nÃo hà / A descarga de efluentes das indÃstrias tÃxteis para corpos aquosos Ã, correntemente, uma das maiores preocupaÃÃes dos ambientalistas em funÃÃo dos corantes sintÃticos usados para colorir os tecidos poluindo assim o ambiente. A aplicaÃÃo de tratamentos biolÃgicos, sobretudo com a utilizaÃÃo de bactÃrias, apresenta-se como um dos mais viÃveis economicamente, sendo um dos sistemas mais utilizados para descolorir efluentes coloridos. Neste sentido, estudos foram realizados testes para a remoÃÃo de cor dos corantes Remazol Brilliant Blue R, Orange G e Orange II utilizando cepas de Escherichia coli e de Aeromonas hydrophila, isoladas e em cultura mista. Primeiramente foi feito o isolamento das cepas bacterianas de trÃs ambientes diferentes. Em seguida, foram feitos testes para verificar qual concentraÃÃo do corante seria limite para o crescimento de cada microrganismo. AlÃm dos testes de descoloraÃÃo tambÃm foram monitorados outros parÃmetros como o pH, biomassa, remoÃÃo de DQO, proteÃnas totais e toxicidade dos metabÃlitos formados. A cepa Escherichia coli, isolada do ambiente marinho, foi capaz de descolorir concentraÃÃes de 2, 5 e 2 mg L-1, respectivamente, para os corante RBBR, Orange G e Orange II, enquanto que a cepa E. coli, isolada do efluente tÃxtil, descoloriu nas concentraÃÃes de 5, 0,5 e 5 mg L-1, respectivamente. A bactÃria Aeromonas hydrophila descoloriu respectivamente nas concentraÃÃes de 10, 5 e 5 mg L-1, enquanto que o consÃrcio das trÃs bactÃrias descoloriu na concentraÃÃo de 5 mg L-1 para os trÃs corantes testados individualmente. Nessas condiÃÃes de cultivo a diminuiÃÃo na taxa de DQO variou entre 45 e 69%, com a menor taxa observada no ensaio contendo A. hydrophila e o corante Orange II (45%) e a maior taxa de remoÃÃo no ensaio contendo o consÃrcio e o corante RBBR (69%). Bioensaios utilizando o microcrustÃceo Artemia salina mostraram que durante o processo de descoloraÃÃo foram produzidos metabÃlitos com caracterÃsticas recalcitrantes. Os resultados demonstram que as bactÃrias Escherichia coli e Aeromonas hydrophila apresentam potencial biotecnolÃgico na descoloraÃÃo de corantes tÃxteis, desde que sejam utilizadas baixas concentraÃÃes dos corantes / The discharge of effluents from textile industries for water bodies is currently a major concern for environmentalists as a function of synthetic dyes used to color fabrics thus polluting the environment. Biological treatments, especially with the use of bacteria, present themselves as the most economically viable and widely used to decolorize colored effluents. Thus, studies were conducted to test the color removal of dyes Remazol Brilliant Blue R, Orange G and Orange II using isolated and in mixed culture strains of Escherichia coli and Aeromonas hydrophila. Firstly, the isolation of bacterial strains from three different environments was made. Then, tests were performed to verify that the dye concentration would limit the growth of each microorganism. In addition to tests of decolorization, other parameters such as pH, biomass, COD removal, total protein and toxicity of metabolites were also monitored. The Escherichia coli strain isolated from the marine environment was able to decolorize concentrations of 2, 5 and 2 mg L-1, respectively, for the RBBR dye, Orange G and Orange II dyes, while the strain E. coli isolated from textile effluent, decolorized in concentrations of 5, 0.5 and 5 mg L-1, respectively. The bacteria Aeromonas hydrophila decolorized, respectively, at 10, 5 and 5 mg L-1, while the consortium of three bacteria decolorized at concentration of 5 mg L-1 for the three dyes tested individually. In these culture conditions the decrease in the rate of COD ranged from 45% to 69% with the lowest rate observed in the assay containing A. hydrophila and dye Orange II (45%) and the highest removal rate in the test containing the dye RBBR and the consortium (69%). Bioassays using Artemia salina showed that during the process of decolorization metabolites were produced with recalcitrant characteristics. The results show that the bacteria Escherichia coli and Aeromonas hydrophila have biotechnological potential in textile dyes, provided that they use low dye concentrations decolorizing

