Interações populacionais mediadas por quorum sensing na microbiota do soro-fermento utilizado na produção do queijo Canastra / Population interactions mediated by quorum sensing in the microbiota of the endogenous culture used in the production of canasta cheese

Bactérias regulam a expressão de diversos fenótipos de acordo com a sua densidade populacional, em um comportamento conhecido como quorum sensing. Em micro-organismos de origem alimentar, o quorum sensing pode influenciar na formação de biofilmes, produção de toxinas e de enzimas hidrolíticas. Em bactérias Gram-negativas a sinalização é normalmente mediada por moléculas de N-acilhomoserina lactona (AHLs), conhecidas por autoindutor 1 (AI-1). Estudos revelam a inibição do quorum sensing nestas bactérias por enzimas que degradam as AHLS, em um processo denominado quorum quenching. Tipicamente brasileiro, o queijo Canastra é um produto artesanal maturado, produzido a partir de leite cru e do pingo, um tipo de soro-fermento coletado e utilizado diariamente na produção. A composição microbiana do pingo é diversificada e característica da região produtora. Essa combinação de bactérias, única em cada queijaria, resulta em aroma e textura típicos. Enquanto a microbiota Gram-positiva contribui para o desenvolvimento de sabor, textura e aroma no produto, bactérias Gram-negativas nesses queijos são geralmente associadas à formação de olhaduras, aromas desagradáveis, má coagulação da massa e até à patogenicidade. Este trabalho visou analisar a interação entre a microbiota Gram-positiva e Gram-negativa presente no pingo pela detecção dos sistemas de quorum sensing e quorum quenching nas amostras. A presença de AHLs foi avaliada em 45 amostras de pingo, a partir da extração em acetato de etila acidificado e da avaliação dos extratos por meio de bioensaios com Agrobacterium tumefaciens WCF47(pCF218)(pCF372) e KYC55(pJZ410)(pJZ372)(pJZ384), resultando em apenas uma amostra positiva. Em seguida, 350 isolados foram obtidos a partir de 11 amostras de pingo, sendo 200 isolados classificados como Gram-positivos e 150 Gram-negativos. Os Gramnegativos foram avaliados quanto à produção de AHLs in vitro através de ensaio em placa utilizando as estirpes biossensoras A. tumefaciens WCF47(pCF218)(pCF372), Chromobacterium violaceum CV026 e Escherichia coli pSB403, resultando em 39 isolados produtores de AHLs, provenientes de 10 pingos diferentes. Já os isolados Gram-positivos foram analisados quanto à capacidade de inibição do QS utilizando as estirpes biossensoras C. violaceum CV026 e A. tumefaciens WCF47(pCF218)(pCF372), em meio suplementado com C6-HSL ou 3-oxo-C12-HSL. Foi detectada a inibição total da resposta ao quórum por 78 isolados testados, enquanto a inibição parcial foi provocada por outros 63. A inibição do crescimento das estirpes biossensoras também foi observada para 24 isolados. Os isolados promotores de inibição parcial foram recultivados em meio mínimo com C6-HSL ou 3-oxo-C12-HSL como únicas fontes de carbono. Foram recuperados 28 isolados, e a ação desses sobre diferentes substratos foi avaliada, resultando em 22 isolados produtores de lactonases e 6 produtores de acilase. Os 39 isolados Gram-negativos e os 28 isolados Gram-positivos finais foram identificados por MALDI-TOF MS, resultando, segundo o conhecimento do autor, no primeiro relato de produção de AHLs por Pseudomonas fulva, Enterobacter xiangfangensis e Lelliottia amnigena, bem como a produção de lactonases por Staphylococcus xylosus e a produção de acilase por S. aureus, Microbacterium maritypicum e Rothia kristinae. Este trabalho mostrou que interações populacionais mediadas por quorum sensing dependente de AHLs na microbiota do soro-fermento são possíveis. Porém, essas interações estão propensas a serem inibidas por meio de lactonases e acilases produzidas por parte das bactérias Gram-positivas

