Dissertationen zum Thema „Environmental aeromonads“

A luminescence-based detection system was developed to study changes in the survival and activity of cells following release from moribund and dead fish. A.salmonicida was chromosomally marked with the genes encoding bacterial luciferase, originally isolated from Vibrio harveyi. Characterisation of the growth and luminescence of the lux-marked strain demonstrated that light was directly proportional to cell biomass concentration during logarithmic growth. The survival of lux-marked and wild type A.salmonicida strains was investigated in sterile sea water at 4°C. The number of culturable cells declined rapidly, but the total number of cells remained relatively constant, suggesting A. salmonicida entered a nonculturable state. The survival of lux-marked A. salmonicida did not significantly differ from that of the wild type strain. A small number of cells remained culturable throughout starvation experiments and luminometry confirmed that the lux-marked cells were metabolically active, possibly surviving by cryptic growth. The viability of putative dormant cells could not be established since these cells could not be reactivated following the addition of a range of substrates. The lux-marked A.salmonicida strain was pathogenic only when injected at high doses. This poor virulence was probably due to loss of the proteinaceous A-layer which is responsible for hydrophobic cell interactions and cell defence against lytic agents. This prevented further studies aimed at determining the virulence of nonculturable cells using this strain. Preliminary experiments indicated the potential of the lux-marked system for studying vertical transmission of A. salmonicida. The main sites for attachment of the lux-marked strain were the gill and skin/mucus regions. Identical results were obtained using a wild type virulent A. salmonicida strain, but significantly higher numbers of cells were recovered from fish tissue.

Doutoramento em Biologia
Espécies de Aeromonas encontram-se distribuídas por diferentes habitats, estando especialmente relacionadas com ambientes aquáticos. O seu papel em complicações na saúde humana e animal é reconhecido. De facto, não só pelo seu potencial de virulência, mas também pelos determinantes genéticos de resistência a antibióticos que possam conter, estes organismos constituem uma preocupação na medicina humana e veterinária. Assim, é essencial o estudo da diversidade de espécies de Aeromonas bem como explorar as suas características fenotípicas e genéticas que podem conduzir a impactos negativos. A água constitui um importante veículo de transmissão de microrganismos e espécies de Aeromonas estão amplamente distribuídas em águas tratadas e não tratadas. Em Portugal é ainda comum o consumo de águas não tratadas cuja qualidade, na maioria das vezes, não é sujeita a monitorização, como acontece por exemplo, em explorações agrícolas de gestão familiar. Neste estudo, investigou-se a presença de Aeromonas em águas não tratadas para consumo. Estabeleceu-se também uma linha horizontal de colheitas de diferentes amostras de origem agrícola com o intuito de avaliar a possibilidade de a água ser uma das vias de contaminação de culturas agrícolas e animais por espécies de Aeromonas. Obtiveram-se 483 isolados que foram discriminados por RAPD-PCR. 169 estirpes distintas foram identificadas ao nível da espécie por análise filogenética baseada no gene gyrB. Verificou-se uma frequente ocorrência bem como uma diversidade considerável de espécies de Aeromonas. Em alguns casos, as relações genotípicas entre isolados de diferentes amostras eram muito próximas. Adicionalmente, a maioria das amostras continha diferentes espécies e estirpes distintas da mesma espécie. A. media e A. hydrophila foram as espécies mais ocorrentes. Um grupo de isolados apresentou variantes moleculares de gyrB diferente das conhecidas até agora, o que indica que poderão constituir espécies não descritas. O perfil de susceptibilidade da colecção de Aeromonas a diferentes antibióticos foi estabelecido, constituindo um perfil típico do género, com algumas excepções. Estirpes multirresistentes foram encontradas. A presença de genes tet e bla foi investigada por estudos de PCR, hibridação e, em alguns casos, de sequenciação. Como era esperado, cphA/imiS foi o mais detectado. A detecção de integrões fez-se por PCR e hibridação e a sua caracterização foi feita por sequenciação de DNA; a sua ocorrência foi reduzida. A maioria das estirpes sintetizou enzimas extracelulares com actividade lipolítica e proteolítica que potencialmente contribuem para virulência. A análise por PCR e hibridação permitiram a detecção de vários determinantes genéticos que codificam moléculas possivelmente envolvidas em processos patogénicos. Diversas espécies de Aeromonas apresentando características