Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.

Análisis de la monitorización de cinco indicadores de calidad del hemocultivo en un hospital universitario en Chile 2009-2011 / Quality indicators for blood culture: three years of monitoring at a university hospital in Chile

Blood culture is considered the "gold standard" for the diagnosis of bacteremia, critical condition with high morbidity and mortality. Because of its importance, it is estimated that the blood culture is a critical test that requires close monitoring on the quality with which the process is performed. The objective of this work is to show the results of the monitoring carried out during the past three years, of 5 quality indicators of blood cultures in the laboratory of the Hospital Clínico de la Pontificia Universidad Católica de Chile, considering pre-analytical, analytical and post-analytical aspects. In the 3 years monitored the mean contamination was 0,7%, 46% of adult bottles had adequate volume, match between Gram stain with final identification was 99.4%, 100% of correct participations were achieved in surveys of external quality control and Gram staining notification before 1 hour was 88.7%. With regard to proposed aims, in 2011 the laboratory complies with all, except the percentage of bottles with appropriate volume of blood inoculated. This indicator is very low and should be corrected as soon as possible since it is known that it is an important condition for optimum performance of blood cultures.

El hemocultivo es el "estándar de oro" para el diagnóstico de bacteriemia, condición grave de alta morbi-mortalidad. Por esto, se estima que el hemocultivo es un examen crítico, que requiere monitorización de su calidad. El objetivo de este trabajo es mostrar los resultados de la monitorización de 3 años de 5 indicadores de calidad del hemocultivo implementados en nuestro hospital. El porcentaje promedio de contaminación de las botellas para hemocultivo fue 0,7%, el porcentaje de botellas de adultos con volumen adecuado fue 46%, la concordancia de la tinción de Gram con la identificación final correspondió a 99,4%, el 100% de las participaciones en encuestas de control de calidad externo fueron correctas y 88,7% de los avisos de valores de alerta de la tinción de Gram fueron realizados antes de 1 hora. En el año 2011 se cumplió con las metas propuestas por el laboratorio para todos los indicadores, excepto con el porcentaje de botellas con volumen apropiado de sangre. Este último, se encuentra muy por debajo de la meta y debe ser mejorado a la brevedad, ya que el volumen de sangre cultivada es el factor más importante para obtener un rendimiento óptimo del hemocultivo.

Estudo comparativo entre um sistema de sonda genética e métodos clássicos na identificação das micobactérias / Gene probes versus classical methods in the identification of mycobacteria

OBJETIVO: O aparecimento da co-infecção tuberculose/HIV e o aumento de casos de doenças provocadas por micobactérias não-tuberculosas (MNT) exigem repostas laboratoriais rápidas tanto no isolamento como na identificação das micobactérias. O objetivo deste trabalho foi avaliar a identificação das micobactérias através de sonda genética em comparação com os métodos bioquímicos clássicos. MÉTODOS: Entre 2002 e 2004, foram analisadas 178 culturas de micobactérias, confirmadas como bacilos álcool-ácido resistentes e obtidas de isolados clínicos de pacientes sintomáticos respiratórios ou com suspeita clínica de tuberculose pulmonar e/ou micobacterioses, atendidos nas Unidades de Saúde da Baixada Santista. RESULTADOS: A sonda genética identificou 137 amostras (77%) como complexo Mycobacterium tuberculosis e 41 (23%) como MNT. A discordância observada de 3% entre os métodos ocorreu apenas no ano de implantação (2002). Ao comparar os métodos, a sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo da sonda genética foram 98%, 93%, 98% e 93%, respectivamente. CONCLUSÕES: Apesar do custo elevado, a identificação de micobactérias pela técnica molecular é mais rápida: máximo de 3 h vs. 28-30 dias para os métodos clássicos. A utilização de sondas genéticas é uma técnica molecular validada, simples e disponível no mercado, com elevada especificidade, sensibilidade e rapidez, o que justifica sua implantação e uso rotineiro em laboratórios de referência, facilitando o diagnóstico e permitindo uma intervenção clínica ágil.