Bacteria regulate the expression of different phenotypes according to their population density, in a behavior known as quorum sensing. In food-borne microorganisms, quorum sensing can influence the formation of biofilms, production of toxins and hydrolytic enzymes. In Gram-negative bacteria, signaling is normally mediated by Nacyl homoserine lactone molecules (AHLs), known as autoinducer 1 (AI-1). Studies reveal the inhibition of quorum sensing in these bacteria by enzymes that degrade AHLS, in a process called quorum quenching. Typically Brazilian, Canastra cheese is a matured artisanal product, produced from raw milk and pingo, a type of endogenous culture collected and used daily in production. The microbial composition of pingo is diverse and characteristic of the producing region. This combination of bacteria, unique in each cheese factory, results in a typical aroma and texture. While the Gram-positive microbiota contributes to the development of flavor, texture and aroma in the product, Gram-negative bacteria in these cheeses are generally associated with the formation of eyes, off-flavors, poor curd coagulation and even pathogenicity. Thus, this work aimed to analyze the interaction between the Gram-positive and Gram-negative microbiota present in this culture by detecting quorum sensing and quorum quenching systems in the samples. The presence of AHLs was evaluated in 45 samples of pingo, with extraction with acidified ethyl acetate and the evaluation of the extracts through bioassays with Agrobacterium tumefaciens WCF47(pCF218)(pCF372) and KYC55(pJZ410)(pJZ372)(pJZ384 ), resulting in only one positive sample. Then, 350 isolates were obtained from 11 endogenous culture samples, with 200 being classified as Gram-positive and 150 Gram-negative. Gram-negatives were evaluated for the production of AHLs in vitro by plaque assay using the biosensor strains A. tumefaciens WCF47(pCF218)(pCF372), Chromobacterium violaceum CV026 and Escherichia coli pSB403, resulting in 39 AHL-producing isolates from 10 different samples. Gram-positive isolates were analyzed for their ability to inhibit quorum sensing using biosensor strains C. violaceum CV026 and A. tumefaciens WCF47(pCF218)(pCF372), in medium supplemented with N-hexanoyl-L-homoserine lactone or 3-oxo-dodecanoyl-Lhomoserine lactone. Total inhibition of the quorum response was detected by 78 tested isolates, while partial inhibition was caused by 63. Growth inhibition of biosensor strains was also observed for 24 isolates. Partial inhibition promoter isolates were recultured on minimal medium with C6-HSL or 3-oxo-C12-HSL as sole carbon sources. Twenty-eight isolates were recovered, and the action of these isolates on different substrates was evaluated, resulting in 22 lactonase producers and 6 acylase producers. The 39 Gram-negative isolates and the final 28 Grampositive isolates were identified by MALDI-TOF MS, resulting, to the best of the author's knowledge, in the first report of AHL production by Pseudomonas fulva, Enterobacter xiangfangensis and Lelliottia amnigena, as well as the lactonase production by Staphylococcus xylosus and acylase production by S. aureus, Microbacterium maritypicum and Rothia kristinae. This work demonstrated that population interactions mediated by AHLs-dependent quorum sensing in Canastra cheese endogenous culture microbiota are possible. However, these interactions are prone to inhibition by lactonases and acylases produced by Gram-positive bacteria

Infecciones por bacilos Gram-negativos multirresistentes en neonatología / Multidrug resistant Gram-negative infections in neonatology

Introducción. Las infecciones por bacilos Gram-negativos multirresistentes (BGN-MR) constituyen un problema creciente en las unidades de cuidado intensivo neonatal. El objetivo del estudio fue conocer las características epidemiológicas, clínicas, microbiológicas, evolutivas y los factores de riesgo de infección por BGN-MR resistentes a carbapenemes en el Servicio de Neonatología de un hospital de alta complejidad. Población y método. Se realizó un estudio de cohorte retrospectivo en dicho Servicio, donde se incluyeron los pacientes con infección documentada por BGN-MR del 24/4/2013 al 29/4/2015. Resultados. Se incluyeron 21 pacientes. La mediana de edad gestacional y peso de nacimiento fue 35 semanas y 2070 gramos, respectivamente. Dieciocho pacientes (86 %) tuvieron hemocultivos positivos y el aislamiento microbiológico más frecuente fue Acinetobacter baumannii (17 pacientes, 81 %), seguido por Klebsiella pneumoniae productora de carbapenemasa (3 pacientes, 14 %) y Enterobacter cloacae (1 paciente, 5 %). La mediana de edad al momento del diagnóstico fue de 28 días y todos tenían factores de riesgo para la infección, como cirugía, asistencia respiratoria mecánica, nutrición parenteral, catéter central y antibióticos. El tratamiento antibiótico definitivo fue colistina en todos los casos, combinado en el 84 %. Cinco pacientes (24 %) fallecieron por la infección. La prematurez y el peso < 2000 g fueron factores de riesgo estadísticamente significativos asociados a la mortalidad (p = 0,03 y 0,01, respectivamente). Conclusión. Las infecciones por BGN-MR se presentaron en pacientes con factores predisponentes. Acinetobacter baumannii fue el primer agente etiológico. La mortalidad fue elevada y relacionada con prematurez y bajo peso al nacer.

Introduction. Multidrug resistant Gramnegative (MDRGN) infections are an increasing problem in neonatal intensive care units. The objective of this study was to establish the epidemiological, clinical, microbiological, and evolutionary characteristics of carbapenem-resistant MDRGN infections and the risk factors for them at the Division of Neonatology of a tertiary care hospital. Population and method. A retrospective cohort study was done in this Division in patients with a documented MDRGN infection between 4/24/2013 and 4/29/2015. Results. Twenty-one patients were included. Their median gestational age and birth weight were 35 weeks and 2070 g, respectively. Eighteen patients (86 %) had a positive blood culture; the most commonly isolated microorganism was Acinetobacter baumannii (17 patients, 81 %), followed by carbapenemase-producing Klebsiella pneumoniae (3 patients, 14 %) and Enterobacter cloacae (1 patient, 5 %). The median age at diagnosis was 28 days and all patients had risk factors for infection, including surgery, assisted mechanical ventilation, parenteral nutrition, central venous line, and antibiotics. The definite antibiotic therapy included colistin in all cases; in combination, in 84 %. Five patients (24 %) died due to the infection. Prematurity and a birth weight < 2000 g were statistically significant risk factors associated with mortality (p = 0.03 and 0.01, respectively). Conclusion. MDRGN infections were observed in patients with predisposing factors. Acinetobacter baumannii was the main etiologic agent. Mortality was high and related to prematurity and a low birth weight.

