Diagnóstico de endoftalmitis por método microbiológico convencional y molecular en pacientes de centros oftalmológicos de Paraguay / Diagnosis of endophthalmitis by molecular and conventional microbiological methods in patients from ophthalmologic centers of Paraguay

La endoftalmitis infecciosa es una condición intraocular grave, aunque poco frecuente, que puede llevar a la pérdida de la visión. El diagnóstico etiológico precoz y el tratamiento adecuado llevan a una mejor evolución. Las técnicas microbiológicas convencionales tienen baja sensibilidad en el diagnóstico de la endoftalmitis infecciosa, en cambio la reacción en cadena de la polimerasa (PCR) es un método de mayor sensibilidad que está siendo utilizado ampliamente. El objetivo fue determinar por cultivo y PCR la etiología de la endoftalmitis infecciosa en pacientes que consultaron en centros oftalmológicos de Paraguay. Se estudiaron muestras de humor vítreo obtenidas por punción de 51 pacientes con endoftalmitis. La muestra se dividió en dos alícuotas; una para el estudio microbiológico convencional y la otra para la PCR anidada. Esta última utiliza un par de cebadores basado en secuencias conservadas del gen 16S ribosomal y cebadores específicos para bacterias gram positivas. Se estandarizó la PCR logrando una sensibilidad de detección de 3.10-5 ng/µl para S. aureus, 2,5.10-3 ng/µl para S. pneumoniae y 6.10-7 ng/µl para E. coli. De las 51 muestras, 12 (23,5%) fueron positivas por métodos microbiológicos convencionales y 26 (51,0%) por la PCR anidada. Por cultivo se identificaron predominantemente bacterias grampositivas 8/11 (73%) y un aislado de hongo filamentoso. La PCR anidada presentó muy buena sensibilidad y de gran utilidad en la identificación del origen bacteriano de la endoftalmitis, mejorando la sensibilidad del cultivo en 27,5%. La discriminación bacteriana en grampositivos y gramnegativos por la PCR se correlacionó con el cultivo en el 100% de los casos(AU)

Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry and real-time PCR in a combined protocol for diagnosis of bloodstream infections: a turnaround time approach

ABSTRACT Bloodstream infections (BSIs) are serious infections associated with high rates of morbidity and mortality. Every hour delay in initiation of an effective antibiotic increases mortality due to sepsis by 7%. Turnaround time (TAT) for conventional blood cultures takes 48 h, forcing physicians to streamline therapy by exposing patients to broad-spectrum antimicrobials. Our objective was (1) to evaluate the accuracy and TAT of an optimized workflow combining direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in-house real-time polymerase chain reaction (PCR) for bacterial identification and antimicrobial resistance profiling directly from positive blood bottles for diagnosing bloodstream infections and (2) to verify the effect of reporting results to medical staff. A total of 103 BSI episodes from 91 patients admitted to three hospitals in São Paulo, Brazil were included. TAT from molecular versus conventional methods was measured and compared. Our protocol showed an overall agreement of 93.5% for genus and 78.5% for species identification; 74.2% for methicillin resistance detection, 89.2% for extended-spectrum β-lactamase profiling, 77.8% for metallo-β-lactamase profiling, and 100% for carbapenemase profile and vancomycin-resistance detection when compared with conventional testing. TAT of molecular sample processing according to our protocol was 38 h shorter than conventional methods. Antimicrobial interventions were possible in 27 BSI episodes. Antimicrobial discontinuation was achieved in 12 BSI episodes while escalation of therapy occurred in 15 episodes. Antimicrobial therapy was inadequate in three (12%) BSI episodes diagnosed using results of molecular testing. Our in-house rapid protocol for identifying both bacteria and antimicrobial resistance provided rapid and accurate results, having good agreement with conventional testing results. These results could contribute to faster antimicrobial therapy interventions in BSI episodes.

