Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum)

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.

Protective effect of carbon dioxide against bacterial peritonitis induced in rats

Carbon dioxide (CO(2)) has been used in the food industry as an antimicrobial agent. This study aimed to investigate whether CO(2) pneumoperitoneum might act similarly as an antimicrobial agent in the infected peritoneal cavity. Peritonitis was induced in 58 rats by intraabdominal injection of an Escherichia coli inoculum (6 x 105 colony-forming units [CFU]/ml). Control rats were injected with saline solution. The rats were randomly divided into four groups: rat control (RC, n = 15), bacterial inoculation control (BIC, n = 10), bacterial inoculation and laparotomy (BIL, n = 17), and bacterial inoculation and CO(2) pneumoperitoneum (BIP, n = 16). The survival rates and histopathologic changes in the abdominal wall muscles, spleen, liver, intestines, and omentum were evaluated, and the samples were classified as ""preserved"" or ""inflamed"" (acute inflammation or tissue regeneration). The survival rates for the four groups were as follows: RC (100%), BIP (75%), BIL (53%), and BIC (30%). With regard to survival rates, statistically significant differences were observed between the following groups: RC and BIC (p = 0.0009), RC and BIL (p = 0.0045), BIP and BIC (p = 0.0332), and RC and BIP (p = 0.0470). No significant differences regarding survival rates were observed between the BIL and BIC groups or between the BIP and BIL groups. With regard to the number of inflamed samples per group, a statistically significant difference was observed between the BIC and RC groups and the BIL and RC groups (p = 0.05). Carbon dioxide pneumoperitoneum has a protective effect against bacterial peritonitis induced in rats.

An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7

Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety.

Initial performance of maize in response to NPK fertilization combined with Herbaspirillum seropedicae

The inoculation with plant growth-promoting bacteria can be a technological approach useful for increasing the production of maize. The objective of this study was to evaluate the initial performance of maize in response to application of doses of NPK combined with the inoculation of the diazotrophic bacteria Herbaspirillum seropedicae in an greenhouse experiment. The experiment consisted of six fertilizer levels: 0, 25, 50, 75, 100 and 200% of the recommended dose of NPK applied to maize inoculated and non-inoculated with H. seropedicae. At 30 days after the treatment application, the growth characteristics and nutritional status of the plants were evaluated. Plant development was influenced by fertilization, but it was enhanced by combination with the bacteria, which resulted in significant increases in the dry mass of shoots (7%) and leaf area (9%) when compared with non-inoculated plants. The results showed increases in the concentration of N (11%), P (30%) and K (17%) of maize plants in response to bacterial inoculation together with NPK compared with plants that were applied fertilize only. The greater consistency and stability response of the host plant to bacterization in the presence of chemical fertilizer indicate a promissory biotechnological approach for improving the initial growth and adaptation of maize to the cultivation environment.

Diversity and capacity to promote maize growth of bacteria isolated from the Amazon region

ABSTRACT Maize plants can establish beneficial associations with plant growth-promoting bacteria. However, few studies have been conducted on the characterization and inoculation of these bacteria in the Amazon region. This study aimed to characterize endophytic bacteria isolated from maize in the Amazon region and to assess their capacity to promote plant growth. Fifty-five bacterial isolates were obtained from maize grown in two types of ecosystems, i.e., a cerrado (savanna) and a forest area. The isolates were characterized by the presence of the nifH gene, their ability to synthesize indole-3-acetic acid (IAA) and solubilize calcium phosphate (CaHPO4), and 16S rRNA partial gene sequencing. Twenty-four bacteria contained the nifH gene, of which seven were isolated from maize plants cultivated in a cerrado area and seventeen from a forest area. Fourteen samples showed the capacity to synthesize IAA and only four solubilized calcium phosphate. The following genera were found among these isolates: Pseudomonas; Acinetobacter; Enterobacter; Pantoea; Burkholderia and Bacillus. In addition, eight isolates with plant growth-promoting capacity were selected for a glasshouse experiment involving the inoculation of two maize genotypes (a hybrid and a variety) grown in pots containing soil. Inoculation promoted the development of the maize plants but no significant interaction between maize cultivar and bacterial inoculation was found. A high diversity of endophytic bacteria is present in the Amazon region and these bacteria have potential to promote the development of maize plants.