relacionadas com resistência a antibióticos e potencialmente de virulência estão frequentemente presentes em produtos para consumo humano e animal em Portugal. ABSTRACT: Aeromonas spp. are present in a wide range of ecological niches, being mainly related to aquatic environments. Their role in human and animal health complications is recognised. In fact, not only for their putative virulence but also for the antibiotic resistance genetic determinants Aeromonas may harbour, these organisms constitute an issue of concern in human and veterinary medicine. Thus, it is essential to get knowledge on Aeromonas sp. diversity and on their genotypic and phenotypic characteristics that may lead to negative impacts. Water constitutes a good contamination route for microorganisms and Aeromonas are widespread in untreated and treated waters from different sources. In Portugal there is still an extensive use of untreated water which is not regularly monitored for quality. This is often the case in family smallholding farms. In this study untreated drinking and mineral waters were assessed for their content in Aeromonas spp. Furthermore, a sampling scheme was designed to investigate the occurrence and diversity of Aeromonas sp. in different agricultural correlated sources and to assess the possibility of water being the transmission vehicle between those sources. 483 isolates were obtained and discriminated by RAPD-PCR. Identification at the species level for 169 distinct strains was done by gyrB based phylogenetic analysis. Results demonstrated the frequent occurrence and considerable diversity of Aeromonas spp. In some cases, genotypic close relations were found between isolates from different sources. Also, most samples contained different species and distinct strains of the same species. A. media and A. hydrophila were the most occurring. A group of isolates displayed gyrB gene sequences distinct from the previously known, indicating that they may constitute representatives of non-described species. The antibiotic susceptibility profile of the aeromonads collection was established and constituted a typical profile of the genus, although few exceptions. Multiresistance patterns were found. The presence of tet and bla genes was investigated by PCR, hybridisation and, in some cases, sequencing analysis. As expected, cphA/imiS was the most detected. Integrons were screened by PCR and hybridisation and characterised by DNA sequencing; low occurrence was recorded. The bulk of strains was able to produce extracellular enzymes with lipolytic and proteolytic activities, which may contribute to virulence. PCR and hybridisation surveys allowed the detection of distinct genetic determinants coding for molecules putatively involved in pathogenic processes. Diverse Aeromonas sp. presenting distinct antibiotic resistance features and putative virulence traits are frequently present in many sources for human and animal consumption in Portugal.

The epidemiology of Aeromonas salmonicida subsp. salmonicida in the marine environment was investigated. Nutrient resuscitation and infectivity studies did not support a previous claim of dormancy in A. salmonicida and validated the use of colony-forming units (cfu) in survival studies. Survival of A. salmonicida in seawater was assessed and found to be of short duration «10 days). Survival of the bacterium in non-sterile sediment, obtained from beneath a salmon cage, appeared to be limited. The minimum infective dose of A. salmonicida to Atlantic salmon in short duration (1-3 days) bath exposure in sea water was 10' cfu ml-I. Prolonged exposure for three weeks resulted in infection with 102 cfu ml- I. Intragastric intubation of the bacterium established infection with doses >105 cfu. Shedding of A. salmonicida from infected salmon was 105-108 cfu/fish/hr. Survival and shedding results were combined in a computer model. A. salmonicida was predicted to travel >6 km suspended within the water column of a sea loch. Covert infection in freshwater farmed salmon was assessed by ELISA and the standard stress test. Results indicated that ELISA may be useful as a routine monitor of furunculosis infection. The efficacy of dot-blot immunoassay was found to be 108 cfu A. salmonicida in fish kidney tissue. Rainbow trout (Oncorhynchus mykiss) and salmon mucus were not found to inhibit the growth of A. salmonicida supporting recent evidence that fish skin is a site of carriage. In vitro studies suggested that trout serum proteins do not confer protection from fish antibody on A. salmonicida in covert infections. Preliminary work was undertaken to develop a specific DNA probe for A. salmonicida which will allow its detection in environmental samples and carrier fish. A gene library of A. salmonicida was constructed in lambda gtll and screened for "A"-protein with antibodies.

