Chapter 5.2.3 --- "Change of UV-Vis Spectra of N,N-Dimethyl-p-phenylene diamine in Different Matrixes at Different pH " --- p.187 / Chapter 5.2.4 --- "UV-Vis Spectra and Standard Curve of Methyl Red, N,N- dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.188 / Chapter 5.2.5 --- "HPLC Separation of Methyl Red, N,N-Dimethyl-p-phenylene diamine and o-Aminobenzoic acid " --- p.189 / Chapter 5.3 --- Methyl Red Degradation by Selected Methyl Red-degrading Microbial Isolates --- p.190 / Chapter 5.3.1 --- Effect of Glucose --- p.194 / Chapter 5.3.2 --- Effect of Ethanol --- p.196 / Chapter 5.3.3 --- Effect of Ammonium Sulphate --- p.198 / Chapter 5.3.4 --- Effect of Yeast Extract --- p.199 / Chapter 5.3.5 --- Effect of Phosphate Buffer (pH 7) --- p.200 / Chapter 5.3.6 --- Effect of pH --- p.201 / Chapter 5.3.7 --- Effect of Temperature at Static and Shaking Conditions --- p.203 / Chapter 5.3.8 --- Study of Degradation Products of Methyl Red by Selected Methyl Red-degrading Isolates by HPLC Analysis --- p.206 / Chapter 5.4 --- Degradation of Other Azo Dyes by Selected Methyl Red-degrading Isolates --- p.207 / Chapter 6. --- Conclusion --- p.209 / Chapter 7. --- References --- p.213 / Chapter 8. --- Appendix 1: Composition of Media --- p.222 / Appendix 2: Composition of Buffers --- p.225 / Appendix 3 --- p.228

A resistência antimicrobiana e fatores de virulência são importantes mecanismos de sobrevivência das bactérias, através deles elas se tornaram capazes de escapar da ação das adversidades do ambiente. O presente trabalho teve como objetivo principal analisar os fatores de virulência e genes de resistência de isolados de Klebsiella sp. e sua participação na manutenção de populações bacterianas resistentes em pontos na Laguna de Tramandaí. As amostragens foram realizadas em agosto de 2014 e janeiro de 2015 em quatro pontos distintos com diferentes graus de impacto ambiental. Foram analisados 272 isolados bacterianos das amostras de água da laguna de Tramandaí. Estes isolados foram submetidos a testes bioquímicos e microbiológicos para identificação e foram encontrados 37 isolados de Klebsiella sp. Estes isolados foram submetidos a testes de susceptibilidade antimicrobiana por disco-difusão, análise fenotípica de fatores de virulência e genes de resistência e pesquisa de genes de resistência (blaCTX-M, blaSHV, blaTEM) através de PCR e similaridade através do ERIC-PCR. Através da análise por MALDI-TOF, os 37 isolados foram identificados como quatro K. oxytoca, seis K. pneumoniae e 27 K. variicola. Todos os isolados foram suscetíveis a imipenem, ertapenem, piperacilina/tazobactam e polimixina B Ampicilina e amoxacilina foram os antimicrobianos com maior índice de resistência, o gene blashv foi o mais encontrado dentre os genes pesquisados. Todos os isolados apresentaram cápsula polissacarídica, 56,7% dos isolados apresentaram fímbrias do tipo 1, 56,7% demonstraram serem capazes de produzir biofilmes, 78,4% foram capazes de inativar os fatores bactericidas do soro e 81,1% sintetizaram sideróforos. A análise por ERIC-PCR e MALDI-TOF geraram dendrogramas de alta heterogeneidade e baixos índices de similaridade, indicando que diferentes populações de Klebsiella estão se mantendo naquele ambiente. Ficou evidenciado neste estudo, através dos isolados encontrados e suas características genéticas e fenotípicas de resistência bacteriana, que eles podem estar contribuindo para a manutenção e disseminação da resistência aos antimicrobianos no ambiente. / Antimicrobial resistance and virulence factors are important mechanisms for the survival of bacteria, through which they have become capable of escaping from the adversities of the environment. The present work had as main objective to analyze the virulence factors and resistance genes of Klebsiella sp. And its participation in the maintenance of resistant bacterial populations in points in Tramandaí Lagoon. Samplings were carried out in August 2014 and January 2015 at four different points with different degrees of environmental impact. A total of 272 bacterial isolates from the Tramandaí lagoon water samples were analyzed. These isolates were submitted to biochemical and microbiological tests for identification and 37 isolates of Klebsiella sp. These isolates were submitted to antimicrobial susceptibility tests by disc-diffusion, phenotypic analysis of virulence factors and and resistance gene (blaCTX-M, blaSHV, blaTEM) search by PCR and similarity through ERIC-PCR. Through the use of MALDI-TOF, the 37 isolates were identified as four of K. oxytoca, six of K. pneumoniae and 27 of K. variicola. All the isolates were suscetible to imipenem, ertapenem, piperacilin/tazobactam e polimixin B Ampicillin and amoxicillin were the most resistant antimicrobials, the blaSHV gene was the most found among the genes studied. All isolates presented a polysaccharide capsule, 56.7% of the isolates had type 1 fimbriae, 56.7% were able to produce biofilms, 78.4% were able to inactivate serum bactericidal factors and 81.1% synthesized siderophores. Analysis by ERIC-PCR and MALDI-TOF generated dendrograms with high diversity and low similarity indexes, indicating that different populations of Klebsiella are remaining in that environment. It was evidenced in this study, through the isolates found and their genetic and phenotypic characteristics of bacterial resistance that they may be contributing to the maintenance and dissemination of antimicrobial resistance.