A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.

RP 59500 prophylaxis of experimental endocarditis due to erythromycin-susceptible and -resistant isogenic pairs of viridans group streptococci.

RP 59500 is a new injectable streptogramin composed of two synergistic components (quinupristin and dalfopristin) which are active against a number of erythromycin-susceptible and -resistant gram-positive bacteria. The following experiments investigate the ability of RP 59500 to prevent experimental endocarditis due to either of two erythromycin-susceptible streptococcal isolates or their constitutively erythromycin-resistant Tn916 delta E transconjugants. RP 59500 had low MICs (0.125 to 0.5 mg/liter) for all four test organisms and was substantially bactericidal in vitro. Rats with catheter-induced aortic vegetations were given single-dose antibiotic prophylaxis 30 to 60 min before bacterial inoculation through a computerized pump system which permitted the simulation of drug kinetics for humans produced by either 7 mg of RP 59500 per kg of body weight or 1 g of vancomycin. Single-dose RP 59500 prophylaxis successfully prevented endocarditis due to both the erythromycin-susceptible parent strains and their erythromycin-resistant derivatives in rats challenged with the minimal inoculum infecting 90% of controls. In addition, RP 59500 also prevented infection in animals challenged with fivefold-larger inocula of the erythromycin-susceptible parent strains. Vancomycin successfully prevented endocarditis due to any of the four test organisms. These results underline the in vivo efficacy of RP 59500 against both erythromycin-susceptible and -resistant streptococci. Such good results against the resistant strains would not be expected with erythromycin or clindamycin, which are the standard macrolidelincosamide-streptogramin antibiotics used for endocarditis prophylaxis in humans. An oral form of RP 59500 which might advantageously replace some of the older prophylactic regimens is currently being developed.

Cultivable mushroom growth-promoting bacteria and their impact on Agaricus blazei productivity

The objective of this work was to identify growth-promoting bacteria isolated from Agaricus blazei and to evaluate their effect on mushroom mycelial growth and productivity. A total of 56 A. blazei-associated bacterial isolates were obtained from casing soil and identified by 16S rRNA gene sequencing. Bacteria were evaluated as to phosphate-solubilization ability, nitrogen-fixation capability, and secretion of cellulase. Superior isolates were tested for their to effect on A. blazei productivity, micelial growth, and on the contents of the polysaccharide-protein complex and of N, P, K, Ca, and Mg. Bacterial isolates were identified as actinobacteria (60%), firmicutes (20%), and proteobacteria (20%). Among them, ten isolates had phosphate-solubilization ability, eight showed nitrogen-fixation capability, and 12 isolates promoted A. blazei mycelium growth. Bacterial inoculation reduces time till harvest in up to 26 days, increases fresh mushroom yield up to 215%, and increases total polysaccharide-protein complex content twofold when compared to the non-inoculated control. The actinobacteria group is the predominant A. blazei-associated phylum.

O exame direto do lavado broncoalveolar para o diagnóstico precoce de pneumonia nosocomial : um modelo experimental em ratos