This study aimed to identify through polyphasic approach (phenotypic and genotypic methods) strains of Aeromonas spp. isolated from surface water and sediment from three different points of the Coco River, Cearà following the salinity gradient of the water. Sampling was conducted between October 2011 and March 2012 each collection consisted of six samples. Aeromonas strains were isolated according to the technique described in the literature Gelatin Phosphate Salt Agar (agar plus GSP 20 μg/mL ampicillin). The physico-chemical parameters (salinity, pH and temperature) of water were on a track that favored not only survival, but also the multiplication of micro-organisms in both sites. Among this sample was isolated 140 strains suspected of Aeromonas and (36.4%) 51 strains were confirmed like Aeromonas spp., wich will be identifiyed until species for fenotipic and genotipic tests. In parallel to the insulation work was done a survey of alternative culture media, where we assessed the efficiency of culture media with different compositions in the characterization of colonies of multiple species of Aeromonas. While the antimicrobial susceptibility profile of Aeromonas strains were resistant 100% penicillin (PEN) and cephalothin (CFL) and 100% antimicrobial susceptibility Amikacin (AMI) and imipenem (IMP) among the nine antimicrobials tested and generally A. caviae was the species that showed resistance to antimicrobials. Among the 27 strains tested (45.8%) were multidrug-resistant by making a multiple resistence antibiotic (MRA) > 2. Five factors of different virulence and all strains were tested had at least one of the five virulence factors tested so that different combinations of virulence factors were found in isolates from the same sample. The results of genetic analysis using primers Aero16S-F and Aero16S-F showed that all isolates identified by conventional microbiological tests were confirmed by PCR and 65.6% of the species identified by conventional biochemical tests were confirmed by Box-PCR method.
Este trabalho teve como objetivo principal identificar atravÃs de abordagem polifÃsica (tÃcnicas fenotÃpicas e genotÃpicas) de cepas de Aeromonas spp. isoladas de Ãgua de superfÃcie e sedimento de trÃs pontos distintos do Rio CocÃ, Fortaleza, Cearà seguindo o gradiente de salinidade da Ãgua. Foram realizadas coletas no perÃodo de outubro de 2011 a marÃo de 2012, sendo cada coleta composta por seis amostras. As cepas de Aeromonas foram isoladas de acordo com a tÃcnica descrita na literatura em Ãgar Gelatina Fosfato Sal (Ãgar GSP acrescido de 20 μg/mL de ampicilina). Os parÃmetros fÃsico-quÃmicos (salinidade, pH e temperatura) da Ãgua estavam em uma faixa que favorecia nÃo sà a sobrevivÃncia, mas tambÃm a multiplicaÃÃo dos micro-organismos em dois pontos de coleta. Foram isoladas 140 cepas suspeitas de Aeromonas, 36 (25,7%) foram identificadas atà espÃcie sendo elas: A. caviae, A. media, A. eucrenophila e A. veronii, sendo A. caviae a espÃcie que apareceu com maior frequÃncia. Em paralelo ao trabalho de isolamento foi feita uma pesquisa com dois meios de cultivo alternativos, Ãgar UNISC e Ãgar Amido Ampicilina (AAA), nos quais foi avaliada a eficiÃncia dos meios de cultura com diferentes composiÃÃes, na caracterizaÃÃo de colÃnias de vÃrias espÃcies de Aeromonas. O perfil de suscetibilidade antimicrobiana das cepas de Aeromonas apresentou resistÃncia de 100% a Penicilina (PEN) e Cefalotina (CFL) e 100% de sensibilidade aos antimicrobianos Amicacina (AMI) e Imipenem (IMP) entre os nove antimicrobianos testados e de forma geral A. caviae (38,9%) foi a espÃcie que mais apresentou resistÃncia. Dentre as cepas avaliadas 27 (45,8%) foram multirresistentes por apresentarem um MAR > 2. Foram testados cinco fatores de virulÃncia diferentes e todas as cepas apresentaram pelo menos um desses fatores, de forma que diferentes combinaÃÃes desses fatores foram observadas em isolados provenientes da mesma amostra. Os resultados da anÃlise genÃtica utilizando os iniciadores Aero16S-F e Aero16S-R mostraram que todos os isolados identificados atravÃs de testes clÃssicos de microbiologia foram confirmados por meio da tÃcnica de PCR e 65,6% das espÃcies identificadas por testes bioquÃmicos convencionais foram confirmadas pelo mÃtodo de Box-PCR.

