Desarrollo de métodos analíticos para la determinación simultánea de antimicrobianos propuestos en el tratamiento de actinomicetoma .html Norma Cecilia Cavazos Rocha.

Tesis (Doctor en Ciencias con especialidad en Química Biomédica) U.A.N.L., FM 2006.

Distribution and bioactive potential of soil actinomycetes from different ecological habitats

Emergence of drug resistance among pathogenic bacteria to currently available antibiotics has intensified the search for novel bioactive compounds from unexplored habitats. In the present study actinomycetes were isolated from two relatively unexplored and widely differing habitats such as mountain and wetlands and their ability to produce antibacterial substances were analyzed. Pure cultures of actinomycetes were identified by morphological and biochemical tests. Various genera of actinomycetes encountered included Nocardia, Pseudonocardia, Streptomyces, Nocardiopsis, Streptosporangium, Micromonospora, Rhodococcus, Actinosynnema, Nocardiodes, Kitasatosporia, Gordona, Intrasporangium and Streptoalloteichus. The frequency of occurrence of each genus was found to vary with sample. About 47% of wetland isolates and 33% of mountain isolates were identified as various species of Nocardia. The isolated strains differed among themselves in their ability to decompose proteins and amino acids and also in enzyme production potential. Antibiotic activities of these actinomycetes were evaluated against 12 test pathogenic bacteria by well diffusion method using agar wells in glycerol-yeast extract agar. About 95% of actinomycete isolates from wetland ecosystem and 75% of highland isolates suppressed in different degrees the growth of test pathogens. Relatively high antibacterial activity among these isolates underlined their potential as a source of novel antibiotics.

SPATIOTEMPORAL VARIATION AND ANTIBACTERIAL ACTIVITY OF ACTINOMYCETES ISOLATED FROM HIGH ALTITUDE GRASSLAND SOILS OF TROPICAL MONTANE FOREST – KERALA, INDIA

TThe invention of novel antibiotics and other bioactive microbial metabolites continues to be an important aim in new drug discovery programmes. Actinomycetes have the potential to synthesize lots of diverse biologically vigorous secondary metabolites and in the last decades actinomycetes became the most productive source for antibiotics. Therefore in the present study we analyze the antibacterial activity of the actinomycetes isolated from grassland soil samples of Tropical Montane forest. A total of 33 actinomycete strains isolated were characterized and screened for antibacterial activities using well diffusion method against six specific pathogenic organisms. Identification of the isolates revealed that the majority of them were belonging to Streptomycetes followed by Nocardia, Micromonospora, Pseudonocardia, Streptosporangium, Nocardiopsis and Saccharomonospora. Among the 33 isolates, Gr1 strain showed antagonistic activity against all checked pathogens. Nine strains showed antibacaterial activity against Listeria, Vibrio cholera, Bacillus cereus, Staphylococcus aureus and Salmonella typhi and only 2 strains (Gr1and Gr25) showed antagonism to E. coli. The overall percentage of activity of actinomycetes isolates against each pathogenic bacterium was also calculated. While 63.63% of the actinomycetes were antagoinistic against Listeria, Vibrio cholerae, and Bacillus cereus, 60.6% of them were antagonistic to Staphylococcus aureus. Very few isolates (6.06%) showed antibacterial activity against E. coli. In general most of the actinomycetes isolates were antagonistic to grampositive bacteria such as Listeria, Bacillus and Staphylococcus than Gram-negative bacteria Vibrio cholerae, E. coli and Salmonella

Polyhydroxyalkanoates production by actinobacteria isolated from soil

Polyhydroxyalkanoates (PHAs) are biodegradable and renewable polymers produced by a wide range of bacterial groups. New microbial bioprospection approaches have become an important way to find new PHA producers and new synthesized polymers. Over the past years, bacteria belonging to actinomycetes group have become known as PHA producers, such as Nocardia and Rhodococcus species, Kineosphaera limosa Liu et a]. 2002, and, more recently, Streptomyces species. In this paper, we disclose that there are more actinobacteria PHA producers in addition to the genera cited. Some unusual genera, such as Streptoalloteichus, and some genera frequently present in soil, such as Streptacidiphilus, have been found. Thirty-four isolates were able to accumulate poly(3-hydroxybutyrate) and a number of these have traces of poly(3-hydroxyvalerate) when cultivated on glucose or glucose and casein as carbon source. Furthermore, some strains showed traces of medium chain length PHA. Transmission electron microscopy demonstrated that the PHA accumulation occurs in hyphae and spores.

