Cultivable bacterial diversity associated with bromeliad roots from ironstone outcrops in central Brazil

Abstract Studies on the bacterial diversity associated with wild plants are rare, especially on those that grow in association with bromeliads. In the present study, we isolated and identified epiphytic and endophytic bacteria from the roots of the bromeliads Dyckia excelsa, Dyckia leptostachya and Deuterocohnia meziana occurring in the cangas in the Pantanal from Mato Grosso do Sul State, Brazil. The epiphytic bacteria were isolated from washed roots, while the endophytic bacteria were isolated from surface disinfested roots. Bacterial representatives corresponding to each BOX-PCR fingerprint, as well as those that did not result in amplicons, were selected for 16S rDNA gene sequence analysis. The BOX-PCR data showed intrageneric and intraspecific diversity and could discriminate strains and identify their phenotypic characteristics. The 16S rDNA gene sequence and phylogeny analysis showed a higher occurrence of strains belonging to the genus Bacillus than Mycobacterium and Brevibacterium, which were found in lower numbers. Species from the Bacillus genus are well known for their sporulation capacity and longer survival in arid locations, such as the cangas. This study clearly showed that the bromeliad species represent a vast reservoir of bacterial community diversity, and the cultivable strains represent a new source for biotechnological prospecting.

Resumo Estudos sobre a diversidade bacteriana associada a plantas silvestres são raros, especialmente naqueles que crescem em associação com bromélias. No presente estudo, isolamos e identificamos bactérias epífitas e endofíticas das raízes das bromélias Dyckia excelsa, D. leptostachya e Deuterocohnia meziana ocorrentes nas cangas no Pantanal do Mato Grosso do Sul, Brasil. As bactérias epifíticas foram isoladas de raízes lavadas, enquanto as bactérias endofíticas foram isoladas de raízes desinfestadas na superfície. Representantes bacterianos correspondentes a cada perfil do BOX-PCR, bem como aqueles que não resultaram em amplificações, foram selecionados para o sequenciamento do gene 16S rDNA. Os dados da BOX-PCR mostraram diversidade intragênica e intraespecífica e puderam discriminar cepas e identificar suas características fenotípicas. A seqüência do gene 16S rDNA e a análise filogenética mostraram uma maior ocorrência de cepas pertencentes ao gênero Bacillus do que as bactérias Mycobacterium e Brevibacterium, encontradas em menor número. Espécies do gênero Bacillus são bem conhecidas por sua capacidade de esporulação e maior sobrevida em locais áridos, como as cangas. Este estudo mostrou claramente que as espécies de bromélias representam um vasto reservatório de diversidade de comunidades bacterianas, e as linhagens cultiváveis podem representar uma nova fonte para a prospecção biotecnológica.

Cultivable bacterial diversity associated with bromeliad roots from ironstone outcrops in central Brazil / Diversidade de bactérias cultiváveis associadas a raízes de bromélias de afloramentos ferruginosos do Brasil central

Studies on the bacterial diversity associated with wild plants are rare, especially on those that grow in association with bromeliads. In the present study, we isolated and identified epiphytic and endophytic bacteria from the roots of the bromeliads Dyckia excelsa, Dyckia leptostachya and Deuterocohnia meziana occurring in the cangas in the Pantanal from Mato Grosso do Sul State, Brazil. The epiphytic bacteria were isolated from washed roots, while the endophytic bacteria were isolated from surface disinfested roots. Bacterial representatives corresponding to each BOX-PCR fingerprint, as well as those that did not result in amplicons, were selected for 16S rDNA gene sequence analysis. The BOX-PCR data showed intrageneric and intraspecific diversity and could discriminate strains and identify their phenotypic characteristics. The 16S rDNA gene sequence and phylogeny analysis showed a higher occurrence of strains belonging to the genus Bacillus than Mycobacterium and Brevibacterium, which were found in lower numbers. Species from the Bacillus genus are well known for their sporulation capacity and longer survival in arid locations, such as the cangas. This study clearly showed that the bromeliad species represent a vast reservoir of bacterial community diversity, and the cultivable strains represent a new source for biotechnological prospecting.(AU)

Estudos sobre a diversidade bacteriana associada a plantas silvestres são raros, especialmente naqueles que crescem em associação com bromélias. No presente estudo, isolamos e identificamos bactérias epífitas e endofíticas das raízes das bromélias Dyckia excelsa, D. leptostachya e Deuterocohnia meziana ocorrentes nas cangas no Pantanal do Mato Grosso do Sul, Brasil. As bactérias epifíticas foram isoladas de raízes lavadas, enquanto as bactérias endofíticas foram isoladas de raízes desinfestadas na superfície. Representantes bacterianos correspondentes a cada perfil do BOX-PCR, bem como aqueles que não resultaram em amplificações, foram selecionados para o sequenciamento do gene 16S rDNA. Os dados da BOX-PCR mostraram diversidade intragênica e intraespecífica e puderam discriminar cepas e identificar suas características fenotípicas. A seqüência do gene 16S rDNA e a análise filogenética mostraram uma maior ocorrência de cepas pertencentes ao gênero Bacillus do que as bactérias Mycobacterium e Brevibacterium, encontradas em menor número. Espécies do gênero Bacillus são bem conhecidas por sua capacidade de esporulação e maior sobrevida em locais áridos, como as cangas. Este estudo mostrou claramente que as espécies de bromélias representam um vasto reservatório de diversidade de comunidades bacterianas, e as linhagens cultiváveis podem representar uma nova fonte para a prospecção biotecnológica.(AU)