The survival and physiological responses of A. hydrophila were investigated in lake water microcosms under starvation and other stress conditions. Longer survival was shown in filtered- autoclaved water than in Whatman-filtered water than in untreated water. Longer survival also occurred at 4° than at 15° than at 25° than at 30° than at 37°C. The enhanced survival times over unamended controls suggesting that protozoa could be involved in regulating the size of the population of A. hydrophila. Nutrient amendments such as synthetic sewage, casein, ammonium sulphate, serine and glutamine also increased survival of A. hydrophila over that in unamended controls. Organic compounds released from Flavobacterium, Anacystis, and a number of algae also increased survival times of A. hydrophila in Filtered-autoclaved lake water microcosms. Cell size was reduced under starvation conditions and the numbers of cells capable of respiration also decreased but was always greater than their viable count especially if cells were starved at 37°C. This suggests that A. hydrophila is capable of entering a viable but non-culturable phase under starvation conditions. The effects of starvation on two enzymes, phosphatase and exoprotease, both important in the scavenging of nutrients were examined. The activity of alkaline phosphatase and exoprotease both increased under starvation stress with the largest increases being seen at higher starvation temperatures although the viable count often decreased below the limits of detection at the same time. Nutrient amendments, such as a variety of carbon and nitrogen sources, led to an increase in activity of both enzymes and also led to induction of alkaline phosphatase activity in cells grown in high phosphate medium to repress alkaline phopsphatase activity. This is an obvious indicator that derepression of alkaline phosphatase and synthesis of the enzyme could occur under these conditions in lake water microcosms. Exoprotease activity was also increased upon the addition of single nutrient source to the microcosms. Osmotic stress coupled with starvation stress also increased alkaline phosphatase activity and the addition of the osmoprotectant, betaine, also increased activity. In both cases activity increased although the viable count decreased, in some cases below the limits of dection. Exoprotease activity increased in osmotically shocked cells and increased further if betaine was added to the starvation medium. Plasmid transfer could still occur between A. hydrophila and Escherichia coli in unamended and lake water microcosms amended with carbon and some nitrogen sources. The transfer of the F group plasmid R1drd19 was temperature dependent with no detectable transfer occurring at temperatures below 15° C even in nutrient broth. Plasmid transfer was dependent upon the size of the donor and recipient populations with no detectable transfer occurring at low population densities but transfer at high densities shows that even under prevailing environmental conditions the transfer of F-plasmids was possible between E. coli and A. hydrophila. The changes in protein fingerprints of cell and periplasm extracts under starvation and other stresses were examined using two-dimensional gel electrophoresis. Several starvation specific proteins were identified on the gels and some proteins which were only transiently produced were noted. The N-terminal sequences of two stress proteins produced in response to starvation and ethanol and heat stress were obtained from the gels. Alkaline phosphatase one of the key proteins in the response to stress was also identified by colourimetric staining of two-dimensional gels. The survival of A. hydrophila under starvation and other stresses is dependent upon the sequential synthesis of many proteins, including alkaline phosphatase.

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.

Thesis (M.S.)--University of Cincinnati, 2008.
Advisor: Margaret Kupferle Ph. D. (Committee Chair), Steven Buchberger Ph. D. (Committee Member), Mark Rodgers Ph. D. (Committee Member), Randy Revetta (Committee Member). Title from electronic thesis title page (viewed Jan. 18, 2009). Includes abstract. Keywords: Aeromonas hydrophila; Biofilm: Biofilm Reactors; Drinking water; Iron coupons; Phospholipid phosphate; Polycarbonate coupons; Water quality. Includes bibliographical references.

