Caracterização molecular de bastonetes Gram positivos irregulares e actinomicetos aeróbios obtidos de espécimes clínicos, de ensaios de esterilidade e de áreas limpas

Os bastonetes Gram positivos irregulares (BGPIs) compõem um grupo de espécies bacterianas com ampla diversidade fenotípica e que podem estar presente no meio ambiente, na microbiota humana e de animais. A identificação acurada de BGPIs em nível de gênero e espécie empregando métodos bioquímicos convencionais é bastante limitada, sendo recomendado, portanto, o uso de técnicas moleculares. No presente estudo, foram identificadas amostras de BGPIs oriundas de espécimes clínicos de humanos, de produtos farmacêuticos e de áreas limpas através da análise de sequencias do gene 16S rRNA e de outros genes conservados (housekeeping genes). Os resultados obtidos pelo sequenciamento dos genes 16S rRNA e rpoB demonstraram C. striatum multi-resistente (MDR) como responsável por surto epidêmico em ambiente hospitalar da cidade do Rio de Janeiro. Quinze cepas de C. striatum foram isoladas em cultura pura a partir de secreção traqueal de pacientes adultos submetidos a procedimentos de entubação endotraqueal. A análise por eletroforese em gel de campo pulsado (PFGE) indicou a presença de quatro perfis moleculares, incluindo dois clones relacionados com cepas MDR (PFGE I e II). Os dados demonstram a predominância de PFGE I entre cepas MDR isoladas de unidades de terapia intensiva e enfermarias cirúrgicas. Uma potencial ligação causal entre a morte e a infecção por C. striatum MDR (PFGE tipos I e II) foi observada em cinco casos. Adicionalmente, acreditamos que este seja o primeiro estudo de identificação de espécies de Nocardia relacionadas com infecções humanas pela análise da sequencia multilocus (MLSA) no Brasil. Diferente dos dados observados na literatura (1970 a 2013) e obtidos pelos testes fenotípicos convencionais, a caracterização molecular de quatro lócus (gyrB-16S-secA1-hsp65) permitiu a identificação das espécies N. nova, N. cyriacigeorgica, N. asiatica e N. exalbida/gamkensis relacionadas com quadros de nocardiose em humanos. Cepas de N. nova isoladas de diferentes materiais clínicos de um único paciente apresentaram padrões de susceptibilidade antimicrobianos idênticos e dois perfis PFGE, indicando a possibilidade de quadros de co-infecção por N. nova em humanos. Em outra etapa da investigação, amostras de BGPIs obtidos de ambientes de salas limpas que não puderam ser identificadas por critérios convencionais foram submetidas a análise da sequência do gene 16S rRNA e caracterizadas 95,83% em nível de gênero e 35,42% em espécies. Para gêneros mais encontrados no estudo, foram analisados os genes rpoB e recA de dezessete cepas de Microbacterium e utilizado o MLSA para a identificação de sete cepas identificadas como Streptomyces. Os ensaios permitiram a identificação de três cepas de Microbacterium e de uma única amostra de Streptomyces ao nível de espécie. A análise da sequencia do gene rpoB também se mostrou eficaz na identificação de espécies de cepas de Corynebacterium. Finalmente, para as cepas ambientais pertencentes à classe Actinobacteria os dados morfológicos, bioquímicos e genotípicos permitiram documentar a cepa 3117BRRJ como representante de uma nova espécie do gênero Nocardioides, para o qual o nome Nocardioides brasiliensis sp. nov. e as cepas 3712BRRJ e 3371BRRJ como representante de um novo gênero e espécie para o qual o nome Guaraldella brasiliensis nov. foi proposto.

Caracterização de Bacilos Gram-Negativos Não Fermentadores não usuais em bacteremias pelas técnicas de Matrix-Assisted Laser Desorption IonizationTime of Flight Mass Spectrometry, sequenciamento de DNA e método fenotípico convencional