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper

ABSTRACT Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out.Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper

Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out.Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.(AU)

Cultivable bacterial diversity associated with bromeliad roots from ironstone outcrops in central Brazil

Abstract Studies on the bacterial diversity associated with wild plants are rare, especially on those that grow in association with bromeliads. In the present study, we isolated and identified epiphytic and endophytic bacteria from the roots of the bromeliads Dyckia excelsa, Dyckia leptostachya and Deuterocohnia meziana occurring in the cangas in the Pantanal from Mato Grosso do Sul State, Brazil. The epiphytic bacteria were isolated from washed roots, while the endophytic bacteria were isolated from surface disinfested roots. Bacterial representatives corresponding to each BOX-PCR fingerprint, as well as those that did not result in amplicons, were selected for 16S rDNA gene sequence analysis. The BOX-PCR data showed intrageneric and intraspecific diversity and could discriminate strains and identify their phenotypic characteristics. The 16S rDNA gene sequence and phylogeny analysis showed a higher occurrence of strains belonging to the genus Bacillus than Mycobacterium and Brevibacterium, which were found in lower numbers. Species from the Bacillus genus are well known for their sporulation capacity and longer survival in arid locations, such as the cangas. This study clearly showed that the bromeliad species represent a vast reservoir of bacterial community diversity, and the cultivable strains represent a new source for biotechnological prospecting.

Resumo Estudos sobre a diversidade bacteriana associada a plantas silvestres são raros, especialmente naqueles que crescem em associação com bromélias. No presente estudo, isolamos e identificamos bactérias epífitas e endofíticas das raízes das bromélias Dyckia excelsa, D. leptostachya e Deuterocohnia meziana ocorrentes nas cangas no Pantanal do Mato Grosso do Sul, Brasil. As bactérias epifíticas foram isoladas de raízes lavadas, enquanto as bactérias endofíticas foram isoladas de raízes desinfestadas na superfície. Representantes bacterianos correspondentes a cada perfil do BOX-PCR, bem como aqueles que não resultaram em amplificações, foram selecionados para o sequenciamento do gene 16S rDNA. Os dados da BOX-PCR mostraram diversidade intragênica e intraespecífica e puderam discriminar cepas e identificar suas características fenotípicas. A seqüência do gene 16S rDNA e a análise filogenética mostraram uma maior ocorrência de cepas pertencentes ao gênero Bacillus do que as bactérias Mycobacterium e Brevibacterium, encontradas em menor número. Espécies do gênero Bacillus são bem conhecidas por sua capacidade de esporulação e maior sobrevida em locais áridos, como as cangas. Este estudo mostrou claramente que as espécies de bromélias representam um vasto reservatório de diversidade de comunidades bacterianas, e as linhagens cultiváveis podem representar uma nova fonte para a prospecção biotecnológica.

Ecotypes and virulence factors of Salmonella spp. detected in shrimp farms in Ceara-Brazil / Ecotipos e fatores de virulência de Salmonella spp. detectadas em fazendas de carcinicultura do Ceará, Brasil

Our objective was to group in ecotypes 12 serovars of Salmonella isolated from shrimp farming environments in the State of Ceara (Northeast Brazil). Grouping was done based on genotypic virulence factors. Two groups based on the similarity of the Box-PCR were identified: a group consisting of three strains (01 S. ser. Madelia serovar and 02 S. ser. enterica subs. houtenae) and another group consisting of nine isolates (02 S. ser. Saintpaul serovars; 03 S. ser. Infantis; 02 S. ser. Panama; 01 S. enterica subs. enterica; and 01 S. enterica subs. houtenae). Distribution pattern of the serovars was not influenced by the origin matrices (water and sediment). Plasmid virulence genes pefA and invA were detected, unrelated to the serovar and environmental origin of the isolates. The presence of virulence genes in the isolates underlines the potential to trigger salmonellosis events via shrimp consumption. Biomonitoring of these sources of contamination should be encouraged as a protective measure, minimizing health risks and economic losses for the industry.(AU)

