Amplificación directa de ADN de Bordetella pertussis purificado de hisopados nasofaríngeos por un método de bajo costo, rápido (60-segundos) y libre de equipos / Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method

RESUMEN Objetivo. Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. Materiales y métodos. Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). Resultados. El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. Conclusión. El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.

ABSTRACT Objective. To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. Results. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. Conclusion. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

Insight into DNA Gyrase inhibition using quantitative structure-Activity relationships and structure-based drug design approaches

The emergence of certain bacterial strains resistant to antibiotics has become a major public health problem, hence the need to develop new antibiotic molecules. Bacterial DNA gyrase, a type II DNA topoisomerase found in all bacteria is a proven target for antibacterial chemotherapy. Our objective is designing novel DNA Gyrase inhibitors using Quantitative StructureActivity Relationships and Structure-Based Drug Design Approaches. We used bioinformatics tools, biological databases like PDB (Protein DataBank), Binding Databases and software's like, MarvinView, MarvinSketch, PyMOL, AutoDockTools-1.5.6. The 3D crystal structure of DNA Gyrase was extracted from PDB (code: 4DHU) and we characterized the active site. Using 83 compounds with different Ki were extracted from Binding Databases, we built and validated a QSAR Model (PLS regression) and we confirmed the interesting correlation between predicted and experimental Ki (R2=0,843). Four molecules were chosen to be docked into DNA Gyrase active site using AutoDockTools. The compound which has the low Ki (Benzimidazole urea analogue 5) shows more binding affinity with score value of ΔG= -8,6 kcal/mol than the others compounds. So, it would be very interesting to synthesis this promising compound and to test in vitro its antibacterial properties.

Mobile phones of hospital workers: a potential reservoir for the transmission of pathogenic bacteria

Background: Mobile phones are increasingly associated with the transmission of pathogenic microbial agents. In the clinical setting where there is usually high exposure to pathogens, these devices may serve as vehicles for the transmission/spread of pathogens. This study determined the prevalence of bacterial contamination of mobile phones of health workers and the predisposing factors, in order to ascertain the risk of transmission of pathogenic bacteria through mobile phones. Methodology: This study was carried out in a private medical center at Mbouda, Cameroon, involving 78 health workers including health professionals (nurses, physicians, laboratory scientists) and hospital support workers (cleaners, cashiers and security guards), recruited by convenient sampling. Sterile swab sticks moistened with physiological saline were used to swab about three quarter of the surface of each phone. The swabs were cultured on MacConkey and Mannitol Salt agar plates which were incubated aerobically at 37oC for 24 hours, while Chocolate agar plate was incubated in a candle extinction jar for microaerophilic condition. The isolates were identified using standard biochemical tests including catalase, coagulase, and the analytical profile index (API) system. Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 20.0. Results: Mobile phones of 75 of the 78 (96.2%) health workers were contaminated, with highest contamination rates for the phones of laboratory scientists (100%, 12/12), followed by support staff (98.9%, 13/14), nurses (97.7%, 43/44) and physicians (87.3%, 7/8), but the difference in contamination rates was not statistically significant (p=0.349). A total of 112 bacteria belonging to 12 genera were isolated, with predominance of Staphylococcus aureus (31.3%, n=35), Micrococcus spp (30.4%, n=34), coagulase negative staphylococci (10.7%, n=12) and Pseudomonas spp (5.4%, n=6). The laboratory (18.8%, 21/112) and medical wards (16.1%, 18/112) had the highest bacterial contamination of mobile phones (p=0.041), and more bacterial species were isolated from smartphones (68.8%, n=77/112) than keypad phones (31.2%, n=35/112) (p=0.032). There was no significant difference between phone contamination rates and the practice of hand hygiene or decontamination of work surfaces (p>0.05). Conclusion: The presence of potentially pathogenic bacteria on cell phones of health-care workers emphasizes the role of fomites in the transmission of infectious diseases. Consequently, good hand hygiene and decontamination practices are encouraged among health workers in order to limit the spread of hospital-acquired infections.

