Fish flesh agar medium - a suitable experimental medium for the detection of spoilage bacteria

The spoilage characteristics of bacterial strains were studied by growing them at 28 _+ 2 °C in agar and broth media prepared with sterile fish and prawn flesh homogenates. The percentage of spoilers found among the bacterial isolates tested, as shown by odour production and halo zone formation, was independent of the source of flesh used. Indole and fluorescent pigment production were also observed in the broth. Pseudomonas, Vibrio and Acinetobacter exhibited faster growth in flesh media than in the usual artificial media. Decrease of protein and lipid concentration in the clear zone of agar media suggests the utilization of the available substrate by spoilage bacteria.

Iron Uptake and Homeostasis in Microorganisms

Preface. Iron is considered to be a minor element employed, in a variety of forms, by nearly all living organisms. In some cases, it is utilised in large quantities, for instance for the formation of magnetosomes within magnetotactic bacteria or during use of iron as a respiratory donor or acceptor by iron oxidising or reducing bacteria. However, in most cases the role of iron is restricted to its use as a cofactor or prosthetic group assisting the biological activity of many different types of protein. The key metabolic processes that are dependent on iron as a cofactor are numerous; they include respiration, light harvesting, nitrogen fixation, the Krebs cycle, redox stress resistance, amino acid synthesis and oxygen transport. Indeed, it is clear that Life in its current form would be impossible in the absence of iron. One of the main reasons for the reliance of Life upon this metal is the ability of iron to exist in multiple redox states, in particular the relatively stable ferrous (Fe2+) and ferric (Fe3+) forms. The availability of these stable oxidation states allows iron to engage in redox reactions over a wide range of midpoint potentials, depending on the coordination environment, making it an extremely adaptable mediator of electron exchange processes. Iron is also one of the most common elements within the Earth’s crust (5% abundance) and thus is considered to have been readily available when Life evolved on our early, anaerobic planet. However, as oxygen accumulated (the ‘Great oxidation event’) within the atmosphere some 2.4 billion years ago, and as the oceans became less acidic, the iron within primordial oceans was converted from its soluble reduced form to its weakly-soluble oxidised ferric form, which precipitated (~1.8 billion years ago) to form the ‘banded iron formations’ (BIFs) observed today in Precambrian sedimentary rocks around the world. These BIFs provide a geological record marking a transition point away from the ancient anaerobic world towards modern aerobic Earth. They also indicate a period over which the bio-availability of iron shifted from abundance to limitation, a condition that extends to the modern day. Thus, it is considered likely that the vast majority of extant organisms face the common problem of securing sufficient iron from their environment – a problem that Life on Earth has had to cope with for some 2 billion years. This struggle for iron is exemplified by the competition for this metal amongst co-habiting microorganisms who resort to stealing (pirating) each others iron supplies! The reliance of micro-organisms upon iron can be disadvantageous to them, and to our innate immune system it represents a chink in the microbial armour, offering an opportunity that can be exploited to ward off pathogenic invaders. In order to infect body tissues and cause disease, pathogens must secure all their iron from the host. To fight such infections, the host specifically withdraws available iron through the action of various iron depleting processes (e.g. the release of lactoferrin and lipocalin-2) – this represents an important strategy in our defence against disease. However, pathogens are frequently able to deploy iron acquisition systems that target host iron sources such as transferrin, lactoferrin and hemoproteins, and thus counteract the iron-withdrawal approaches of the host. Inactivation of such host-targeting iron-uptake systems often attenuates the pathogenicity of the invading microbe, illustrating the importance of ‘the battle for iron’ in the infection process. The role of iron sequestration systems in facilitating microbial infections has been a major driving force in research aimed at unravelling the complexities of microbial iron transport processes. But also, the intricacy of such systems offers a challenge that stimulates the curiosity. One such challenge is to understand how balanced levels of free iron within the cytosol are achieved in a way that avoids toxicity whilst providing sufficient levels for metabolic purposes – this is a requirement that all organisms have to meet. Although the systems involved in achieving this balance can be highly variable amongst different microorganisms, the overall strategy is common. On a coarse level, the homeostatic control of cellular iron is maintained through strict control of the uptake, storage and utilisation of available iron, and is co-ordinated by integrated iron-regulatory networks. However, much yet remains to be discovered concerning the fine details of these different iron regulatory processes. As already indicated, perhaps the most difficult task in maintaining iron homeostasis is simply the procurement of sufficient iron from external sources. The importance of this problem is demonstrated by the plethora of distinct iron transporters often found within a single bacterium, each targeting different forms (complex or redox state) of iron or a different environmental condition. Thus, microbes devote considerable cellular resource to securing iron from their surroundings, reflecting how successful acquisition of iron can be crucial in the competition for survival. The aim of this book is provide the reader with an overview of iron transport processes within a range of microorganisms and to provide an indication of how microbial iron levels are controlled. This aim is promoted through the inclusion of expert reviews on several well studied examples that illustrate the current state of play concerning our comprehension of how iron is translocated into the bacterial (or fungal) cell and how iron homeostasis is controlled within microbes. The first two chapters (1-2) consider the general properties of microbial iron-chelating compounds (known as ‘siderophores’), and the mechanisms used by bacteria to acquire haem and utilise it as an iron source. The following twelve chapters (3-14) focus on specific types of microorganism that are of key interest, covering both an array of pathogens for humans, animals and plants (e.g. species of Bordetella, Shigella, , Erwinia, Vibrio, Aeromonas, Francisella, Campylobacter and Staphylococci, and EHEC) as well as a number of prominent non-pathogens (e.g. the rhizobia, E. coli K-12, Bacteroides spp., cyanobacteria, Bacillus spp. and yeasts). The chapters relay the common themes in microbial iron uptake approaches (e.g. the use of siderophores, TonB-dependent transporters, and ABC transport systems), but also highlight many distinctions (such as use of different types iron regulator and the impact of the presence/absence of a cell wall) in the strategies employed. We hope that those both within and outside the field will find this book useful, stimulating and interesting. We intend that it will provide a source for reference that will assist relevant researchers and provide an entry point for those initiating their studies within this subject. Finally, it is important that we acknowledge and thank wholeheartedly the many contributors who have provided the 14 excellent chapters from which this book is composed. Without their considerable efforts, this book, and the understanding that it relays, would not have been possible. Simon C Andrews and Pierre Cornelis