O gênero Aeromonas está amplamente distribuído em ambientes aquáticos, e estudos recentes incluem o gênero no grupo de patógenos emergentes, devido à sua freqüente associação com infecções locais e sistêmicas em humanos. Este trabalho foi realizado com o objetivo de pesquisar por meio da PCR e confirmar por meio de seqüenciamento, a ocorrência dos genes de virulência act, alt e ast, e resistência cphA, blaIMP, blaVIM, blaSPM-1, blaCTXM, blaTEM e blaSHV, verificando também o perfil de resistência a partir de antibiogramas, e a ocorrência de plasmídios nas cepas estudadas. A partir dos resultados observou-se que das 100 cepas selecionadas inicialmente, 87 pertenciam às espécies A. jandaei (46) e A. hydrophila (41). Dentre as quais pôde-se observar a ocorrência de act, alt e ast, respectivamente em 70,7 por cento (29), 97,6 por cento (40) e 26,8 por cento (11) das cepas de A. hydrophila, e em 4,4 por cento (2), 0 por cento (0) e 32,6 por cento (15) nas cepas de A. jandaei. Os genes blaIMP, blaVIM, blaSPM-1, blaCTX-M, e blaSHV não foram encontrados em nenhuma cepa. O gene cphA foi encontrado em 97,6 por cento (40) e 100 por cento (46) das cepas de A. hydrophila e A. jandaei, respectivamente e o gene blaTEM foi encontrado em 97,6 por cento (40) das cepas de A. hydrophila e em 85 por cento (39) das cepas de A. jandaei. Foi verificada a presença de plasmídio em 10/41 (24,4 por cento) das cepas de A. hydrophila e em 16/46 (34,9 por cento) das cepas de A. jandaei
The genus Aeromonas is widely distributed in aquatic environments, and recent studies include the genus in the emergent pathogens group, due to its frequent association with local and systemic human infections. This work was carried out aiming the investigation and sequencing virulence (act, alt and ast) and resistance (cphA, blaIMP, blaVIM, blaSPM-1, blaCTX-M, blaTEM e blaSHV) genes, also verifying the resistance profile of the strains using antibiograms and the occurrence of plasmids. From the 100 strains analyzed in this study, 87 belonged to A. jandaei (46) and A. hydrophila (41) species. Out of which it was observed the occurrence of act, alt and ast, respectively in 70.7 per cent (29), 97.6 per cent (40) and 26.8 per cent (11) of A. hydrophila strains, and in 4.4 per cent (2), 0 per cent (0) e 32.6 per cent (15) of A. jandaei strains. The genes blaIMP, blaVIM, blaSPM-1, blaCTX-M e blaSHV were not found. CphA gene was found in 97.6 per cent (40) and 100 per cent (46) of A. hydrophila and A. jandaei strains, respectively and blaTEM gene was found in 97.6 per cent (40) of A. hydrophila strains and in 85 per cent (39) of A. jandaei strains. Presence of plasmid was found in 10/41 (24.4 per cent) of A. hydrophila strains and in 16/46 (34,9 per cent) of A. jandaei strains

This research work was designed to detect the presence of Aeromonas in the Cocà River estuary, Fortaleza, Cearà State, Brazil. The database consisted of 30 samples of the riverâs surface water and 30 samples of the riverâs sooil, in the period from September, 2007 to April, 2008.. They were amenable, simultaneously, to counting of bacteria on Agar Gelatin Phosphate Salt (GSP) plus 20μg/mL of ampicilim (UFC/mL or UFC/g) The results showed dissemination of Aeromonas in the estuary. The counts for the water samples varied from 10 to 7,050 and from 25 to 38,500 UFC/mL at points A and B respectively; and from 100 to 37,500 UFC/g, and 1,200 to 43500 UFC/g at points A and B respectively. At point C, the counts for water and sediment were smaller than 10 UFC per ml or gram in all samples The occurrence of the greatest indices of Aeromonas in April, at the height of the rainy season, and the lowest in Septemer, at the height of the dry season, suggests there to be a probable seasonality of bacteria density in the studied environment. Among the 41 isolated strains were found the species A. caviae, A. sobria, A. trota, A. salmonicida and A. allossacharophyla. All the strains of Aeromonas sp. were found to be sensitive to cloranfenicol and ceftriaxona except for ampicillim, to which they showed 100% resistance.All the stirps showed resistance to two out of the nine tested antibiotics. After the Curingâ technique, the eritromicina resistance seems to be of plasmidian origin.
