Rapid identification of Burkholderia cepacia complex species including strains of the novel Taxon K, recovered from cystic fibrosis patients by intact cell MALDI-ToF mass spectrometry.

Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepacia complex (Bcc) species, Burkholderia contaminans belonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminans spectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients.

Triclosan-loaded poloxamine micelles for enhanced topical antibacterial activity against biofilm.

Our research group is interested in the study of different technological approaches to treat hospital biofilm as a means to constrain nosocomial-acquired infections. The present work investigated the effect of the incorporation of the antibacterial agent triclosan (TS) into polymeric micelles of poloxamine T1107 (MW=15 kDa, 70 wt% PEO). The aggregation phenomenon was primarily investigated by means of Critical Micellar Concentration in a broad range of pH. Then, the effect of the polymer concentration on the micellar size was evaluated by Dynamic Light Scattering. Solubility levels increased up to 4 orders of magnitude. The drug inclusion affected the micellization, resulting in size increase and micellar fusion. This phenomenon was only apparent in TS-saturated systems. TS-loaded aggregates proved to be active in vitro against a broad spectrum of bacteria but more importantly, also against two representative clinical pathogens: methicilin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VREF). While the former was sensitive to even very low TS levels attainable in poloxamine-free aqueous media, the later was inhibited only when exposed to higher drug levels affordable exclusively using an inclusion system. These findings indicated the release of the drug from the reservoir. Finally, the activity of a TS-containing 5% poloxamine combination of pH 7.4 was assessed on biofilms of Staphylococcus epidermidis. Results showed a significant decrease (p<0.001) in the number of Colony-Formation Units when the biofilm was exposed to the TS/poloxamine as compared to the limited activity of the polymer-free TS control.

Sol-gel immobilisation of Saccharomyces cerevisiae enhances viability in organic media.

The polyhydroxylated silane network of a sol-gel protected immobilised Saccharomyces cerevisiae against the effects of five organic solvents. The viability of immobilised yeast directly correlated with the logarithm of the partition coefficient of the solvent in an octanol/water two phase system increasing the decimal reduction time (D) and reaching the maximum with octanol, the most hydrophobic solvent assayed. The D value increased from 0.16 min for free yeast to 1.9 and to 22 min for immobilised yeast exposed to ethanol and 1-octanol respectively.

Antifungal properties of clove oil (Eugenia caryophylata) in sugar solution

No estudo avaliou-se o efeito da temperatura, concentração e tempo de contato na atividade antifúngica do óleo essencial do cravo disperso em solução concentrada de açúcar. Os ensaios foram feitos a 21ºC e a 37ºC, utilizando suspensão de óleo essencial de 0.2 a 0.8 per center v/v. Os microorganismos utilizados foram Candida albicans, Penicillium citrinum, Aspergillus niger e Trichophyton mentagrophytes. A melhor atividade fungicida ocorreu a 37ºC, onde um minuto de contato foi suficiente para matar uma população de 106 u.f.c./ml de Candida albicans. Não obstante, P. citrinum, A. niger, T. mentagrophytes foram mais resistentes, embora uma importante ação letal da essência tenha sido observada. Em 60 minutos, o óleo essencial de cravo (0,4 per center v.v.) em solução concentrada de açúcar reduziu em 99,6 per center um inóculo de 106 u.f.c./ml de T. mentagrophytes. O efeito letal do óleo essencial de cravo sobre C. albicans, após 2 minutos de contato, foi semelhante ao de desinfectantes comumente usados em hospitais, como povidona-iodo e cloroxilenol.

Influencia de la materia orgánica sobre la acción de desinfectantes / Organic material influence on the desinfectants action

En la práctica de la desinfección, el antagonismo existente entre material orgánico y los desinfectantes, dio lugar a un estudio tendiente a determinar el grado de tolerancia que pueden presentar estos compuestos frente a diferentes concentraciones de sustancia orgánica. Dicho estudio se llevó a cabo con veinte soluciones de desinfectantes normalmente utilizados en el área hospitalaria, determinándose su actividad sobre los microorganismos habitualmente contaminantes o infectantes. Como microorganismos de ensayo se utilizaron: Escherichia coli, Staphylococcus aureus y Pseudomonas aeruginosa. La sustancia orgánica antagonista, constituida por una mezcla de suero equino con levadura de cerveza, se usó en concentraciones finales del 5, 10, 15 y 20 por ciento. Los resultados permiten considerar un comportamiento variable en el grado de antagonismo en dos aspectos: 1) con los diferentes compuestos y, 2) con las diferentes especies microbianas utilizadas. Con respecto al primero, se demostró que algunos compuestos no fueron inhibidos por una elevada concentración de sustancias orgánicas (20 por ciento), tal como el hipoclorito de sodio con 6000 mg por litro de cloro activo. Sin embargo, no todos los desinfectantes considerados de alto nivel alcanzaron esa barrera, tal el caso del glutaraldehido y del formaldehido que en las concentraciones utilizadas del 1 por ciento al 8 por ciento respectivamente, fueron inhibidos por esa concentración de sustancia orgánica. En el segundo aspecto, se observó que en presencia de material orgánico, tanto Staphylococcus aureus como Pseudomonas aeruginosa, mostraron mayor resistencia a los desinfectantes que Escherichia coli. Los resultados obtenidos permiten concluir que, entre los criterios de selección de un desinfectante, debe asignarse un rol primordial al contenido de materia orgánica en los elementos a desinfectar. De esta forma, se incrementará la probabilidad de procedimientos exitosos