Nosso objetivo foi agrupar em ecotipos 12 sorovares de Salmonella isolados em ambientes decarcinicultura no Estado do Ceará. O agrupamento foi feito a partir da pesquisa de fatores genotípicos devirulência. Constatou-se a formação de dois grupos baseados na similaridade do Box-PCR: um grupo comtrês estirpes (01 sorovar S. ser. Madelia e 02 sorovares S. enterica subs. houtenae) e outro constituído pornove isolados (02 sorovares S. ser. Saintpaul, 03 sorovares S. ser. Infantis, 02 sorovares S. ser. Panama, 01sorovar S. enterica subs. enterica e 01 sorovar S. enterica subs. houtenae). O padrão de distribuição dos sorovaresnão sofreu influência das matrizes de origem (água e sedimento). Os genes de virulência plasmidial pefA einvA foram detectados independente do sorovar e da origem ambiental dos isolados. A presença dessesgenes de virulência nos isolados de carcinicultura evidencia o potencial para desencadear eventos desalmonelose relacionados ao consumo de camarão. O biomonitoramento dessas fontes de contaminaçãodeve ser incentivado como medida protetiva, minimizando os riscos do ponto de vista sanitário e das perdaseconômicas para o setor da carcinicultura.(AU)

Characterization of rhizobia isolates obtained from nodules of wild genotypes of common bean

This study aimed to evaluate the tolerance to salinity and temperature, the genetic diversity and the symbiotic efficiency of rhizobia isolates obtained from wild genotypes of common bean cultivated in soil samples from the States of Goiás, Minas Gerais and Paraná. The isolates were subjected to different NaCl concentrations (0%, 1%, 2%, 4% and 6%) at different temperatures (28 °C, 33 °C, 38 °C, 43 °C and 48 °C). Genotypic characterization was performed based on BOX-PCR, REP-PCR markers and 16S rRNA sequencing. An evaluation of symbiotic efficiency was carried out under greenhouse conditions in autoclaved Leonard jars. Among 98 isolates about 45% of them and Rhizobium freirei PRF81 showed a high tolerance to temperature, while 24 isolates and Rhizobium tropici CIAT899 were able to use all of the carbon sources studied. Clustering analysis based on the ability to use carbon sources and on the tolerance to salinity and temperature grouped 49 isolates, R. tropici CIAT899 and R. tropici H12 with a similarity level of 76%. Based on genotypic characterization, 65% of the isolates showed an approximately 66% similarity with R. tropici CIAT899 and R. tropici H12. About 20% of the isolates showed symbiotic efficiency similar to or better than the best Rhizobium reference strain (R. tropici CIAT899). Phylogenetic analysis of the 16S rRNA revealed that two efficient isolates (ALSG5A1 and JPrG6A8) belong to the group of strains used as commercial inoculant for common bean in Brazil and must be assayed in field experiments.(AU)

Rhizobial characterization in revegetated areas after bauxite mining

Little is known regarding how the increased diversity of nitrogen-fixing bacteria contributes to the productivity and diversity of plants in complex communities. However, some authors have shown that the presence of a diverse group of nodulating bacteria is required for different plant species to coexist. A better understanding of the plant symbiotic organism diversity role in natural ecosystems can be extremely useful to define recovery strategies of environments that were degraded by human activities. This study used ARDRA, BOX-PCR fingerprinting and sequencing of the 16S rDNA gene to assess the diversity of root nodule nitrogen-fixing bacteria in former bauxite mining areas that were replanted in 1981, 1985, 1993, 1998, 2004 and 2006 and in a native forest. Among the 12 isolates for which the 16S rDNA gene was partially sequenced, eight, three and one isolate(s) presented similarity with sequences of the genera Bradyrhizobium, Rhizobium and Mesorhizobium, respectively. The richness, Shannon and evenness indices were the highest in the area that was replanted the earliest (1981) and the lowest in the area that was replanted most recently (2006).(AU)

Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat

The main objective of the present study was to isolate phytohormone-producing, phosphate-solubilizing strains of Azospirillum from wheat to be used as inoculants for plant growth promotion. Five Azospirillum strains were isolated from the rhizosphere of field-grown wheat (Triticum aestivum L.), and it was confirmed by BOX-polymerase chain reaction (PCR) that the isolates were different and not re-isolates of the same strain. Sequence analysis of the PCR-amplified 16S rRNA gene indicated that four isolates showed maximum similarity to Azospirillum brasilense and one isolate showed maximum similarity to Azospirillum zeae. This is the first report indicating the presence of an A. zeae like isolate in the wheat rhizosphere in Pakistan. The bacterial isolates were characterized for their plant growth-promoting traits, phosphate solubilization, and indole-3-acetic acid (IAA) production. None of the isolates showed phosphate solubilization activity in the commonly used Pikovskaya medium. However, all strains (except AzoK4) exhibited ability to solubilize tricalcium phosphate (TCP) in modified Pikovskaya medium in which sucrose was replaced by Na-malate, as well as in TCP-supplemented Luria-Bertani (LB) medium. Organic acids, such as acetic, citric, lactic, malic, and succinic acids, were detected in culture supernatants of the tested Azospirillum strains. All strains exhibited ability to produce IAA in the growth medium, except Azospirillum sp. AzoK1. Among the strains tested, the maximum IAA production (30.49 ± 1.04 mg L-1) and phosphate solubilization (105.50 ± 4.93 mg L-1) were shown by a pure culture of Azospirillum sp. AzoK2. In pot experiments, single-strain inocula of Azospirillum sp. AzoK1 and AzoK2 improved wheat plant growth.(AU)

