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.

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.

Water samples were collected at four sites on the White River, Muncie, Indiana, on a biweekly basis from April 1980 to April 1981, to determine seasonal variation in A. hydrophila density. In conjunction with water sampling, temperature, dissolved oxygen, conductivity and pH were determined at each site. During this period a yearly mean of 589 colony forming units (CFU) per ml was obtained with a range of 0-6350. Peaks in mean A. hydrophila numbers occurred during the summer (1808 CFU ml-1) and early spring (3946 CFU ml-1) with lows occurring in winter (42 CFU ml-1). No significant correlations could be found between CFU values and the physico-chemical parameters studied over the calendar year. However, the compiling of data from May 1979 to April 1981 did yield a significant correlation between A. hydrophila density values and the following water quality parameters: temperature, conductivity, and dissolved oxygen.Fish were collected during June through October 1980 at various sites along the White River, Delaware County, Indiana, for the determination of past or present exposure to A. hydrophila infection by serological and direct isolation methods. Of 102 different fish sera tested, 36% (37/102) had a detectable titer to A. hydrophila (isolate #113). However, the direct isolation method only yielded seventeen percent (8/47) of the fishes sampled to be positive forA. hydrophila systemically. Agglutinins to A. hydrophila were demonstrated in eleven different fish species, whereas five different species revealed no detectable antibody to the specific particulate antigen employed. Estimated CFU ml-1 of A. hydrophila was the only parameter which correlated with the presence of agglutinating antibody to this organism in river fish populations.Ball State UniversityMuncie, IN 47306

Temperature, conductivity, and Aeromonas hydrophila density were determined bimonthly at six sites in the White River drainage system, Delaware County, Indiana, from April, 1984 through December, 1984. Fish were collected from four of the six sites. Fish were identified to species, examined for gross pathology, and their blood collected. Titers against A. hydrophila antigen 157 were determined for all carp (Cyprinus carpio) captured. Certain carp serum samples were also tested against three additional A. hydrophila antigens.Aerononas hydrophila densities appeared to be related to temperature and conductivity, primary producers, and runoff/effluent. Aeromonas hydrophila densities were higher in areas affected by urban runoff/ effluent than in areas affected by rural runoff/effluent. Aeromonas hydrophila densities increased as one progressed through the city of Muncie.Only five percent of all fish captured exhibited signs of gross pathology. Diseased fish typically demonstrated low titers against antigen 157. Several A. hydrophila serotypes were apparently infectious for the carp studied. Thirty-two percent of the carp sampled exhibited a positive titer response against antigen 157. Considerable variation in titer response existed between individuals.Ball State UniversityMuncie, IN 47306

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.

The bacterium Aeromonas hydrophila is a known fish and opportunistic human pathogen commonly occurring in surface waters supplying drinking water distribution systems. The major concern of government and drinking water providers is that A. hydrophila may invade and become established in the biofilm of a distribution system, thus potentially leading to outbreaks of disease. The purpose of this study was to survey source water, distribution system biofilm, and to establish a simulated distribution system to explore the possibility of A. hydrophila invading and becoming established under normal and disrupted treatment conditions. A. hydrophila (AH) medium and the API-20E system were used for identification, while Ampicillin-Dextrin Agar (ADA) was used for enumeration. Presumptive counts were high in source water approaching 103 CFU/ml during summer months. Biofiim from an actual distribution system showed the presence of A. hydrophila in 10 % of the samples. In the simulated distribution system A. hydrophila was never found in the bulk water or biofilm under normal treatment condition, showing disinfectant efficiency. Under disrupted treatment conditions A. hydrophila was not able to colonize a pre-established biofilm over a 14 week period. / Department of Biology

Recently the EPA placed Aeromonas hydrophila on the Candidate Contaminant List (CCL). It has long been known to be a pathogen of cold blooded animals and now is a suspected human opportunistic pathogen as well. Among the various virulence factors produced by A. hydrophila, the cytolytic enterotoxin (AHCYTOEN) is by far one of the most important contributors to the pathogenicity of the organism. This factor is also produced by other pathogenic Aeromonas spp. In this study, PCR technology was used to detect AHCYTOEN gene from a simulated water distribution system. A set of primers was selected to amplify the unique sequence of a pathogenic island, AHCYTOEN gene. To examine the sensitivity of the PCR, serial dilutions of pure A. hydrophila culture were tested. The PCR technique used was sensitive enough to detect samples containing less than 10.0 cells/ml. Source water, bulk water, and simulated distribution biofilm samples were examined for the gene. Biofilm and bulk water samples exposed to raw source water were collected on 4 occasions during a 24-day period. PCR technology detected the AHCYTOEN gene from 100 % of the bulk water samples and 85% of tightly bound biofilm (TB) samples from a simulated water distribution system while no positive results were observed in loosely bound biofilm samples (LB). After the inlet line of the system was changed to normally treated distribution water, 11 biofilm samples were collected on 3 occasions during 15 day sampling period along with bulk water samples. No positive results were observed from the bulk water and LB samples while 91% of TB samples tested for the presence of the gene. No significant difference was observed in detection by PCR from biofilm samples before and after the switch to chloraminated water. / Department of Biology