Biosynthesized silver nanoparticles: are they effective antimicrobials?

BACKGROUND Silver nanoparticles (AgNPs) are increasingly being used in medical applications. Therefore, cost effective and green methods for generating AgNPs are required. OBJECTIVES This study aimed towards the biosynthesis, characterisation, and determination of antimicrobial activity of AgNPs produced using Pseudomonas aeruginosa ATCC 27853. METHODS Culture conditions (AgNO3 concentration, pH, and incubation temperature and time) were optimized to achieve maximum AgNP production. The characterisation of AgNPs and their stability were evaluated by UV-visible spectrophotometry and scanning electron microscopy. FINDINGS The characteristic UV-visible absorbance peak was observed in the 420-430 nm range. Most of the particles were spherical in shape within a size range of 33-300 nm. The biosynthesized AgNPs exhibited higher stability than that exhibited by chemically synthesized AgNPs in the presence of electrolytes. The biosynthesized AgNPs exhibited antimicrobial activity against Escherichia coli, P. aeruginosa, Salmonella typhimurium, Staphylococcus aureus, methicillin-resistant S. aureus, Acinetobacter baumannii, and Candida albicans. MAIN CONCLUSION As compared to the tested Gram-negative bacteria, Gram-positive bacteria required higher contact time to achieve 100% reduction of colony forming units when treated with biosynthesized AgNPs produced using P. aeruginosa.

Phenol degradation and genotypic analysis of dioxygenase genes in bacteria isolated from sediments

Abstract The aerobic degradation of aromatic compounds by bacteria is performed by dioxygenases. To show some characteristic patterns of the dioxygenase genotype and its degradation specificities, twenty-nine gram-negative bacterial cultures were obtained from sediment contaminated with phenolic compounds in Wuhan, China. The isolates were phylogenetically diverse and belonged to 10 genera. All 29 gram-negative bacteria were able to utilize phenol, m-dihydroxybenzene and 2-hydroxybenzoic acid as the sole carbon sources, and members of the three primary genera Pseudomonas, Acinetobacter and Alcaligenes were able to grow in the presence of multiple monoaromatic compounds. PCR and DNA sequence analysis were used to detect dioxygenase genes coding for catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and protocatechuate 3,4-dioxygenase. The results showed that there are 4 genotypes; most strains are either PNP (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is positive) or PNN (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is negative). The strains with two dioxygenase genes can usually grow on many more aromatic compounds than strains with one dioxygenase gene. Degradation experiments using a mixed culture representing four bacterial genotypes resulted in the rapid degradation of phenol. Determinations of substrate utilization and phenol degradation revealed their affiliations through dioxygenase genotype data.

Membrane permeabilization of colistin toward pan-drug resistant Gram-negative isolates

Abstract Pan-drug resistant Gram-negative bacteria, being resistant to most available antibiotics, represent a huge threat to the medical community. Colistin is considered the last therapeutic option for patients in hospital settings. Thus, we were concerned in this study to demonstrate the membrane permeabilizing activity of colistin focusing on investigating its efficiency toward those pan-drug resistant isolates which represent a critical situation. We determined the killing dynamics of colistin against pan-drug resistant isolates. The permeability alteration was confirmed by different techniques as: leakage, electron microscopy and construction of an artificial membrane model; liposomes. Moreover, selectivity of colistin against microbial cells was also elucidated. Colistin was proved to be rapid bactericidal against pan-drug resistant isolates. It interacts with the outer bacterial membrane leading to deformation of its outline, pore formation, leakage of internal contents, cell lysis and finally death. Furthermore, variations in membrane composition of eukaryotic and microbial cells provide a key for colistin selectivity toward bacterial cells. Colistin selectively alters membrane permeability of pan-drug resistant isolates which leads to cell lysis. Colistin was proved to be an efficient last line treatment for pan-drug resistant infections which are hard to treat.

