The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR / A influência da escolha de iniciadores na filogenia de Archaea em ensaios de PCR para o gene rRNA 16S

Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

Resumo A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu subsequente sequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).

The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR / A influência da escolha de iniciadores na filogenia de Archaea em ensaios de PCR para o gene rRNA 16S

Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu sub sequentesequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).

The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR

Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

Resumo A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu subsequente sequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).

Trichoderma: biological control efficiency and perspectives for the Brazilian Midwest states and Tocantins.

Brazil is one of the world leaders in the agribusiness sector tending to directly influence a growing dependence on imported inputs, specifically synthetic agrochemicals. At the state level, in 2013, Tocantins stood out in first place in the ranking of agrochemical consumers, however, these products can cause several problems, such as poisoning to humans, environmental contamination, and increased resistance to phytopathogens. Biological control is an alternative to the use of agrochemicals towards eliminating pests naturally by using living organisms called Biological Control Agents (BCA). Currently, fungi of the Trichoderma genus are some of the most used organisms in biological pest control for their relevant characteristics that favor them in terms of survival in the environment, such as high capacity to adapt to ecological conditions, potential to colonize the rhizosphere of plants, mycoparasitism, production of volatile and non-volatile metabolites. In addition, it works on plant growth and productivity. In general, the use of Trichoderma favors the control of soil pathogens, such as Rhizoctonia, Pythium, Sclerotinia, and nematodes. Thus, this review aims to demonstrate the importance of using Trichoderma in biological control, as well as to present an overview and perspectives of research developed by respondents in the Brazilian Midwest region and Tocantins state.

The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR.

Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

Impact of Age, Education Level and Occupation on the Ability to Use the ICIQ-bladder Diary: Results From A Community Cohort Study Using a Predefined Clinical History.

OBJECTIVE: To assess difficulties in filling the International Consultation on Incontinence Questionnaire Bladder Diary (ICIQ-BD), determining the most troublesome items and patients' characteristics (age, education level, and professional activity) related to these difficulties. METHODS: A text composed of a clinical history of a fictitious patient with Lower Urinary Tract Symptoms (LUTS) was designed. 95 healthy volunteers (49 females, 46 males, mean age 45.6 ± 17.3 years) were asked to fill in the ICIQ-BD according to the data from the fictitious clinical history. Filling errors were analyzed, and patients' demographic characteristics tested as predictors. RESULTS: Bladder Sensation Score is the most problematic item of the ICIQ-BD, with only 38% of patients accurately filling this item. No association was found between total number of errors and age or education level, but participants working in less specialized professions had more difficulty in filling the BD (mean: 7.00 vs 3.94 errors, P= .002). CONCLUSION: ICIQ-BD is a reliable tool to assess for LUTS, but filling the Bladder Sensation Score item was more likely to be incorrectly filled by manual workers and volunteers with less intellectually demanding current professions.

A dual-electrochemical cell to study the biocorrosion of stainless steel.

The presence of microorganisms on metal surfaces can alter the local physical/chemical conditions and lead to microbiologically influenced corrosion (MIC). The goal of the present work was to study the effect of a mixed aerobic-anaerobic biofilm on the behaviour of stainless steel (316 L) in underground conditions. Rather than testing different bacteria or consortia, investigations were based on the mechanisms of MIC. Mixed biofilms were simulated by the addition of glucose oxidase to reproduce the aerobic conditions and by sulphide or sulphate-reducing bacteria (SRB) for the anaerobic conditions. A double thermostated electrochemical cell has been developed to study the coupling between aerobic and anaerobic conditions. Results suggested a transfer of electrons from the stainless steel sample of the anaerobic cell to the stainless steel sample of the aerobic one. Inorganic sulphide was replaced by SRB in the anaerobic cell revealing an increase of the galvanic current which may be explained by an effect of lactate and/or acetate on the anodic reaction or by a high sulphide concentration in the biofilm. The results of this study underline that the dual-electrochemical cell system is representative of phenomena present in natural environments and should be considered as an option when studying MIC.

Interaction of Desulfovibrio desulfuricans biofilms with stainless steel surface and its impact on bacterial metabolism.

AIMS: To study the influence of some metallic elements of stainless steel 304 (SS 304) on the development and activity of a sulfate-reducing bacterial biofilm, using as comparison a reference nonmetallic material polymethylmethacrylate (PMMA). METHODS AND RESULTS: Desulfovibrio desulfuricans biofilms were developed on SS 304 and on a reference nonmetallic material, PMMA, in a flow cell system. Steady-state biofilms were metabolically more active on SS 304 than on PMMA. Activity tests with bacteria from both biofilms at steady state also showed that the doubling time was lower for bacteria from SS 304 biofilms. The influence of chromium and nickel, elements of SS 304 composition, was also tested on a cellular suspension of Des. desulfuricans. Nickel decreased the bacterial doubling time, while chromium had no significant effect. CONCLUSIONS: The following mechanism is hypothesized: a Des. desulfuricans biofilm grown on a SS 304 surface in anaerobic conditions leads to the weakening of the metal passive layer and to the dissolution in the bulk phase of nickel ions that have a positive influence on the sulfate-reducing bacteria metabolism. This phenomenon may enhance the biocorrosion process. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the interactions between metallic surfaces such as stainless steel and bacteria commonly implied in the corrosion phenomena which is primordial to fight biocorrosion.