Bacteriological analysis of necrotic pulp and fistulae in primary teeth

Objectives: Primary teeth work as guides for the eruption of permanent dentition, contribute for the development of the jaws, chewing process, preparing food for digestion, and nutrient assimilation. Treatment of pulp necrosis in primary teeth is complex due to anatomical and physiological characteristics and high number of bacterial species present in endodontic infections. The bacterial presence alone or in association in necrotic pulp and fistula samples from primary teeth of boys and girls was evaluated. Material and Methods: Necrotic pulp (103) and fistula (7) samples from deciduous teeth with deep caries of 110 children were evaluated. Bacterial morphotypes and species from all clinical samples were determined. Results: A predominance of gram-positive cocci (81.8%) and gram-negative coccobacilli (49.1%) was observed. In 88 out of 103 pulp samples, a high prevalence of Enterococcus spp. (50%), Porphyromonas gingivalis (49%), Fusobacterium nucleatum (25%) and Prevotella nigrescens (11.4%) was observed. Porphyromonas gingivalis was detected in three out of seven fistula samples, Enterococcus spp. in two out of seven samples, and F. nucleatum, P. nigrescens and D. pneumosintes in one out of seven samples. Conclusions: Our results show that Enterococcus spp. and P. gingivalis were prevalent in necrotic pulp from deciduous teeth in boys from 2 to 5 years old, and that care of the oral cavity of children up to five years of age is important. .

La evolución de la resistencia bacteriana en México, 1973-2013 / Evolution of bacterial resistance to antibiotics in México, 1973-2013

Introducción. La resistencia bacteriana a los antibióticos es un problema de salud mundial. Las investigaciones relacionadas con este problema emergente son indispensables para reconocer y desarrollar programas para su vigilancia y control. Objetivo. Revisar y comentar las contribuciones de los investigadores mexicanos en el área de la resistencia bacteriana a los antibióticos. Materiales y métodos. Se realizó una búsqueda de la literatura científica relacionada con la resistencia bacteriana a los antibióticos producida por investigadores mexicanos y registrada en Medline-PubMed entre 1973 y julio de 2013. Resultados. En 66 publicaciones, las contribuciones de investigadores mexicanos incluyeron datos sobre la resistencia de agentes patógenos entéricos como Salmonella Typhi, múltiples contribuciones sobre la producción de betalactamasas de espectro extendido, de metalobetalactamasas y de carbapenemasas, los mecanismos de resistencia en Pseudomonas aeruginosa y la evolución de la resistencia en cocos Gram positivos como Streptococcus pneumoniae , Staphylococcus aureus y Enterococcus spp., entre otros. Conclusiones. Los datos publicados en los últimos 40 años son fuente adecuada para entender la evolución de la resistencia bacteriana a los antibióticos y desarrollar programas para su control.

Introduction: Bacterial resistance to antibiotics is a worldwide public health concern. Research priorities for the study and control of this emerging problem include country-wide surveillance. Objective: To review and comment on the contributions by Mexican investigators towards a greater understanding of the mechanisms of bacterial antibiotic resistance. Materials and methods: A comprehensive search of the medical literature on Medline/PubMed between 1973 and July 2013 was performed. Results: The contributions of Mexican investigators have included descriptions of resistance in enteric pathogens, such as Salmonella Typhi, publications on the production of extended spectrum beta-lactamases, metallo-beta-lactamases, and carbapenemases, resistance mechanisms of Pseudomonas aeruginosa , and the evolution of resistance in Gram-positive pathogens, including Streptococcus pneumoniae , Staphylococcus aureus , and Enterococcus spp. Conclusion: The Mexican literature on mechanisms of bacterial resistance is relevant for the development of plans to control the antibiotic resistance crisis.

Performance of Kiestra Total Laboratory Automation Combined with MS in Clinical Microbiology Practice

BACKGROUND: Microbiological laboratories seek technologically innovative solutions to cope with large numbers of samples and limited personnel and financial resources. One platform that has recently become available is the Kiestra Total Laboratory Automation (TLA) system (BD Kiestra B.V., the Netherlands). This fully automated sample processing system, equipped with digital imaging technology, allows superior detection of microbial growth. Combining this approach with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) (Bruker Daltonik, Germany) is expected to enable more rapid identification of pathogens. METHODS: Early growth detection by digital imaging using Kiestra TLA combined with MS was compared to conventional methods (CM) of detection. Accuracy and time taken for microbial identification were evaluated for the two methods in 219 clinical blood culture isolates. The possible clinical impact of earlier microbial identification was assessed according to antibiotic treatment prescription. RESULTS: Pathogen identification using Kiestra TLA combined with MS resulted in a 30.6 hr time gain per isolate compared to CM. Pathogens were successfully identified in 98.4% (249/253) of all tested isolates. Early microbial identification without susceptibility testing led to an adjustment of antibiotic regimen in 12% (24/200) of patients. CONCLUSIONS: The requisite 24 hr incubation time for microbial pathogens to reach sufficient growth for susceptibility testing and identification would be shortened by the implementation of Kiestra TLA in combination with MS, compared to the use of CM. Not only can this method optimize workflow and reduce costs, but it can allow potentially life-saving switches in antibiotic regimen to be initiated sooner.