by Li Ho Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 213-245). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT --- p.viii / TABLE OF CONTENTS --- p.xi / Chapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Effector Cells Mediating Anti-Tumour Immunity --- p.5 / Chapter 1.2.1 --- Cytotoxic T Lymphocytes --- p.6 / Chapter 1.2.2 --- Natural Killer Cells --- p.7 / Chapter 1.2.3 --- Lymphokine-Activated Killer Cells --- p.9 / Chapter 1.2.4 --- Macrophages --- p.10 / Chapter 1.2.5 --- Tumour-Infiltrating Lymphocytes --- p.12 / Chapter 1.3 --- Current Modalities for Cancer Treatment --- p.13 / Chapter 1.3.1 --- Surgery --- p.13 / Chapter 1.3.2 --- Radiation Therapy --- p.14 / Chapter 1.3.3 --- Chemotherapy --- p.14 / Chapter 1.3.4 --- Biologic Therapy --- p.15 / Chapter 1.4 --- Biological Response Modifiers as Cancer Therapeutics --- p.17 / Chapter 1.4.1 --- Monoclonal Antibodies --- p.20 / Chapter 1.4.2 --- Differentiating Agents --- p.21 / Chapter 1.4.3 --- Cytokines --- p.24 / Chapter 1.4.4 --- Bioactive Plant Polysaccharides --- p.30 / Chapter 1.5 --- Bacterial Polysaccharides as Potential Immunomodulators and Anti-Tumour Agents --- p.36 / Chapter 1.5.1 --- General Properties of Bacterial Polysaccharides --- p.36 / Chapter 1.5.2 --- Structure and Function of Bacterial Polysaccharides --- p.37 / Chapter 1.5.3 --- Immunomodulatory and Anti-Tumour Activities of Bacterial Polysaccharides --- p.39 / Chapter 1.5.4 --- General Properties of Klebsiella K7 Capsular Antigen --- p.41 / Chapter 1.6 --- Aims and Scopes of the Investigation --- p.44 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.46 / Chapter 2.1 --- Materials --- p.47 / Chapter 2.1.1 --- Animals --- p.47 / Chapter 2.1.2 --- Klebsiella pneumoniae Serotype7 --- p.47 / Chapter 2.1.3 --- Agar Medium for Cultivation of Klebsiella pneumoniae Serotype7 --- p.47 / Chapter 2.1.4 --- Cell Lines --- p.47 / Chapter 2.1.5 --- "Buffers, Culture Medium and Other Reagents" --- p.49 / Chapter 2.1.6 --- Antibodies --- p.54 / Chapter 2.1.7 --- Radioisotopes --- p.55 / Chapter 2.1.8 --- Recombinant Cytokines --- p.55 / Chapter 2.1.9 --- Oligonucleotide Primers and Internal Probes --- p.56 / Chapter 2.1.10 --- Reagents and Solutions for Gene Expression Analysis --- p.59 / Chapter 2.2 --- Methods --- p.64 / Chapter 2.2.1 --- "Extraction, Purification and Characterization of Klebsiella K7 Capsular Antigen" --- p.54 / Chapter 2.2.1.1 --- Extraction and Purification of K7 Capsular Antigen --- p.64 / Chapter 2.2.1.2 --- Gel Filtration of K7 Capsular Antigen --- p.65 / Chapter 2.2.1.3 --- Characterization of K7 Capsular Antigen --- p.67 / Chapter 2.2.1.4 --- Determination of the Bio-Toxicity of K7 Capsular Antigen --- p.67 / Chapter 2.2.1.5 --- In Vitro Cytotoxicity of K7 Capsular Antigen on Splenocytes --- p.53 / Chapter 2.2.2 --- Assay for the Hematopoietic and Mitogenic Activities of Klebsiella K7 Capsular Antigen --- p.69 / Chapter 2.2.2.1 --- Isolation and Preparation of Cells --- p.69 / Chapter 2.2.2.2 --- In Vitro Lymphocyte Transformation Assay --- p.70 / Chapter 2.2.2.3 --- In Vitro Assay of Thymocyte Proliferation --- p.70 / Chapter 2.2.2.4 --- Depleting Mouse T Cells by Anti-Thy-1.2 Antibody Plus Complement Treatment --- p.71 / Chapter 2.2.2.5 --- Depleting Mouse B Cells by Anti-mouse B Cell Antibody Plus Complement Treatment --- p.71 / Chapter 2.2.2.6 --- Depleting Macrophages from Spleen Cell Suspension --- p.71 / Chapter 2.2.2.7 --- Flow Cytometric Analysis of Different Cell Populations from Splenocytes --- p.72 / Chapter 2.2.2.8 --- In Vitro Assay of rmIL-3-Stimulated Proliferation of Murine Bone Marrow Cells --- p.73 / Chapter 2.2.2.9 --- In Vitro Cytotoxicity of K7 Capsular Antigen on Bone Marrow Cells --- p.73 / Chapter 2.2.2.10 --- Colony Assay of Murine Bone Marrow Cells --- p.73 / Chapter 2.2.2.11 --- Assay of Differentiation of Murine Bone Marrow Cells --- p.74 / Chapter 2.2.3 --- Assay for Macrophage Activating Activities of Klebsiella K7 Capsular Antigen --- p.75 / Chapter 2.2.3.1 --- Preparation of Murine Peritoneal Exudate Cells (PEC) --- p.75 / Chapter 2.2.3.2 --- Assay of Phagocytic Activity of Peritoneal Macrophages --- p.75 / Chapter 2.2.3.3 --- In Vitro Macrophage-Mediated Cytostatic Activity --- p.76 / Chapter 2.2.3.4 --- Nitric Oxide (NO) Production of Peritoneal Macrophages --- p.76 / Chapter 2.2.3.5 --- In Vivo Migration of Macrophages --- p.77 / Chapter 2.2.3.6 --- Tumour Necrosis Factor (TNF) Production by Peritoneal Macrophages --- p.77 / Chapter 2.2.3.7 --- TNF Bioassay --- p.78 / Chapter 2.2.3.8 --- Gene Expression Analysis in Peritoneal Macrophages --- p.78 / Chapter 2.2.3.8.1 --- Preparation of Cell Lysate --- p.78 / Chapter 2.2.3.8.2 --- RNA Isolation --- p.79 / Chapter 2.2.3.8.3 --- Reverse Transcription of RNA --- p.80 / Chapter 2.2.3.8.4 --- Polymerase Chain Reaction (PCR) --- p.80 / Chapter 2.2.3.8.5 --- Agarose Gel Electrophoresis --- p.82 / Chapter 2.2.3.8.6 --- 3' End Labelling of Oligonucleotide Probes --- p.82 / Chapter 2.2.3.8.7 --- Dot Blot Hybridization --- p.83 / Chapter 2.2.3.8.8 --- DIG Chemiluminescent Detection --- p.83 / Chapter 2.2.4 --- Assay for Anti-Tumour Activities of Klebsiella K7 Capsular Antigen --- p.85 / Chapter 2.2.4.1 --- Assay of Tumour Cell Proliferation --- p.85 / Chapter 2.2.4.2 --- Assay of Anti-Tumour Activity In Vivo --- p.85 / Chapter 2.2.4.3 --- Assay of Lymphokine-Activated Killer Cell Activity --- p.85 / Chapter 2.2.4.4 --- Assay of Natural Killer Cell Activity --- p.87 / Chapter 2.2.4.5 --- Assay for Differentiation-Associated Characteristics of Myeloid Leukemia Cells --- p.88 / Chapter 2.2.4.5.1 --- Determination of the Viability of Myeloid Leukemia JCS cells --- p.88 / Chapter 2.2.4.5.2 --- Assessment of Cell Morphology --- p.88 / Chapter 2.2.4.5.3 --- Surface Antigen Immunophenotyping --- p.88 / Chapter 2.2.4.5.4 --- Assay of Non-specific Esterase Activity --- p.89 / Chapter 2.2.4.5.5 --- Assay of Phagocytosis --- p.90 / Chapter 2.2.5 --- Statistical Analysis --- p.90 / Chapter CHAPTER 3: --- "EXTRACTION, PURIFICATION & CHARACTERIZATION OF KLEBSIELLA K7 CAPSULAR ANTIGEN" --- p.91 / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Extraction and Purification of K7 Capsular Antigen from Klebsiella pneumoniae Serotype7 --- p.94 / Chapter 3.2.2 --- Gel Filtration Chromatography of K7 Capsular Antigen --- p.94 / Chapter 3.2.3 --- Characterization of K7 Capsular Antigen --- p.97 / Chapter 3.2.4 --- Determination of Bio-toxicity and Cellular Toxicity of K7 Capsular Antigen --- p.97 / Chapter 3.3 --- Discussion --- p.101 / Chapter CHAPTER 4: --- HEMATOPOIETIC AND MITOGENIC ACTIVITIES OF KLEBSIELLA K7 CAPSULAR ANTIGEN --- p.103 / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Results --- p.107 / Chapter 4.2.1 --- Effect of K7 Capsular Antigen on the In Vitro Proliferation of Murine Bone Marrow Cells --- p.107 / Chapter 4.2.2 --- Effect of K7 