The main aim of the study was to analyse the impact of physico-chemical parameters on drinking water quality, biofilm formation and antibiotic resistant bacteria in the drinking water distribution system in Mafikeng, North West Province, South Africa. Another objective was to isolate and characterise Pseudomonas and Aeromonas species from drinking water distribution system and detect the virulence gene determinants in the isolates by PCR analysis. The physico-chemical data obtained were subjected to statistical analysis using Excel 2007 (Microsoft) and SPSS (version 14.0) programmes. Pearson’s correlation product of the moment was used to determine the correlation between EC, TDS, pH and temperature. The two tailed test of significance (p<0.05) was used in order to determine the significance of the result. Antibiotic susceptibility tests were performed using Kirby-Bauer disk diffusion method. Cluster analysis based on the antibiotic inhibition zone diameter data of different organisms isolated from different sites was determined and was expressed as dendograms using Wards algorithm and Euclidean distance of Statistica version 7. Specific PCR was used to determine the identities of presumptive Pseudomonas and Aeromonas species through amplification of the gyrB, toxA and the ecfX gene fragments. Virulence gene determinants for the confirmed Pseudomonas and Aeromonas species were detected by amplifying the exoA, exoS and exoT genes and the aerA and hylH gene fragments, respectively. A Gene Genius Bio imaging system (Syngene, Synoptics; UK) was used to capture the image using GeneSnap (version 3.07.01) software (Syngene, Synoptics; UK) to determine the relative size of amplicons. Physico-chemical parameters were monitored from three drinking water sources three times a week and bacteriological quality was monitored weekly for four months from raw and treated drinking water. Water samples were analysed for pH, temperature, total dissolved solids (TDS) and electric conductivity (EC). Bacterial consortia from drinking water samples were isolated using selective media and enumerated. The results revealed a good chemical quality of water. However, the microbial quality of the water is not acceptable for human consumption due to the presence of Pseudomonas, Aeromonas, faecal coliforms (FC), total coliforms (TC) and Heterotrophic bacteria. The results showed that the drinking water is slightly alkaline with pH value ranging between7.7 to 8.32. What is of concern was the microbial quality of the water. Pseudomonas sp., faecal coliforms (FC), total coliforms (TC) and heterotrophic bacteria were present in some of the treated water samples. The most significant finding of this study is that all drinking water samples were positive for Pseudomonas sp.(>100/100ml), but also that when one considers the TDS it demonstrates that water from the Modimola Dam has an impact on the quality of the mixed water. The prevalence and antibiotic resistance profiles of planktonic and biofilm bacteria isolated from drinking water were determined. The susceptibility of these isolates was tested against 11 antibiotics of clinical interest and the multiple antibiotic resistance (MAR) patterns were compiled. The most prevalent antibiotic resistance phenotype observed was KF-AP-C-E-OT-K-TM-A. All isolates from all samples were susceptible to ciprofloxacin. However, all faecal coliforms and Pseudomonas spp. were susceptible to neomycin and streptomycin. On the contrary all organisms tested were resistant to erythromycin (100%) trimethoprim and amoxycillin. Cluster analysis based on inhibition zone diameter data could not differentiate the various isolated into sample types. The highest prevalence of antibiotic resistant isolates was observed in Modimola Dam and Molopo eye. Biofilms were investigated in both raw water and treated drinking water sources for the presence of faecal coliforms, total coliforms, Pseudomonas spp., Aeromonas spp. and heterotrophic bacteria based on conventional microbiology and molecular methods. Drinking water biofilms were grown twice and the biofilm developing device containing copper and galvanized steel coupons were utilized. The Mini Tap filter, a home water treatment device which can be used at a single faucet, under constant flow was used during the second collection of treated water samples from cold water taps. Scanning electron micrograph revealed the existence of biofilms in all the sites investigated and the highest density was obtained on galvanized steel coupons. Isolates were tested against the antibiotics ampicillin (10μg), cephalothin (5μg), streptomycin (10μg), erythromycin (15μg), chloramphenicol (30μg), neomycin (30 μg), amoxycillin (10 μg), ciprofloxacin (5 μg), trimethoprim (25μg), kanamycin (30μg), and oxytetracycline (30μg). The multiple antibiotic resistance profiles and the presence of virulence related genes were determined. Various types of drug resistance and presence of virulence genes were observed. The most prevalent resistance phenotype observed was KF-AP-C-E-OT-TM-A. In conclusion, the results indicated the occurrence of faecal indicator bacteria in the drinking water destined for human consumption. Faecal indicator bacteria are the major contributors of poor drinking water quality and may harbour opportunistic pathogens. This highlighted survival of organisms to treatment procedures and the possible regrowth as biofilms in plumbing materials. The detection of large proportion of MAR Aeromonas and Pseudomonas species which possessed virulent genes was a cause of concern as these could pose health risks to humans. The data obtained herein may be useful in assessing the health risks associated with the consumption of contaminated water. / PhD (Microbiology), North-West University, Potchefstroom Campus, 2014