Alguns Bastonetes Gram-negativos não fermentadores (BGNNF) costumam ser considerados clinicamente pouco significantes e a sua implicação em infecções é subestimada. Devido à similaridade fenotípica, mudanças taxonômicas, baixa reatividade bioquímica e limitações nos bancos de dados em sistemas comerciais, a identificação de BGNNF é frequentemente equivocada, culminando com a denominação de diferentes micro-organismos apenas como BGNNF, por falta de melhor diferenciação. O objetivo desse estudo foi avaliar, por métodos fenotípico convencional, proteômico e molecular, a identificação de BGNNF incomuns isolados em hemoculturas de pacientes atendidos em um hospital universitário no Rio de Janeiro. Foram selecionadas 78 amostras isoladas de hemoculturas caracterizadas no laboratório clinico como BGNNF para a identificação por sequenciamento dos genes 16S RNA e recA, por um conjunto amplo de testes fenotípicos manuais e por MALDI-TOF MS. Os micro-organismos predominantes na amostragem foram genotipados pela técnica de eletroforese em gel de campo pulsado (PFGE). Pelo sequenciamento do gene 16S rRNA, a maioria das amostras (n=31; 40%) foi incluída no gênero Burkholderia, seguido de Pseudomonas stutzeri (10%) e Delftia acidovorans (4%). Os demais isolados foram agrupados em 27 diferentes espécies. O sequencimento do gene recA identificou a maioria das espécies de Burkholderia como Burkholderia contaminans (n=19; 24%). Os testes fenotípicos incluíram as 31 amostras apenas no CBc e para as outras 47 amostras, a concordância com o sequenciamento do gene 16S rRNA em nível de espécie foi de 64% (n=30) e apenas em gênero a concordância foi de 17% (n=8). A análise comparativa geral da identificação por MALDI-TOF MS com o sequenciamento do gene16S rRNA mostrou que 42% (n=33) das 78 amostras foram concordantes em nível de espécie e 45% (n=35) apenas em gênero. Excluindo as amostras do CBc, houve um aumento da concordância em nível de espécie para 60%. As discordâncias parecem ser devido às diferenças nos perfis proteicos das amostras em relação às amostras-referência do banco de dados do equipamento e podem ser aprimorados com a atualização de perfis no sistema. A análise do polimorfismo genético de B. contaminans mostrou a ausência de um clone disseminado causando surto, além da provável origem ambiental das infecções. Os setores de nefrologia e hemodiálise contribuíram com maior número de pacientes com amostras positivas (5 pacientes e 9 amostras). Os grupos clonais BcoD e BcoE foram encontrados em pacientes assistidos no mesmo setor com diferença de quatro meses (BcoD, nefrologia) e 1,5 ano (BcoE, hemodilálise), entre as culturas, respectivamente. As discordâncias entre as técnicas ocorreram principalmente devido a dificuldade de identificação das espécies do CBc. Os BGNNF incomuns são de difícil caracterização independente da metodologia usada e nenhum método por si só foi capaz de identificar todas as amostras.

Achromobacter xylosoxidans na fibrose cística: infecção crônica e disseminação clonal

A mortalidade na Fibrose Cística (FC) é decorrente de infecções pulmonares causadas comumente por: Pseudomonas aeruginosa, Staphylococcus aureus e espécies do Complexo Burkholderia cepacia (CBc). Mais recentemente, tem sido observada a emergência de BGN-NF raros, como Achromobacter xylosoxidans, porém, sua prevalência, potencial de transmissão e significado clínico são desconhecidos. O objetivo deste trabalho foi verificar a ocorrência de colonização crônica por A. xylosoxidans e avaliar a possibilidade de transmissão cruzada entre os pacientes acompanhados em dois centros de referência na cidade do Rio de Janeiro. Foram incluídos 39 pacientes com FC, com pelo menos uma cultura positiva para o gênero Achromobacter spp., em um total de 897 analisadas, do período de janeiro de 2003 a dezembro de 2008. A frequência de isolamento de Achromobacter spp. nas culturas analisadas foi de 14,5% (130 em 897 culturas). A maioria (n=122; 93,8%) foi identificada como A. xylosoxidans por testes fenotípicos e pelo sequenciamento do gene rrs que codifica o 16S rRNA. A análise do polimorfismo genético dos isolados de A. xylosoxidans pela técnica de PFGE, mostrou 22 grupos clonais. Destes, sete foram compartilhados entre pacientes distintos sugerindo transmissão cruzada. Apenas o clone G foi amplamente disseminado entre 56,4% dos pacientes estudados, sugerindo a possibilidade de um surto. Os 15 clones restantes constituíram-se em clones exclusivos por pacientes. Os cinco pacientes colonizados cronicamente por A. xylosoxidans mostraram a prevalência de clones únicos. Até o momento, este é o primeiro caso da ocorrência de surto por A. xylosoxidans em pacientes com Fibrose Cística. A. xylosoxidans é um microrganismo que vem se destacando em frequência e como um possível patógeno pulmonar nesses pacientes. Entretanto, até o momento os dados são insuficientes para avaliar a sua contribuição para a evolução da doença pulmonar. Estudos que busquem elucidar as características de A. xylosoxidans que o permitem colonizar persistentemente o pulmão dos pacientes com FC, bem como seu potencial de virulência, são necessários.