"CARACTERIZAÇÃO DO PERFIL FENOTÍPICO E GENOTÍPICO DE RESISTÊNCIA A ANTIMICROBIANOS DE ISOLADOS DE Escherichia coli EM UMA CADEIA PRODUTIVA DE FRANGO DE CORTE"

O objetivo do estudo foi caracterizar o perfil de resistência de isolados de Escherichia coli obtidos de amostras da cadeia produtiva de frangos corte. Foram coletadas 495 amostras em um abatedouro de aves regularmente fiscalizado pelo Serviço de Inspeção Federal, localizado no estado do Paraná (Brasil). O isolamento de E. coli foi realizado em ágar MacConkey e as colônias com morfologia típica de E. coli foram confirmadas bioquimicamente. Os isolados confirmados seguiram para os testes de diluição em ágar, realizado como triagem, onde os antimicrobianos testados foram: amoxicilina - AMO (32 g), ceftiofur CTF (8 g), cloranfenicol - CLO (32 g), tetraciclina - TET (16 g), ciprofloxacina - CIP (1 g) e sulfametaxazol + trimetropim - SUT (76/4 g). Para o teste disco-difusão em ágar os antimicrobianos utilizados foram AMO (10 g); CTF (30 g); aztreonam - ATM (30 g); imipenem IPM (10 g); CIP (5 g); TET (30 g); gentamicina - GEN (10 g), SUT (23,75/1,25 g), CLO (30 g) e azitromicina - AZI (15 g). Para a detecção de de E. coli produtora de beta-lactamases de espectro estendido (ESBL), foram utilizados quatro antimicrobianos: amoxicilina com ácido clavulânico (20/10 g), ceftazidima 30 g, cefotaxima 30 g e cefepima 30 g. Para caracterização molecular, os genes blaTEM; aac(3)-II; tetA; qnrS; dhfrI; sul1; cmIA5 e floR foram avaliados pela técnica de reação em cadeia da polimerase (PCR). Um total de 1128 isolados de E. coli foram identificados nas amostras de carcaça de frango após a sangria, após depenagem, após evisceração e após a saída do chiller, água residual da escaldagem e chiller, caixa de transporte das aves e fezes funcionário. Nenhum isolado foi detectado na água industrial. A resistência mais comumente detectada no Breakpoint foi para os antimicrobianos SUT, TET e AMO. Para o teste de disco-difusão em ágar, 701 isolados foram avaliados, onde uma alta resistência foi detectada para a CIP (94,1%), TET (56,3%), SUT (54,3%) e AMO (48,1%), sendo um resultado preocupante, já que alguns desses antimicrobianos são utilizados para tratamento de casos graves em humanos e também de forma ampla na avicultura. Dentre os isolados, 578 foram multidroga resistente e apenas 15 isolados (2,2%) foram ESBL positivas. A resistência antimicrobiana entre os isolados de amostras dos funcionários e as demais amostras da cadeia avícola foram muito semelhantes, sendo um sinal de alerta, já que a exposição a esses antimicrobianos podem selecionar micro-organismos e resistentes. Um total de 74 isolados (42%) apresentaram a presença de um ou mais genes de resistência antimicrobiana. Os genes mais detectados nos isolados foram cmIA5, sul1 e dhfrl, totalizando 21,6%, 21% e 12,5% respectivamente. Em relação a correlação dos resultados fenotípicos e genotípicos observados pelo teste Kappa, amoxicilina demostrou uma concordância pobre, já os demais antimicrobianos uma concordância leve. O assunto envolvendo a resistência antimicrobiana deve ser tratado com cuidado, procurando maneiras para redução do uso de antimicrobianos, já que os efeitos adversos do uso incorreto e exagerado pode contribuir para disseminação de genes de resistência entre os micro-organismos e pelo ecossistema.