Atividade antimicrobiana da Terminalia arjuna Wight & Arn.: uma planta etnomedicinal contra patógenos causadores de infecções no ouvido / Antimicrobial activity of Terminalia arjuna Wight & Arn.: an ethnomedicinal plant against pathogens causing ear infection

Infecções de ouvido representam uma das doenças mais comuns do mundo. Diferentes agentes etiológicos são responsáveis por tais infecções. OBJETIVO: Avaliar o potencial antimicrobiano de extratos de folhas e da casca de Terminalia arjuna contra Staphylococcus aureus, Acinetobacter sp., Proteus mirabilis, Escherchia coli, Pseudomonas aeruginosa e Candida albicans - patógenos que causam infecções de ouvido e como esses extratos se comparam às medicações tópicas atualmente disponíveis. MATERIAIS E MÉTODOS: Metanol, etanol, acetona, extratos aquosos (quentes e frios) de folhas e casca da T. arjuna foram testados para avaliação de suas atividades antimicrobianas. RESULTADOS: Dos três solventes orgânicos avaliados, o extrato acetônico das folhas foi o que teve o melhor resultado contra S. aureus. O extrato orgânico da casca exibiu quase o mesmo grau de ação antimicrobiana entre todas as bactérias gram-negativas, exceto contra P. aeruginosa. Entretanto, o extrato aquoso da casca de T. arjuna exibiu boa atividade contra S. aureus. Nenhum dos extratos exibiu atividade antifúngica. CONCLUSÃO: O extrato orgânico obtido a partir da casca e folhas de T. arjuna pode ser usado para tratar infecções bacterianas de ouvido, especialmente aquelas causadas por S. aureus, que exibiu maiores zonas de inibição do que gotas herbáceas; entretanto, ainda necessitamos de estudos mais detalhados - como ensaios in vivo e avaliações de propriedades farmacocinéticas para avaliarmos sua utilidade terapêutica no tratamento de infecções de ouvido.

Ear infection is one of the common diseases occurring throughout the world. Different etiological agents are responsible for ear infections. AIM: To assess the antimicrobial potential of Terminalia arjuna leaves and bark extracts against Staphylococcus aureus, Acinetobacter sp., Proteus mirabilis, Escherchia coli, Pseudomonas aeruginosa and Candida albicans, pathogens causing ear infections and their comparison with locally available ear drops. MATERIALS AND METHODS: Methanol, ethanol, acetone, aqueous (hot and cold) extracts from the leaves and bark of T. arjuna were tested for their antimicrobial activity. RESULTS: Of the three organic solvents evaluated, acetonic leaf extract was found to be best against S. aureus. Organic bark extract showed almost equal inhibition of all tested Gram negative bacteria except P. aeruginosa. However, aqueous extract of T. arjuna bark exhibited good activity against S. aureus. All the extracts were unable to exhibit any antifungal activity. CONCLUSION: Organic extract obtained from the T. arjuna bark and leaves may be used to treat the bacterial ear pathogens especially S. aureus, which has shown greater inhibition zones than the herbal drops, however, we still need more detailed studies as in vivo testing and pharmacokinetics properties for their therapeutic utility in treating ear infections.