New alk genes detected in Antarctic marine sediments

Alkane monooxygenases (Alk) are the key enzymes for alkane degradation. In order to understand the dispersion and diversity of alk genes in Antarctic marine environments, this study analysed by clone libraries the presence and diversity of alk genes (alkB and alkM) in sediments from Admiralty Bay, King George Island, Peninsula Antarctica. The results show a differential distribution of alk genes between the sites, and the predominant presence of new alk genes, mainly in the pristine site. Sequences presented 53.10-69.60% nucleotide identity and 50.90-73.40% amino acid identity to alkB genes described in Silicibacter pomeroyi, Gordonia sp., Prauserella rugosa, Nocardioides sp., Rhodococcus sp., Nocardia farcinica, Pseudomonas putida, Acidisphaera sp., Alcanivorax borkumensis, and alkM described in Acinetobacter sp. This is the first time that the gene alkM was detected and described in Antarctic marine environments. The presence of a range of previously undescribed alk genes indicates the need for further studies in this environment.

Identificação e caracterização de actinomicetos isolados de processo de compostagem

Atualmente, o isolamento e a caracterização de actinomicetos tem recebido atenção especial, pois, juntamente com os fungos, eles são os principais responsáveis pela degradação de substâncias de difícil decomposição durante o processo de compostagem. Portanto este trabalho tem por objetivo identificar os actinomicetos isolados durante o processo de compostagem através de métodos de microbiologia clássica e pela amplificação do 16S DNAr. Para a realização deste trabalho foram realizadas seis coletas na Central de Triagem e Compostagem de Resíduos Sólidos de Sapiranga (CETRISA) e três numa composteira da UFRGS. Para o isolamento dos actinomicetos foi utilizada a diluição de 10-3 da amostra e a mesma foi semeada nos meios Jaunsen, 72C e Agar Amido Caseína e incubada nas temperaturas de 37oC, 50ºC e a temperatura ambiente por um período de 10 a 14 dias. A identificação foi realizada através de análise taxonômica dos microcultivos dos isolados e de provas bioquímicas. Foram isolados 153 actinomicetos, destes 73 foram isolados da CETRISA e 80 da composteira da UFRGS. Na primeira, houve o predomínio do gênero Nocardia e na segunda, do gênero Streptomyces. Após a identificação dos actinomicetos o trabalho teve prosseguimento com a amplificação da região 16S do DNAr e digestão dos produtos obtidos com endonucleases de restrição. Foram testadas oito endonucleases de restrição, porém somente a Msp1 e a HinfI produziram resultados favoráveis que puderam separar os isolados em nível de gênero.

Nocardiosis: an overview and additional report of 28 cases in cattle and dogs

A caracterização fenotípica, perfil de sensibilidade aos antimicrobianos e aspectos clínico-epidemiológicos foram avaliados em 28 linhagens de Nocardia isoladas de 19 casos de mastite, oito lesões tegumentares e um caso de pneumonia em cão. Foram utilizados no diagnóstico métodos microbiológicos, bioquímicos, citológicos e microscopia eletrônica de varredura. Nocardia asteroides tipo IV, N. otitidiscaviarum,N. nova (tipo III) e N. farcinica (tipo V) foram isoladas do leite de vacas com mastite, de material de lavado transtraqueal e de lesões cutâneas de cães. Nocardiose mamária bovina foi diagnosticada predominantemente sob a forma clínica, em propriedades com precárias condições de higiene na pré e pós-ordenha, e inadequado procedimento de terapia intramamária. Nocardiose canina foi diagnosticada comumente em animais co-infectados com o vírus da cinomose. Sulfametoxazole/trimetoprim (92,8%), amicacina (92,8%) e ceftiofur (92,8%) foram os antimicrobianos mais efetivos frente às linhagens de Nocardia. Resistência múltipla a três ou mais e cinco ou mais antimicrobianos foram observadas, respectivamente, em dez (35,7%) e três (10,7%) linhagens, notadamente frente à cloxacilina, cefoperazona e ampicilina. A caracterização de espécies (tipo), aspectos clínico-epidemiológicos, diagnóstico, resistência múltipla aos antimicrobianos e reflexos em saúde pública de linhagens de Nocardia isoladas de bovinos e cães no Brasil foram discutidos. Foi destacada a similaridade entre as espécies de Nocardia isoladas de animais e do homem, e a primeira descrição no Brasil de N. otitidiscaviarum na etiologia da mastite bovina.

Sensitive voltammetric sensor for fast detection of mycolic acids present in mycobacteria

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

Mastite infecciosa equina: uma visão geral da doença

Mastits is considered uncommon disease in mares. Streptococcus equi, Staphylococcus sp., Corynebacterium sp., Actinobacillus sp., Nocardia sp. and enterobacterias are major microorganisms involved in equine mammary infections. The disease is commonly related to traumatic lesions in mammary glands and teats. Edema, fibrosis, masses to palpation of glands, and viscous to seropurulent milk are mainly clinical signs observed in affected animals. The diagnosis is based on clinical exam of mammary glands and microbiological culture of the milk. There are no standard to use of indirect exams on diagnosis, including California Mastits Test and Somatic Cell Count. Systemic antimicrobials are recommended in therapy, based on previous “in vitro” susceptibility microbiological test. No specific control measures are indicated in equine mastits. The present study reviewed the mastits in mares, with emphasis to etiology, epidemiology findings, clinical manifestation, diagnosis, treatment and control aspects.