Capsular Antigen on rmIL-3-Stimulated Proliferation of Murine Bone Marrow Cells --- p.107 / Chapter 4.2.3 --- Effect of K7 Capsular Antigen on the In Vitro Differentiation of Murine Bone Marrow Cells --- p.112 / Chapter 4.2.4 --- Effect of K7 Capsular Antigen on the In Vitro Murine Bone Marrow Colony Formation --- p.112 / Chapter 4.2.5 --- Effect of K7 Capsular Antigen on the Viability of Splenocytes In Vitro --- p.117 / Chapter 4.2.6 --- In Vitro Mitogenic Effect of K7 Capsular Antigen on Splenocytes and Thymocytes --- p.117 / Chapter 4.2.7 --- Characterization of the Lymphocyte Population(s) Responding to K7 Capsular Antigen --- p.121 / Chapter 4.2.8 --- Effect of Polymyxin B Sulphate on the Mitogenic Activity of K7Capsular Antigen --- p.125 / Chapter 4.2.9 --- In Vivo Mitogenic Activity of K7 Capsular Antigen --- p.125 / Chapter 4.2.10 --- Flow Cytometric Analysis of Splenocytes from K7 Capsular Antigen-Treated Mice --- p.128 / Chapter 4.2.11 --- In Vitro Co-mitogenic Activity of K7 Capsular Antigen --- p.128 / Chapter 4.3 --- Discussion --- p.132 / Chapter CHAPTER 5: --- ACTIVATION OF MACROPHAGES BY KLEBSIELLA K7CAPSULAR ANTIGEN --- p.136 / Chapter 5.1 --- Introduction --- p.137 / Chapter 5.2 --- Results --- p.140 / Chapter 5.2.1 --- Effect of K7 Capsular Antigen on the Phagocytic Activity of Macrophages In Vitro --- p.140 / Chapter 5.2.2 --- Effect of K7 Capsular Antigen on the In Vivo Migration of Macrophages --- p.140 / Chapter 5.2.3 --- Effect of K7 Capsular Antigen on the In Vitro Cytostatic Activity of Picolinic Acid (PLA)-Activated Macrophages --- p.144 / Chapter 5.2.4 --- Effect of K7 Capsular Antigen on Nitric Oxide (NO) Production by Macrophages In Vitro --- p.144 / Chapter 5.2.5 --- Effect of K7 Capsular Antigen on the In Vitro Production of Tumour Necrosis Factor (TNF) by Macrophages --- p.148 / Chapter 5.2.6 --- Effect of K7 Capsular Antigen on the In Vitro Induction of Gene Expression in Macrophages --- p.148 / Chapter 5.3 --- Discussion --- p.166 / Chapter CHAPTER 6: --- ANTI-TUMOUR ACTIVITIES OF KLEBSIELLA K7 CAPSULAR ANTIGEN --- p.171 / Chapter 6.1 --- Introduction --- p.172 / Chapter 6.2 --- Results --- p.174 / Chapter 6.2.1 --- Effect of K7 Capsular Antigen In Vitro Growth of Various Tumour Cell Lines --- p.174 / Chapter 6.2.2 --- Effect of K7 Capsular Antigen on the In Vitro Growth of EAT Cells --- p.174 / Chapter 6.2.3 --- Effect of K7 Capsular Antigen on the Activation of LAK cells --- p.185 / Chapter 6.2.4 --- Effect of K7 Capsular Antigen on the Activation of NK Cells --- p.185 / Chapter 6.2.5 --- Effect of K7 Capsular Antigen on the Induction of Monocytic Differentiation of the Murine Myeloid Leukemia JCS Cells --- p.185 / Chapter 6.2.5.1 --- Effect of K7 Capsular Antigen on the Induction of Morphological Changes of JCS Cells --- p.188 / Chapter 6.2.5.2 --- Effect of K7 Capsular Antigen on the Expression of Macrophage Differentiation Antigen on Leukemia JCS Cells --- p.192 / Chapter 6.2.5.3 --- Effect of K7 Capsular Antigen on the Induction of Non-specific Esterase Activity in Leukemia JCS Cells --- p.192 / Chapter 6.2.5.4 --- Effect of K7 Capsular Antigen on the Stimulation of Phagocytic Activity in Leukemia JCS Cells --- p.192 / Chapter 6.3 --- Discussion --- p.198 / Chapter CHAPTER 7: --- CONCLUSIONS & FUTURE PERSPECTIVES --- p.202 / REFERENCES --- p.213