Assessing the pharmacodynamic profile of intravenous antibiotics against prevalent Gram-negative organisms collected in Colombia

OBJECTIVES: This study was designed to simulate standard and optimized dosing regimens for intravenous antibiotics against contemporary populations of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa using MIC distribution data to determine which of the tested carbapenem regimens provided the greatest opportunity for obtaining maximal pharmacodynamic (PD) activity. METHODS: The isolates studied were obtained from the COMPACT-COLOMBIA surveillance program conducted between February and November 2009. Antimicrobial susceptibility testing was conducted by broth microdilution method according to the CLSI guidelines. Doripenem, imipenem-cilastatin, and meropenem, were the modeled antibiotics. A 5,000 patient Monte Carlo simulation was performed for each regimen and PD targets were defined as free drug concentrations above the MIC for at least 40 percent of the dosing interval. RESULTS: All carbapenem regimens obtained optimal exposures against E. coli, unlike the other Enterobacteriaceae tested. Against P. aeruginosa, only a prolonged infusion of doripenem exceeded the 90 percent cumulative fraction of response (CFR) threshold. Worrisomely, no regimens for any of the drugs tested obtained optimal CFR against A. baumannii. For P. aeruginosa intensive care unit (ICU) isolates, CFR was approximately 20 percent lower for isolates collected in the respiratory tract compared with bloodstream or intra-abdominal for imipenem and meropenem. Noteworthy, all doripenem and meropenem regimens achieved greater than 90 percent CFR against bloodstream and respiratory isolates of K. pneumoniae. CONCLUSIONS: Our data suggests that higher dosing and prolonged infusion of doripenem or meropenem may be suitable for empirically treating ICU P. aeruginosa, while none of the carbapenems achieved optimal cumulative fraction of response against A. baumannii. Standard dosing regimens of all the carbapenems tested achieved optimal CFR against E. coli isolates, but higher carbapenem dosages might be required for empiric treatment of K. pneumoniae, particularly from an intra-abdominal source. Non-standard dosage regimens studied in this modeling should be proven effective in prospective clinical trials.

Criterios de ensayo, interpretación e informe de las pruebas de sensibilidad a los antibióticos en los bacilos gram negativos no fermentadores de importancia clínica: recomendaciones de la Subcomisión de Antimicrobianos de la Sociedad Argentina de Bacteriología, Micología y Parasitología Clínicas, Asociación Argentina de Microbiología / Antimicrobial susceptibility testing in clinically relevant non-fermenting gram-negative bacilli: recommendations from the Antimicrobial Agents Subcommittee of the Sociedad Argentina de Bacteriología, Micología y Parasitología Clínicas, Asociación Argentina de Microbiología

En este documento se dan a conocer una serie de recomendaciones para el ensayo, la lectura, la interpretación y el informe de las pruebas de sensibilidad a los antimicrobianos para los bacilos gram negativos no fermentadores (BGNNF) que se aíslan en humanos. Se adoptaron como base las recomendaciones internacionales, las de la Subcomisión de Antimicrobianos de la Sociedad Argentina de Bacteriología, Micología y Parasitología Clínicas y las de un grupo de expertos invitados. Se incluye, además, la nomenclatura actualizada de los BGNNF y la descripción de algunas de sus características individuales, de sus resistencias naturales o habituales a los antimicrobianos de uso clínico y de los mecanismos responsables de tales resistencias. También se indican los agentes antimicrobianos que se deberían ensayar frente a las distintas especies, con la especificación de cuáles deberían ser informados, y su ubicación estratégica en las placas de cultivo para poder detectar los mecanismos de resistencia más frecuentes y relevantes. Por último, se detallan los métodos de detección y de confirmación fenotípica de la presencia de b-lactamasas emergentes en Argentina, como las carbapenemasas clases A y B.

This document contains the recommendations for antimicrobial susceptibility testing of the clinically relevant non-fermenting gram-negative bacilli (NFGNB), adopted after conforming those from international committees to the experience of the Antimicrobial Agents Subcommittee members and invited experts. This document includes an update on NFGNB classification and description, as well as some specific descriptions regarding natural or frequent antimicrobial resistance and a brief account of associated resistance mechanisms. These recommendations not only suggest the antimicrobial drugs to be evaluated in each case, but also provide an optimization of the disk diffusion layout and a selection of results to be reported. Finally, this document also includes a summary of the different methodological approaches that may be used for detection and confirmation of emerging b-lactamases, such as class A and B carbapenemases.

Growth and biosurfactant synthesis by Nigerian hydrocarbon-degrading estuarine bacteria

The ability of microorganisms to degrade petroleum hydrocarbons is important for finding an environmentally-friendly method to restoring contaminated environmental matrices. Screening of hydrocarbon-utilizing and biosurfactant-producing abilities of organisms from an estuarine ecosystem in Nigeria, Africa, resulted in the isolation of five microbial strains identified as Corynebacterium sp. DDv1, Flavobacterium sp. DDv2, Micrococcus roseus DDv3, Pseudomonas aeruginosa DDv4 and Saccharomyces cerevisae DDv5. These isolates grew readily on several hydrocarbons including hexadecane, dodecane, crude oil and petroleum fractions. Axenic cultures of the organisms utilized diesel oil (1.0 % v/v) with generation times that ranged significantly (t-test, P < 0.05) between 3.25 and 3.88 day, with concomitant production of biosurfactants. Kinetics of growth indicates that biosurfactant synthesis occurred predominantly during exponential growth phase, suggesting that the bioactive molecules are primary metabolites. Strains DDv1 and DDv4 were evidently the most metabolically active in terms of substrate utilization and biosurfactant synthesis compared to other strains with respective emulsification index of 63 and 78 %. Preliminary biochemical characterization indicates that the biosurfactants are heteropolymers consisting of lipid, protein and carbohydrate moieties. The hydrocarbon catabolic properties coupled with biosurfactant-producing capabilities is an asset that could be exploited for cleanup of oil-contaminated matrices and also in food and cosmetic industries. Rev. Biol. Trop. 56 (4): 16031611. Epub 2008 December 30.