The objective of the study was to characterize the resistance profile of Escherichia coli isolates obtained from samples of the broiler production chain. 495 samples were collected in a poultry slaughterhouse regularly inspected by the Federal Inspection Service, located in the state of Paraná (Brazil). E. coli isolation was performed on MacConkey agar and colonies with typical E. coli morphology were confirmed biochemically. The confirmed isolates went to the agar dilution tests, performed as a screening, where the tested antimicrobials were: amoxicillin - AMO (32 g), ceftiofur - CTF (8 g), chloramphenicol - CLO (32 g), tetracycline - TET (16 g), ciprofloxacin - CIP (1 g) and sulfametaxazole + trimethoprim - SUT (76/4 g). For the disk-diffusion test on agar, the antimicrobials used were AMO (10 g); CTF (30 g); aztreonam - ATM (30 g); imipenem - IPM (10 g); CIP (5 g); TET (30 g); gentamicin - GEN (10 g), SUT (23.75 / 1.25 g), CLO (30 g) and azithromycin - AZI (15 g). For the detection of E. coli producing extended-spectrum beta-lactamases (ESBL), four antimicrobials were used: amoxicillin with clavulanic acid (20/10 g), ceftazidime - 30 g, cefotaxime - 30 g and cefepime - 30 g. For molecular characterization, the blaTEM genes; aac (3) -II; theta; qnrS; dhfrI; sul1; cmIA5 and floR were evaluated using the polymerase chain reaction (PCR) technique. A total of 1128 E. coli isolates were identified in the chicken carcass samples after bleeding, after plucking, after evisceration and after the chiller left, scalding and chiller residual water, poultry and employee feces transport box. No isolates were detected in industrial water. The most commonly detected resistance at agar dilution tests was for the antimicrobials SUT, TET and AMO. For the agar disc-diffusion test, 701 isolates were evaluated, where a high resistance was detected for CIP (94.1%), TET (56.3%), SUT (54.3%) and AMO (48.1%), being a worrying result, since some of these antimicrobials are used to treat severe cases in humans and also in a wide way in poultry. Among the isolates, 578 were multidrug resistant and only 15 isolates (2.2%) were ESBL positive. The antimicrobial resistance between isolates from employee samples and other samples from the poultry chain were very similar, being a warning sign, since exposure to these antimicrobials can select microorganisms and resistant. A total of 74 isolates (42%) presented the presence of one or more antimicrobial resistance genes. The most detected genes in the isolates were cmIA5, sul1 and dhfrl, totaling 21.6%, 21% and 12.5% respectively. Regarding the correlation between the phenotypic and genotypic results observed by the Kappa test, amoxicillin showed a poor agreement, while the other antimicrobials showed a slight agreement. The issue involving antimicrobial resistance should be treated with care, looking for ways to reduce the use of antimicrobials, since the adverse effects of incorrect and exaggerated use can contribute to the spread of resistance genes among microorganisms and the ecosystem.

Thermal manipulation mid-term broiler chicken embryogenesis: effect on muscle growth factors and muscle marker genes

Thermal manipulation (TM) during broiler chicken embryogenesis has been shown to promote muscle development and growth. However, the molecular bases of promoting broiler muscle development and growth are not fully understood. The aim of this study was to investigate the molecular bases of muscle growth and development in broiler chickens subjected to TM. This included the investigating of the changes in mRNA expression levels of muscle marker genes, namely MyoD, myogenin, paired box transcription factor (Pax7) and proliferating cell nuclear antigen (PCNA), and muscle growth factors namely insulin-like growth factor 1 (IGF-1), myostatin and growth hormone (GH) during embryogenesis and on posthatch days 10 and 28. Fertile Cobb eggs (n=1500) were divided into four groups. Eggs in the first group (control) were incubated at 37.8°C and 56% RH, whereas, eggs in the second group (TM1), third group (TM2), and fourth group (TM3) were subjected to 39 ºC and 65% RH daily during embryonic days (ED) 12-18 for 9, 12, and 18 hours, respectively. Body weight (BW) during embryogenesis and posthatch days (1, 3, 5, 7, 14, 21, 28 and 35) was recorded. mRNA expression levels of muscle marker genes and muscle growth factor genes during ED 12, 14, 16 and 18 and on posthatch days 10 and 28 were analyzed using real-time RT-PCR. TM upregulated the mRNA expressions of muscle marker and growth factors genes. This upregulation was accompanied by improvement of body weight near and at market age.

Thermal manipulation mid-term broiler chicken embryogenesis: effect on muscle growth factors and muscle marker genes

Thermal manipulation (TM) during broiler chicken embryogenesis has been shown to promote muscle development and growth. However, the molecular bases of promoting broiler muscle development and growth are not fully understood. The aim of this study was to investigate the molecular bases of muscle growth and development in broiler chickens subjected to TM. This included the investigating of the changes in mRNA expression levels of muscle marker genes, namely MyoD, myogenin, paired box transcription factor (Pax7) and proliferating cell nuclear antigen (PCNA), and muscle growth factors namely insulin-like growth factor 1 (IGF-1), myostatin and growth hormone (GH) during embryogenesis and on posthatch days 10 and 28. Fertile Cobb eggs (n=1500) were divided into four groups. Eggs in the first group (control) were incubated at 37.8°C and 56% RH, whereas, eggs in the second group (TM1), third group (TM2), and fourth group (TM3) were subjected to 39 ºC and 65% RH daily during embryonic days (ED) 12-18 for 9, 12, and 18 hours, respectively. Body weight (BW) during embryogenesis and posthatch days (1, 3, 5, 7, 14, 21, 28 and 35) was recorded. mRNA expression levels of muscle marker genes and muscle growth factor genes during ED 12, 14, 16 and 18 and on posthatch days 10 and 28 were analyzed using real-time RT-PCR. TM upregulated the mRNA expressions of muscle marker and growth factors genes. This upregulation was accompanied by improvement of body weight near and at market age.(AU)