Preliminary X-ray diffraction analysis of YqjH from Escherichia coli: a putative cytoplasmic ferri-siderophore reductase

YqjH is a cytoplasmic FAD-containing protein from Escherichia coli; based on homology to ViuB of Vibrio cholerae, it potentially acts as a ferri-siderophore reductase. This work describes its overexpression, purification, crystallization and structure solution at 3.0 A resolution. YqjH shares high sequence similarity with a number of known siderophore-interacting proteins and its structure was solved by molecular replacement using the siderophore-interacting protein from Shewanella putrefaciens as the search model. The YqjH structure resembles those of other members of the NAD(P)H:flavin oxidoreductase superfamily.

Geographical distribution of American cutaneous leishmaniasis and its phlebotomine vectors (Diptera: Psychodidae) in the state of Sao Paulo, Brazil

Background: American cutaneous leishmaniasis (ACL) is a re-emerging disease in the state of Sao Paulo, Brazil. It is important to understand both the vector and disease distribution to help design control strategies. As an initial step in applying geographic information systems (GIS) and remote sensing (RS) tools to map disease-risk, the objectives of the present work were to: (i) produce a single database of species distributions of the sand fly vectors in the state of Sao Paulo, (ii) create combined distributional maps of both the incidence of ACL and its sand fly vectors, and (iii) thereby provide individual municipalities with a source of reference material for work carried out in their area. Results: A database containing 910 individual records of sand fly occurrence in the state of Sao Paulo, from 37 different sources, was compiled. These records date from between 1943 to 2009, and describe the presence of at least one of the six incriminated or suspected sand fly vector species in 183/645 (28.4%) municipalities. For the remaining 462 (71.6%) municipalities, we were unable to locate records of any of the six incriminated or suspected sand fly vector species (Nyssomyia intermedia, N. neivai, N. whitmani, Pintomyia fischeri, P. pessoai and Migonemyia migonei). The distribution of each of the six incriminated or suspected vector species of ACL in the state of Sao Paulo were individually mapped and overlaid on the incidence of ACL for the period 1993 to 1995 and 1998 to 2007. Overall, the maps reveal that the six sand fly vector species analyzed have unique and heterogeneous, although often overlapping, distributions. Several sand fly species - Nyssomyia intermedia and N. neivai - are highly localized, while the other sand fly species - N. whitmani, M. migonei, P. fischeri and P. pessoai - are much more broadly distributed. ACL has been reported in 160/183 (87.4%) of the municipalities with records for at least one of the six incriminated or suspected sand fly vector species, while there are no records of any of these sand fly species in 318/478 (66.5%) municipalities with ACL. Conclusions: The maps produced in this work provide basic data on the distribution of the six incriminated or suspected sand fly vectors of ACL in the state of Sao Paulo, and highlight the complex and geographically heterogeneous pattern of ACL transmission in the region. Further studies are required to clarify the role of each of the six suspected sand fly vector species in different regions of the state of Sao Paulo, especially in the majority of municipalities where ACL is present but sand fly vectors have not yet been identified.