The influence of nickel on the adhesion ability of Desulfovibrio desulfuricans.

The build-up of biofilms on metals surfaces may lead to severe corrosion, especially in the presence of sulphate-reducing bacteria (SRB). To prevent the deterioration of material caused by biofilms it is necessary to understand the processes governing biofilm development including mechanisms of cell adhesion. Additionally, corrosion of metallic surfaces due to bacteria may lead to the dissolution of metallic elements that may further affect adhesion and biofilm development. A study was carried out to evaluate how the presence of nickel in the substrata affects the adhesion ability of Desulfovibrio desulfuricans. The substrata tested were stainless steel 304 (SS), metallic nickel (Ni) and polymethylmetacrylate (PMMA), a non-metallic material used as control. The influence of nickel on SRB growth and its relation to adhesion was also checked. A statistically significant difference in the number of adhered cells to the materials tested was detected, with higher bacterial number on nickel, followed by SS and finally by PMMA. The higher number of SRB adhered to steel compared with PMMA may be explained by differences in hydrophobicity, in roughness and in the electron-acceptor character of the substrata. Additionally, bacterial growth was found to be positively affected by the presence of nickel as revealed by a significant increase in the specific growth rate of SRB in the presence of increased nickel concentrations.

The mother's participation in infant carrying in captive groups of Leontopithecus chrysomelas and Callithrix jacchus.

Callithrix and Leontopithecus exhibit ecological differences that have implications for the patterns of infant care. In C. jacchus, which uses a small home range because it depends mainly on plant exudates, infants can forage independently early in their life. L. chrysomelas, which feeds mainly on fruits and insects, needs larger home ranges and, therefore, its infants have a more extensive period of dependence. Three families of C. jacchus and four families of L. chrysomelas were studied in captivity. The animals were observed starting from the birth of the infants up to their 8th week of age. Our results suggest that the pattern of infant transfer in L. chrysomelas did not follow the one reported for L. rosalia in that transfers from the mother occurred much earlier. L. chrysomelas infants were carried for about 15% of total time during the 8th week of life against less than 1% in C. jacchus in the same week. Infant care seems to be more extensive in L. chrysomelas than in C. jacchus, and the period of exclusive mother carrying in L. chrysomelas is shorter than that observed in L. rosalia.

Behavior and progesterone levels in Callithrix jacchus females.

In callitrichids, and especially in the Callithrix and Saguinus genera, reproductive suppression maintains the breeding exclusivity of dominant females. It has been suggested that dominance may be inferred from agonistic behavior of females. The purpose of this study was to assess the adequacy of behaviors as indicators of hormonal status in Callithrix jacchus females. Two pairs of twin females were observed for affiliative, agonistic and sexual behaviors for 20 weeks, in four stages of increasingly potential competition for one male. During the same period, blood samples were taken regularly for progesterone determination by ELISA. The two pairs differed markedly as to behaviors and hormonal status: pair 1 showed a well established behavioral dominance and only one ovulating female from the beginning of the observation period; pair 2 showed competition for dominance until the last stage of observation, when one of the females started ovulating. Correlations between behaviors and progesterone levels showed differences between subordinate and dominant females mostly in pair 2. Although limited in number of animals studied, these results suggest that there is a relationship between behavior and hormones in female marmosets, but this relationship may be useful in the identification of the physiological status of females only in groups where the dominance is not well established.

Behavior and progesterone levels in Callithrix jacchus females

In callitrichids, and especially in the Callithrix and Saguinus genera, reproductive suppression maintains the breeding exclusivity of dominant females. It has been suggested that dominance may be inferred from agonistic behavior of females. The purpose of this study was to assess the adequacy of behaviors as indicators of hormonal status in Callithrix jacchus females. Two paires of twin females were observed for affiliative, agonistic and sexual behaviors for 20 weeks, in four stages of increasingly potential competition for one male. During the same period, blood samples were taken regularly for progesterone determination by ELISA. The two pairs differed markedly as to behaviors and hormonal status: pair 1 showed a well established behavioral dominance and only one ovulating female from the beginning of the observation period; pair 2 showed competition for dominance until the last stage of observation, when one of the females started ovulating. Correlations between behaviors and progesterone levels showed differences between subordinate and dominant females mostly in pair 2. Although limited in number of animals studied, these results suggest that there is a relationship between behavior and hormones in female marmosets, but this relationship may be useful in the identification of the physiological status of females only in groups where the dominance is not well estblished