Historical knowledge of sharks: ancient science, earliest American encounters, and American science, fisheries, and utilization

In western civilization, the knowledge of the elasmobranch or selachian fishes (sharks and rays) begins with Aristotle (384–322 B.C.). Two of his extant works, the “Historia Animalium” and the “Generation of Animals,” both written about 330 B.C., demonstrate knowledge of elasmobranch fishes acquired by observation. Roman writers of works on natural history, such as Aelian and Pliny, who followed Aristotle, were compilers of available information. Their contribution was that they prevented the Greek knowledge from being lost, but they added few original observations. The fall of Rome, around 476 A.D., brought a period of economic regression and political chaos. These in turn brought intellectual thought to a standstill for nearly one thousand years, the period known as the Dark Ages. It would not be until the middle of the sixteenth century, well into the Renaissance, that knowledge of elasmobranchs would advance again. The works of Belon, Salviani, Rondelet, and Steno mark the beginnings of ichthyology, including the study of sharks and rays. The knowledge of sharks and rays increased slowly during and after the Renaissance, and the introduction of the Linnaean System of Nomenclature in 1735 marks the beginning of modern ichthyology. However, the first major work on sharks would not appear until the early nineteenth century. Knowledge acquired about sea animals usually follows their economic importance and exploitation, and this was also true with sharks. The first to learn about sharks in North America were the native fishermen who learned how, when, and where to catch them for food or for their oils. The early naturalists in America studied the land animals and plants; they had little interest in sharks. When faunistic works on fishes started to appear, naturalists just enumerated the species of sharks that they could discern. Throughout the U.S. colonial period, sharks were seldom utilized for food, although their liver oil or skins were often utilized. Throughout the nineteenth century, the Spiny Dogfish, Squalus acanthias, was the only shark species utilized in a large scale on both coasts. It was fished for its liver oil, which was used as a lubricant, and for lighting and tanning, and for its skin which was used as an abrasive. During the early part of the twentieth century, the Ocean Leather Company was started to process sea animals (primarily sharks) into leather, oil, fertilizer, fins, etc. The Ocean Leather Company enjoyed a monopoly on the shark leather industry for several decades. In 1937, the liver of the Soupfin Shark, Galeorhinus galeus, was found to be a rich source of vitamin A, and because the outbreak of World War II in 1938 interrupted the shipping of vitamin A from European sources, an intensive shark fishery soon developed along the U.S. West Coast. By 1939 the American shark leather fishery had transformed into the shark liver oil fishery of the early 1940’s, encompassing both coasts. By the late 1940’s, these fisheries were depleted because of overfishing and fishing in the nursery areas. Synthetic vitamin A appeared on the market in 1950, causing the fishery to be discontinued. During World War II, shark attacks on the survivors of sunken ships and downed aviators engendered the search for a shark repellent. This led to research aimed at understanding shark behavior and the sensory biology of sharks. From the late 1950’s to the 1980’s, funding from the Office of Naval Research was responsible for most of what was learned about the sensory biology of sharks.

Studies on fish epidemics from Assam

Investigations conducted since July 1988 on ulcerative fish epidemics in Assam, India, indicated that mainly four species of fishes belonging to the genera Puntius, Channa, Macrognathus and Mystus were widely affected by the disease. Results indicated that outbreak of the disease may not be due to organic pollution of water or radioactive and heavy-metallic contamination. Bacterial culture revealed colonies of Escherichia coli and Pseudomonas aeruginosa in the surface muscular lesions and gill tissues while preliminary electron microscopic studies indicated the presence of viruses in the muscles and gills of diseased fishes.

Environmental parameters and incidence of white spot disease in Penaeus monodon (Fab.) farming

An investigation was carried out to monitor management practices and to find out whether there is any relationship with occurrence of deadly white spot disease and environmental parameters. Three semi-intensive and a improved traditional shrimp farms were selected in which mass mortality of shrimp (Penaeus monodon) by white spot disease occurred previously. The farms were situated at two different geographical locations. Two ponds from each farm at random were selected for the study. Out of eight investigated ponds, 6 ponds in three farms were affected by the disease during investigation period. The non-affected ponds had relatively lower stocking density, lightly different management practice and were located at different geographical area. There was no significant variation in water quality parameters among the affected and non-affected ponds. No significant variations were recorded in pond preparation, source of Post Larvae (PL), water and feed management among the affected and non-affected ponds. The observation indicated that pond micro-organisms in a farm may not the only cause of the disease but some external factors also might be responsible for the outbreak of this disease.