Lemon juice as a natural biocide for disinfecting drinking water

The natural biocidal activity of lemon juice was studied in order to explore its possible use as a disinfectant and inhibitor of Vibrio cholerae in drinking water for areas lacking water treatment plants. Fron January through July 1993, water samples of varying alkalinity and hardness were prepared artificially, and underground and surface water samples were obtained from a number of different rural and urban areas in Argentina's Buenos Aires Province. After measuring the latter samples' hardness and alkalinity, a range of concentrations of lemon juice and other acidifiers were added to each sample, and the resulting pH as well as the samples' ability to destroy V. cholerae were determined. The results show that lemon juice can actively prevent survival of V. cholerae but that such activity is reduced in markedly alkaline water. For example, treatment of underground drinking water, which is characterized as having the greatest degree of alkalinity in our area, will typically destroy V. cholerae if the alkalinity of the water is the equivalent of that produced by 200 mg CaCO3 per liter, if enough lemon juice is added to bring the lemon juice concentration to 2 percent, and if the lemon juice is allowed to act for 30 minutes. All this points up the need to determine the alkalinity of water from any local source to be treated in the process of assessing the minimum concentration of lemon juice required

El limón como biocida natural para desinfectar las aguas de consumo / Lemon as a natural biocidal agent for disinfecting drinking water

Se estudió la actividad biocida natural del zumo de limón para examinar su posible uso como desinfectante e inhibidor de Vibrio cholerae en las aguas de consumo de lugares sin plantas de tratamiento. De enero a julio de 1993 se prepararon artificialmente aguas de distinta dureza y alcalinidad y se obtuvieron muestras de aguas subterráneas y superficiales de zonas rurales y urbanas de la provincia de Buenos Aires, Argentina. Después de medir su dureza y alcalinidad, se le añadió a cada una zumo de limón en diferentes concentraciones y se determinaron el grado de acidez obtenido y la actividad del limón contra V. cholerae. Los resultados demostraron que el zumo de limón es activo contra V. cholerae, pero que esta actividad se reduce en aguas muy alcalinas. Por consiguiente, el tratamiento de las aguas de consumo subterráneas, que son las de mayor alcalinidad, puede destruir V. cholerae solo a una concentración mínima de limón de 2 por ciento y a un tiempo de acción mínimo de 30 minutos. Se recomienda, sin embargo, conocer la alcalinidad del agua local que se busca tratar antes de fijar la concentración mínima de limón

El limón como biocida natural para desinfectar las aguas de consumo / Lemon as a natural biocidal agent for disinfecting drinking water

Se estudió la actividad biocida natural del zumo de limón para examinar su posible uso como desinfectante e inhibidor de Vibrio cholerae en las aguas de consumo de lugares sin plantas de tratamiento. De enero a julio de 1993 se prepararon artificialmente aguas de distinta dureza y alcalinidad y se obtuvieron muestras de aguas subterráneas y superficiales de zonas rurales y urbanas de la provincia de Buenos Aires, Argentina. Después de medir su dureza y alcalinidad, se le añadió a cada una zumo de limón en diferentes concentraciones y se determinaron el grado de acidez obtenido y la actividad del limón contra V. cholerae. Los resultados demostraron que el zumo de limón es activo contra V. cholerae, pero que esta actividad se reduce en aguas muy alcalinas. Por consiguiente, el tratamiento de las aguas de consumo subterráneas, que son las de mayor alcalinidad, puede destruir V. cholerae solo a una concentración mínima de limón de 2 por ciento y a un tiempo de acción mínimo de 30 minutos. Se recomienda, sin embargo, conocer la alcalinidad del agua local que se busca tratar antes de fijar la concentración mínima de limón

Edición en inglés se publica en Bull. PAHO Vol. 28(4):324-30, 1994

Lemon juice as a natural biocide for disinfecting drinking water

The natural biocidal activity of lemon juice was studied in order to explore its possible use as a disinfectant and inhibitor of Vibrio cholerae in drinking water for areas lacking water treatment plants. Fron January through July 1993, water samples of varying alkalinity and hardness were prepared artificially, and underground and surface water samples were obtained from a number of different rural and urban areas in Argentina's Buenos Aires Province. After measuring the latter samples' hardness and alkalinity, a range of concentrations of lemon juice and other acidifiers were added to each sample, and the resulting pH as well as the samples' ability to destroy V. cholerae were determined. The results show that lemon juice can actively prevent survival of V. cholerae but that such activity is reduced in markedly alkaline water. For example, treatment of underground drinking water, which is characterized as having the greatest degree of alkalinity in our area, will typically destroy V. cholerae if the alkalinity of the water is the equivalent of that produced by 200 mg CaCO3 per liter, if enough lemon juice is added to bring the lemon juice concentration to 2 percent, and if the lemon juice is allowed to act for 30 minutes. All this points up the need to determine the alkalinity of water from any local source to be treated in the process of assessing the minimum concentration of lemon juice required