OBJECTIVE: The emergence of tuberculosis/HIV co-infection and the increase in the number of cases of infection with nontuberculous mycobacteria (NTM) require rapid laboratory test results in the isolation and identification of mycobacteria. The objective of this study was to evaluate the identification of mycobacteria by means of gene probes in comparison with that obtained using classical biochemical methods. METHODS: Between 2002 and 2004, 178 mycobacterial cultures, all testing positive for acid-fast bacilli, were analyzed. Samples were obtained from clinical specimens of patients with respiratory symptoms or with clinical suspicion of pulmonary tuberculosis/mycobacteriosis who were treated in the greater metropolitan area of Santos. RESULTS: The gene probe identified 137 samples (77%) as Mycobacterium tuberculosis complex and 41 (23%) as NTM. Discordant results between the methods (3%) were obtained only in the year of implementation (2002). When comparing the methods, the sensitivity, specificity, positive predictive value and negative predictive value of the gene probe method were 98%, 93%, 98% and 93%, respectively. CONCLUSIONS: Despite the cost, the identification of mycobacteria using the molecular technique is faster: maximum 3 h vs. 28-30 days for classical methods. The use of gene probes is a validated molecular technique. It is fast, easy to use and readily available on the market. It has high specificity and sensitivity, which justifies its implementation and routine use in referral laboratories, since it facilitates the diagnosis providing agile clinical interventions.

Diagnóstico bacteriológico de la tuberculosis en Argentina: resultados de una encuesta nacional / Bacteriological diagnosis of tuberculosis in Argentina: results of a national survey

Con el objetivo de evaluar recursos, prácticas y medidas de bioseguridad utilizados en el diagnóstico bacteriológico de tuberculosis y determinar la utilidad y alcance de las técnicas empleadas, se llevó adelante una encuesta transversal a laboratorios del sector público de Argentina utilizando un cuestionario estructurado. El análisis de los resultados mostró que la densidad de centros de microscopía y cultivo se encuentra al nivel o encima de lo recomendado por Organización Mundial de la Salud. El programa de control de calidad de microscopía cubre mayoritariamente los laboratorios con alta carga de trabajo. El promedio de baciloscopías/paciente fue bajo (1,6). El 25 por ciento de los laboratorios de cultivo empleaban protección respiratoria inadecuada. El aporte del cultivo a la confirmación de casos pulmonares en las jurisdicciones estuvo asociado a la proporción de muestras cultivadas. Globalmente, el porcentaje de pacientes pulmonares baciloscopía-negativa que fue confirmado bacteriológicamente fue de 18.9 por ciento; mientras que la may-oría de los casos extrapulmonares se confirmó sólo por cultivo (71,3 por ciento). Es prioritario aún el incremento del número de esputos estudiados por baciloscopía y cultivo, así como aumentar la cobertura de programa de control de calidad de microscopía; es fundamental proveer al personal de laboratorio de adecuada protección respiratoria y evaluación médica regular.

In order to assess laboratory resources, practices, and biosafety measures during mycobacterial testing and determine the usefulness and scope of mycobacterial techniques, a cross-sectional survey of public laboratories was conducted in Argentina using a structured questionnaire. Sputum smear analysis showed that both smear and culture testing centers are being provided at or above the WHO recommended density for such facilities. The microscopy quality assessment program covered most high-demand laboratories. Mean number of sputum smears per patient was low (1.6). The use of inadequate personal respiratory protection was identified in 25 percent of culture laboratories. Jurisdictions that cultured a higher proportion of their smear-tested sputa identified a higher proportion of smear-negative cases. The percentage of smear negatives among all bacteriologically confirmed pulmonary cases was 18.9 percent, while most extrapulmonary cases were confirmed by culture (71.3 percent). In conclusion, increasing the number of sputa studied by microscopy and culture (while expanding the coverage of the quality assessment program) is a priority; adequate respiratory protection and regular medical evaluation of laboratory staff is still needed in some laboratories.

Inactivation of Escherichia coli O157: H7 in hamburgers by gamma irradiation

Escherichia coli O157:H7 causes bloody diarrhoea, haemorrhagic colitis and life-threatening complications like haemolytic uremic syndrome and thrombotic thrombocitopenic purpura. Among foods associated with outbreaks caused by this pathogen, hamburger is the most common one. The aim of this research was to determine the radiation dose to reduce the population of E. coli O157:H7 in hamburgers to non-detectable levels in order to render a safer product. Hamburgers, inoculated with Escherichia coli O157:H7, were exposed to gamma radiation (60Co) treatment, with doses ranging from 0 to 0.7 kGy. The average temperature during the process was 5.6§C. Non-inoculated hamburgers were submitted to sensory evaluation after being exposed to irradiation doses of 0.8 kGy and 1.0 kGy. The D10 for the pathogen varied from 0.17 kGy to O.27 kGy in hamburger. Considering the highest D10 value in hamburger, a dose of 1.08 kGy would be sufficient to reduce E. coli O157:H7 contamination in 4 log cycles, without affecting the sensory attributes of the product.