Culturable diversity of halophilic bacteria in foreshore soils

Halophilic bacteria are commonly found in natural environments containing significant concentration of NaCl such as inland salt lakes and evaporated sea-shore pools, as well as environments such as curing brines, salted food products and saline soils. Dependence on salt is an important phenotypic characteristic of halophilic bacteria, which can be used in the polyphasic characterization of newly discovered microorganisms. In this study the diversity of halophilic bacteria in foreshore soils of Daecheon, Chungnam, and Saemangeum, Jeonbuk, was investigated. Two types of media, namely NA and R2A supplemented with 3%, 5%, 9%, 15%, 20% and 30% NaCl were used. More than 200 halophilic bacteria were isolated and BOX-PCR fingerprinting analysis was done for the typing of the isolates. The BLAST identification results showed that isolated strains were composed of 4 phyla, Firmicutes (60%), Proteobacteria (31%), Bacteriodetes (5%) and Actinobacteria (4%). Isolates were affiliated with 16 genera and 36 species. Bacillus was the dominant genus in the phylum Firmicutes, comprising 24% of the total isolates. Halomonas (12%) and Shewanella (12%) were also found as the main genera. These findings show that the foreshore soil of Daecheon Beach and Saemangeum Sea of Korea represents an untapped source of bacterial biodiversity.

Symbiotic nitrogen-fixing bacterial populations trapped from soils under agroforestry systems in the Western Amazon

Cowpea (Vigna unguiculata) is an important grain-producing legume that can forego nitrogen fertilization by establishing an efficient symbiosis with nitrogen-fixing bacteria. Although inoculating strains have already been selected for this species, little is known about the genotypic and symbiotic diversity of native rhizobia. Recently, Bradyrhizobium has been shown to be the genus most frequently trapped by cowpea in agricultural soils of the Amazon region. We investigated the genetic and symbiotic diversity of 148 bacterial strains with different phenotypic and cultural properties isolated from the nodules of the trap species cowpea, which was inoculated with samples from soils under agroforestry systems from the western Amazon. Sixty non-nodulating strains indicated a high frequency of endophytic strains in the nodules. The 88 authenticated strains had varying symbiotic efficiency. The SPAD (Soil Plant Analysis Development) index (indirect measurement of chlorophyll content) was more efficient at evaluating the contribution of symbiotic N2-fixation than shoot dry matter under axenic conditions. Cowpea-nodulating bacteria exhibited a high level of genetic diversity, with 68 genotypes identified by BOX-PCR. Sequencing of the 16S rRNA gene showed a predominance of the genus Bradyrhizobium, which accounted for 70 % of all strains sequenced. Other genera identified were Rhizobium, Ochrobactrum, Paenibacillus, Bosea, Bacillus, Enterobacter, and Stenotrophomonas. These results support the promiscuity of cowpea and demonstrate the high genetic and symbiotic diversity of rhizobia in soils under agroforestry systems, with some strains exhibiting potential for use as inoculants. The predominance of Bradyrhizobium in land uses with different plant communities and soil characteristics reflects the adaptation of this genus to the Amazon region.

Symbiotic nitrogen-fixing bacterial populations trapped from soils under agroforestry systems in the Western Amazon

Cowpea (Vigna unguiculata) is an important grain-producing legume that can forego nitrogen fertilization by establishing an efficient symbiosis with nitrogen-fixing bacteria. Although inoculating strains have already been selected for this species, little is known about the genotypic and symbiotic diversity of native rhizobia. Recently, Bradyrhizobium has been shown to be the genus most frequently trapped by cowpea in agricultural soils of the Amazon region. We investigated the genetic and symbiotic diversity of 148 bacterial strains with different phenotypic and cultural properties isolated from the nodules of the trap species cowpea, which was inoculated with samples from soils under agroforestry systems from the western Amazon. Sixty non-nodulating strains indicated a high frequency of endophytic strains in the nodules. The 88 authenticated strains had varying symbiotic efficiency. The SPAD (Soil Plant Analysis Development) index (indirect measurement of chlorophyll content) was more efficient at evaluating the contribution of symbiotic N2-fixation than shoot dry matter under axenic conditions. Cowpea-nodulating bacteria exhibited a high level of genetic diversity, with 68 genotypes identified by BOX-PCR. Sequencing of the 16S rRNA gene showed a predominance of the genus Bradyrhizobium, which accounted for 70 % of all strains sequenced. Other genera identified were Rhizobium, Ochrobactrum, Paenibacillus, Bosea, Bacillus, Enterobacter, and Stenotrophomonas. These results support the promiscuity of cowpea and demonstrate the high genetic and symbiotic diversity of rhizobia in soils under agroforestry systems, with some strains exhibiting potential for use as inoculants. The predominance of Bradyrhizobium in land uses with different plant communities and soil characteristics reflects the adaptation of this genus to the Amazon region.