Prevalence of Bartonella species in shelter cats and their ectoparasites in southeastern Brazil / Prevalência de espécies de Bartonella em gatos de abrigos e seus ectoparasitas no Sudeste do Brasil

Abstract Feline Bartonella can be transmitted to humans through cat scratches or bites, and between cats, by the flea Ctenocephalides felis. The study was carried out in order to investigate the occurrence of Bartonella DNA in cats living in shelters and their ectoparasites and the relationship between the infection status of cats and ectoparasites they host. Bartonella DNA was detected in 47.8% of the cat blood samples, 18.3% of C. felis fleas, 13.3% of flea egg pools and 12.5% of lice pools. B. henselae and B. clarridgeiae DNA were detected in cat fleas, while B. henselae, B. clarridgeiae and B. koehlerae were found in blood samples from bacteremic cats. Cats infested by positive ectoparasites showed approximately twice the odds of being infected. Our results indicate that shelter cats have high prevalence of Bartonella species that are known to be human pathogens. This highlights the importance of controlling infestations by ectoparasites to avoid cat and human infection.

Resumo Algumas espécies de Bartonella têm os felinos como principais hospedeiros reservatórios. Tais patógenos são transmitidos ao homem por intermédio da arranhadura ou mordedura de gatos e entre os gatos, por meio da pulga Ctenocephalides felis. O objetivo deste estudo foi investigar a ocorrência de DNA de Bartonella spp. em gatos de abrigos e seus ectoparasitas e a relação entre o estado de infecção dos gatos e dos ectoparasitas albergados por estes. Material genético bacteriano foi detectado em 47,8% das amostras de sangue de gatos, 18,3% das pulgas C. felis, 13,3% dos "pools" de ovos de pulgas e 12,5% dos "pools" de piolhos. DNA de B. henselae e B. clarridgeiae foi detectado em pulgas, e B. henselae, B. clarridgeiae e B. koehlerae, em amostras de sangue de gatos. Gatos infestados por ectoparasitas que carreavam DNA de Bartonella spp. demonstraram aproximadamente o dobro de chance de estarem infectados. Esses resultados indicam que os gatos de abrigos têm alta prevalência de infecção por espécies de Bartonella, capazes de causar doenças no homem. E também destacam a importância do controle e prevenção da infestação por ectoparasitas, no intuito de prevenir a infecção em gatos e humanos.

Change in intestinal flora after treatment in children with focal epilepsy / 中国当代儿科杂志

OBJECTIVES@#To study the difference in intestinal flora between children with focal epilepsy and healthy children and the change in intestinal flora after treatment in children with epilepsy.@*METHODS@#A total of 10 children with newly diagnosed focal epilepsy were recruited as the case group and were all treated with oxcarbazepine alone. Their clinical data were recorded. Fecal specimens before treatment and after 3 months of treatment were collected. Fourteen aged-matched healthy children were recruited as the control group. Total bacterial DNA was extracted from the fecal specimens for 16S rDNA sequencing and bioinformatics analysis.@*RESULTS@#After 3 months of carbamazepine treatment, the seizure frequency was reduced by >50% in the case group. At the phylum level, the abundance of Actinobacteria in the case group before treatment was significantly higher than that in the control group (P<0.05), and it was reduced after treatment (P<0.05). At the genus level, the abundances of Escherichia/Shigella, Streptococcus, Collinsella, and Megamonas in the case group before treatment were significantly higher than those in the control group (P<0.05), and the abundances of these bacteria decreased significantly after treatment (P<0.05).@*CONCLUSIONS@#There is a significant difference in intestinal flora between children with focal epilepsy and healthy children. Oxcarbazepine can significantly improve the symptoms and intestinal flora in children with epilepsy.

Estructura genética de cepas de Mycobacterium tuberculosis farmacorresistentes en Perú con base en haplotipos obtenidos de un ensayo de sonda lineal / Genetic structure of drug-resistant strains of Mycobacterium tuberculosis in Peru based on haplotypes obtained from a linear probe assay