A pneumonia nosocomial é a principal causa de infecção nosocomial em unidades de tratamento intensivo e possui alta morbi/mortalidade. A incidência cumulativa varia, conforme os autores, entre limites amplos desde 8% até 51%, dependendo do tipo de paciente e do uso de instrumentação das vias respiratórias. Nos casos específicos das pneumonias de má resolução e da pneumonia associada à ventilação mecânica, o diagnóstico é problemático devido à ausência de uma padronização definitiva, decorrente não só da grande variabilidade fisiopatológica como também da pouca acurácia dos critérios clínicos, microbiológicos e radiológicos. Estes fatos ensejaram a utilização progressiva de técnicas invasivas sofisticadas para coleta de amostras respiratórias endobrônquicas. Entretanto, a validação dessas técnicas para uso sistemático depende ainda de estudos que avaliem não só o seu custo/benefício em cenários clínicos diversos como também a comparação de seu desempenho para o diagnóstico com um padrão-ouro histopatológico. Além disso, o rendimento das técnicas invasivas é freqüentemente limitado quando são aplicadas em pacientes sob antibioticoterapia, que constituem a maioria dos casos em unidades de cuidados intensivos. A otimização desses testes, discutindo suas indicações e avaliando sua capacidade técnica para a confirmação ou exclusão de pneumonia, é justificada pela necessidade da instituição precoce e correta do tratamento, pois disto resulta menor mortalidade, redução de custos e permanência no hospital. Entre os testes que podem auxiliar no diagnóstico encontra-se o exame direto do lavado broncoalveolar, que proporciona resultados precoces e úteis para a tomada de decisão, mas não suficientemente testados em diferentes situações clínicas ou experimentais. Com o objetivo de avaliar o rendimento diagnóstico do exame direto precoce e das culturas quantitativas obtido por lavado broncoalveolar, estudou-se sua utilização em um modelo experimental de pneumonia provocada através de inoculação bacteriana intrabrônquica em ratos. O estudo comparou a acurácia do exame direto e dos exames culturais em três grupos de animais: Grupo A com pneumonia pneumocócica (37 animais); Grupo P com pneumonia por P. aeruginosa (26 animais) e Grupo B controle (10 animais), utilizando a histopatologia dos pulmões como teste de referência para o diagnóstico. Os Grupos A e P foram ainda randomizados em dois subgrupos cada, para tratamento ou não com antibióticos, usando penicilina no grupo pneumococo e amicacina no grupo Pseudomonas. Como resultado, observou-se que nos animais com pneumonia e ausência de antibióticos a pesquisa de bactéria fagocitada (BIC) no exame direto mostrou um rendimento elevado para o diagnóstico, sendo superior ao das culturas quantitativas. No grupo com pneumonia pneumocócica a BIC mostrou: S:94,4% a 100%, E:100%, VPP:100% e VPN:100%; e as culturas quantitativas mostraram: S:77,8%, E:100%, VPP:100%, VPN:40%. Nos com pneumonia por Pseudomonas a BIC obteve: S: 69%, E:100%; VPP:100% e VPN:71,4%); e as culturas quantitativas mostraram valores muito baixos: S:28,6%, E:100%, VPP:100% e VPN:50%). Nos animais com pneumonia pneumocócica sob tratamento antibiótico havia uma queda acentuada de sensibilidade das culturas quantitativas (S:21%) e, em menor grau da BIC (S:57,9%), mas sem perda da especificidade de ambos os exames. Ao contrário, nos animais com pneumonias por Pseudomonas sob tratamento não havia alteração no rendimento dos dois exames, cujos resultados foram semelhantes aos dos animais sem tratamento. Não havia diferenças de leitura da BIC para o diagnóstico, contando a sua positividade em macrófagos ou em neutrófilos infectados. A avaliação global dos casos estudados, reunindo todos os grupos (tratados e não-tratados) em ambos os modelos de pneumonia, mostrou que a acurácia do exame direto, representado pela pesquisa da BIC, foi superior (66%) ao das culturas quantitativas (53%). As conclusões principais do estudo foram: 1) o exame direto do lavado broncoalveolar (LBA) mostrou-se um teste útil e de alto rendimento para o diagnóstico de pneumonia quando realizado na ausência de antibióticos; 2) o LBA feito na vigência de antibióticos efetivos para a pneumonia perde mais de 50% de sua acurácia, mas não é afetado quando o antibiótico for ineficaz ou houver resistência ao mesmo; 3) a pesquisa de BIC no LBA é um exame de resultado precoce, de alta especificidade e com melhor rendimento do que as culturas quantitativas.