OCCURRENCE OF PATHOGENIC MICROORGANISMS IN FISH SOLD IN SAO PAULO, BRAZIL

This study investigated the presence of potentially human pathogenic strains of Vibrio spp., Aeromonas spp., Escherichia coli, Salmonella spp. and Staphylococcus aureus in fish commercialized in street markets of Sao Paulo city, Brazil. Twenty fish of different species were analyzed for foodborne pathogens using conventional methods. High levels of fecal contamination were detected in 25% of samples. S. aureus was isolated from 10% of samples. All were negative for Salmonella. Vibrio species, including Vibrio cholerae non-O1/non-O139, were observed in 85% of samples although Vibrio parahaemolyticus was not found in this study. Aeromonas spp., including A. hydrophila, was isolated from 50% of fish samples. The occurrence of these pathogens suggests that the fish commercialized in Sao Paulo may represent a health risk to the consumers.

Dynamics of Aeromonas species isolated from wastewater treatment system

Aeromonas are widely distributed in the aquatic environment, and are considered to be emerging organisms that can produce a series of virulence factors. The present study was carried out in a sanitary sewage stabilization pond treatment system, located in Lins, State of Sao Paulo, Brazil. Most probable number was applied for estimation of the genus Aeromonas. Colony isolation was carried out on blood agar ampicillin and confirmed by biochemical characterization. Aeromonas species were isolated in 72.4% of influent samples, and in 55.2 and 48.3% of effluent from anaerobic and facultative lagoons, respectively. Thirteen Aeromonas species were isolated, representing most of the recognized species of these organisms. Even though it was possible to observe a tendency of decrease, total elimination of these organisms from the studied system was not achieved. Understanding of the pathogenic organism`s dynamics in wastewater treatment systems with a reuse potential is especially important because of the risk it represents.

Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N-2-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of Sao Sebastiao (Sao Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n = 76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n = 7), V. alginolyticus (n = 8), V. campbellii (n = 3), and V parahaemolyticus (n = 1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N, might explain why they are so abundant in the mucus of different coral species. (C) 2008 Published by Elsevier GmbH.

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the eft operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the eft operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

Protein Assembly onto Cationic Supported Bilayers

Cationic supported bilayers on latex are useful to isolate and immobilize oppositely charged proteins as a monomolecular layer over a range of low protein concentrations and particle number densities. Cholera toxin (CT) from Vibrio cholerae, an 87 kDa AB(5) hexameric protein and bovine serum albumin (BSA) self-assembled on dioctadecyldimethylammonium bromide (DODAB) supported bilayers with high affinity yielding highly organized and monodisperse particulates at 5 x 10(9) particles/mL, over a range of low protein concentrations (0-0.025 mg/mL BSA or CT). Protein association onto the bilayer-covered polystyrene sulfate (PSS) was determined from adsorption isotherms, dynamic light scattering for size distributions and zeta-potential analysis revealing a monomolecular, thin and highly organized protein layer surrounding each particle with potential for biospecific recognition such as antigen-antibody, receptor-ligand, hybridization of oligonucleotide sequences, all of them important in immunodiagnosis, selective biomolecular chromatographic separations, microarrays design and others.