Verrucosispora maris sp nov., a novel deep-sea actinomycete isolated from a marine sediment which produces abyssomicins

Verrucosispora isolate AB-18-032(T), the abyssomicin- and proximicin-producing actinomycete, has chemotaxonomic and morphological properties consistent with its classification in the genus Verrucosispora. The organism formed a distinct phyletic line in the Verrucosispora 16S rRNA gene tree sharing similarities of 99.7%, 98.7% and 98.9% with Verrucosispora gifhornensis DSM 44337(T), Verrucosispora lutea YIM 013(T) and Verrucosispora sediminis MS 426(T), respectively. It was readily distinguished from the two latter species using a range of phenotypic features and from V. gifhornensis DSM 44337(T), its nearest phylogenetic neighbor, by a DNA G+C content of 65.5 mol% obtained by thermal denaturation and fluorometry and DNA:DNA relatedness values of 64.0% and 65.0% using renaturation and fluorometric methods, respectively. It is apparent from the combined genotypic and phenotypic data that strain AB-18-032(T) should be classified in the genus Verrucosispora as a new species. The name Verrucosispora maris sp. nov. is proposed for this taxon with isolate AB-18-032(T) (= DSM 45365(T) = NRRL B-24793(T)) as the type strain.

Virological and pathological findings in Bluetongue virus serotype 8 infected sheep

Twenty-seven sheep of the four most common Swiss breeds and the English breed Poll Dorset were experimentally infected with a northern European field strain of bluetongue virus serotype 8 (BTV-8). Animals of all breeds developed clinical signs, viremia and pathological lesions, demonstrating that BTV-8 is fully capable of replicating and inducing bluetongue disease (BT) in the investigated sheep. Necropsy performed between 10 and 16 days post-infectionem (d.p.i.) revealed BT-typical hemorrhages, effusions, edema, erosions and activation of lymphatic tissues. Hemorrhages on the base of the Arteria pulmonalis and the left Musculus papillaris subauricularis were frequently present. Histology confirmed the macroscopical findings. Using a score system, clinical manifestation and pathology were found to be significantly related. Furthermore, clinical signs and fever were shown to be indicative for the concurrent presence of high amounts of viral ribonucleic acid (RNA) in blood. Spleen, lung, lymph nodes and tonsils from all animals were analyzed regarding viral RNA loads and infectivity using real-time reverse transcriptase PCR (rRT-PCR) and virus isolation in cell culture, respectively. The highest amount of viral RNA was detected in spleen and lung and rRT-PCR revealed to be a more sensitive method for virus detection compared to virus isolation. A long-term follow-up was performed with three sheep showing that BTV-8 viral RNA in blood was present up to 133 d.p.i. and in certain tissues even on 151 d.p.i. No significant breed-related differences were observed concerning clinicopathological picture and viremia, and the Swiss sheep were as susceptible to BTV-8 infection as Poll Dorset sheep, demonstrating a remarkably high virulence of BTV-8 for indigenous sheep breeds.

Nocardial brain abscess: observation of treatment strategies and outcome in Switzerland from 1992 to 1999

BACKGROUND Brain abscesses caused by Nocardia spp. are rare, but life-threatening infections that are notoriously difficult to diagnose and treat and which occur mainly in immunocompromised patients. Standard treatment guidelines are not available. METHODS A systematic search for nocardial brain abscesses from 1992 to 1999 was conducted in Switzerland for the comparison of clinical presentation, treatment strategies and outcome. RESULTS Seven cases were found, for which data of six were available. In 4/6 patients antimicrobial therapy led to a decrease in the size of abscesses. Four of six patients died. The cause of death was likely due to underlying co-morbidities, rather than the nocardial infection. CONCLUSION The finding that treatment was different in each case underscores the lack of therapeutic guidelines.

Plötzlicher Tod infolge eines persistierenden Ductus arteriosus. Fall eines 4 Tage alt gewordenen Säuglings

Ein persistierender Ductus arteriosus (Ductus Botalli) ist seit Jahren als Ursache eines plötzlichen und unerwarteten Neugeborenentods und pulmonaler Hämorrhagien identifiziert. Insbesondere Frühgeborene weisen ein erhöhtes Risiko zur Entwicklung eines hämodynamisch signifikanten persistierenden Ductus arteriosus auf. Vorgestellt wird der plötzliche und unerwartete Todesfall eines 4 Tage alt gewordenen Säuglings, der 2 Wochen vor dem errechneten Termin geboren worden war. Bei der Obduktion zeigte sich ein sehr muskelkräftiger arterieller Gefäßkurzschluss zwischen der A. pulmonalis und dem Aortenbogen mit einem Durchmesser knapp unter dem der A. pulmonalis. Als Folge der pulmonalen Hyperperfusion fanden sich eine massive akute Blutstauung und kongestives Herzversagen infolge des „shunting“ von der systemischen in die pulmonale Zirkulation.