Este projeto teve como objetivo principal a pesquisa de Aeromonas spp. em Ãgua de superfÃcie e sedimento em trÃs pontos distintos ao longo do rio CocÃ, CearÃ. Foram realizadas coletas no perÃodo de outubro de 2007 a abril de 2008 gerando um total de 30 amostras de Ãgua e 30 de sedimento. A quantificaÃÃo da comunidade bacteriana pertencente ao gÃnero Aeromonas foi feita atravÃs de plaqueamento direto sobre Agar Gelatina Fosfato Sal (Agar GSP acrescido de 20μg/mL de ampicilina). Nas amostras de Ãgua, os valores obtidos variaram de 10 a 7.050 UFC/mL e de 25 a 38.500 UFC/mL nos pontos A e B, respectivamente. Nas amostras de sedimento, as contagens variaram de 100 a 37500 UFC/g e 1.200 a 43.500 UFC/g nos pontos A e B, respectivamente. Nas amostras de Ãgua e sedimento do ponto C, os valores foram menores que 10 UFC (por mL ou g) em todas as coletas. As maiores contagens foram verificadas no mÃs de abril, perÃodo de chuva, e as menores no mÃs de setembro, perÃodo de estiagem. Foram feitos isolamentos e apÃs identificaÃÃo as estirpes foram submetidas à teste de antibiograma e à tÃcnica da âcuraâ do plasmÃdio. Dentre as 41 cepas isoladas, foram identificadas as espÃcies A. caviae, A. sobria, A. trota, A. salmonicida e A. allossacharophyla. Todas as cepas se mostraram sensÃveis ao cloranfenicol e ceftriaxona. Todas as estirpes apresentaram resistÃncia a, pelo menos, dois dos nove antibiÃticos testados. ApÃs a tÃcnica de cura, a maior parte da resistÃncia a eritromicina ficou caracterizada como de origem plasmidial.Conclui-se que o estuÃrio do Rio Cocà està contaminado por Aeromonas e que muitas delas apresentam resistÃncias a antibiÃticos denotando um ambiente poluÃdo e de risco para a populaÃÃo que usa suas Ãguas para lazer, pesca ou outra atividade qualquer.

SILVA, Camila Magalhães. Detecção de Aeromonas spp. em amostras de água superficial e sedimento ao longo de um gradiente de salinidade no estuário do Rio Cocó - Ceará. 2009. 86 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2009
Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-11T15:14:11Z No. of bitstreams: 1 2009_dis_cmsilva.pdf: 1804188 bytes, checksum: cd5c4f372d190f7b3e2f64821e983185 (MD5)
Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-11T15:14:50Z (GMT) No. of bitstreams: 1 2009_dis_cmsilva.pdf: 1804188 bytes, checksum: cd5c4f372d190f7b3e2f64821e983185 (MD5)
Made available in DSpace on 2016-07-11T15:14:50Z (GMT). No. of bitstreams: 1 2009_dis_cmsilva.pdf: 1804188 bytes, checksum: cd5c4f372d190f7b3e2f64821e983185 (MD5) Previous issue date: 2009
This research work was designed to detect the presence of Aeromonas in the Cocó River estuary, Fortaleza, Ceará State, Brazil. The database consisted of 30 samples of the river’s surface water and 30 samples of the river’s sooil, in the period from September, 2007 to April, 2008.. They were amenable, simultaneously, to counting of bacteria on Agar Gelatin Phosphate Salt (GSP) plus 20μg/mL of ampicilim (UFC/mL or UFC/g) The results showed dissemination of Aeromonas in the estuary. The counts for the water samples varied from 10 to 7,050 and from 25 to 38,500 UFC/mL at points A and B respectively; and from 100 to 37,500 UFC/g, and 1,200 to 43500 UFC/g at points A and B respectively. At point C, the counts for water and sediment were smaller than 10 UFC per ml or gram in all samples The occurrence of the greatest indices of Aeromonas in April, at the height of the rainy season, and the lowest in Septemer, at the height of the dry season, suggests there to be a probable seasonality of bacteria density in the studied environment. Among the 41 isolated strains were found the species A. caviae, A. sobria, A. trota, A. salmonicida and A. allossacharophyla. All the strains of Aeromonas sp. were found to be sensitive to cloranfenicol and ceftriaxona except for ampicillim, to which they showed 100% resistance.All the stirps showed resistance to two out of the nine tested antibiotics. After the Curing’ technique, the eritromicina resistance seems to be of plasmidian origin.