by Ho Cheong Yip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 152-164). / ACKNOWLEDGMENTS --- p.I / ABBREVIATIONS --- p.II / ABSTRACT --- p.VI / CHAPTER / Chapter 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Immunomodulators of biological origin --- p.1 / Chapter 1.2 --- Effector cells involved in anti-tumor immunity --- p.3 / Chapter 1.2.1 --- Cytotoxic T lymphocytes --- p.3 / Chapter 1.2.2 --- Macrophages --- p.4 / Chapter 1.2.3 --- Natural killer cells --- p.5 / Chapter 1.2.4 --- Lymphokine-activated killer cells --- p.7 / Chapter 1.2.5 --- Tumor-infiltrating lymphocytes --- p.9 / Chapter 1.3 --- Cytokines involved in immunomodulation and anti-tumor immunity --- p.11 / Chapter 1.3.1 --- Interleukin-1 --- p.11 / Chapter 1.3.2 --- Interleukin-2 --- p.13 / Chapter 1.3.3 --- Interleukin-3 --- p.14 / Chapter 1.3.4 --- Tumor necrosis factor-alpha --- p.15 / Chapter 1.3.5 --- Interferons --- p.18 / Chapter 1.4 --- Carbohydrates used as potential immunopotentiating agents --- p.19 / Chapter 1.5 --- General properties of K1 capsular antigen --- p.20 / Chapter 2 --- AIM AND SCOPE OF THIS DISSERTATION --- p.22 / Chapter 3 --- MATERIALS AND METHODS --- p.24 / Chapter 3.1 --- Materials --- p.24 / Chapter 3.2 --- Methods --- p.33 / Chapter (I) --- "Extraction, purification and characterization of K1 capsular antigen from Klebsiella pneumoniae" / Chapter 3.2.1 --- Extraction and purification of Kl capsular antigen --- p.33 / Chapter 3.2.2 --- Gel filtration of K1 capsular antigen --- p.35 / Chapter 3.2.3 --- Characterization of K1 capsular antigen --- p.35 / Chapter 3.2.3.1 --- Determination of carbohydrate and protein contents --- p.35 / Chapter 3.2.3.2 --- Determination of uronic acid content --- p.35 / Chapter 3.2.4 --- Determination of bio-toxicity of K1 capsular antigen --- p.35 / Chapter 3.2.5 --- Treatment of K1 capsular antigen with sodium periodate --- p.36 / Chapter 3.2.6 --- Treatment of K1 capsular antigen with sodium hydroxide followed by acetic acid neutralization --- p.36 / Chapter (II) --- Isolation and preparation of cells / Chapter 3.2.7 --- Murine splenocytes --- p.37 / Chapter 3.2.8 --- Removal of red blood cells and dead cells using Ficoll-paque gradient method --- p.37 / Chapter 3.2.9 --- Murine thymocytes --- p.37 / Chapter 3.2.10 --- Murine peritoneal exudate cells (PEC) --- p.38 / Chapter 3.2.11 --- Bone marrow cells --- p.38 / Chapter (III) --- Assays of immunomodulatory activities of K1 capsular antigen on lymphocytes / Chapter 3.2.12 --- In vitro mitogenic assay --- p.39 / Chapter 3.2.13 --- In vivo mitogenic assay --- p.39 / Chapter 3.2.14 --- "In vitro co-mitogenic assay of K1 capsular antigen with Polymyxin B sulfate, LPS or Con A" --- p.40 / Chapter 3.2.15 --- In vitro mitogenic effect of K1 capsular antigen on lymphocyte sub-populations --- p.40 / Chapter 3.2.16 --- Assay of murine interleukin-2 --- p.41 / Chapter 3.2.17 --- Assay of murine interleukin-1 --- p.41 / Chapter (IV) --- Assays of immunomodulatory activities of K1 capsular antigen on macrophages and bone marrow cells / Chapter 3.2.18 --- In vivo migration of macrophages --- p.42 / Chapter 3.2.19 --- Assay of interleukin-1 produced from murine macrophages --- p.42 / Chapter 3.2.20 --- Assay of nitric oxide produced from murine macrophages --- p.43 / Chapter 3.2.21 --- Assay of proliferation of murine bone marrow cells --- p.44 / Chapter 3.2.22 --- Assay of differentiation of murine bone marrow cells --- p.44 / Chapter (V) --- Assays of anti-tumor activities of K1 capsular antigen / Chapter 3.2.23 --- In vitro