Non-staphylococcal infections of cardiac implantable electronic devices

Background. The number of infections of cardiac implantable electronic devices (CIED) continues to escalate out of proportion to the increase rate of device implantation. Staphylococcal organisms account for 70% to 90% of all CIED infections. However, little is known about non-staphylococcal infections, which have been described only in case reports, small case series or combined in larger studies with staphylococcal CIED infections, thereby diluting their individual impact. ^ Methods. A retrospective review of hospital records of patients admitted with a CIED-related infections were identified within four academic hospitals in Houston, Texas between 2002 and 2009. ^ Results. Of the 504 identified patients with CIED-related infection, 80 (16%) had a non-staphylococcal infection and were the focus of this study. Although the demographics and comorbities of subjects were comparable to other reports, our study illustrates many key points: (a) the microbiologic diversity of non-staphylococcal infections was rather extensive, as it included other Gram-positive bacteria like streptococci and enterococci, a variety of Gram-negative bacteria, atypical bacteria including Nocardia and Mycobacteria, and fungi like Candida and Aspergillus; (b) the duration of CIED insertion prior to non-staphylococcal infection was relatively prolong (mean, 109 ± 27 weeks), of these 44% had their device previously manipulated within a mean of 29.5 ± 6 weeks; (c) non-staphylococcal organisms appear to be less virulent, cause prolonged clinical symptoms prior to admission (mean, 48 ± 12.8 days), and are associated with a lower mortality (4%) than staphylococcal organisms; (d) thirteen patients (16%) presented with CIED-related endocarditis; (e) although not described in prior reports, we identified 3 definite and 2 suspected cases of secondary Gram-negative bacteremia seeding of the CIED; and (f) inappropriate antimicrobial coverage was provided in approximately 50% of patients with non-staphylococcal infections for a mean period of 2.1 days. ^ Conclusions. Non-staphylococcal CIED-related infections are prevalent and diverse with a relatively low virulence and mortality rate. Since non-staphylococcal organisms are capable of secondarily seeding the CIED, a high suspicion for CIED-related infection is warranted in patients with bloodstream infection. Additionally, in patients with suspected CIED infection, adequate Gram positive and -negative antibacterial coverage should be administered until microbiologic data become available.^

Diversity and identification of heterotrophic bacteria from Antarctic Rocks of the McMurdo Dry Valleys (Ross Desert)

Diversity of endolithic Dry Valley rock microorganisms was studied by evaluating the presence of morphotypes in enrichments. Storage of rock samples for 16 h over dry ice affected the diversity of endolithic organisms, especially that of algae and fungi. Diversity in various samples depended on rock location and exposure, on the rock type, and to some extent on the pH of the pulverized rock samples. In most cases sandstone contained more morphotypes than dolerite or granite. Presence of many different phototrophs resulted in greater diversity of the heterotrophs in the enrichments. Samples from Linnaeus Terrace and Battleship Promontory had higher morphotype (MT) numbers than those from more exposed sites such as New Mountain, University Valley, Dais, or Mt. Fleming. Beacon sandstone (13 samples) from Linnaeus Terrace varied greatly with respect to MT numbers, although the pH values ranged only from 4.2-5.3. The highest MT number of 24 per sample was obtained from the upper surface of a flat boulder tilted to the North. Only two MT's were found in a hard sandstone sample from the wind-exposed and more shaded east side of the Terrace. 15 sandstone samples from Battleship Promontory contained more diverse populations: there occurred a total of 131 different MT's in these samples as compared to only 68 in Linnaeus Terrace samples. Cysts of colorless flagellates were found in some Battleship Promontory samples; rnost samples were populated with a wealth of different cyanobacteria. Studies on the distribution of actinomycete morphotypes in Linnaeus Terrace sandstone revealed great differences between individual boulders. Identification tests and lipid analyses made with representative strains of the isolated 1500 pure cultures led to genus names such as Caulobacter, Blastobacter, Hyphomicrobium, Micrococcus, Arthrobacter, Brevibacterium, Corynebacterium, Bifidobacterium, Mycobacterium, Nocardia (Amycolata), Micromonospora, Streptomyces, Blastococcus, and Deinococcus. Our data demonstrate the great diversity of Antarctic endolithic microbial populations.