Comparison of electrophoretic protein profiles of campylobacter jejuni subsp. jejuni isolated from different animal species

Electrophoretic protein profiles of Campylobacter jejuni subsp. jejuni strains isolated from feces of seven animal species, including man, were compared. Fourteen strains (two from each species) plus two human strains and the reference one, were ruptured by ultrasound and their total soluble proteins were analyzed by SDS-PAGE technique in a 12(per cent) polyacrylamide gel with computerized densitometric reading by the molecular analyst software. All the strains had bands in common that correspond to 45 and 66 Kda molecular weight. The disagreement corresponded to a 97 to 200 Kda molecular weight region. From the 17 strains, 13 (76.5 per cent), were classified as biotype I, three (17.6 per cent) as biotype II and one (5.8 per cent) as biotype III. Since protein extracts were obtained from cells grown under identical conditions, and thus, able to express the same phenotype, this disagreement region could be related to different genotypes or serotypes.

A comparative study of bacteriostatic activity of synthetic hydroxylated flavonoids

Among other properties, flavonoids present a notable bacteriostatic activity. In this paper, minimal inhibitory concentrations (MICs) of 5,7,4'-trihydroxyflavanone (naringenin), 5,7-dihydroxyflavone and 2',4',4- trihydroxychalcone (isoliquitirigenin) against Staphylococcus aureus ATCC 25 923 were determined and compared to values obtained for other chalcones and flavanones previously investigated. Specific growth rates and MICs were determined by a turbidimetric kinetic method. The observed sequence MICflavanone (inactive) >MIC7-hidroxyflavanone (197.6 µgml-1)>MIC5,7,4'-trihydroxyflavanone (120 µgml-1) showed that the introduction of an electron donating group (-OH) causes an increase in bioactivity. On the other hand, the comparisons MIC5,7,4'-trihydroxyflavanone (120 µgml-1) >>> MIC2',4',4-trihydroxychalcone (29 µgml-1) and MIC5,7-dihydroxyflavone (105 µgml-1) >>> MIC2',4'-dihydroxychalcone (28.8 µgml-1) indicated that the chalcone structure is the most favourable for bacteriostatic activity within the flavonoid family.

Experimental Salmonella Gallinarum infection in light laying hen lines

Although the epidemiology of fowl typhoid in chickens supposedly involves a vertical transmission stage, a previous work run by the authors has suggested that this did not happen in a commercial line of laying hens highly susceptible to systemic disease with Salmonella Gallinarum. A new experiment was carried out in two other lines of commercial layers, considerably more resistant than those used in the previous study. Clinical fowl typhoid was not observed, but Salmonella Gallinarum was isolated from spleen and liver four weeks after infection and, sporadically, from the ovary.

Molecular characterization of Listeria monocytogenes isolated from foods

A total of 30 strains of "Listeria monocytogenes" isolated from different foods (16 of different kinds of sausage, 14 cheese) purchased at groceries of São Paulo City were ribotyped and analysed for the presence and expression of hemolysin gene and production of phosphatidylinositol-specific phospholipase C-PI-PLC enzyme. The "L. monocytogenes" strains were differentiated into six ribotype classes. A total of 13 (43.3(per cent)) from these strains belong to the same ribotype (ribotype I), and was coincident to the ribotype of the standard "L. monocytogenes" prototype strain (ATCC-15313). The hemolytic activity was observed in 29 (96.7(per cent)) strains when incubated at 37ºC, but not at 4ºC. The direct colony hibridizatioon method for hemolysin gene detection showed a positive reaction with other "Listeria" spp. The PI-PLC was produced by (90(per cent)) of the strains analysed. There was no correlation between the six identified ribotypes and the virulence facors (hemolysin and PI-PLC) studied

Occurrence of Listeria monocytogenes in salami

Eighty-one samples of four different types of salami (Friolan, Hamburguese, Italian and Milanese), belonging to five brands, and purchased at Rio de Janeiro market, were evaluated for the occurrence of "Listeria monocytogenes, The pathogen was detected in 13.3(per cent) of Italian type samples of salami, while "L. innocua" occurred in 6.5(per cent) of the Italian type and 16.6(per cent) of the Milanese type. The remaining samples were negative for "Listeria" spp

Pathogenicity characteristics of filamentous fungi strains isolated from processed oat