Resistencia a los antibacterianos en América Latina: consecuencias para la infectología / Antibacterial drug resistance in Latin America: consequences for infectiousdisease control

Antibacterial drug resistance is a particularly significant issue in Latin America. This article explores antimicrobial resistance in three classes of clinically important bacteria: gram-positive bacteria, enterobacteria, and nonfermenting gram-negativebacilli. The gram-positive bacteria frequently responsible for infections in humans are for the most part cocci: staphylococci, streptococci (including pneumococci), and enterococci,in both community and hospital settings. This situation is no different in theRegion of the Americas. Among the gram-positive bacteria, the causative agents of bacteremia are most commonly strains of coagulase-negative Staphylococcus, followed by enterococci. This report explores the resistance of these species to different antimicrobial drugs, resistance mechanisms in community and hospital strains, and new drugs for treating infections caused by these bacteria. In Latin America, antimicrobialresistance in Enterococcus strains is still a minor problem compared to the situation in the United States. The strains of the genus Streptococcus isolated from respiratory infections are still sensitive to penicillin. Furthermore, the resistance of enterobacteriais extremely important in the Region, particularly because of the broad dissemination of CTX-M extended-spectrum beta-lactamases (ESBL), some of which originated in Latin America. This article analyzes the resistance of Streptococcus pneumoniae, betahemolytic streptococci, and viridans group streptococci. Among the nonfermentinggram-negative bacilli, while Pseudomonas aeruginosa strains remain the leading cause of bacteremia, infections caused by strains of Acinetobacter spp. have proliferatedextensively in some areas. With regard to antibiotics, several options are available for treating gram-positive bacterial infections...

La resistencia a los fármacos antibacterianos tiene particular importancia en América Latina. En este artículo se analiza la resistencia a los antimicrobianos de tres clases de bacterias de importancia clínica: bacterias grampositivas, enterobacterias y bacilos gramnegativos no fermentadores.Las bacterias grampositivas que producen infecciones humanas frecuentes son, en su mayoría, cocos: estafilococos, estreptococos (incluidos neumococos) y enterococos, tanto en elmedio comunitario como en el nosocomial. Esta situación no es diferente en la Región de las Américas. Entre las bacterias grampositivas, las que causan bacteriemia con mayor frecuencia corresponden a cepas de estafilococos coagulasa negativos, seguidas de las de enterococos. Eneste informe se analiza la resistencia de estas especies a distintos antimicrobianos, los mecanismosde resistencia para las cepas de origen hospitalario y comunitario y los nuevos medicamentos para tratar las infecciones por estas bacterias. La resistencia a los antimicrobianos delas cepas de Enterococcus en América Latina todavía es un problema menor en relación con la situación en los Estados Unidos de América. Las cepas del género Streptococcus aisladasde infecciones respiratorias aún son sensibles a penicilina. Por otra parte, la resistencia de las enterobacterias es de gran importancia en la Región, particularmente por la gran difusión debetalactamasas de espectro extendido (BLEE) de tipo CTX-M, algunas de las cuales se originaron en América Latina. En el presente artículo se analizan la situación de la resistencia de las cepas de Streptococcus pneumoniae, y de los estreptococos betahemolítico y del grupo viridans. Entre los bacilos gramnegativos no fermentadores, si bien las cepas de Pseudomonasaeruginosa siguen siendo la causa principal de bacteriemias, la proliferación de infecciones por cepas de Acinetobacter spp. tiene en algunas partes gran magnitud...

Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm³ to the occurrence of bacteraemia as detected by the four methods (p < 0.05).

Glass bead transformation method for gram-positive bacteria

A simple, inexpensive and reproducible transformation method was developed for Gram-positive bacteria. It was based on agitation of bacterial protoplasts with glass beads in the presence of DNA and polyethylene glycol. By using this method, introduction of pGK12 into protoplasts of several strains of Gram-positive bacteria was achieved.

Nosocomial bloodstream infections: organisms, risk factors and resistant phenotypes in the Brazilian University Hospital

Bacteremia is one of the most frequent and challenging hospital-acquired infection and it is associated with high attributable morbidity and mortality and additional use of healthcare resources. The objective of this work was to determine the frequencies of its occurrence, organisms and resistance phenotypes associated to nosocomial acquired bloodstream infections. A total number of 51 nosocomial bacteremia by Gram-negative and 99 by Gram-positive were evaluated and compared during a 15-month period. The risk factors associated with these bacteremias were analyzed and antibiotic use and surgery were associated with bacteremia by Gram-negative and > 2 invasive devices with Gram-positive. The resistance phenotypes ESBL (extended-spectrum beta-lactamases) (23.5 percent) and AmpC/others (17.6 percent) correspond to 41.2 percent with predominance of E. agglomerans among AmpC (44.4 percent) and K. pneumoniae among ESBLs (38.5 percent). Among S. aureus bacteremia, approximately 40 percent were associated to MRSA (methicillin-resistant Staphylococcus aureus).

Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from "Bacillus thuringiensis"

The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a 'cry' gene from "Bacillus thuringiensis", envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria "Gluconacetobacter diazothophicus" strain BR11281 and "Herbaspirillum seropedicae" strain BR11335 were used as models. The 'cry3A' gene was transferred by conjugation using a suicide plasmid and recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the 'cry' gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of (delta)-endotoxin by recombinant "H. seropedicae" strain was detected by dot blot while for "G. diazotrophicus" the Western-blot technique was used. In both cases, a specific antibody raised against the "B. thuringiensis" toxin was applied. The (delta)-endotoxin production showed by the "G. diazotrophicus" recombinant strain was dependent on the nitrogen fixing conditions since the 'cry3A' gene was fused to a 'nif' promoter. In the case of "H. seropedicae" the (delta)-endotoxin expression was not affected by the promoter ('rhi') used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests

Azurexidina, proteína de neutrófilo com funçäo antibiótica: Expressäo, purificaçäo e caracterizaçäo da proteína recombinante / Azurocidin, protein of neutrophilic with function antibiotical: expression, purification and characterization of recombinant protein

Os leucócitos polimorfonucleares têm papel crítico na destruiçäo, digestäo de patógenos, geraçäo e regulaçäo do processo inflamatório. Proteínas que säo estocadas dentro dos grânulos citoplasmáticos têm sido implicadas nos mecanismos microbicidas, independentes de oxigênio, bem como na degradaçäo de proteínas. Os grânulos azurófilos e específicos de polimorfonucleares neutrófilos contêm pelo menos 10 proteínas que podem destruir microorganismos. Dentre estas proteínas, 3 säo serina proteinases: catepsina G, proteinase 3 e elastase. Estas serina proteinases têm atividade antibiótica e também degradam proteínas do tecido conjuntivo. Azurocidina apresenta diversas semelhanças na sequência de aminoácidos e de DNA com as serina proteinases, também possui atividade antibiótica, porém näo apresenta atividade proteolítica, devido a mutaçäoes ocorridas na molécula de DNA, alterando o sítio catalítico típico desta família de proteínas. Esta família de proteínas, incluindo a azurocidina, é denominada de serprocidinas (serina proteinases com atividade microbicida). Azurocidina tem peso molecular de 29 kD e seu DNA codifica 225 aminoácidos da proteína madura e 24 resíduos de aminoácidos do sinal peptídico. O presente trabalho de tese diz respeito à expressäo e produçäo de azurocidina recombinante no sistema de expressäo baculovírus-células de inseto. Azurocidina recombinante expressada e produzida neste sistema foi caracterizada bioquimicamente e sua atividade microbicida conta bactérias Gram negativas e Gram positivas, bem como contra fungo, foi demonstrada. Azurocidina recombinante possui similaridades com a molécula nativa no peso molecular, 29 kD, padräo de glicosilaçäo, ambas possuem manose como resíduo de açucar terminal, sequência N-terminal de aminoácidos e com a atividade microbicida. A LD50 de azurocidina recombinante comtra E. coli, S. faecalis e C. albicans foi 1,2 (mais ou menos) 0,1, 2,9 (mais ou menos) 0,4 e 8,2 (mais ou menos) 0,2 ug/ml, respectivamente e a LD50 de azurocidina nativa foi de 1,3 (mais ou menos) 0,05, 2,3 (mais ou menos) 0,2 e 8 (mais ou menos) 0,8 ug/ml. Também foi demonstrado, com experimentos de deleçäo na molécula de DNA de azurocidina, que as sequências de aminoácidos localizadas entre as posiçöes 114 a 129 e/ou entre 154 a 186, säo necessárias para que azurocidina recombinante seja secretada pelo mio de cultura das células de inseto infectadas com o baculovírus recombinante. Associado a este trabalho de tese, desenvolvemos...