The main aim of the study was to analyse the impact of physico-chemical parameters on drinking water quality, biofilm formation and antibiotic resistant bacteria in the drinking water distribution system in Mafikeng, North West Province, South Africa. Another objective was to isolate and characterise Pseudomonas and Aeromonas species from drinking water distribution system and detect the virulence gene determinants in the isolates by PCR analysis. The physico-chemical data obtained were subjected to statistical analysis using Excel 2007 (Microsoft) and SPSS (version 14.0) programmes. Pearson’s correlation product of the moment was used to determine the correlation between EC, TDS, pH and temperature. The two tailed test of significance (p<0.05) was used in order to determine the significance of the result. Antibiotic susceptibility tests were performed using Kirby-Bauer disk diffusion method. Cluster analysis based on the antibiotic inhibition zone diameter data of different organisms isolated from different sites was determined and was expressed as dendograms using Wards algorithm and Euclidean distance of Statistica version 7. Specific PCR was used to determine the identities of presumptive Pseudomonas and Aeromonas species through amplification of the gyrB, toxA and the ecfX gene fragments. Virulence gene determinants for the confirmed Pseudomonas and Aeromonas species were detected by amplifying the exoA, exoS and exoT genes and the aerA and hylH gene fragments, respectively. A Gene Genius Bio imaging system (Syngene, Synoptics; UK) was used to capture the image using GeneSnap (version 3.07.01) software (Syngene, Synoptics; UK) to determine the relative size of amplicons. Physico-chemical parameters were monitored from three drinking water sources three times a week and bacteriological quality was monitored weekly for four months from raw and treated drinking water. Water samples were analysed for pH, temperature, total dissolved solids (TDS) and electric conductivity (EC). Bacterial consortia from drinking water samples were isolated using selective media and enumerated. The results revealed a good chemical quality of water. However, the microbial quality of the water is not acceptable for human consumption due to the presence of Pseudomonas, Aeromonas, faecal coliforms (FC), total coliforms (TC) and Heterotrophic bacteria. The results showed that the drinking water is slightly alkaline with pH value ranging between7.7 to 8.32. What is of concern was the microbial quality of the water. Pseudomonas sp., faecal coliforms (FC), total coliforms (TC) and heterotrophic bacteria were present in some of the treated water samples. The most significant finding of this study is that all drinking water samples were positive for Pseudomonas sp.(>100/100ml), but also that when one considers the TDS it demonstrates that water from the Modimola Dam has an impact on the quality of the mixed water. The prevalence and antibiotic resistance profiles of planktonic and biofilm bacteria isolated from drinking water were determined. The susceptibility of these isolates was tested against 11 antibiotics of clinical interest and the multiple antibiotic resistance (MAR) patterns were compiled. The most prevalent antibiotic resistance phenotype observed was KF-AP-C-E-OT-K-TM-A. All isolates from all samples were susceptible to ciprofloxacin. However, all faecal coliforms and Pseudomonas spp. were susceptible to neomycin and streptomycin. On the contrary all organisms tested were resistant to erythromycin (100%) trimethoprim and amoxycillin. Cluster analysis based on inhibition zone diameter data could not differentiate the various isolated into sample types. The highest prevalence of antibiotic resistant isolates was observed in Modimola Dam and Molopo eye. Biofilms were investigated in both raw water and treated drinking water sources for the presence of faecal coliforms, total coliforms, Pseudomonas spp., Aeromonas spp. and heterotrophic bacteria based on conventional microbiology and molecular methods. Drinking water biofilms were grown twice and the biofilm developing device containing copper and galvanized steel coupons were utilized. The Mini Tap filter, a home water treatment device which can be used at a single faucet, under constant flow was used during the second collection of treated water samples from cold water taps. Scanning electron micrograph revealed the existence of biofilms in all the sites investigated and the highest density was obtained on galvanized steel coupons. Isolates were tested against the antibiotics ampicillin (10μg), cephalothin (5μg), streptomycin (10μg), erythromycin (15μg), chloramphenicol (30μg), neomycin (30 μg), amoxycillin (10 μg), ciprofloxacin (5 μg), trimethoprim (25μg), kanamycin (30μg), and oxytetracycline (30μg). The multiple antibiotic resistance profiles and the presence of virulence related genes were determined. Various types of drug resistance and presence of virulence genes were observed. The most prevalent resistance phenotype observed was KF-AP-C-E-OT-TM-A. In conclusion, the results indicated the occurrence of faecal indicator bacteria in the drinking water destined for human consumption. Faecal indicator bacteria are the major contributors of poor drinking water quality and may harbour opportunistic pathogens. This highlighted survival of organisms to treatment procedures and the possible regrowth as biofilms in plumbing materials. The detection of large proportion of MAR Aeromonas and Pseudomonas species which possessed virulent genes was a cause of concern as these could pose health risks to humans. The data obtained herein may be useful in assessing the health risks associated with the consumption of contaminated water. / PhD (Microbiology), North-West University, Potchefstroom Campus, 2014

In Southeast Asia, the family Pangasiidae is important for commercial fisheries and aquaculture. Pangasianodon hypophthalmus (striped catfish) is the most economically important species farmed in Vietnam, with a total export value of 1.7 billion USD in 2012. Intensive aquaculture can lead to problems with major outbreaks of disease and Edwardsiella ictaluri and Aeromonas hydrophila represent two important bacterial pathogens in P. hypophthalmus aquaculture. Immunostimulants have proven to be a very useful food additive for the aquaculture industry, since they can be easily fed to fish to enhance their immune response at times of stress and to improve resistance to disease. The immune system of pangasius catfish has not been fully described, despite the recent growth in aquaculture for this species, and little is known about the effects of immunostimulants on disease resistance. Understanding the immune response is very important in order to evaluate the health status of the fish and assist in control of disease (including prevention) so that production levels by the aquaculture industry can be sustained. The aims of this thesis were to develop and standardise methods to elucidate and measure immune responses in P. hypophthalmus and then to use these with relevant disease models (A. hydrophila and E. ictaluri) and immunomodulators (β-glucans from different sources and at different doses) to determine if bacterial diseases can be controlled, and which functional immune responses and immune genes could be correlated with disease resistance. As a variety of different species from family Pangasiidae are economically important for aquaculture, initial work focused on the characterisation of the immunoglobulin IgM molecule in these species, and anti-P. hypophthalmus IgM mAbs were tested to determine if they cross-reacted between different Pangasiidae species (Chapter 2). Although affinity purification of IgM from the different fish species resulted in a purer preparation ammonium sulphate precipitation (14% w/w), the latter proved faster and easier to perform. The heavy (H) and light (L) chains of IgM from P. hypophthalmus were estimated to be 70-72 kDa and 25-26 kDa, respectively, using SDS-PAGE (12.5%). The L chains of IgM in the other Asian fish species examined were similar in molecular weight to P. hypophthalmus, while the H chains varied (P. gigas and P. larnaudii 76kDa, P. sanitwongsei 69kDa, H. filamentus 73kDa, P. borcoti and H. wyckioides 75kDa, C. bactracus 74kDa, C. macrocephalus 73kDa and C. carpio 70kDa), as did the native IgM molecules. Sedimentation velocity ultracentrifugation was used to determine the molecular weight of the whole IgM molecule from P. hypophthalmus as an alternative to the more commonly used native gels that are run under non-denaturing conditions, although this technique proved more complex. Anti–P. hypophthalmus IgM monoclonal antibodies (mAbs) cross reacted with all of the Pangasiidae species and were successfully applied in an enzyme-linked immunosorbent assay (ELISA) using mAb 23 to measure serum antibody response of P. hypoophthalmus following experimental infection with A. hydrophila by interperitoneal (I.P.) injection in Chapter 3 and E. ictaluri by immersion in Chapter 4. As P. hypophthalmus is a relatively new aquaculture species, there are few reports evaluating its immune response to pathogens. Thus, functional assays were standardised to evaluate both innate and adaptive immune responses of this species and then these assays used to compare immune response following stimulation with live and killed A. hydrophila. (Chapter3). Four treatment groups of 40 fish per group (53.2 ± 14.8g.) consisting of an untreated control group, a