RESUMEN Objetivo. Determinar la estructura genética de las cepas drogorresistentes de Mycobacterium tuberculosis que circularon en todo el Perú durante los años 2011-2015 a través de haplotipos obtenidos de un ensayo con sondas en línea. Materiales y métodos. Se analizaron 6589 muestras que ingresaron al Instituto Nacional de Salud para el diagnóstico rutinario mediante el ensayo GenoType® MTBDRplus v2, durante el periodo de estudio. Se crearon haplotipos resistentes mediante la concatenación de 21 sitios polimórficos de los genes evaluados por el ensayo con sondas en línea, y se realizó el análisis de asociación con fenotipos obtenidos por el método de proporciones agar 7H10. Resultados. Las mutaciones de mayores frecuencias fueron: rpoB S531L (55,4%) y rpoB D516V (18,5%) para la resistencia a rifampicina, y katG S315T (59,5%) e inhA c-15t (25,7%) para la resistencia a isoniacida. Se obtuvieron 13 haplotipos representativos (87,8% de muestras analizadas) de los cuales seis correspondieron al genotipo multidrogorresistente, cuatro al genotipo monorresistente a isoniacida y tres al genotipo monorresistente a rifampicina. Dieciocho departamentos, y la provincia del Callao, presentaron una alta diversidad haplotípica; cuatro presentaron moderada diversidad y dos presentaron baja diversidad. Conclusiones. Existe una alta diversidad haplotípica en la mayoría de los departamentos, además de una concentración de las cepas de Mycobacterium tuberculosis drogorresistentes en las ciudades de Lima y Callao. Asimismo, las cepas de Mycobacterium tuberculosis con perfil drogorresistente que circulan en el Perú contienen principalmente los marcadores genéticos de mayor prevalencia a nivel mundial asociados con la resistencia frente a rifampicina e isoniacida.

ABSTRACT Objective. To determine the genetic structure of drug-resistant strains of Mycobacterium tuberculosis that circulated throughout Peru during the years 2011-2015, by using haplotypes obtained from a line probe assay. Materials and methods. A total of 6589 samples that were admitted to the Instituto Nacional de Salud for routine diagnosis using the GenoType® MTBDRplus v2 assay were analyzed during the study period. Resistant haplotypes were created by concatenating 21 polymorphic sites of the evaluated genes using the line probe assay; and the association analysis was carried out with phenotypes obtained by the 7H10 agar ratio method. Results. The most frequent mutations were: rpoB S531L (55.4%) and rpoB D516V (18.5%) for rifampicin resistance, and katG S315T (59.5%) and inhA c-15t (25.7%) for isoniazid resistance. We obtained 13 representative haplotypes (87.8% of analyzed samples), 6 corresponded to the multidrug-resistant genotype, 4 to the isoniazid mono-resistant genotype and 3 to the rifampicin mono-resistant genotype. Eighteen regions and the province of Callao showed high haplotype diversity; four showed moderate diversity and two showed low diversity. Conclusions. Most regions showed high haplotype diversity; in addition, most drug-resistant strains of Mycobacterium tuberculosis were concentrated in the cities of Lima and Callao. Likewise, drug-resistant Mycobacterium tuberculosis strains circulating in Peru mainly contain the genetic markers with the highest prevalence worldwide, which are associated with resistance to rifampicin and isoniazid.

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates / Otimização de um método baseado em PCR em tempo real para rastreamento de bactérias em concentrados de plaquetas

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.

The prevalence of novel periodontal pathogens and bacterial complexes in Stage II generalized periodontitis based on 16S rRNA next generation sequencing

Abstract Objective: To define the subgingival microbial profile associated with Stage II generalized periodontitis using next-generation sequencing and to determine the relative abundance of novel periodontal pathogens and bacterial complexes. Methodology: Subgingival biofilm samples were collected from 80 subjects diagnosed with Stage II generalized periodontitis. Bacterial DNA was extracted, and 16S rRNA-based bacterial profiling via next-generation sequencing was carried out. The bacterial composition and diversity of microbial communities based on the age and sex of the patients were analyzed. The bacterial species were organized into groups: bacterial complexes (red, orange, purple, yellow, and green), novel periodontal pathogens, periodontal health-related species, and unclassified periodontal species. The results were analyzed and statistically evaluated. Results: The highest number of bacteria belonged to the phylum Bacteroidetes and Firmicutes. In terms of relative abundance, the orange complex represented 18.99%, novel bacterial species (Fretibacterium spp. and Saccharibacteria spp.) comprised 17.34%, periodontal health-related species accounted for 16.75% and unclassified periodontal species represented (Leptotrichia spp. and Selenomonas spp.) 15.61%. Novel periodontal pathogens had outweighed the periodontal disease-related red complex (5.3%). The one-sample z-test performed was statistically significant at p<0.05. The Beta diversity based on the unweighted UniFrac distance at the species level demonstrated a total variance of 15.77% based on age and 39.19% on sex, which was not statistically significant. Conclusion: The bacterial species corresponding to the disease-related orange complex and novel periodontal pathogens are predominant in Stage II generalized periodontitis.

Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of / 生物医学与环境科学（英文）

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of

Cultivable bacterial diversity associated with bromeliad roots from ironstone outcrops in central Brazil / Diversidade de bactérias cultiváveis associadas a raízes de bromélias de afloramentos ferruginosos do Brasil central

Abstract Studies on the bacterial diversity associated with wild plants are rare, especially on those that grow in association with bromeliads. In the present study, we isolated and identified epiphytic and endophytic bacteria from the roots of the bromeliads Dyckia excelsa, Dyckia leptostachya and Deuterocohnia meziana occurring in the "cangas" in the Pantanal from Mato Grosso do Sul State, Brazil. The epiphytic bacteria were isolated from washed roots, while the endophytic bacteria were isolated from surface disinfested roots. Bacterial representatives corresponding to each BOX-PCR fingerprint, as well as those that did not result in amplicons, were selected for 16S rDNA gene sequence analysis. The BOX-PCR data showed intrageneric and intraspecific diversity and could discriminate strains and identify their phenotypic characteristics. The 16S rDNA gene sequence and phylogeny analysis showed a higher occurrence of strains belonging to the genus Bacillus than Mycobacterium and Brevibacterium, which were found in lower numbers. Species from the Bacillus genus are well known for their sporulation capacity and longer survival in arid locations, such as the "cangas". This study clearly showed that the bromeliad species represent a vast reservoir of bacterial community diversity, and the cultivable strains represent a new source for biotechnological prospecting.

Resumo Estudos sobre a diversidade bacteriana associada a plantas silvestres são raros, especialmente naqueles que crescem em associação com bromélias. No presente estudo, isolamos e identificamos bactérias epífitas e endofíticas das raízes das bromélias Dyckia excelsa, D. leptostachya e Deuterocohnia meziana ocorrentes nas "cangas" no Pantanal do Mato Grosso do Sul, Brasil. As bactérias epifíticas foram isoladas de raízes lavadas, enquanto as bactérias endofíticas foram isoladas de raízes desinfestadas na superfície. Representantes bacterianos correspondentes a cada perfil do BOX-PCR, bem como aqueles que não resultaram em amplificações, foram selecionados para o sequenciamento do gene 16S rDNA. Os dados da BOX-PCR mostraram diversidade intragênica e intraespecífica e puderam discriminar cepas e identificar suas características fenotípicas. A seqüência do gene 16S rDNA e a análise filogenética mostraram uma maior ocorrência de cepas pertencentes ao gênero Bacillus do que as bactérias Mycobacterium e Brevibacterium, encontradas em menor número. Espécies do gênero Bacillus são bem conhecidas por sua capacidade de esporulação e maior sobrevida em locais áridos, como as "cangas". Este estudo mostrou claramente que as espécies de bromélias representam um vasto reservatório de diversidade de comunidades bacterianas, e as linhagens cultiváveis podem representar uma nova fonte para a prospecção biotecnológica.

Detection of Helicobacter pylori in oral yeasts from students of a Chilean university

SUMMARY INTRODUCTION: Nearly 73% of the Chilean population is infected with Helicobacter pylori (H. pylori), a factor predisposing for gastric cancer. Recent studies have demonstrated the presence of this pathogen within yeasts, suggesting that this fact can directly influence the failure of a treatment, transmission, and reinfection. AIM: To detect the presence of H. pylori inside oral yeasts isolated from students of the University of Concepción (Chile). METHODS: 72 samples, obtained from the oral cavity using cotton swabs were incubated in YPD broth for 48h at 37°C and posteriorly seeded in Sabouraud Dextrose agar plus chloramphenicol at the same temperature and for the same time. Yeasts isolated were observed microscopically (wet mounting and Gram-stained) and identified using microbiological techniques. Intracellular H. pylori detection was performed by the amplification of 16S rDNA by PCR. RESULTS: Oral yeasts were detected in 24 samples (33.3%), being C. albicans (79.2%) the most frequent species, followed by C. dubliniensis (12.4%), C. krusei (4.2%), and C. tropicalis (4.2%). When analyzed by PCR, 15 of the 24 oral yeasts 62.5 % were positive for H. pylori 16S rDNA. From the 15 individuals positive for yeast harboring H. pylori, 81% of them reported stomach discomfort, and the presence of the bacteria was diagnosed at some moment in 20% of them. CONCLUSION: The intracellular presence of the H. pylori in oral yeasts suggests an endosymbiotic relationship of these microorganisms, which could favor H. pylori transmission and reinfection in the gastrointestinal tract.