Published in Spanish in the BOSP. 117(4), 1994

Depuracion de aguas contaminadas por medio de microorganismos ambientales

Los objetivos del trabajo fueron: a)Seleccionar microorganismos ambientales degradadores de contaminantes tales como fenoles y derivados b)estudiar la capacidad de degradacion de dichos microorganismos en presencia de sustancias interferentes de su metabolismo c)estudiar el comportamiento de los mismos,en aguas del Riachuelo y efluentes industriales

Isolamento e caracterização de uma nova raça de Pseudomonas capaz de degradar p-clorofenol / Isolation and characterization of a new para-chlorophenol degrading strain of Pseudomonas from samples of natural waters

A partir de amostras de águas naturais foram isoladas cinco cepas bacterianas capazes de degradar o p-clorofenol, as quais foram caracterizadas como bacilos Gram-negativos e oxidase-positivas. Uma das cepas (CS2) foi caracterizada como pertencente ao gênero Pseudomonas, logrando uma mineralização rápida e completa. Para esta cepa autóctone, se estudou a influência de diferentes parâmetros, tais como a concentração de p-clorofenol, toxidade, biodegradação de compostos xenofóbicos associados com a velocidade de crescimento. A velocidade de crescimento e a remoção do substrato em estudo foram determinadas de forma simultânea. A forma não dissociada do p-clorofenol não mostrou efeitos tóxicos quando utilizado em diferentes pH. A remoção do substrato em misturas contendo fontes de carbono alternativas demonstrou a possibilidade de um crescimento concorrente. A pré-exposição das populações microbianas ao p-clorofenol, indicam mudanças nas velocidades de biodegradação observadas.

Viabilidad de Bacillus subtilis auxotrofos en ausencia de sus metabolitos esenciales / Viability of Bacillus subtilis auxotrophs in the absence of their essential metabolites

Se estudió el efecto de la privación de triptofano y uracilo sobre la viabilidad del tansformante Bacillus subtilis BSA trp ura y de las cepas parentales Bacillus subtilis PB 168 trp-C y Bacillus subtilis PB 3308 ura. Los ensayos se llevaron a cabo en las condiciones descriptas para el desarrollo de competencia para transformación, durante 16 h. Los resultados obtenidos demostraron que Bacillus subtilis BSA 170 trp ura es resistente a la muerte por carencia de triptofano durante el tiempo del ensayo y a la muerte por carencia de uracilo durante 3h. Luego ocurre una disminución de la recuperación de UFC, acompañada por una reduccción de las absorbancias de los cultivos. La muerte por carencia de uracilo es prevenida en ausencia de triptofano, sugiriendo que reuiere síntesis de proteínas. Las cepas parentales mostraron un comportamiento semejante. Bacillus subtilis PB 168 trp-C mostró ser resistente a la muerte por carencia de triptofano y Bacillus subtilis PB 3308 uta sufrió una reducción de la capacidad para formar colonias, debida a la privación de uracilo, comparable a la de la cepa transformante

Action of Halogenated Compounds on Aspergillus Conidiospores.

The fungicidal activity of two halogenated compounds against conidiospores of four Aspergillus strains ( A. flavus and A. sydowi isolated from a poultry farm, A. parasiticus NRRL 2999 and A. niger 29-CCM-A 41) was studied. Accordingly, the sodium salt of a synthetic organic compound derived from trichloroisocyanuric acid and an organic complex of iodine (iodophor) were used at 20°C at their recommended dilution (0.1%). More than 99.9% of the exposed spore population of all strains was inactivated within 30 min of contact with either product. During the first minute of contact, the iodophor solution was more effective than the chlorinated one. Among parameters tested on A. niger conidiospores, a 10°C temperature rise slightly increased antimicrobial activity, which was substantially affected by dilution, the active principle being exhausted when using 0.05% concentration. In addition, organic matter (1% human serum) practically neutralized the fungicidal effect of both compounds, whereas acid pH (5.33) notably increased the antimicrobial capacity of the chlorinated derivative.

Bacillus subtilis rec Assay for Quantification of Aflatoxins.

The Bacillus subtilis 1791 rec- assay was used to quantify genotoxic mycotoxins. This assay is based on detection of mycotoxin-produced DNA alterations arising from recombinational deficiency in rec- cells. Aflatoxin B1 showed a linear dose-response relationship when the inhibition halo was taken as a parameter for the evaluation procedure. Assays carried out with or without hepatic microsomal activation exhibited a similar response.