Pathology of mixed infections of saprolegniasis-myxosporidiosis in Indian major carp (Catla catla Ham.)

An outbreak of saprolegniasis in Catla catla in composite carp culture ponds were recorded during winter season. The typical cotton wool growths were observed on whole body surfaces of catla along with sporadic mortality. The fungal invasion was only restricted to skin and no fungal elements were visible in any internal organs after periodic acid schiff staining. On histology, periportal accumulation of mononuclear cells in liver, presence of myxosporidean cysts in antieror kidney, eosinophilic granular cells reaction in submucosa of stomach and intestine, dilated and engorged blood vessels of brain along with sloughing of epidermis and hyperplasia at gill lamellar base were pronounced changes. The possible role of release of Saprolegnia toxin in producing internal organs pathology has been discussed.

A study on performances of intensification in the farming system of a shrimp farm at Cox's Bazar, Bangladesh

This study examines the harvest and mean production in relation to the stocking of P. monodon fry during the period between March 1992 and October 1994, at the farm owned by M/s Monugung Sea Food Ltd., Cox's Bazar. The analysis shows that production figures were initially up to expectation, but after harvesting 4 crops within 16 months, production sharply decreased. The unexpected high mortality of the growing stock was due to outbreak of an uncontrollabe disease (Vibriosis). Significantly higher variations in production, survival and growth were also noted among the different treatments.

Incidence of ulcer type of disease in wild fishes of Bangladesh

Disease occurred in wild fish species investigated in different water bodies like canals, ditches, beel, haor, flood plain in 17 districts of Dhaka division. Haemorrhagic lesions were observed on the body surface of affected fishes. Incidence of the disease in the investigated water bodies ranged from 0 to 100%. In total 19 fish species were found to be affected and prevalence of infection ranged from 0.0 to 100.0%. Channa punctatus and Puntius ticto were severely affected in all locations. Percentage of infection in these fishes ranged from 0.0 to 100.0. The highest infection was observed in Netrokona, Kishoreganj, and Mymensingh districts. Bacterial genera isolated from the lesions of these affected fishes were Aeromonas, Pseudomonas, Flavobacterium, Micrococcus, and Staphylococcus. Among these isolates Aeromonas was the dominant. Abundance of Aeromonas in the lesions among the investigated bacteria ranged from 75 to 90%. Five identified Aero monas lrydrophila were examined for their pathogenicity and were able to infect the experimental fish, silver barb (Puntius gonionotus). The pathogen Aeromonas hydrophila was thus considered to have an association with the outbreak of ulcer type of disease in the investigated fish species.

Disease problems in shrimp sector in Khulna district of Bangladesh: a question of sustainability

Disease is the main restraining factor for the development of shrimp sector in Bangladesh. Both brackish water shrimp bagda (Penaeus monodon) and freshwater prawn golda, (Macrobrachium rosenbergii) farming in Bangladesh have been facing disease problem. A study on disease outbreak in both brackish water and freshwater small-scale shrimp farms (gher) in Khulna district was carried out through interviewing randomly selected 3-5% of shrimp farmers with a structured questionnaire during March to December, 2002. The study showed that 97% bagda farming ghers and 80% golda farming ghers were affected by disease. White spot disease was the severe disease for bagda, whereas, antenna rot was the main disease for freshwater prawn. Change of water and liming were carried out as control measures of disease for bagda shrimp farming, while it was only liming for golda farming. A small number of shrimp farmers (15%) used chemicals for treatment of shrimp diseases. Development of suitable farming technology to prevent disease contamination and innovation of proper treatments for diseases are required to overcome the disease problems for sustainable shrimp farming in Bangladesh.

Identification of causes of Thai pangas mortality in ponds and their control strategies

Mass mortality of Thai pangas (Pangasius hypophthalmus) is reported to be a big threat to monoculture of the species in Bangladesh. Twenty affected and twenty control Thai pangas ponds were investigated around Mymensingh district in order to identify the causes of pangas mortality. Sixty affected and sixty unaffected fish samples were examined and compared to find the fish-level variables associated with the disease. A range of haemorrhagic signs on snout, skin and fins were recorded during examination with naked eyes. Aeromonas spp. and Edwardsiella spp. were isolated from 87% and 80% of the affected fish, respectively. Even 4% of the seemingly healthy fish carried Aeromonas spp. on their skin. Among the four water quality parameters monitored, remarkably higher total ammonia (1.5 ppm) was found in water of the affected ponds compared to that of the unaffected ones (0.4 ppm). High ammonia in affected water caused by excessive organic decomposition and poor pond management might have reduced the immunity of fish, which predisposed them for bacterial invasion and consequent disease outbreak.