Nineteen strains of filamentous fungi isolated from processed oat were tested for pathogenecity factores, based on three parameters: growth at 37ºC, production of phospholipase and urease. "Aspergillus niveus", "Oidiidendron gryseum and "Spotothrix cyanescens" were positive for the three parameters. The other species were positive only for one or two of them

An experimental study of nanoflagellate bacterivory

Heterotrophic nanoflagellate "Pseudobodo tremulans" (4.8 to 7.0 (micro)m) and heterotrophic bacteria, isolated from coastal waters in Ubatuba, SP, Brazil, were used in experiments to analyze quantitatively the relationships between bacteria and nanoflagellates. The meaning of these results for the role of heterotrophic nanoflagellates in the Ubatuba coastal ecosystem is discussed

Actinomicose facial: relato de um caso / Facial actinomycosis: case report

Actinomicose é um processo infeccioso bacteriano. Seu diagnóstico é difícil e demanda um alto nível de suposição. Relatamos um caso de actinomicose facial cujo exame histológico confirmou a hipótese diagnosticada

Procesamiento bacteriológico y micológico de muestras de infecciones de vías respiratorias superiores: otitis media (OMA) y sinusitis / Processing specimens of upper respiratory tract infections for bacteria and fungi: otitis media and sinusitis

Las infecciones del tracto respiratorio superior son la causa infecciosa más frecuente de consulta al médico. En el caso de la otitis media y sinusitis aguda, los agentes etiológicos más frecuentes son S. pneumoniae, H. influenzae no "b" y M. catarrhalis; en tanto que en las formas crónicas aumenta la incidencia de anaerobios, bacilos gram negativos y S. aureus. La punción de oído medio y de senos paranasales se recomienda para casos puntuales como inmunocomprometidos, fracaso terapéutico, complicaciones supurativas, neonatos con otitis media, pacientes intubados con sinusitis y tal vez pacientes provenientes de áreas con alto porcentaje de cepas resistentes

Identificación bioquímica de streptococcus agalactiae / Biochemical identification of streptococcus agalactiae

Se evaluaron las características bioquímicas: hidrólisis del hipurato por dos procedimientos diferentes, test de CAMP y producción de pigmento para el diagnóstico de laboratorio de streptococcus agalactiae. La totalidad de las 28 cepas pudieron ser identificadas eficientemente por sus características bioquímicas. Especialmente adecuado para el diagnóstico de este microorganismo, es el test de hidrólisis rápida del hipurato de sodio

Incidencia de cepas de Stretococcus bovis y estreptococos del grupo "viridans" bilis-esculina positivas en materiales clínicos: propuesta de un esquema mínimo de pruebas para su diferenciación / Incidence of Streptococcus bovis and \"viridans\" group bile-esculin positive streptococci in clinical specimens: identification of species by using minimal schedule test

Streptococcus bovis comparte muchas características bioquímicas con algunas especies del grupo "viridans". En ese sentido, algunas de éstas son capaces de dar positiva la prueba de bilis-esculina (BE+). Debido a la implicancia clínica que tiene la correcta diferenciación de estos microorganismos, se estudió su incidencia en materiales clínicos y se evaluó una marcha mínima de pruebas bioquímicas para su identificación. Sobre 172 aislamientos sucesivos, tipificados según un esquema descripto previamente por los autores, se encontraron 59 cepas (34 por ciento) BE+, 23 (13 por ciento) fueron S. bovis y 36 (21 por ciento) fueron especies del grupo "viridans". La distribución de cepas BE+ dentro de las mismas fue: S. anginosus, 22 por ciento (9/41), S. sanguis, 26 por ciento (6/23), S. mutans, 72 por ciento (13/18), S. vestibularis 4/7 y S. salivarius 4/6 cepas. El 41 por ciento de las 59 cepas BE+ se aisló de hemocultivos. El esquema propuesto identificó correctamente todas las cepas BE+. Utilizando sólo las pruebas de bilis-esculina y desarrollo en caldo CINa (6,5 por ciento), se cometería un error del 60 por ciento en la identificación presuntiva de S. bovis. Creemos que este estudio sugiere la necesidad de incluir pruebas adicionales a las de identificación presuntiva de este microorganismo. Considerando la comprobada asociación de S. bovis con patología gastrointestinal, el esquema propuesto podría ser de interés para el laboratorio de bacteriología clínica, ya que permite llegar al nivel de especie con bajo riesgo de error