Análise do perfil de expressão dos genes da cana-de-açúcar envolvidos na interação com Leifsonia xyli subsp: xyli

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

Effects of Antibacterial Agents on Dental Pulps of Monkeys Mechanically Exposed and Contaminated.

The purpose of this study was to compare the effectiveness of antibacterial agents and mineral trioxide aggregate in the healing of bacterial contaminated primate pulps. Study Design: The experiment required four adult male primates (Cebus opella) with 48 teeth prepared with buccal penetrartions into the pulpal tissues. The preparations (Cebus opella) with 48 teeth prepared with buccal penetrations into the exposed to cotton pellets soaked in a bacterial mixture consisting of microorganisms normally found in human pulpal abscesses obtained from the Endodontic Clinic of UNESP. Following bacterial inoculation (30 minute exposure), the pulpal tissue was immediately treated with either sterile saline, Cipro HC Otic solution (12), diluted Buckley formecresol solution (12) or Otosporin otic solution (12) for 5 minutes. After removal of the pellet, hemostasis was obtained and a ZOE base applied to the DFC treated pulps and the non-treated controls (12). After hemostasis, the other exposed pulps were covered with mineral trioxide aggregate (ProRoot). The pulpal bases were all covered with a RMGI (Fuji II LC). The tissue samples were collected at one day, two days, one week and over four weeks (34 days). Results: Following perfusion fixation, the samples were demineralized, sectioned, stained and histologically graded. After histologic analysis, presence of neutrophilic infiltrate and areas of hemorrhage with hyperemia were observed . The depth of the neutrophilic infiltrate depended on the agent or material used. The pupal tissue treated with Otic suspensions demonstrated significantly less inflammation (Kruskal Wallis non parametric analysis, H=9.595 with 1 degree of freedom; P=0.0223) than the formocresol and control groups. The hard tissue bridges formed over the exposure sites were more organized in the MTA treatment groups than in the control and ZOE groups (Kruskal Wallis non parametric analysis, H=18.291 with 1 degree of freedom; P=0.0004). Conclusions: Otic suspensions and MTA are effective in treating bacterial infected pulps and stimulate the production of a hard tissue bridge over the site of the exposure.

Rehydration of corn grain with acid whey improves the silage quality

Our objective was to investigate the effects of rehydration with acid whey or water at three moisture levels, as well as the effects of bacterial inoculation, on the fermentation, chemical composition and aerobic stability of corn grain silages. The trial was conducted in a completely randomized design with four replicates in a factorial arrangement as follows: 3 (rehydration with three different moisture levels: 300,350 and 400 mL/kg of corn grain)x 2 (silage inoculated with bacteria or not inoculated (control))x 2 (liquid used in the rehydration: acid whey or water). Overall, corn grain silages rehydrated with acid whey produced more lactic acid than the silages rehydrated with water (13.8 vs. 12.6 g/kg of dry matter (DM), respectively). In addition, increases in the rehydration of corn grain silages promoted decreases (linear) in lactic acid concentration as well as in production of total acids. Although inoculated silages had higher pH as consequence of the rehydration using water at the three levels, these treatments presented high DM recovery. In general, neutral detergent fiber (aNDFom) decreased if inoculant was applied in corn grain silages rehydrated with acid whey. After silos opening, silages rehydrated with 350 or 400 mL/kg (independent of the liquid) had lower aerobic stability than silages rehydrated with 300 mL/kg. Overall, we found that the inoculant did not promote significant changes in the composition of the corn grain silage. In contrast, the potential of the use of acid whey in ensiling corn grain is high, as its addition leads to improvements in the fermentation process and aerobic stability of the silages. (C) 2014 Elsevier B.V. All rights reserved.

Energy deficit does not affect immune responses of experimentally infected pacu (Piaractus mesopotamicus)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Eficácia do levamisol como imunoestimulante, redutor de estresse e antiparasitário no pacu

Pós-graduação em Aquicultura - FCAV

Chemical products induce resistance to Xanthomonas perforans in tomato

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