Resistotipagem e transferência genética de plasmídios de resistência entre coliformes e vibrios potencialmente patogênicos para o homem isolados de camarão (litopenaeus vannamei) comercializado em Natal-Rio Grande do Norte

Cinquenta amostras de camarão fresco e refrigerado (Litopenaeus vannamei) foram coletadas em diferentes pontos de comercialização na cidade de Natal RN. As amostras foram maceradas em um gral estéril e 25 gramas semeadas em 225mL de APA contendo 1% de NaCl e 25g em 225mL de CL incubadas a 35ºC - 24 horas. O crescimento em APA foi semeado em placas de Ágar TCBS, incubadas a 35ºC-24h para isolamento de Vibrio e Aeromonas. O crescimento do CL foi semeado em Agar EAM, para isolamento de coliformes. Dos 102 isolados, 91 (89,2%) pertenciam ao gênero Vibrio e 11 (10,8%) ao gênero Aeromonas, com predominância de V. cholerae não O1/não O139, V. alginolyticus, V. carchariae e V. parahaemolyticus K- e A. veronii biogrupo sobria , A. jandaei, A. schubertii, A. veronii biogrupo veronii e A. hydrophila. A menor eficiência entre os antimicrobianos foi da AMP (57,8% de resistência) seguida da AMK (29,4%) e TCY (21,6%). As 39 cepas de Vibrio e Aeromonas multirresistentes se distribuíram em 10 perfis distintos, sendo que um revelou cinco marcos (AMP, CHL, NIT, SXT e TCY) em um isolado de V. carchariae de camarão, adquirido em supermercados. O índice MAR, nas 39 cepas variou de 0,28 a 0,42, sugerindo que são de risco na transferência e difusão da resistência na cadeia alimentar. Após a cura plasmidial pelo tratamento com AO de 24 cepas multirresistentes e com resistência intermediária de víbrio e aeromonas escolhidas aleatoriamente, 13 perderam totalmente a resistência e 7 perderam parcialmente, sendo que o maior percentual de perda da resistência ocorreu nas cepas de V. cholerae não O1 e não O139 (6 cepas), se concentrando nos marcos de resistência a AMP (13), AMK (11), TCY(8) e CIP(3). Os resultados da conjugação realizada entre amostras de Vibrio xvi curadas e a E. coli K12C600 demonstraram que 78,5% das culturas de Vibrio testadas revelaram capacidade de transferência para o gene que confere resistência a AMP e 28,5% para a TCY. Dos coliformes, E. coli foi a mais frequente, seguida de Citrobacter spp, isoladas em 40,3% e 27,5% das amostras respectivamente. AMP foi o antimicrobiano menos eficaz, seguido de TCY. As 11 cepas multirresistentes se distribuíram em 9 perfis distintos, um deles constituído de cinco marcos (AMP, NIT, TCY, CHL, SXT), albergados em uma cepa de Klebsiella spp, oriunda de camarão adquirido em supermercado, similar ao resultado obtido em V. carchariae. Conclui-se que, os camarões marinhos frescos e refrigerados, comercializados em Natal-RN evidenciaram contaminação com coliformes, víbrios e aeromonas multirresistentes a antimicrobianos comumente utilizados na terapia médica e veterinária, e que, possivelmente, a transferência de genes de resistência entre bactérias se constitui um sério problema de saúde pública

Tropical dermatology: Marine and aquatic dermatology

Dermatoses caused by marine organisms are frequently seen in dermatology clinics worldwide. Cutaneous injuries after exposure to marine environments include bacterial and fungal infections and lesions caused by aquatic plants and protists. Some of these diseases are well known by dermatologists, such as Vibrio vulnificus septicemia and erysipeloid, but others are uncommon, such as envenomation caused by ingestion or contact with certain dinoflagellates or cyanobacteria, which are associated with rashes that can begin Within minutes after exposure. Many marine/aquatic invertebrates, such as sponges, cnidarians, echinoderms, crustaceans, and mollusks, are associated with different kinds of dermatologic lesions that call vary from irritant or allergic contact dermatitis to physical trauma and envenomations. These cutaneous lesions May result in mild local reactions or can be associated with severe systemic reactions. Invertebrate animals, such as cnidarians, sea urchins, and worms, and aquatic vertebrates, such as venomous fishes and stingrays, are commonly associated with skin lesions in many countries, where they call constitute occupational dermatoses among fishermen and scuba divers, but they can also be observed among persons who contact these animals in kitchens or beaches. The presence of unusual lesions, a recent travel history, and/or a report of contact with an aquatic environment (including ownership of a marine or freshwater aquarium) should alert the dermatologist to the etiology of the cutaneous problems. (J Am Acad Dermatol 2009;61:733-50.)