cytostatic effect of K1 capsular antigen on murine tumor cell lines --- p.45 / Chapter 3.2.24 --- In vivo anti-tumor activities of K1 capsular antigen --- p.45 / Chapter 3.2.25 --- In vitro stimulation of TNF-α-like factor release from thioglycollate-elicited murine peritoneal macrophages by K1 capsular antigen --- p.46 / Chapter 3.2.26 --- Effects of K1 capsular antigen on TNF-α production in normal mice in vivo --- p.47 / Chapter 3.2.27 --- Effects of K1 capsular antigen on TNF-α production as well as EAT growth in vivo --- p.48 / Chapter 3.2.28 --- In vitro induction of macrophage-mediated cytostasis on tumor cells --- p.48 / Chapter 3.2.29 --- In vitro induction of macrophage-mediated cytolysis on tumor cells --- p.49 / Chapter 3.2.30 --- In vivo induction of alloreactive cytotoxic T cells --- p.49 / Chapter 3.2.31 --- In vitro induction of lymphokine-activated killer cell activity --- p.50 / Chapter 3.2.32 --- In vivo induction of lymphokine-activated killer cell activity --- p.51 / Chapter 3.2.33 --- In vivo induction of lymphokine-activated killer cell activity in tumor-bearing mice --- p.52 / Chapter 3.2.34 --- In vivo induction of lymphokine-activated killer cell activity in tumor-bearing mice with in vitro culture --- p.52 / Chapter 3.2.35 --- Phenotypic characterization of LAK cells by flow cytometry --- p.53 / Chapter 3.2.36 --- Winn-type tumor-inhibition of LAK cells --- p.54 / Chapter 3.2.37 --- Adoptive transfer of LAK cells to tumor-bearing mice --- p.54 / Chapter 3.2.38 --- In vivo stimulation of tumor-infiltrating lymphocytes --- p.55 / Chapter 3.2.39 --- Statistical analysis --- p.57 / Chapter 4 --- "EXTRACTION, PURIFICATION AND CHARACTERIZATION OF K1 CAPSULAR ANTIGEN FROM KLEBSIELLA PNEUMONIAE" / Introduction --- p.58 / Results --- p.59 / Chapter 4.1 --- Extraction and purification of Kl capsular antigen from Klebsiella pneumoniae --- p.59 / Chapter 4.2 --- Gel filtration of Kl capsular antigen --- p.59 / Chapter 4.3 --- Characterization of Kl capsular antigen --- p.62 / Chapter 4.4 --- Determination of toxicity (LC50) of Kl capsular antigen by brine shrimp assay --- p.62 / Discussion --- p.62 / Chapter 5 --- IMMUNOMODULATORY ACTIVITIES OF K1 CAPSULAR POLYSACCHARIDE ANTIGEN FROM KLEBSIELLA PNEUMONIAE / Introduction --- p.67 / Results --- p.69 / Chapter 5.1 --- Mitogenic activity of Kl capsular antigen on murine lymphocytes in vitro --- p.69 / Chapter 5.2 --- Mitogenic activity of K1 capsular antigen on murine lymphocytes in vivo --- p.69 / Chapter 5.3 --- Evidences to support the mitogenic activity of K1 capsular antigen is due to its polysaccharide structure rather than due to the contamination by LPS --- p.74 / Chapter 5.4 --- Effect of Kl capsular antigen on IL-2 production from murine lymphocytes in vitro --- p.79 / Chapter 5.5 --- Effect of K1 capsular antigen on IL-1 -like factor production from murine thymocytes in vitro --- p.79 / Chapter 5.6 --- Immunopotentiating activities of Kl capsular antigen on macrophages --- p.85 / Chapter 5.6.1 --- In vivo migration of macrophages in K1-treated mice --- p.85 / Chapter 5.6.2 --- Effect ofKl capsular antigen on the macrophage EL-1-like factor production in vitro --- p.85 / Chapter 5.6.3 --- Effect ofKl capsular antigen on the macrophage nitric oxide (NO) production in vitro --- p.85 / Chapter 5.7 --- Effects of Kl capsular antigen on the proliferation and differentiation of murine bone marrow cells --- p.89 / Discussion ´ب --- p.94 / Chapter 6 --- ANTI-TUMOR ACTIVITIES OF K1 CAPSULAR POLYSACCHARIDE ANTIGEN FROM KLEBSIELLA PNEUMONIAE / Introduction --- p.100 / Results --- p.102 / Chapter 6.1 --- In vitro cytostatic effects of Kl capsular antigen on murine tumor cell lines --- p.102 / Chapter 6.2 --- In vivo anti-tumor activities of Kl capsular antigen --- p.102 / Chapter 6.3 --- Effects of Kl capsular antigen on TNF-α production and on EAT growth in vivo --- p.110 / Chapter 6.4 --- In vitro induction of macrophage-mediated cytostatic effect on tumor cells by K1 capsular antigen --- p.110 / Chapter 6.5 --- In vitro induction of macrophage-mediated cytolytic effect on tumor cells by K1 capsular antigen --- p.115 / Chapter 6.6 --- Effect of Kl capsular antigen on the activation of alloreactive cytotoxic T cells --- p.115 / Chapter 6.7 --- Effect of Kl capsular antigen on the activation of lymphokine- activated killer (LAK) cells in vitro and in vivo --- p.115 / Chapter 6.8 --- Phenotypic characterization of LAK cells by flow cytometry --- p.123 / Chapter 6.9 --- Winn-type tumor inhibition assay of LAK cells --- p.126 / Chapter 6.10 --- Adoptive transfer of LAK cells to tumor-bearing mice --- p.132 / Chapter 6.11 --- Effect ofKl capsular antigen on the activation of tumor- infiltrating lymphocytes (TILs) in tumor-bearing mice --- p.132 / Discussion --- p.136 / Chapter 7 --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.146 / BIBLIOGRAPHY --- p.152

by Chen Paul. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 141-150). / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Immunomodulation --- p.1 / Chapter 1.2 --- Effector cells mediating anti-tumour immunity --- p.1 / Chapter 1.2.1 --- Cytotoxic T Lymphocytes --- p.3 / Chapter 1.2.2 --- Macrophages --- p.4 / Chapter 1.2.3 --- Natural Killer Cells --- p.5 / Chapter 1.2.4 --- Lymphokine-activated Killer (LAK) --- p.6 / Chapter 1.3 --- Cytokines as immunomodulators in cancer therapy --- p.7 / Chapter 1.3.1 --- Tumour Necrosis Factor-α (TNF-α) --- p.7 / Chapter 1.3.2 --- Interleukin-1 (IL-1) --- p.9 / Chapter 1.3.3 --- Interleukin-2 (IL-2) --- p.9 / Chapter 1.3.4 --- Granulocytes/Macrophages Colony-Stimulating Factors --- p.10 / Chapter 1.4 --- Polysaccharides as potential immunostimulating agents --- p.11 / Chapter 1.5 --- General properties of Klebsiella pneumoniae --- p.12 / Chapter 2. --- AIM AND SCOPE OF THIS DISSERTATION --- p.16 / Chapter 3. --- MATERIALS AND METHODS --- p.18 / Chapter 3.1 --- Materials --- p.18 / Chapter 3.1.1 --- Animals --- p.18 / Chapter 3.1.2 --- Klebsiella pneumoniae K24 --- p.18 / Chapter 3.1.3 --- Cell lines --- p.18 / Chapter 3.1.4 --- "Buffer, Culture media and Chemicals" --- p.19 / Chapter 3.2 --- Methods --- p.27 / Chapter 3.2.1 --- Extraction and Characterization of Klebsiella pneumoniae K24 Capsular Polysaccharide (K24 CPS) --- p.27 / Chapter 3.2.2 --- Assays of Immunomodulatory Activities of K24 CPS on Lymphocytes --- p.30 / Chapter 3.2.3 --- Assays of Immunomodulatory Effect of K24 CPS on Macrophages --- p.34 / Chapter 3.2.4 --- Assays of Anti-Tumour Activities of K24 CPS --- p.39 / Chapter 3.2.5 --- Assays of the Effects of K24 CPS on the Proliferation and Differentiation of Murine Bone Marrow Cells --- p.54 / Chapter 3.2.6 --- Assays of the Immunorestorative Activities of K24 CPS --- p.56 / Chapter 4. --- EXTRACTION AND CHARACTERIZATION OF KLEBSIELLA PNEUMONIAE K24 CAPSULAR POLYSACCHARIDE (K24 CPS) --- p.59 / Chapter 