Endo-and exoglucanase activities in bacteria from mangrove sediment

The mangrove ecosystem is an unexplored source for biotechnological applications. In this unique environment, endemic bacteria have the ability to thrive in the harsh environmental conditions (salinity and anaerobiosis), and act in the degradation of organic matter, promoting nutrient cycles. Thus, this study aimed to assess the cellulolytic activities of bacterial groups present in the sediment from a mangrove located in Ilha do Cardoso (SP, Brazil). To optimize the isolation of cellulolytic bacteria, enrichments in two types of culture media (tryptone broth and minimum salt medium), both supplemented with 5% NaCl and 1% of cellulose, were performed. Tests conducted with the obtained colonies showed a higher occurrence of endoglycolytic activity (33 isolates) than exoglycolytic (19 isolates), and the degradation activity was shown to be modulated by the presence of NaCl. The isolated bacteria were clustered by BOX-PCR and further classified on the basis of partial 16S rRNA sequences as Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes or Bacteroidetes. Therefore, this study highlights the importance of studies focusing on the endemic species found in mangroves to exploit them as novel biotechnological tools for the degradation of cellulose.(AU)

Investigação molecular de Leptospira spp em rins de bovinos / Molecular Research of Leptospira spp in Kidneys of Cattle

Background: Leptospirosis is a zoonotic bacterial disease caused by Leptospira spp. that affects wild and domestic animals and humans. It has a worldwide distribution and causes public health problems and economic losses in livestock. The genus Leptospira is divided basically into two species: the Leptospira interrogans that is pathogenic and Leptospira biflexa that is considered saprobic. Cattle are an important source of infection, mainly to humans who work with these animals such as breeders, cowboys, slaughtermen, veterinarians and agricultural technicians. The temperature and rainfall indices of tropical and subtropical climate regions encourage the continuing disease outbreaks. In humans the symptoms are headache, fever, myalgia, nausea and vomiting. The identification of the disease is possible with some tests such as ELISA, immunofluorescence, bacterial culture, and Polymerase Chain Reaction (PCR). The aim of this work was to perform the molecular research for the presence of Leptospira spp. in kidneys of cattle refuted at a slaughterhouse. Materials, Methods & Results: Two hundred fragments of cattle kidney were collected at slaughterhouse located at Ilhéus, Bahia, Brazil. The macroscopic kidney refutation causes were nefritis, presence of cysts, congestion and presence of whitening and hemorrhagic areas. The kidney samples were placed in plastic containers identified and packed in an isothermal box until arrival at the laboratory, where they were kept at -20°C until the DNA extraction with phenol/chloroform/ isoamyl alcohol (25:24:1). The DNA was quantified by spectrophotometer at 260 nm wavelength. The integrity of DNA was evaluated with 1% agarose gel stained with ethidium bromide and visualized at an ultraviolet light transilluminator. DNA amplification was performed with primers G1- [forward] 5'-CTGAATCGCTGTATAAAAGT-3' and G2- [reverse] 5'-GGAAAACAAATGGTCGGAAG-3' for pathogenic and non-pathogenic Leptospira. Aliquots of 50 ng/µL of extracted DNA were used for PCR reaction. Out of 200 tissue samples, 16 (8%) were positive and the amplified fragments had molecular weight compatible with Leptospira spp. Discussion: The amplicons presented molecular weight compatible with pathogenic and non-pathogenic strains of Leptospira spp. wich may represent a health risk for the ones who may contact animals or consume uncoocked products. Leptospira molecular diagnose may allow the pathogen identification in asymptomatic animals from small DNA amounts. Primers G1 and G2 amplified bacterial 16S rRNA gene area, allowing the identification. The renal tissue alterations identified during inspection, may be associated with Leptospira spp. presence showing the fundamental importance of correct identification and discard of affected organs in order to reduce the bacterial transmission risk. Many articles have being showing the Leptospira cattle prevalence and human risks by contacting these animals. The results presented in this article emphasizes the importance of the cattle as possible transmitters of the pathogen for slaughterhouses workers and meat inspectors, veterinarians as well as rural workers and their families, being critical to conduct orientation and conscientization programs about the problem and ways to combat and control it.

Phenotypic and genotypic diversity of Pseudomonas aeruginosa strains isolated from hospitals in siedlce (Poland)

A total of 62 Pseudomonas aeruginosa strains isolated from two hospitals in Siedlce (Poland) were studied by repetitive element based PCR (rep-PCR) using BOX primer. BOX-PCR results revealed the presence of 7 numerous genotypes and 31 unique patterns among isolates. Generally, the strains of P. aeruginosa were characterized by resistance to many antibiotics tested and by differences in serogroups and types of growth on cetrimide agar medium. However, the P. aeruginosa strains isolated from faeces showed much lower phenotypic and genotypic variations in comparison with strains obtained from other clinical specimens. It was observed that genetic techniques supported by phenotypic tests have enabled to conduct a detailed characterization of P. aeruginosa strains isolated from a particular environment at a particular time.