group injected I.P. with adjuvant (Montanide ISA 760 VG) only, a group injected with heat-killed A. hydrophila (1 x109 cfu ml-1 mixed with adjuvant), and a group injected with a subclinical dose of live A. hydrophila 2.7 x105 cfu ml-1 were used in the study. Samples were collected 0, 1, 3, 7, 14 and 21 days post injection (d.p.i.) to assess the immune response of fish. The results indicated that challenge with live or/and dead bacteria stimulated the immune response in P. hypophthalmus significantly above control groups with respect to specific antibody titre, lysozyme activity, phagocytosis and plasma peroxidase at 7 or/and 14 d.p.i. Moreover, on 21 d.p.i. total IgM, specific antibody titre and lysozyme activity from both live and dead A. hydrophila challenge groups were significantly different to the control groups. Differential immune responses between live and dead bacterial challenges were also observed as only live A. hydrophila significantly stimulated WBC counts and plasma peroxidase at 3 d.p.i. with the greatest increase in WBC counts noted at 21 d.p.i. and in phagocytosis at 14 d.p.i. By 21 d.p.i. only the macrophages from fish challenged with dead A. hydrophila showed significantly stimulated respiratory burst activity. Immunostimulants are food additives used by the aquaculture industry to enhance the immune response, and β-glucan is now commonly used for this purpose in aquaculture. In Chapter 4 the effect of the prebiotic β-glucan on the immune response and disease resistance of P. hypophthalmus was evaluated. The fish (60.3 ± 11.7 g.) were fed with a basal diet (control) or diets supplemented with fungal derived β-glucan at concentrations of 0.05 %, 0.1 %, or 0.2 % g/kg for four weeks. Fish fed 0.1 % commercial yeast derived β-glucan were also included as a positive control group. Samples were collected from fish on Days 0, 1, 3, 7, 14, 21 and 28. The results showed that fish fed with the highest two levels of fungal derived β-glucan had enhanced immune responses compared to the control group, with respiratory burst activity on all days examined and lysozyme activity on 7 days post feeding (d.p.f.) being significantly elevated (P<0.05) in the group fed with 0.2 % fungal derived β-glucan, while plasma anti-protease activity on 21 d.p.f., natural antibody titre on 3 d.p.f. and complement activity 7 d.p.f. and 14 d.p.i. were significantly enhanced (P<0.05) in the group fed 0.1 % fungal derived β-glucan. The lowest dose of fungal derived β-glucan (0.05 %) appeared insufficient to effectively stimulate the fish’s immune response. WBC count, respiratory burst, lysozyme activity and complement were useful as an early indication of immunostimulation (1 to 7 days). Four weeks after feeding with the different diets, the fish were experimentally infected with E. ictaluri by immersion using 8 x104 cfu ml-1 for 1 h and mortalities were monitored for 14 days. There was a great deal of variation in the level of mortalities within the four replicate tanks for each dietary group. Although the in vivo challenge results showed no statistical differences between the groups fed on the different diets, the highest mortalities were observed in group fed with the control diet and the lowest mortalities were observed in the groups fed with commercial yeast derived β-glucan and 0.2 % fungal derived β glucan. Immune gene expression following stimulation with β-glucan and challenge with E. ictaluri was investigated in Chapter 5.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Bacterial infections are an increasing problem for human health. In fact, an increasing number of infections are caused by bacteria that are resistant to most antibiotics and their combinations. A new solution to fight bacteria and infectious diseases, without promoting antimicrobial resistance, is required. A promise strategy is the disruption or attenuation of bacterial Quorum Sensing (QS), a refined system that bacteria use to communicate. In a QS event, bacteria produce and release specific small chemicals, signal molecules - autoinducers (AIs) - into the environment. AIs regulate gene expression as a function of cell population density. Phenotypes mediated by QS (QS- phenotypes) include virulence factors, toxin production, antibiotic resistance and biofilm formation. In this work, two polymeric materials (linear polymers and molecularly imprinted nanoparticles) were developed and their ability to attenuate QS was evaluated. Both types of polymers should be able to adsorb bacterial signal molecules, limiting their availability in the extracellular environment, with expected disruption of QS. Linear polymers were composed by methyl methacrylate as backbone and itaconic acid or methacrylic acid as functional monomer. IA and MAA monomers were identified by computer modelling to have strong interactions with the AIs produced by Gram-negative bacteria. Cont/d.