Este projeto teve como objetivo principal a pesquisa de Aeromonas spp. em água de superfície e sedimento em três pontos distintos ao longo do rio Cocó, Ceará. Foram realizadas coletas no período de outubro de 2007 a abril de 2008 gerando um total de 30 amostras de água e 30 de sedimento. A quantificação da comunidade bacteriana pertencente ao gênero Aeromonas foi feita através de plaqueamento direto sobre Agar Gelatina Fosfato Sal (Agar GSP acrescido de 20μg/mL de ampicilina). Nas amostras de água, os valores obtidos variaram de 10 a 7.050 UFC/mL e de 25 a 38.500 UFC/mL nos pontos A e B, respectivamente. Nas amostras de sedimento, as contagens variaram de 100 a 37500 UFC/g e 1.200 a 43.500 UFC/g nos pontos A e B, respectivamente. Nas amostras de água e sedimento do ponto C, os valores foram menores que 10 UFC (por mL ou g) em todas as coletas. As maiores contagens foram verificadas no mês de abril, período de chuva, e as menores no mês de setembro, período de estiagem. Foram feitos isolamentos e após identificação as estirpes foram submetidas à teste de antibiograma e à técnica da “cura” do plasmídio. Dentre as 41 cepas isoladas, foram identificadas as espécies A. caviae, A. sobria, A. trota, A. salmonicida e A. allossacharophyla. Todas as cepas se mostraram sensíveis ao cloranfenicol e ceftriaxona. Todas as estirpes apresentaram resistência a, pelo menos, dois dos nove antibióticos testados. Após a técnica de cura, a maior parte da resistência a eritromicina ficou caracterizada como de origem plasmidial.Conclui-se que o estuário do Rio Cocó está contaminado por Aeromonas e que muitas delas apresentam resistências a antibióticos denotando um ambiente poluído e de risco para a população que usa suas águas para lazer, pesca ou outra atividade qualquer.

碩士
國立臺灣大學
農業化學研究所
89
Abstract Aeromonas hydrophila is gram negative, anaerobic bacterium which frequently isolated from aquatic environment. It has been increasingly recognized as an enteric pathogen and has been associated with a wide variety of animals and human infections. A hydrophila produces a variety of biologically active extracellular substrates, including hemolysin, lipase, and protease etc. Such extracellular substrates have been studied in relation to the patogenicity of the organisms. It has been demonstrated that hemolysin, especiallyβ-hemolysin, produced by A. hydrophila was one of the most important enterotoxigenic factor. In this study, we research on the hemolytic properties of the hemolysin produced by A. hydrophila environmental strain P1611. Extracellular hemolysin produced by A. hydrophila P1611 was collected after 6 h incubation at 30℃ with DY medium. The purification of hemolysin then has been achieved by combination of acid precipitation, ion-exchange and gel filtration. The purified hemolysin has a molecular weight of 51 kD by SDS-PAGE and 93.3 kD by SephacrylTM S-200 HR gel filtration. According to these results, it was predicted that the protein posses a dimer structure to keep in a stable form. The hemolysin can be separated into six hemolytic proteins of 6.0、6.2、6.5、6.9、7.2、7.3 by isoelectrofocusing electrophoresis, proteins of 7.2、7.3 were the stronger active compounds. N-Terminal sequences of the hemolysin analysed by HPLC is AEPVYPDQVG and is homologous to several other hemolysins of Aeromonas. The hemolysin is thermo-labile and can lyse erythrocytes in a very short time. The lysis mechanism of hemolysin is predicted by binding on the membrane to form a pore on the membrane of erythrocytes resulting to leaking of cytoplasm and the enterance of extracellular components. The hemolysin shows proteolytic activity which can lyse the protein in the membrane of erythrocytes including spectrin, the main protein of membrane skeleton.

Celem badań podjętych w ramach niniejszej rozprawy była (i) szczegółowa analiza fizjologiczna, genomiczna, i funkcjonalna dwóch szczepów DARB: Aeromonas sp. O23A i Shewanella sp. O23S wyizolowanych z kopalni złota i arsenu w Złotym Stoku, określenie (ii) roli tych szczepów w procesach mobilizacji i immobilizacji arsenu i (iii) czynników wpływających na te przemiany, oraz (iv) oszacowanie możliwości zastosowania analizowanych szczepów w systemach bioremediacji.
The aim of the research described in this dissertation was: (i) a detailed genomic, physiological and functional analysis of two DARB strains: Aeromonas sp. O23A and Shewanella sp. O23S, isolated from the gold and arsenic mine in Zloty Stok, and determination of (ii) the role of these strains in the processes of mobilization and immobilization of arsenic, (iii) factors affecting these transformations, and (iv) estimation of the application potential of the analyzed strains in bioremediation systems.