La capacidad de los microorganismos para degradar hidrocarburos del petróleo es de gran importancia para hallar un método aceptable y ambientalmente amigable para la restauración de terrenos ambientalmente contaminados. Al investigar las capacidades de los organismos de un ecosistema de estuario que utilizan hidrocarburos y producen biosurfactantes, se produjo como resultado el aislamiento de cinco cepas microbianas identificadas como Corynebacterium sp. DDv1, Flavobacterium sp. DDv2, Micrococcus roseus DDv3, Pseudomonas aeruginosa y DDv4 Saccharomyces cerevisiae DDv5. Estas cepas crecieron fácilmente en varios hidrocarburos incluyendo hexadecanos, dodecanos, petróleo crudo y fracciones de petróleo. Los cultivos axénicos de organismos utilizaron diesel (1.0% v/v) con períodos por generación con ámbitos significativos (t-test, P <0.05) de entre 3.25 y 3.88 días, con la consiguiente producción de bio-surfactantes. La cinética del crecimiento indica que la síntesis de bio-surfactante se produjo principalmente durante la fase de crecimiento exponencial, lo que sugiere que las moléculas bioactivas son metabolitos primarios. Las cepas DDv1 y DDv4 fueron evidentemente las más metabólicamente activas en términos de utilización del sustrato y la síntesis de bio-surfactantes en comparación con otras cepas con índices respectivos de emulsificación de 63 y 78%. La caracterización bioquímica preliminar indica que los bio-surfactantes son heteropolímeros constituidos de fracciones de lípidos, proteínas y carbohidratos. Las propiedades catabólicas de los hidrocarburos, junto con las capacidades de producción de bio-surfactantes, es una ventaja que puede ser aprovechada para la limpieza de terrenos contaminados con petróleo y también en la industria alimentaria y cosmética.

Diversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags

The diversity and load of heterotrophic bacteria and fungi associated with the mangrove soil from Suva, Fiji Islands, was determined by using the plate count method. The ability of the bacterial isolates to produce various hydrolytic enzymes such as amylase, gelatinase and lipase were determined using the plate assay. The heterotrophic bacterial load was considerably higher than the fungal load. There was a predominance of the gram positive genus, Bacillus. Other genera encountered included Staphylococcus, Micrococcus, Listeria and Vibrio. Their effectiveness on the degradation of commercial polythene carry bags made of high density polyethylene (HDPE) and low density polyethylene (LDPE) was studied over a period of eight weeks in the laboratory. Biodegradation was measured in terms of mean weight loss, which was nearly 5 % after a period of eight weeks. There was a significant increase in the bacterial load of the soil attached to class 2 (HDPE) polythene. After eight weeks of submergence in mangrove soil, soil attached to class 1 and class 3 polythene mostly had Bacillus (Staphylococcus predominated in class 2 polythene). While most of the isolates were capable of producing hydrolytic enzymes such as amylase and gelatinase, lipolytic activity was low. Class 2 HDPE suffered the greatest biodegradation. Rev. Biol. Trop. 55 (3-4): 777-786. Epub 2007 December, 28.

Se determinó la diversidad y la carga de bacterias heterotróficas, así como los hongos asociados al suelo del manglar de Suva, Islas Fiji, utilizando el método de conteo de placas, usado también para medir la capacidad de bacterias aisladas para producir enzimas hidrolíticas como amilasa, gelatinasa y lipasa. La carga bacteriana heterotrófica resultó ser considerablemente más alta que la carga funguicida. Hubo predominancia de bacterias "Gram-positivas" del género de Bacillus. Otros géneros encontrados fueron Staphylococcus, Micrococcus, Listeria y Vibrio. La eficacia de esta microflora en la degradación del polietileno comercial de bolsas hechas de polietileno de alta densidad (HDPE) y de baja densidad (LDPE) fue estudiada en el laboratorio por un periodo de ocho semanas. La biodegradación fue medida en términos de pérdida de peso, la cual indicó una disminución del 5 %. Después de ocho semanas en el suelo de un manglar, el polietileno clase 1 y clase 3 contenía fundamentalmente Bacillus, pero en el polietileno clase 2 predominó el género Staphylococcus. Mientras que la mayoría de bacterias aisladas fueron capaces de producir enzimas hidrolíticas como la amilasa y la gelatinasa, la actividad lipolítica fue muy baja. La clase 2 (HDPE) experimentó la mayor biodegradación.