Submitted by Raphaela Sanches de Oliveira null (raphasol84@hotmail.com) on 2017-02-13T11:57:31Z No. of bitstreams: 1 Dissertação Pronta.pdf: 4209987 bytes, checksum: 54ceb01d6a0e30fcb6560f6689be5b46 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-02-16T15:59:08Z (GMT) No. of bitstreams: 1 oliveira_rs_me_sv.pdf: 4209987 bytes, checksum: 54ceb01d6a0e30fcb6560f6689be5b46 (MD5) / Made available in DSpace on 2017-02-16T15:59:08Z (GMT). No. of bitstreams: 1 oliveira_rs_me_sv.pdf: 4209987 bytes, checksum: 54ceb01d6a0e30fcb6560f6689be5b46 (MD5) Previous issue date: 2017-01-18 / O desenvolvimento urbano em áreas costeiras tem ocorrido intensamente e o aumento das descargas de esgotos domésticos é uma das consequências desta expansão e motivo de preocupação para a saúde pública. Assim a qualidade microbiológica das águas, sedimentos e alimentos de origem marinha devem ser monitoradas, principalmente os organismos filtradores como os mexilhões, Perna perna. moluscos bivalves, por filtrarem a água para obtenção de alimento e oxigênio, concentram material em suspensão nos seus tecidos, inclusive bactérias patogênicas resistentes a substâncias antimicrobianas, o que os torna uma fonte potencial de contaminação humana por patógenos. O objetivo do presente estudo foi avaliar a densidade e resistência a antimicrobianos de Enterococcus sp, Escherichia coli e Aeromonas sp isolados dos tecidos de mexilhão Perna perna, água e sedimento coletados em Santos e Itanhaém, Estado de São Paulo. Como resultado observa-se que as densidade de Enterococcus sp e Escherichia coli encontradas na água dos dois pontos de coleta estão dentro das normas da Resolução Conama 274/2000, já as densidades encontradas nos sedimentos e em tecidos moles dos mexilhões foram elevadas. Para Aeromonas sp não existe padrão, pois as mesma não estão presentes na legislação. Observou-se também altas frequências de resistência bacteriana aos antimicrobianos. Os resultados obtidos sugerem que pode haver uma questão importante para a saúde pública, pois foram encontrados microrganismos apresentando múltipla resistência aos antibióticos analisados, sendo que apenas a ciprofloxacina se mostrou mais eficiente para os três microrganismos analisados. Tendo em vista os resultados obtidos foi possível concluir a existência de situação preocupante, pois os microrganismos testados se mostram altamente resistentes a múltiplos antimicrobianos, gerando um grande problema de saúde pública.

Since the passage of the Clean Water Act, concern about surface water quality has increased. Reducing exposure to pathogens and adverse impacts on human health because of contact with surface waters has become the focus of many regulatory agencies. Fecal pollution is often a cause of surface water impairment. Fecal indicators, such as fecal coliforms and Escherichia coli, are used as surrogates to evaluate the presence or absence of fecal pollution. However, a growing body of research has shown that these species lack key characteristics necessary to be adequate indicators. As such, explorations into the efficacy of indicator species in predicting fecal pollution in water are necessary. Sinking Creek is a tributary of the Watauga River Watershed, located in Northeast Tennessee. Approximately ten miles of Sinking Creek have been placed on the national 303(d) list for fecal pollution, denoting the presence of fecal contamination exceeding the regulatory limit. Salmonella and Aeromonas are two enteric pathogens that would be expected to be detected in fecally contaminated waters. The primary objective of this study was to detect the presence of Salmonella and Aeromonas in Sinking Creek. The secondary objective was to evaluate their relationship with fecal coliforms, E. coli, and water quality parameters. Six study sites along Sinking Creek were sampled and standard methods were used to enumerate Salmonella and Aeromonas. Samples for Salmonella were collected for 8 months, while samples for Aeromonas were collected for seven. Salmonella and Aeromonas were present in Sinking Creek. Salmonella had the highest concentration at site 2 (the most downstream site), and was detected during all months of the study except for November. Salmonella concentrations varied by site. Aeromonas was present only during colder months, and had the highest concentration at site 2. Both Salmonella and Aeromonas show qualitative relationships with water quality parameters, such as dissolved oxygen and conductivity. However, statistically significant correlations of Salmonella and Aeromonas with water quality parameters were not observed. The lack of statistical significance is partially due to large variability and a small data set. Neither Salmonella or Aeromonas exhibited a relationship with fecal coliforms or E. coli. Therefore, fecal coliforms and E. coli may not be adequate indicator species for the presence of Salmonella, Aeromonas and possibly other waterborne pathogens. Traditional indicator species may inflate risk of pathogen exposure. Thus, many water bodies may be unnecessarily deemed as impaired. The results from this study can be used to guide further research regarding covariates influencing pathogen densities at fecally contaminated sites, as well as to guide decisions regarding impaired surface waters and management techniques.