4.1 --- Preparation of Klebsiella pneumoniae K24 CPS Capsular Polysaccharide (K24 CPS) --- p.59 / Chapter 4.2 --- Acetic Acid Treatment of K24 CPS --- p.59 / Chapter 4.3 --- Gel Filtration --- p.59 / Chapter 4.4 --- Carbohydrate and Protein contents of K24 CPS --- p.61 / Chapter 4.5 --- Cytotoxicity Assay using Artemia franciscana (Brine Shrimp) --- p.61 / Chapter 5. --- IMMUNOMODULATORY EFFECTS OF K24 CPS --- p.68 / Chapter 5.1 --- The Effect of K24 CPS in vitro Mitogenic Assay of K24 CPS using Murine Splenocytes --- p.68 / Chapter 5.2 --- The in vivo Mitogenic Effect of K24 CPS on Murine Splenic Lymphocytes --- p.73 / Chapter 5.3 --- The Effect of K24 CPS on the Production of Interleukin-2 (IL-2)-like substance by Murine Splenocytes --- p.73 / Chapter 5.4 --- The effect of K24 CPS on the in vitro Stimulation of Murine Macrophage Nitric Oxide (NO) Production --- p.73 / Chapter 5.5 --- The effect of K24 CPS on the in vitro Stimulation of Macrophage Interleukin-1-like Production --- p.77 / Chapter 5.6 --- The effect of K24 CPS on in vivo Migration of Macrophage --- p.82 / Chapter 5.7 --- The effect of K24 CPS in vitro Stimulation of Macrophage Tumour Necrosis Factor- a (TNF-a) Production --- p.82 / Chapter 6. --- IN VITRO ANTI-TUMOUR EFFECT OF K24 CPS --- p.89 / Chapter 6.1 --- The in vitro Cytostatic effect of K24 CPS on the Suppression of EAT growth --- p.89 / Chapter 6.2 --- The effect of K24 CPS on cell cycle of EAT cells --- p.89 / Chapter 6.3 --- Study of the cytostatic effect of K24 CPS on EAT cells using Western Analysis --- p.93 / Chapter 6.3.1 --- Pattern of Phosphotyrosine Proteins --- p.93 / Chapter 6.3.2 --- Pattern of Phosphoserine Proteins --- p.96 / Chapter 6.3.3 --- Pattern of Phosphothreonine Proteins --- p.96 / Chapter 6.3.4 --- Level of c-fos --- p.99 / Chapter 6.3.5 --- Level of c-jun --- p.99 / Chapter 6.3.6 --- Level of c-myc --- p.102 / Chapter 7. --- THE IN VIVO ANTI-TUMOUR ACTIVITIES OF K24 CPS --- p.103 / Chapter 7.1 --- The effect of K24 CPS on the In vivo Suppression of EAT growth --- p.103 / Chapter 7.2 --- The effect of K24 CPS on the survival of EAT-bearing mice --- p.103 / Chapter 7.3 --- The effect of K24 CPS on the in vivo induction of Natural Killer (NK) Cell Cytotoxicity --- p.111 / Chapter 7.4 --- The effect of K24 CPS in vitro induction of Lymphokine-activated Killer (LAK) Cell Cytotoxicity --- p.111 / Chapter 7.5 --- The effect of K24 CPS on the in vivo Induction of Lymphokine-activated Killer (LAK) Cell Cytotoxicity --- p.114 / Chapter 7.6 --- The effect of K24 CPS on the endogenous production of TNF-α --- p.114 / Chapter 7.7 --- The effect of K24 CPS on the endogenous TNF-α production and EAT growthin vivo --- p.117 / Chapter 8. --- THE IMMUNORESTORATIVE ACTIVITIES OF K24 CPS --- p.122 / Chapter 8.1 --- The in vivo Immunorestorative Activities of K24 CPS in EAT-bearing Mice --- p.122 / Chapter 8.2 --- The in vitro Immunorestorative Activities of K24 CPS in Mice bearing 10-day-old- EAT --- p.122 / Chapter 9. --- THE EFFECT OF K24 CPS IN VITRO INDUCTION OF MURINE BONE MARROW CELLS PROLIFERATION AND DIFFERENTIATION --- p.126 / Chapter 9.1 --- The effect of K24 CPS in vitro induction of Murine Bone Marrow Cells Proliferation --- p.126 / Chapter 9.2 --- The effect of K24 CPS in vitro induction of Murine Bone Marrow Cells Differentiation --- p.126 / Chapter 10. --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.135 / Chapter 11. --- BIBLIOGRAPHY --- p.141