Diversidad bacteriana en un biorreactor de lecho fluidificado durante el tratamiento de agua contaminada con nafta / Bacterial diversity in a fluidized bed bioreactor (FBR) treating gasoline-contaminated groundwater

El objetivo principal de esta investigación fue determinar la diversidad bacteriana del proceso de biorremediación de agua contaminada con nafta en un biorreactor de lecho fluidificado en el Recinto Universitario de Mayagüez, de la Universidad de Puerto Rico. El aislamiento y la caracterización de las colonias bacterianas del sistema de biorremediación fueron realizados en medio R2A. Las pruebas morfológicas incluyeron la determinación de la morfología celular y de las colonias, y la reacción frente a la coloración de Gram. Las propiedades fisiológicas se determinaron usando el sistema Biolog® y sobre la base de la habilidad para desarrollar en medio mínimo con nafta como única fuente de carbono. La caracterización molecular se llevó a cabo por BOX-PCR y por análisis de secuencia del ADNr 16S mediante la técnica de ARDRA (amplified ribosomal DNA restriction analysis). De los 162 morfotipos de colonias aislados, 75% fueron bacilos gram-negativos, 19% bacilos gram-positivos, 5% cocos gram-negativos y 1% cocos gram-positivos. Según el análisis ARDRA, estos morfotipos se distribuyeron en 90 grupos genéticos, de los cuales 53% incluyeron cepas con crecimiento en nafta. Las 86 cepas que crecieron en nafta presentaron 52 patrones de amplificación, los que a través de BOX-PCR se agruparon en 50 grupos metabólicamente no relacionados. El alto nivel de diversidad microbiana observado en el reactor permitió la remoción del contaminante y, al parecer, fue importante para la operación estable y eficiente del sistema.

The main objective of this research project was to determine the bacterial diversity during the process of bioremediation of water contaminated with gasoline in a fluidized bed reactor at Mayagüez, PR. Isolation and characterization of bacterial populations from the bioremediation system was performed on R2A medium. Morphological tests included cellular and colonial shape and reaction to Gram coloration. Physiological properties were determined by using carbon utilization profiles (Biolog®) and by the ability of axenic cultures to use gasoline as the sole carbon source. Molecular characterization was performed by BOX-PCR and 16S rDNA sequence analysis (ARDRA). From a total of 162 distinctive isolates, 75% were gram-negative bacilli, 19% gram-positive bacilli, 5% gram-negative cocci and 1% gram-positive cocci. The 162 axenic cultures corresponded to 90 different genetic groups; 53% of which included strains with growth in gasoline as sole carbon source. The 86 strains capable of growing in gasoline corresponded to 52 different amplification patterns in BOX-PCR; which were not metabolically related (Biolog® system). The high degree of microbial diversity in the FBR allowed efficient and stable hydrocarbon removal throughout the operation of the system.

Microbial flora associated with submerged mangrove leaf litter in India

We studied the microbial flora in decomposing mangrove leaves in relation to changes in nitrogen and tannin levels, and in penaeid prawn assemblages. Senescent leaves of two mangrove species (Rhizophora apiculata and Avicennia marina) kept in nylon bags, were separately immersed for 80 days in five tanks full of mangrove water. A known amount of decomposing leaves was collected every ten days from each tank for microorganism counts, total nitrogen and tannin measurement, and juvenile penaeid prawn counts. Five genera of total heterotrophic bacteria (THB), three species of azotobacters and 19 species of fungi were identified. The azotobacters showed a significant peak around 40-50 days after the beginning of of decomposition, similar to the trend for total nitrogen and for prawn assemblages. Rev. Biol. Trop. 55 (2): 393-400. Epub 2007 June, 29.

Se estudió la flora microbiana en hojas en descomposición de mangles, considerando nitrógeno, taninos y camarones peneidos jóvenes. Colocamos hojas viejas de dos especies de mangle (Rhizophora apiculata y Avicennia marina) en bolsas de nylon y las sumergimos en agua de manglar durante 80 días usando cinco tanques separados. Cada diez días extrajimos una cantidad conocida de hojas en descomposición de cada tanque. Hallamos cinco géneros de bacterias heterotróficas totales (THB), tres especies de azotobacterias y 19 especies de hongos. Las azotobacterias presentaron un pico significativo de abundancia alrededor de los 40-50 días de descomposición, un patrón similar a los del nitrógeno total y los camarones.