Ballast water: a review of the impact on the world public health

Since the nineteenth century ships have been using ballast water (BW) for safety, stability, propulsion and maneuverability, as well as to redress loss of fuel weight and water consumption, and to maintain structural stress at acceptable levels. Ballast water has been spreading many non-native species around the globe, but little is known about the extent and potential significance of ship-mediated transfer of microorganisms. The global movements of ballast water by ships create a long-distance dispersal mechanism for human pathogens that may be important in the worldwide distribution of microorganisms, as well as for the epidemiology of waterborne diseases. Only a few studies have been carried out on this subject, most of them involving ballast water containing crustacean larvae and phytoplankton. Specialized microbiological studies on these waters are necessary to avoid a repeat of what happened in 1991, when epidemic cholera was reported in Peru and rapidly spread through Latin America and Mexico. In July of 1992, Vibrio cholerae was found in the USA and the Food and Drug Administration (FDA) determined that it came from ballast water of ships whose last port of call was in South America. In Brazil, just a few studies about the subject have been performed. An exploratory study by the Brazilian National Health Surveillance Agency (Agencia Nacional de Vigilancia Sanitaria - ANVISA) found in ballast water different microorganisms, such as fecal coliforms, Escherichia coli, Enterococcus faecalis, Clostridium perfringens, coliphages, Vibrio cholerae O1 and Vibrio cholerae non-O1. Until now, Brazil has been focusing only on organisms transported to its territory from other countries by ballast water, to avoid their establishment and dissemination in Brazilian areas. Studies that can assess the probability that water ballast carries pathogenic microorganisms are extremely important, as is the examination of ships that arrive in the country. Treatment of the human infections caused by BW exists but none is completely safe and efficient.

Ribonuclease production by Aspergillus species

Ribonuclease production by Aspergillus flavipes. A sulphureus and A. fischeri in semisynthetic medium, after 24-144 hours at 30 degrees C under shaking, was studied. After cultivation, the medium was separated from micelia by filtration and the resultant solution was used as enzymatic extract. The highest amount of biomass and RNase was obtained after 96 hours of cultivation. The enzymes produced by three species presented similar characteristics, with optimum temperature at 55 degrees C and two peaks of activity at pH 4.5 and 7.0. A. flavipes RNases were more sensitive to temperature: 50% of the initial activity was lost after 1 hour at 70 degrees C. After this heat treatment, RNase of A. sulphureus lost 30% of this activity and that of A. fischeri only 16%. The nucleotides released by enzimatic hydrolysis of RNA were separated by ion exchange chromatography in a AG-1X8-formiate column and identified by paper chromatography. This procedure indicated that the raw enzymatic extract of Aspergillus flavipes is able to hydrolyze RNA, releasing 3'-nucleotides monophosphate at pH 4.5 and 3' and 5'-nucleotides monophosphate at pH 7.0 and 8.5. This result suggests that this strain produces two different types of RNase, one acidic and other alcaline, with different specificities.

Microbiological quality of salmon (Salmo salar) sold in cities of the state of São Paulo, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Novos registros de Lutzomyia longipalpis (Lutz & Neiva, 1912)(Diptera: Psychodidae) na região Centro-Leste do estado de São Paulo, Brasil

A incidência das leishmanioses tegumentar (LTA) e visceral (LVA) americanas, especialmente essa última, em hospedeiros caninos e humanos, encontra-se em expansão no Estado de São Paulo. Na vigilância epidemiológica dessas endemias, torna-se fundamental o conhecimento da distribuição e ecologia das diferentes espécies de flebotomíneos. Assim, a divulgação de novos registros de seus vetores é fundamental para apontar novas áreas de risco para a transmissão dessas doenças. Neste estudo, realizaram-se capturas de flebotomíneos em ambiente de mata, em diferentes localidades dos municípios de Ipeúna, Itirapina e Analândia, entre agosto e setembro de 2007. Foram capturados 248 flebotomíneos de nove espécies diferentes, em Ipeúna, seis e sete espécimes de duas espécies distintas coletados respectivamente em Itirapina e Analândia. A espécie mais abundante em Ipeúna foi Pintomyia pessoai (37,5%), seguida de P. fischeri (33,06%) e Migonemyia migonei (16,53%). Essas três espécies são consideradas importantes vetores de LTA no território paulista. O registro de Lutzomyia longipalpis pela primeira vez em Ipeúna e Analândia e a confirmação de sua presença em Itirapina indicam risco de essabelecimento da LVA na área e a necessidade de mais estudos locais sobre sua ecologia, sobretudo em relação à ocupação de ambientes antrópicos.