RESUMO INTRODUÇÃO: Quase 73% da população chilena estão infectadas pelo Helicobacter pylori (H. pylori), fator predisponente ao câncer gástrico. Estudos recentes demonstraram a presença desse patógeno em leveduras, sugerindo que esse fato pode influenciar diretamente a falha de um tratamento, transmissão e reinfecção. OBJETIVO: Detectar a presença de H. pylori em leveduras orais isoladas de estudantes da Universidade de Concepción (Chile). MÉTODOS: 72 amostras, obtidas da cavidade oral utilizando cotonetes, foram incubadas em caldo YPD por 48h a 37°C e posteriormente sementes em ágar Sabouraud Dextrose mais cloranfenicol na mesma temperatura e ao mesmo tempo. Leveduras isoladas foram observadas microscopicamente (montagem úmida e corada por Gram) e identificadas utilizando técnicas microbiológicas. A detecção intracelular de H. pylori foi realizada pela amplificação do 16S rDNA por PCR. RESULTADOS: Leveduras orais foram detectadas em 24 amostras (33,3%), sendo C. albicans (79,2%), a espécie mais frequente seguida por C. dubliniensis (12,4%), C. krusei (4,2%) e C. tropicalis (4,2 %) Quando analisadas por PCR, 15 das 24 leveduras orais 62,5% foram positivas para o H. pylori 16S rDNA. Dos 15 indivíduos positivos para leveduras que abrigam H. pylori, 81% deles relataram desconforto estomacal e a presença da bactéria foi diagnosticada em algum momento em 20% deles. CONCLUSÃO: A presença intracelular do H. pylori em leveduras orais sugere uma relação endossimbiótica desses microrganismos, o que poderia favorecer a transmissão e a reinfecção do H. pylori no trato gastrointestinal.

Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication

Abstract Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.

Is there any association between Helicobacter pylori and otitis media with effusion? / Há associação entre Helicobacter pylori e otite média com efusão?

Abstract Introduction: It is proposed that Helicobacter pylori can be responsible for the development of otitis media with effusion. Objective: The aim of this study is to investigate the prevalence of H. pylori in the adenoid tissue and fluid of the middle ear in patients who suffer from adenoid hyperplasia and otitis media with effusion in comparison with those who suffer from adenoid hyperplasia without otitis media with effusion. Methods: This is a case-control study that was carried out in 50 children of age 2-7 years old who were admitted with adenoid hyperplasia. Patients were divided into case and control groups. The study group included 25 patients with adenoid hyperplasia and otitis media with effusion and the control group included 25 patients with adenoid hyperplasia without otitis media with effusion. The patients in both groups underwent surgical adenoidectomy. For the case group we carried out myringotomy and placement of tympanostomy tube, and fluid samples were collected under sterile conditions. The samples were sent to the laboratory for polymerase chain reactions. Results: In the case group H. pylori was found to be positive in 18 samples of the middle ear fluid (70%) and in 1 polymerase chain reaction adenoid tissue sample (4%). In the control group H. pylori was positive in 3 samples of adenoid tissues (12%). There was no gender difference. Conclusion: H. pylori is one of the important bacteria that plays a role in the pathogenesis of otitis media with effusion. Whether adenoid tissue may be a reservoir for H. Pylori is unclear.