Biogeochemical processes in the continental slope of Bay of Bengal: I. Bacterial solubilization of inorganic phosphate

Microorganisms play a vital role in the biogeochemical cycles of various marine environments, but studies on occurrence and distribution of such bacteria in the marine environment from India are meager. We studied the phosphate solubilizing property of bacteria from the deep sea sediment of Bay of Bengal, India, to understand their role in phosphorous cycle (and thereby the benthic productivity of the deep sea environment). Sediment samples were obtained from 33 stations between 10 degrees 36'N-20 degrees 01' N and 79 degrees 59' E-87 degrees 30' E along 11 transects at 3 different depths i.e. ca. 200 m, 500 m, 1000 m in each transect. Total heterotrophic bacterial (THB) counts ranged from 0.42 to 37.38 x 10(4) CFU g(-1) dry sediment weight. Of the isolates tested, 7.57% showed the phosphate solubilizing property. The phosphate solubilizing bacterial genera were Pseudomonas, Bacillus, Vibrio, Alcaligenes, Micrococcus, Corynebacterium and Flavobacterium. These strains are good solubilizers of phosphates which ultimately may play a major role in the biogeochemical cycle and the benthic productivity of the Exclusive Economic Zone (EEZ) of Bay of Bengal, because this enzyme is important for the slow, but steady regeneration of phosphate and organic carbon in the deep sea.

Microbial degradation of palm (Elaeis guineensis )biodiesel

The kinetics of biodegradation of palm-derived fatty methyl and ethyl esters (Elaeis guineensis biodiesel) by a wild-type aerobic bacterial population was measured at 20 °C, as the rate of oxygen uptake by a manometric technique. The methyl and ethyl biodiesels were obtained by potassium-hydroxide catalysed transesterification of palm oil, respectively. The bacterial flora included the genera Bacillus, Proteus, Pseudomonas, Citrobacter and Enterobacter. The rate of oxygen uptake for palm biodiesel is similar to the quantity observed in the biodegradation of 1.0 mM solutions of simple substrates such as carbohydrates or amino acids.Palm methyl or ethyl biodiesel is subjected to facile aerobic biodegradation by wild-type bacteria commonly present in natural open environments. This result should lessen any environmental concern for its use as alternative fuel, solvent or lubricant.

La cinética de la biodegradación de los ésteres metílicos y etílicos derivados de palma (biodiesel) por una población silvestre de bacterias aeróbicas fue medida a 20 °C, como medición manométrica del consumo de oxígeno. Los ésteres metílicos y etílicos se obtuvieron por transesterificación del aceite de palma con metanol y etanol,respectivamente. La flora bacteriana incluyó a los géneros Bacillus, Proteus, Pseudomonas, Citrobacter y Enterobacter. Las velocidades de consumo de oxígeno para las muestras de biodiesel fueron similares a lo observado en la biodegradación de disoluciones 1.0 mM de sustratos sencillos solubles en agua, tales como carbohidratos, aminoácidos y albúmina de huevo.

Avances en el desarrollo de terapias neutralizantes en la sepsis / Advances in the development of neutralizing therapies for sepsis

El lipopolisacarido bacteriano (LPS), tambien denominado endotoxina, es el constituyente mayoritario de la membrana externa de bacterias Gram negativas. Esta molecula es liberada de la bacteria a la circulacion exhibiendo una amplia variedad de efectos toxicos y pro-inflamatorios, los cuales estan asociados al lipido A y a su vez estan relacionados a la patogenesis de la sepsis. Muchos de los fenomenos fisiologicos producidos por el LPS resultan de la capacidad de esta molecula de activar las celulas del sistema inmune del huesped, entre ellas monocitos, macrofagos y leucocitos polimorfonucleares. Este proceso produce una inflamacion local, proceso beneficioso para el huesped. Sin embargo, si la cantidad de LPS liberado excede cierta concentracion critica umbral, la exacerbada liberacion de citoquinas inflamatorias como Factor de Necrosis Tumoral (TNF-alfa) e interleuquinas (IL) resulta en sepsis grave, lo que hace necesario encontrar nuevas opciones terapeuticas capaces de neutralizar la endotoxina circulante. En este articulo se presenta una revision actualizada de los resultados experimentales obtenidos in vivo e in vitro empleando proteinas y peptidos sinteticos con la finalidad de neutralizar el LPS, y las perspectivas que en este area ofrece el uso de lipoproteinas, en particular la apolipoproteina A-I y formas mutantes o peptidos derivados de esta proteina.

Lipopolisaccharide (LPS), also called endotoxin, is the major component of the external membrane in Gram negative bacteria. This molecule is released to circulation by the bacteria, producing a large variety of toxic and pro-inflammatory effects which are associated with lipid A as well as with sepsis pathogenesis. Many physiological henomena produced by LPS arise from this molecule's capacity to activate cells in the host immune system such as monocytes, macrophages and polymorphonuclear leukocytes. This process leads to a local inflammation, and it is beneficial for the host. However, if the amount of LPS released exceeds the critical concentration thresholdan augmented release of inflammatory cytokines as TNF-alfa, and interleukines (IL) produce a severe sepsis. This fact led us to find therapeutical alternatives able to neutralize circulating endotoxin. This work is focused on the experimental results obtained in vivo and in vitro using synthetic proteins and peptides in order to neutralizeLPS, and on future perpectives in this research area that offer the use of lipoprotein and in particular apolipoprotein A-I and mutants or peptides derived from this protein.