MSc (Microbiology)
Department of Microbiology
Background: The occurrence of microorganisms and endocrine disrupting chemicals (EDCs) in water poses a serious concern due to their effects on humans, animals and environment. In recent years, EDCs have been increasingly reported in rivers that receive large amounts of wastewater effluents. Of all the EDCs, natural and synthetic hormones are among those that are recognized for their potential to mimic or interfere with normal hormonal functions of humans and animals. The present study aimed at assessing the occurrence of these hormones in relation to the molecular diversity of Aeromonas and evaluating the resistance of Aeromonas to antibiotics as well as to assess anti-bacterial activity of two selected traditional medicinal plants. Methods: Wastewater, water and fish samples were collected from various sources (rivers, wastewater treatment plants, taps, and dams) for the detection of hormones and isolation of Aeromonas species. The analysis of hormones from various organs of the fish and from water samples was conducted, after extraction using enzymelinked immunosorbent assays (ELISA). Different types of hormones including Estriol, Estradiol, Ethinylesradiol and Testosterone were detected, and their concentrations determined. Aeromonas spp were isolated rom the samples using microbiological methods and Conventional PCR was used for genotyping as well as for detection of the beta-lactamase genes. Kirby-bauer method was used to determine the susceptibility profiles of Aeromonas to different antibiotics. Microdilution assay was used to determine the Anti-bacterial activity of the plant (Annoniceae and Zornia milneana) extracts against Aeromonas species. Results: A total of 144 samples were collected from 23 different locations in two countries: South Africa and Zambia. These included wastewater and treated wastewater, River water, fish and tap water. 17α-ethinylestradiol (EE2) was detected in most of the samples (92.7%) with concentrations varying from 0.59 ng/ml to 65 ng/ml. The hormones were also detected from drinking water, with testosterone detected at high concentrations of up to 140 ng/ml in tap water. Most sewage treatment plants were not able to remove the EE2 from the wastewater as the concentration of this hormone in the final effluent was almost always higher than that in the influent. These homones were also detected in drinking water at high concentrations of up to 53.49 ng/ml in the tap water for EE2 and 1777 ng/ml for E2. The overall detection of Aeromonas species in the samples was 84.5%. A. caviae was the most prevalent species accounting for 73.6%, followed by A. veronii with 64.6%. The bacteria were completely resistant to cefuroxime accounting for 100% resistance. Aeromonas isolates also showed high resistance to trimethroprim (88.7% for A. hydrophila), cefazolin (highest 97.8% for A. cavie), and ceftazidime (83.9% for A. sobria). TEM was the most prevalent beta-lactamase gene with detection rate of 87%. All isolates lacked the presence of the CTX-M3 gene. Also, wastewater had the highest prevalence of A. veronni and A. caviae accounting for 87.5% and 82.5% respectively. Multiple antibiotic resistance was also observed with the Aeromonas isolates being resistant to up to 11 antibiotics. High prevalence of 77.1% of Aeromonas hydrophila was observed in the presence of ethinylestradiol (EE2). Aeromonas veronii and Aeromonas caviae were the most predominant species in the presence of total estriol, A. veronii had a prevalence of 57.1% and A. caviae had a prevalence of 52.8%. Aeromonas hydrophila and Aeromonas caviae had the lower prevalence in the presence of hormones with the percentages of 26.1% and 27.8% respectively. The methanol extracts of both Zornia milneana and Annona species showed good activity against the Aeromonas spp with the lowest MIC of 0.078 mg/ml. Ethyl acetate extracts were the least effective. Conclusion: This study has shown high occurrence of steroid hormones in all types of environmental samples tested. These included tap water, river water, wastewater and fish both in Zambia and South Africa. Therefore, steroid hormones constitute and important health problem in the Southern African Sub-Region. The incapacity of the wastewater treatment plants to remove EE2 is an important problem that needs to be tackled immediately. The prevalence of Aeromonas species is very high in our environmental water as well as in drinking water, with the highest prevalence observed in fish and wastewater. It was also revealed that there is relationship between steroid hormones and Aeromonas species, with the hormones supporting the growth of Aeromonas species. The presence of beta-lactamase genes which causes Aeromonas to be resistant to antibiotics was also noted. Methanol extracts of Zornia milneana and Annona spp were the most effective against Aeromonas spp and could serve as primary sources for the isolation of lead compounds.
NRF