Rizóbios nativos do Rio Grande do Sul simbioticamente eficientes em Lotus glaber

Forage legumes besides contributing to increase the supply of high protein content forage are also good cover for the soil surface. Among the legumes with high potential forage, the genus Lotus has been outstanding. This research aimed to selectnative rhizobia efficient in fixing nitrogen with Lotus glaber, from soil samples of five localities of Rio Grande do Sul. A total of 259 rhizobia isolates were obtained, which were evaluated based on colony morphology and in vitro melanin production. Among them, 15 isolates were selected for the evaluation of the symbiotic nitrogen fixation efficiency in greenhouse experiments and genetically characterized by genomic DNA fingerprinting, PCR amplification with BOX and ERIC primers. Only five isolates produced melanin. Nine isolates were more efficient than strain SEMIA 830, which is authorized for the production of commercial inoculants of L. glaber in Brazil. In relation to the genetic characterization, the isolates have shown no similarity with the commercial strains, indicating that in the soils of Rio Grande do Sul there are autochthonous rhizobia capable of efficient symbiotic nitrogen fixation with L. glaber, which might be recommended for field evaluations, aiming a future production of inoculants for this legume in Brazil.

As leguminosas forrageiras, além de contribuírem para o aumento da oferta de forragem com alto teor de proteína, também são importantes como cobertura vegetal de solos. Entre as leguminosas com grande potencial forrageiro, espécies do gênero Lotus têm se destacado. Este trabalho visou à seleção de rizóbios nativos eficientes em Lotus glaber a partir de amostras de solo de cinco localidades do Rio Grande do Sul. Obtiveram-se 259 isolados de rizóbios, que foram avaliados quanto à morfologia colonial e produção de melanina. Destes, 15 isolados foram selecionados para avaliação da eficiência na fixação simbiótica de nitrogênio em experimento em casa de vegetação e caracterizados geneticamente por comparação do perfil eletroforético dos produtos de amplificação do DNA genômico, por PCR com os oligonucleotídeos iniciadores BOX e ERIC. Apenas cinco isolados produziram melanina. Nove isolados foram mais eficientes do que a estirpe SEMIA 830, que é autorizada para a produção de inoculante para L. glaber no país. Na caracterização genética, observou-se que nenhum isolado apresentou identidade com as estirpes recomendadas, o que demonstra que, nos solos do Rio Grande do Sul, existem rizóbios autóctones eficientes na fixação simbiótica de nitrogênio com L. glaber, podendo ser recomendados para estudos a campo, visando a uma futura produção de inoculantes para estas leguminosas no Brasil.

Rizóbios nativos do Rio Grande do Sul simbioticamente eficientes em Lotus glaber

Forage legumes besides contributing to increase the supply of high protein content forage are also good cover for the soil surface. Among the legumes with high potential forage, the genus Lotus has been outstanding. This research aimed to selectnative rhizobia efficient in fixing nitrogen with Lotus glaber, from soil samples of five localities of Rio Grande do Sul. A total of 259 rhizobia isolates were obtained, which were evaluated based on colony morphology and in vitro melanin production. Among them, 15 isolates were selected for the evaluation of the symbiotic nitrogen fixation efficiency in greenhouse experiments and genetically characterized by genomic DNA fingerprinting, PCR amplification with BOX and ERIC primers. Only five isolates produced melanin. Nine isolates were more efficient than strain SEMIA 830, which is authorized for the production of commercial inoculants of L. glaber in Brazil. In relation to the genetic characterization, the isolates have shown no similarity with the commercial strains, indicating that in the soils of Rio Grande do Sul there are autochthonous rhizobia capable of efficient symbiotic nitrogen fixation with L. glaber, which might be recommended for field evaluations, aiming a future production of inoculants for this legume in Brazil.

As leguminosas forrageiras, além de contribuírem para o aumento da oferta de forragem com alto teor de proteína, também são importantes como cobertura vegetal de solos. Entre as leguminosas com grande potencial forrageiro, espécies do gênero Lotus têm se destacado. Este trabalho visou à seleção de rizóbios nativos eficientes em Lotus glaber a partir de amostras de solo de cinco localidades do Rio Grande do Sul. Obtiveram-se 259 isolados de rizóbios, que foram avaliados quanto à morfologia colonial e produção de melanina. Destes, 15 isolados foram selecionados para avaliação da eficiência na fixação simbiótica de nitrogênio em experimento em casa de vegetação e caracterizados geneticamente por comparação do perfil eletroforético dos produtos de amplificação do DNA genômico, por PCR com os oligonucleotídeos iniciadores BOX e ERIC. Apenas cinco isolados produziram melanina. Nove isolados foram mais eficientes do que a estirpe SEMIA 830, que é autorizada para a produção de inoculante para L. glaber no país. Na caracterização genética, observou-se que nenhum isolado apresentou identidade com as estirpes recomendadas, o que demonstra que, nos solos do Rio Grande do Sul, existem rizóbios autóctones eficientes na fixação simbiótica de nitrogênio com L. glaber, podendo ser recomendados para estudos a campo, visando a uma futura produção de inoculantes para estas leguminosas no Brasil.