Moderate mutation rate in the SARS coronavirus genome and its implications

Background: The outbreak of severe acute respiratory syndrome (SARS) caused a severe global epidemic in 2003 which led to hundreds of deaths and many thousands of hospitalizations. The virus causing SARS was identified as a novel coronavirus (SARS-CoV) an

Managing environmental impacts in aquaculture

The article presents the environmental impacts of aquaculture and the ways to manage them. The environmental impacts include issues of recreation and aesthetics, and the usage of resources and discharge of nutrients and organic matter into the environment. Likewise, the results are also discussed, such as reduced production, disease outbreak in cultured and wild populations, and an increase of regulatory restrictions being placed on aquaculture operations.

On-going generation of multiple forms of HIV-1 intersubtype recombinants in the Yunnan Province of China

Objectives: To investigate the molecular epidemiology of HIV in China's Yunnan Province, where the initial HIV-1 outbreak among injecting drug users (IDU) occurred in 1989, and to analyse the genesis and interrelationship of the epidemic with that in surrounding areas. Design: A molecular epidemiological investigation was conducted among IDU in three prefectures in Yunnan Province, including Wenshan (east), Honghe (southeast) and Dehong (west). Methods: Thirty-nine specimens were collected from consenting IDU in 2000-2001. The nucleotide sequences of 2.6 kb gag-RT and 340 base pair (bp) env (C2/V3) regions were determined. Phylogenetic tree and recombination breakpoint analyses were performed. Results: The circulating recombinant form (CRF), CRF08_BC, predominated in east Yunnan near Guangxi Province (89% in Wenshan and 81% in Honghe), whereas it was not detected in Dehong(0/14) in the west. In contrast, 71% (10/14) of the Dehong isolates were unique recombinant forms (URF), mostly between subtypes B' (Thailand variant of subtype B) and C, with distinct profiles of recombination breakpoints. The subtype B' accounts for the remaining 29% (4/14) of Dehong isolates. Interestingly, two Honghe isolates (2/16) shared some of the precise B'/C recombination breakpoints with CRF07_BC. Conclusion: New recombinant strains are arising continually in west Yunnan near the Myanmar border. Some appeared to be secondary recombinants derived from CRF07_BC that had further recombined with other strains. The uneven distribution of subtypes, CRF and URF, suggests the presence of independent transmission networks and clusters among IDU in Yunnan. (C) 2002 Lippincott Williams Wilkins.

First description of a novel Weissella species as an opportunistic pathogen for rainbow trout Oncorhynchus mykiss (Walbaum) in China

Six strains of Gram-positive, catalase-negative, non-motile, irregular short rod-shaped Weissella bacteria, with width and length of 0.5-0.6 and 1.2-2.7 mu m were isolated from diseased rainbow trout Oncorhynchus mykiss (Walbaum) in winter of 2007 at a commercial fishery in Jingmen, Hubei province, China. The diseased rainbow trout exhibited hemorrhage in eyes, anal region, intestine and abdomen wall, petechia of liver, some fish with hydrocele in stomach. Six isolates had identical biochemical reactions, phylogenetic analysis of 16S rDNA sequences, amplified ribosomal DNA restriction analysis (ARDRA), enzymatic profile analysis and antimicrobial susceptibility results, indicating as a single clonal outbreak. But all were different from any other validated twelve Weissella species in the term of physiological and biochemical characters. It is indicated that isolates are phylogenetically closer to Weissella halotolerans, Weissella viridescens and Weissella minor on 16S rDNA phylogenetic analysis result, than to W halotolerans and W viridescens on the result of ARDRA study and enzymatic profile analysis. Antimicrobial susceptibility testing was used to scan effective drugs for the therapy of this disease. Experimental infection assays with one isolate were conducted and pathogenicity (by intraperitoneal injection) was demonstrated in rainbow trout O. mykiss (Walbaum) and crucian carp (Carassius auratus gibelio) fingerlings. Because no Weissella was detected in fish feedstuffs and pond water, the source of this pathogen remains unknown, and Weissella isolates were regarded as an opportunistic pathogen for rainbow trout. This is the first report of Weissella strains which can cause disease of cultured fish in the world. (C) 2009 Elsevier B.V. All rights reserved.