Resumo Introdução: Propõe-se que o Helicobacter pylori possa ser responsável pelo desenvolvimento de otite média com efusão. Objetivo: Investigar a prevalência de H. pylori no tecido adenoideano e no fluido da orelha média em pacientes com hiperplasia de adenoide e otite média com efusão em comparação àqueles com hiperplasia de adenoide sem otite média com efusão. Método: Este é um estudo de caso-controle feito em 50 crianças de 2 a 7 anos, com sinais e sintomas de hiperplasia de adenoide. Os pacientes foram divididos em grupo de estudo e grupo controle. O grupo de estudo incluiu 25 pacientes com hiperplasia de adenoide e otite média com efusão e o grupo controle incluiu 25 pacientes com hiperplasia de adenoide sem otite média com efusão. Os pacientes dos dois grupos foram submetidos a adenoidectomia e, no grupo de estudo, realizou-se também miringotomia com colocação de tubo de ventilação e amostras de fluidos foram coletadas sob condições estéreis. As amostras foram enviadas para o laboratório, para investigação por reação de polimerase em cadeia. Resultados: No grupo de estudo, houve positividade para H. pylori em 18 amostras do fluido de orelha média (70%) e uma amostra de tecido adenoideano foi positiva na reação de polimerase em cadeia (4%). No grupo controle, houve positividade para H. pylori em 3 amostras de tecido adenoideano (12%). Não houve diferença entre os gêneros. Conclusão: H. pylori é uma das bactérias importantes que desempenham um papel na patogênese da otite médica com efusão. Se o tecido adenoideano pode ou não representar um reservatório para H. pylori ainda necessita ser esclarecido.

Desarrollo de marcadores moleculares del tipo SCAR para la identificación de Azospirillum brasilense Az39 / Development of sequence characterized amplified región markers for identification of Azospirillum brasilense Az39

Resumen Azospirillum brasilense Az39 es utilizada por empresas productoras de inoculantespara la formulación de bioinsumos en América del Sur desde hace más de 30 a˜nos. Esta cepapuede promover el crecimiento, desarrollo, así como la capacidad de tolerar diferentes tiposde estrés en las plantas inoculadas, lo que determina un aumento de la productividad de culti-vos de interés agronómico. En la actualidad, no existen protocolos en Argentina que permitanconfirmar la identidad de Az39 en productos comerciales a nivel de laboratorios de control decalidad de inoculantes. Por ello, el objetivo de este trabajo fue desarrollar una metodología enbase molecular que permita la identificación certera de A. brasilense Az39. Con la secuenciacompleta del genoma y mediante herramientas bioinformáticas, se pudieron reconocer frag-mentos de ADN presentes únicamente en el genoma de Az39. Se dise˜naron cebadores dirigidosa amplificar por PCR dichas secuencias. Como resultado se observaron los productos específicosúnicamente en la presencia de la cepa de interés. La reacción pudo detectar un título mínimode 105UFC/ml (4,5 ng/l ADN) o de 102UFC/ml (0,88 ng/l ADN) o una concentración mínimade 0,098 ng/l ADN, dependiendo del método de extracción utilizado. Los cebadores fueronevaluados en el análisis de productos comerciales obtenidos del mercado nacional, arrojandoresultados positivos, tanto en muestras directas como así también en pruebas confirmatoriasa partir de colonias aisladas de tales productos. La metodología desarrollada en este trabajo,permite la detección certera de A. brasilense Az39 en cultivos puros o mezclas complejas demicroorganismos.

Abstract Azospirillum brasilense Az39 has been used since more than 30 years by several companies in South America for biofertilizers production. This strain may promote plants growth and development, as well as the ability of inoculated plants to tolerate environmental stresses, which determines an increase in the productivity under field conditions. At present, there are no protocols in Argentina to confirm the identity of Az39 in commercial products; however, such biofertilizers are formulated almost exclusively with this strain. Therefore, the objective of this paper was to develop a molecular methodology that allows the accurate identification of A. brasilense Az39. Using the complete genome sequence and several bioinformatics tools, fragments of DNA present only in the Az39 genome were recognized. A set of PCR primers to amplify these sequences were designed, and the specific products were observed only in the strain of our interest. The sensitivity of the methodology was evaluated, where the strain could be detected up to a titer of 105 CFU/ml (4.5 ng/pl ADN) or 102 CFU/ml (0.88 ng/pl DNA) or in a minimal concentration of 0.098 ng/pl DNA, depending on the DNA extraction methodology used. Primers were tested against direct samples of commercial inoculants and cultures, in both cases there were specifics products, both in direct samples and in confirmatory tests from isolated colonies from those products. The procedure presented in this paper allows the accurate identification of A. brasilense Az39 in pure cultures, mixtures of microorganisms, and commercial biofertilizers.