The initial aim of this project was to uncover novel cellulolytic organisms or enzymes from the diverse microbial source, activated sludge. Two isolation methods were used; either directly inoculating the sludge material onto filter paper as a carbon source, or using the Evolver� technology as an enrichment device. In both cases, as expected, cellulase activity was evident, however attributing this activity to one species was difficult in either case. This highlighted the complex interrelationships that existed between the many microorganisms present as the cellulosic carbon sources were degraded. In one instance, a Cellvibrio sp. was isolated. This genus of bacteria is known to possess both types of cellulase activity (exo- and endo- acting) and was therefore likely to contribute to the degradation of the cellulose. However, the isolate, once purified, did not display significant cellulolytic ability as compared to the unpurified consortium of microorganisms. Therefore, in each case, microorganisms responsible for the cellulolytic activity were not uncovered. It was suspected that the microorganisms responsible for some of the cellulolytic activity were protists. During the isolation of microorganisms, an Aeromonas sp. bearing the novel phenotype (for this genus) of CMCase activity was isolated. This activity was at first suspected to contribute to the degradation of the filter paper that was seen during isolation. However, tests with the pure isolate suggested that the Aeromonas sp. CMCase was not used for cellulose catabolism. Ironically, the enzyme may instead function in the production of a cellulose-like exopolysaccharide by the bacterium. Part of a cellulose synthase operon was found in the genome of the Aeromonas sp. isolate, including a gene coding for an endoglucanase that gives a predicted molecular weight enzyme similar to the 39 kDa CMCase purified from the bacterium. The CMCase enzyme, operating as part of of a synthetic operon is expected to be important in terms of the biofilm forming ability of this Aeromonas strain. Such capabilities of the bacterium were investigated here, including observing motility behaviour of the organism on agar surfaces. Studying the biofilm forming ability of this genus in general will be important in understanding how the fish and human pathogens persist in aquatic environments

The type II secretion system is used by many pathogenic and non-pathogenic bacteria for the extracellular secretion of enzymes and toxins. <i>Aeromonas hydrophila</i> is a Gram-negative pathogen that secretes proteins via the type II secretion system.<p> In the studies described here, a series of yeast two-hybrid assays was performed to identify protein-protein interactions in the type II secretion system of <i>A. hydrophila</i>. The periplasmic domains of ExeA and ExeB were assayed for interactions with the periplasmic domains of Exe A, B, C, D, K, L, M, and N. Interactions were observed for both ExeA and ExeB with the secretin ExeD in one orientation. In addition, a previously identified interaction between ExeC and ExeD was observed. In order to further examine and map these interactions, a series of eight two-codon insertion mutations in the amino terminal domain of ExeD was screened against the periplasmic domains of ExeA and ExeB. As a result, the interactions were verified and mapped to subdomains of the ExeD periplasmic domain. To positively identify the region of ExeD involved in the interactions with ExeA, B, C and D, deletion mutants of ExeD were constructed based on the two-codon insertion mutation mapping of subdomains of the ExeD periplasmic domain, and yeast two-hybrid assays were carried out. The results showed that a fragment of the periplasmic domain of ExeD, from amino acid residue 26 to 200 of ExeD, was involved in the interactions with ExeA, B and C. As an independent assay for interactions between ExeAB and the secretin, His-tagged derivatives of the periplasmic domains of ExeA and ExeB were constructed and co-purification on Ni-NTA agarose columns was used to test for interactions with untagged ExeD. These experiments confirmed the interaction between ExeA and ExeD, although there was background in the co-purification test.<p> These results provide support for the hypothesis that the ExeAB complex functions to organize the assembly of the secretin through interactions between both peptidoglycan and the secretin that result in its multimerization into the peptidoglycan and outer membrane layers of the envelope.