Orientador: Helio Langoni / Resumo: EBERHARDT, B.G. Avaliação de micro-organismos zoonóticos em filés de tilápia do Nilo (Oreochromis niloticus). Botucatu, 2018. 71p. Tese (Doutorado) – Faculdade de Medicina Veterinária e Zootecnia, Campus de Botucatu, Universidade Estadual Paulista. RESUMO Cinquenta filés de tilápia do Nilo (Oreochromis niloticus) obtidos em mercado de peixes no município de Ourinhos, Estado de São Paulo, foram analisados quanto à prevalência para Aeromonas hydrophila, Edwardsiella tarda, Mycobacterium spp. e Cianobactérias. Amostras de músculo foram avaliadas por PCR para Aeromonas hydrophila, Edwardsiella tarda e Mycobacterium spp., enquanto que as amostras para cianobactérias foram analisadas por PCR em Tempo Real (qPCR). Os resultados obtidos demonstraram ausência de Aeromonas hydrophila e Edwardsiella tarda nas amostras de filés. A prevalência para Mycobacterium spp. foi de 100% (50/50). Realização posterior de sequenciamento revelou Mycobacterium gordonae. Esta bactéria é considerada um colonizador comum, normalmente não patogênico, porém, há relatos de literatura que demonstram risco de infecção em indivíduos imunossuprimidos e até mesmo imunocompetentes. A taxa de prevalência para cianobactérias foi de 48% (24/50). As cianobactérias (algas azuis) produzem grande quantidade de metabólitos bioativos ou mesmo tóxicos, incluindo toxinas associadas a problemas ambientais e de saúde pública. Considerando a natureza e o papel das cianobactérias como patógenos emergentes, a elevada prevalência... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Submitted by Bruno Giorno Eberhardt (bruno.giorno@agricultura.gov.br) on 2018-04-21T01:40:57Z No. of bitstreams: 1 TESE DOUTORADO - BRUNO GIORNO EBERHARDT.pdf: 3876871 bytes, checksum: 3c2c7eac21349ce401f54a1cb03fae4f (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-04-23T12:55:33Z (GMT) No. of bitstreams: 1 eberhardt_bg_dr_botfca.pdf: 2911225 bytes, checksum: 9f9ad5ea743405838741796482e9ab66 (MD5) / Made available in DSpace on 2018-04-23T12:55:33Z (GMT). No. of bitstreams: 1 eberhardt_bg_dr_botfca.pdf: 2911225 bytes, checksum: 9f9ad5ea743405838741796482e9ab66 (MD5) Previous issue date: 2018-03-01 / EBERHARDT, B.G. Avaliação de micro-organismos zoonóticos em filés de tilápia do Nilo (Oreochromis niloticus). Botucatu, 2018. 71p. Tese (Doutorado) – Faculdade de Medicina Veterinária e Zootecnia, Campus de Botucatu, Universidade Estadual Paulista. RESUMO Cinquenta filés de tilápia do Nilo (Oreochromis niloticus) obtidos em mercado de peixes no município de Ourinhos, Estado de São Paulo, foram analisados quanto à prevalência para Aeromonas hydrophila, Edwardsiella tarda, Mycobacterium spp. e Cianobactérias. Amostras de músculo foram avaliadas por PCR para Aeromonas hydrophila, Edwardsiella tarda e Mycobacterium spp., enquanto que as amostras para cianobactérias foram analisadas por PCR em Tempo Real (qPCR). Os resultados obtidos demonstraram ausência de Aeromonas hydrophila e Edwardsiella tarda nas amostras de filés. A prevalência para Mycobacterium spp. foi de 100% (50/50). Realização posterior de sequenciamento revelou Mycobacterium gordonae. Esta bactéria é considerada um colonizador comum, normalmente não patogênico, porém, há relatos de literatura que demonstram risco de infecção em indivíduos imunossuprimidos e até mesmo imunocompetentes. A taxa de prevalência para cianobactérias foi de 48% (24/50). As cianobactérias (algas azuis) produzem grande quantidade de metabólitos bioativos ou mesmo tóxicos, incluindo toxinas associadas a problemas ambientais e de saúde pública. Considerando a natureza e o papel das cianobactérias como patógenos emergentes, a elevada prevalência deste organismo em um alimento popular como o filé de tilápia do Nilo desperta preocupação, uma vez que os métodos tradicionais de inspeção são incapazes de detectar o patógeno, pelo fato de não provocar alterações macroscópicas nos produtos, bem como pelo potencial de toxicidade para humanos. Entretanto, estudos adicionais são necessários a fim de entender se estes compostos tóxicos estão presentes nos peixes e, caso estejam, se podem sofrer bioacumulação em níveis suficientes para afetar a saúde humana. Palavras-chave: Aeromonas hydrophila, Edwardsiella tarda, Mycobacterium spp, Mycobacterium gordonae, cianobactéria, cianotoxina, tilápia do Nilo, Oreochromis niloticus, diagnóstico. / Fifty fillets of Nile tilapia (Oreochromis niloticus) from a Ourinhos fish market, Sao Paulo State (Southeast Brazil) were analyzed for the prevalence of Aeromonas hydrophila, Edwardsiella tarda, Mycobacterium spp. and cyanobacteria. Muscle samples were evaluated by PCR for Aeromonas hydrophila, Edwardsiella tarda and Mycobacterium spp. Samples for cyanobacteria were analyzed by real time PCR. Both Aeromonas hydrophila and Edwardsiella tarda were not present in fish samples. The prevalence of Mycobacterium spp. was 100% (50/50). Sequencing revealed Mycobacterium gordonae. This agent is usually a ubiquitous and commonly nonpathogenic colonizing organism, although many research publications have reported disease in immunocompromised or even in immunocompetent patients. Prevalence for cyanobacteria was 48% (24/50). Cyanobacteria (blue-green algae) produce a diversity of toxic or otherwise bioactive metabolites, including a number of toxins that have been associated with human and environmental health concerns. Considering the nature and role of cyanobacteria as a pathogen of emerging importance, the high prevalence of this organism in a popular food item such as Nile tilapia raises concern, since no macroscopic alterations can be detected through regular food inspection methods. However, further studies are necessary in order to understand whether these compounds are present in fish and, if so, if they could accumulate sufficiently to affect human health.