Rodents as potential reservoirs for Borrelia spp. in northern Chile / Roedores como potenciais reservatórios de Borrelia spp. no norte do Chile

Abstract Small mammals play an essential role in the transmission and maintenance cycles of Borrelia spirochetes. In Chile, recent studies have characterized novel Borrelia genotypes in ticks collected from small mammals, a fact that suggests these vertebrates are hosts for spirochetes from this genus. Considering this evidence, the goal of this study was to determine the presence of Borrelia DNA in small mammals inhabiting northern Chile. In winter of 2018, 58 small mammals were captured in five localities. Blood samples were collected from rodents and DNA was extracted to determine the presence of Borrelia DNA by PCR targeting the flaB gene and rrs-rrlA intergenic spacer (IGS). From three individuals (5%), belonging to two rodent species of Cricetidae family (Phyllotis xanthopygus and Oligoryzomys longicaudatus), we retrieved three flaB and two IGS Borrelia genotypes. Phylogenetic analyses performed with both Maximum Likelihood and Bayesian inferences showed that our sequences grouped with homologous genotypes from the relapsing fever and Lyme borreliosis groups. Our findings suggest that P. xanthopygus and O. longicaudatus rodents may play a role as reservoirs for borrelial spirochetes in Chile.

Resumo Pequenos mamíferos possuem um papel essencial na transmissão e manutenção de espiroquetas do gênero Borrelia. No Chile, estudos recentes têm descrito novos genótipos de Borrelia em carrapatos, parasitando pequenos mamíferos. Isso sugere que esses vertebrados podem atuar como possíveis reservatórios dessas espiroquetas. Considerando-se essa evidência, o objetivo deste estudo foi determinar a presença de DNA de Borrelia em pequenos mamíferos da região norte do Chile. Durante o inverno de 2018, 58 pequenos mamíferos foram capturados em cinco localidades. Amostras de sangue obtidas a partir dos indivíduos capturados foram submetidas à extração de DNA e ensaios de PCR, para a detecção de Borrelia spp. baseados no gene flaB e espaçador intergênico rrs-rrlA (IGS). A partir de três espécimes (5%) pertencentes a duas espécies de roedores da família Cricetidae (Phyllotis xanthopygus e Oligoryzomys longicaudatus) obtiveram-se três genótipos de Borrelia para o gene flaB e dois para IGS. Análises filogenéticas inferidas, usando-se os métodos Bayesiano e de Máxima Verossimilhança, indicaram que as sequências geradas neste estudo agrupam-se com borrelias do grupo da Febre Recorrente e Borreliose de Lyme. Os achados deste estudo sugerem que roedores P. xanthopygus e O. longicaudatus poderiam atuar como possíveis reservatórios para Borrelia spp. no Chile.

Qualitative and quantitative molecular analysis of bacteria in root canals of primary teeth with pulp necrosis

Abstract Information about bacterial diversity, such as the number of each species in the root canals of primary teeth, contributes to improving our effective management of infections of endodontic origin in primary teeth. This study made a qualitative and quantitative assessment of the bacteria in the root canals of primary teeth with necrotic pulp, using the fluorescence in situ hybridization (FISH) technique. Thirty-one primary teeth with pulp necrosis from 31 children were evaluated using the FISH technique, to detect the presence and density of Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Enterococcus faecalis, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Streptococcus, Streptococcus mutans, Streptococcus sobrinus, Tannerella forsythia and Treponema denticola. Descriptive measures explained the data related to density, and Student's t-test assessed the differences among the densities of each bacterium, according to signs and symptoms. The bacterial density was paired and correlated. All bacteria tested were detected and identified in all the samples. The average number of bacterial individuals from each species ranged from 1.9 x 108 cells/mL (S. mutans) to 3.1 x 108 cells/mL (F. nucleatum) (p > 0.05). The sum of the mean counts of each bacterium represented almost 80% of the entire microbial community. Patients with pain had significantly more T. denticola, and those with edema showed a greater density of Streptococcus and P. nigrescens (p < 0.05). This study revealed that all 12 bacteria evaluated were found in all primary teeth with pulp necrosis. There was no predominance among the species studied; all species had a similar number of individuals.