Prevalence of extended spectrum beta lactamase producing gram negative bacteria in a tertiary care hospital.

BACKGROUND & OBJECTIVES: Extended spectrum beta lactamase (ESBL) producing Gram negative bacteria are increasingly being associated with hospital infections thereby rendering all beta lactams, except carbapenems ineffective in the treatment of infections related to these organisms. A knowledge about their prevalence is essential to guide the appropriate antibiotic treatment of severe infections in hospitalized patients. The present work was carried out to study the prevalence of ESBL producing Gram negative bacteria in a tertiary care hospital. METHODS: A total of 678 Gram negative bacteria were included in the study. They were isolated from various clinical samples obtained from indoor patients admitted to the All India Institute of Medical Sciences, New Delhi during March to June 2001. These isolates were screened for ESBL production by the inhibitor based test recommended by the National Committee for Clinical Laboratory Standards (NCCLS) using Klebsiella pneumoniae ATCC 700603 as positive control and Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 as negative controls. RESULTS: Out of the 678 strains tested, 458 (68%) were found to be ESBL producers. Among the bacterial species, ESBL production was most common in Klebsiella spp. (80%). The proportion of ESBL positive isolates was highest from intensive care units (79%), followed by Medical Oncology (75%), Medical (54%) and Surgical wards (50%). INTERPRETATION & CONCLUSION: A high prevalence of ESBL positive isolates was found in our hospital. This has important implications as carbapenems remain the only choice of treatment for infections caused by these organisms. The control measures include judicious use of antibiotics and implementation of appropriate infection control measures to control the spread of these strains in the hospital.

Evaluación in vitro de sustancias antibacterianas producidas por bacterias aisladas de diferentes organismos marinos / In vitro evaluation of antibacterial substances produced by bacteria isolated from different marine organisms

Bacteria from several groups of marine organisms were isolated and, using direct antibiograms, identified those that produce antibacterial substances, using a human pathogenic strain of Staphylococcus aureus ATCC6538 as revealing microorganism. Bacteria which produce substances that inhibited S. aureus growth were identified through morphological, physiological and biochemical tests. Out of 290 bacteria, 54 (18.6) inhibited the growth of S. aureus, but only 27 survived for identification. Bivalves, sponges and corals were the most represented from which 41.2, 33.3 and 29.7, respectively, produced antibacterial substances of the isolated bacteria in each group. The marine species with highest proportions of these bacteria were the hard coral Madracis decactis (62.5), the sponges Cliona sp. (57.1) and the octocoral Plexaura flexuosa (50.0). Out of the 27 strains that produced antibacterial substances, 51.8 were Aeromonas spp. and 14.8 Vibrio spp. Marine bacteria that produce antibacterial substances are abundant, most belong in the Vibrionacea group and were isolated mainly from corals and bivalve mollusks.

Neutrofilos humanos (PMN) inducen la liberacion de lipopolisacaridos (LPS) de bacterias gram negativas / Human polymorphonuclear neutrophils (PMN) induce lipopolysaccharide (LPS) liberation from Gram-negative bacteria

Los lipopolisacáridos bacterianos (LPS) son los principales componentes de las membranas de las bacterias gram negativas. Los LPS constituyen poderosos estímulos para las células del sistema inmune y están asociados, frecuentemente, al desencadenamiento del síndrome séptico o al shock séptico. Los polimorfonucleares neutrófilos humanos (PMN) son una de las células más importantes involucradas en la depuración de los LPS. El conocimiento de los mecanismos de detoxificación de los LPS es crucial para el control del síndrome séptico o del shock séptico causados por bacterias Gram negativas. En este trabajo estudiamos la capacidad de los PMN en la detoxificación de los LPS en dos situaciones diferentes: a) cuando el LPS es ofertado a los PMN como una molécula aislada; b) cuando el LPS ofrecido a los PMN es parte constitutiva de las bacterias Gram negativas (E. coli 0111:B4). Nuestros resultados demuestran que los PMN son capaces de inhibir la actividad biológica de los LPS de generar el factor de necrosis tumoral alfa (TNF-alpha). Sin embargo, cuando los LPS se ofrecen como componentes de las bacterias enteras, los PMN inducen la liberación de los LPS bacterianos sin modificar su actividad biológica. Esto determina que la capacidad depuradora de los PMN esté condicionada por la forma en la cual los LPS son presentados a estas células.

La microtécnica en el diagnóstico bacteriológico / Microtechnic in bacteriological diagnosis

Por primera vez en nuestro medio, se prueba un método microtécnico para la detección de ureasa producida por microorganismos entéricos gramnegativos, procedentes de productos patológicos de origen humano, con el cual es posible realizar esta investigación bioquímica a 96 cepas bacterianas diferentes a la vez, en un tiempo máximo de 60 minutos y un área de incubación mínima. Se investigaron 1.940 cepas. Al comparar los resultados de la microtécnica con los obtenidos por el método convencional, se observó una coincidencia en el 100%