The U.S. aquaculture industry has become well established in the last three decades, and channel catfish aquaculture is the most significant component of this industry. Virulent Aeromonas hydrophila has been a serious disease problem since 2009 in the U.S. catfish aquaculture, and Aeromonas veronii and Edwardsiella piscicida are emerging pathogens of catfish. Therefore, this study aims to address fundamental questions on virulence mechanisms of these three fish pathogens, which I expect to support the development of control measures for preventing these diseases. In this study, E. piscicida and virulent Aeromonas hydrophila (vAh) genomes were sequenced, and comparative analyses were conducted using the genome sequences. Average nucleotide identity (ANI) calculations showed that E. piscicida strains share high sequence identity, yet they are from diverse host species and geographic regions. vAh isolates share very high sequence identity, while the other A. hydrophila genomes are more distantly related to this clonal group. We applied several comparative genomics approaches to evaluate E. piscicida genomes and E. ictaluri genomes, providing valuable information about unique and shared features of these two important pathogens in the Edwardsiella genus. Comprehensive secretion system analysis of 55 A. hydrophila genomes and deletion of tssD and tssI core elements of T6SS from vAh isolate ML09-119 has provided new knowledge. We sequenced the genome of virulent Aeromonas veronii strain ML09-123 from catfish indicated that it was highly similar to an A. veronii strain from China. Evaluation of all 41 A. veronii genomes available in the National Center for Biotechnology Information (NCBI) provides a base platform to investigate in detail the molecular mechanism of A. veronii biology and virulence. Lastly, we constructed deletion mutants vAhΔsia, vAhΔent, vAhΔcol, vAhΔhfq1, vAhΔhfq2, and vAhΔhfq1Δhfq2 to determine roles of A. hydrophila secreted proteins and regulatory proteins on virulence in catfish. Results showed that sialidase (vAhΔsia) and enterotoxin (vAhΔent) mutants were significantly attenuated.