Gene expression profile of Scardovia spp. in the metatranscriptome of root caries

Abstract A few investigations of caries biofilms have identified Scardovia spp.; however, little is known about its involvement in caries pathogenesis. The purpose of this study was to assess the gene expression profile of Scardovia spp. in root caries, and compare it with other microorganisms. Clinical samples from active root caries lesions were collected. Microbial mRNA was isolated and cDNA sequenced. The function and composition of the Scardovia were investigated using two methods: a) de novo assembly of the read data and mapping to contigs, and b) reads mapping to reference genomes. Pearson correlation was performed (p < 0.05). Proportion of Scardovia inopinata and Scardovia wiggsiae sequences ranged from 0-6% in the root caries metatranscriptome. There was a positive correlation between the transcriptome of Lactobacillus spp. and Scardovia spp. (r = 0.70; p = 0.03), as well as with other Bifidobacteriaceae (r = 0.91; p = 0.0006). Genes that code for fructose 6-phosphate phosphoketolase (the key enzyme for "Bifid shunt"), as well as ABC transporters and glycosyl-hydrolases were highly expressed. In conclusion, "Bifid shunt" and starch metabolism are involved in carbohydrate metabolism of S. inopinata and S. wiggsiae in root caries. There is a positive correlation between the metabolism abundance of Lactobacillus spp., Bifidobacteriaceae members, and Scardovia in root caries.

Prioritisation of potential drug targets against Bartonella bacilliformis by an integrative in-silico approach

BACKGROUND Carrion's disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis. METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery. FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion's disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.

Identification of plasmid IncQ1 and NTEKPC-IId harboring bla KPC-2 in isolates from Klebsiella pneumoniae infections in patients from Recife-PE, Brazil

Abstract INTRODUCTION: This study investigated the genetic environment of bla KPC-2 in Klebsiella pnemoniae multi-drug resistant clinical isolates. METHODS: Four carbapenemase gene isolates resistant to carbapenems, collected from infected patients from two hospitals in Brazil, were investigated using polymerase chain reaction and plasmid DNA sequencing. RESULTS: The bla KPC-2 gene was located between ISKpn6 and a resolvase tnpR in the non-Tn4401 element (NTEKPC-IId). It was detected on a plasmid belonging to the IncQ1 group. CONCLUSIONS To our knowledge, this is the first report of the presence of the bla KPC-2 gene in the NTEKPC-IId element carried by plasmid IncQ1 from infections in Brazil.

Prevalence of Klebsiella pneumoniae carbapenemase - and New Delhi metallo-beta-lactamase-positive K. pneumoniae in Sergipe, Brazil, and combination therapy as a potential treatment option

Abstract INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae infection lacks treatment options and is associated with prolonged hospital stays and high mortality rates. The production of carbapenemases is one of the most important factors responsible for this multi-resistance phenomenon. METHODS: In the present study, we analyzed the presence of genes encoding carbapenemases in K. pneumoniae isolates circulating in one of the public hospitals in the city of Aracaju, Sergipe, Brazil. We also determined the best combination of drugs that display in vitro antimicrobial synergy. First, 147 carbapenem-resistant K. pneumoniae isolates were validated for the presence of blaKPC, bla GES, bla NDM, bla SPM, bla IMP, bla VIM, and bla OXA-48 genes using multiplex polymerase chain reaction. Thereafter, using two isolates (97 and 102), the role of double and triple combinational drug therapy as a treatment option was analyzed. RESULTS: Seventy-four (50.3%) isolates were positive for bla NDM, eight (5.4%) for bla KPC, and one (1.2%) for both bla NDM and bla KPC. In the synergy tests, double combinations were better than triple combinations. Polymyxin B and amikacin for isolate 97 and polymyxin B coupled with meropenem for isolate 102 showed the best response. CONCLUSIONS: Clinicians in normal practice use multiple drugs to treat infections caused by multi-resistant microorganism; however, in most cases, the benefit of the combinations is unknown. In vitro synergistic tests, such as those described herein, are important as they might help select an appropriate multi-drug antibiotic therapy and a correct dosage, ultimately reducing toxicities and the development of antibiotic resistance.