A robust, cost-effective method for DNA, RNA and protein co-extraction from soil, other complex microbiomes and pure cultures.

The soil microbiome is inherently complex with high biological diversity, and spatial heterogeneity typically occurring on the submillimetre scale. To study the microbial ecology of soils, and other microbiomes, biomolecules, that is, nucleic acids and proteins, must be efficiently and reliably co-recovered from the same biological samples. Commercial kits are currently available for the co-extraction of DNA, RNA and proteins but none has been developed for soil samples. We present a new protocol drawing on existing phenol-chloroform-based methods for nucleic acids co-extraction but incorporating targeted precipitation of proteins from the phenol phase. The protocol is cost-effective and robust, and easily implemented using reagents commonly available in laboratories. The method is estimated to be eight times cheaper than using disparate commercial kits for the isolation of DNA and/or RNA, and proteins, from soil. The method is effective, providing good quality biomolecules from a diverse range of soil types, with clay contents varying from 9.5% to 35.1%, which we successfully used for downstream, high-throughput gene sequencing and metaproteomics. Additionally, we demonstrate that the protocol can also be easily implemented for biomolecule co-extraction from other complex microbiome samples, including cattle slurry and microbial communities recovered from anaerobic bioreactors, as well as from Gram-positive and Gram-negative pure cultures.

Características moleculares de Staphylococcus aureus susceptível à vancomicina poderia ajudar a prever falhas no tratamento devido a reduzida susceptibilidade à vancomicina / Molecular characteristics of vancomycin-susceptible Staphylococcus aureus could help to predict treatment failure due to reduced vancomycin susceptibility / Características moleculares de Staphylococcus aureus susceptible a la vancomicina podría ayudar a predecir fallas en el tratamiento debido a la reducida susceptibilidad a la vancomicina

Justificativa e Objetivos: Staphylococcus aureus resistente à meticilina (MRSA) é uma das causas mais frequentes de infecções relacionadas à assistência à saúde e comunitárias, e com seu avanço, a vancomicina tornou-se a principal opção terapêutica. Entretanto, o seu uso indiscriminado favoreceu o surgimento de MRSA com reduzida suscetibilidade à vancomicina, comumente associados com falhas no tratamento, bacteremia persistente, hospitalização prolongada e desfechos clínicos adversos. Este estudo avaliou a ocorrência de MRSA com reduzida suscetibilidade à vancomicina e determinou algumas características moleculares em comparação com MRSA suscetível à vancomicina (VS-MRSA). Métodos: Determinação do perfil de suscetibilidade aos antimicrobianos, a concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) para vancomicina, tolerância à vancomicina, tipagem do SCCmec e agr foram realizadas em um total de 177 MRSA. Posteriormente, foram triados para hVISA por BHIA-3V e BHIA-6V e confirmados com a Análise do Perfil Populacional - Área Abaixo da Curva (PAP-AUC). Resultados: Os fenótipos VT-MRSA e hVISA foram encontrados em 13,6% e 5,1% dos isolados clínicos de MRSA, respectivamente, e a presença de hVISA foi estatisticamente significativa entre os isolados de VT-MRSA (p<0,05). Em VT-MRSA, SCCmec tipo II foi significativamente mais frequente do que em não-VT-MRSA, assim como a presença do agr grupo II. Conclusão: Características moleculares encontradas em MRSA são importantes para a epidemiologia, bem como para demonstrar um padrão em isolados com reduzida suscetibilidade à vancomicina. Testes não-convencionais para detecção destas características podem ser realizados para evitar a identificação errada de VS-MRSA que, consequentemente, resulta em falhas no tratamento com vancomicina.(AU)

Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of healthcare-associated and community-acquired infections and with its advancement, vancomycin became the main therapeutic option. However, its indiscriminate use favored the emergence of MRSA with reduced susceptibility to vancomycin, commonly associated with vancomycin treatment failure, persistent bacteremia, prolonged hospitalization and adverse clinical outcome. This study evaluated the occurrence of MRSA with reduced vancomycin susceptibility and determined some molecular characteristics in comparison with vancomycin-susceptible MRSA (VS-MRSA). Methods: Determination of antimicrobial susceptibility profile, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for vancomycin, vancomycin-tolerance, SCCmec and agr typing were performed in a total of 177 MRSA. Thereafter, they were screened for hVISA by BHIA-3V and BHIA-6V and confirmed with population analysis profile - area under the curve method (PAP-AUC). Results: VT-MRSA and hVISA phenotypes were found in 13.6% and 5.1% of clinical isolates of MRSA, respectively, and the presence of hVISA was statistically significant among VT-MRSA isolates (p<0.05). In T-MRSA, SCCmec type II was significantly more frequent than in non-VT-MRSA, as well as the presence of agr group II. Conclusion: Molecular characteristics found in MRSA are important for epidemiology, as well as demonstrate a pattern in reduced vancomycin susceptibility isolates. Non-conventional tests for detection of these characteristics might be performed to prevent misidentification of VS-MRSA that, consequently, results in vancomycin treatment failures.(AU)

Justificación y objetivos: Staphylococcus aureus resistente a la meticilina (MRSA) es una de las causas más frecuentes de infecciones relacionadas con la asistencia sanitaria y comunitarias, y con su avance, a la vancomicina se ha convertido en la principal opción terapéutica. Sin embargo, su uso indiscriminado favoreció el surgimiento de MRSA con reducida susceptibilidad a la vancomicina, comúnmente asociados con fallas en el tratamiento, bacteriemia persistente, hospitalización prolongada y resultados clínicos adversos. Este estudio evaluó la ocurrencia de MRSA con reducida susceptibilidad a la vancomicina y determinó algunas características moleculares en comparación con MRSA susceptible a la vancomicina (VS-MRSA). Métodos: Determinación del perfil de susceptibilidad a los antimicrobianos, la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM) para vancomicina, tolerancia a la vancomicina, tipificación del SCCmec y agr se realizaron en un total de 177 MRSA. Resultados: Los fenotipos VT-MRSA y hVISA se encontraron en el 13,6% y el 5,1% de los aislados clínicos de MRSA, respectivamente, y la presencia de hVISA fue estadísticamente significativa entre los aislados de VT-MRSA (p<0.05). En VT-MRSA, SCCmec tipo II fue significativamente más frecuente que en no-VT-MRSA, así como la presencia del agr grupo II. Conclusión: Características moleculares encontradas en MRSA son importantes para la epidemiología, así como para demostrar un patrón en aislados con reducida susceptibilidad a la vancomicina. Pruebas no convencionales para la detección de estas características pueden realizarse para evitar la identificación errónea de VS-MRSA que, consecuentemente, resulta en fallas en el tratamiento con vancomicina.(AU)

Evaluation of a selective chromogenic medium for detecting vancomycin-resistant enterococci

ABSTRACT Rapid identification of vancomycin-resistant enterococci (VRE) can assist in choosing the appropriate treatment and preventing VRE spread. The performance of chromIDTM VRE agar was evaluated using 184 clinical isolates of Enterococcus spp. and reference strains. The test had a sensitivity of 95.52% but a low specificity of 30%.

Evaluation of a selective chromogenic medium for detecting vancomycin-resistant enterococci.

Rapid identification of vancomycin-resistant enterococci (VRE) can assist in choosing the appropriate treatment and preventing VRE spread. The performance of chromID™ VRE agar was evaluated using 184 clinical isolates of Enterococcus spp. and reference strains. The test had a sensitivity of 95.52% but a low specificity of 30%.

Draft Genome Sequence of Enterococcus faecalis Strain F165 Isolated from a Urinary Tract Infection.

We report here a draft genome sequence of Enterococcus faecalis strain F165 isolated from a urine specimen in South Brazil. The genome size was 3,049,734 bp, with a G+C content of 37.38%, and genes related to antimicrobial resistance and adherence were found in the strain. These findings are consistent with pathogenesis of E. faecalis species.

Draft Genome Sequence of Brazilian Escherichia coli Uropathogenic Strain E2.

Escherichia coli is a common pathogen recovered from cystitis infections. In this report, we announce the draft genome sequence of strain E2 isolated from the urine specimen from a female patient in South Brazil. The genome assembly has 5,081,209 bp, a G+C content of 50.57%, and virulence factors associated with both enteroaggregative and uropathogenic E. coli strains.

Is prediffusion test an alternative to improve accuracy in screening hVISA strains and to detect susceptibility to glycopeptides/lipopeptides?

The characterization of heteroresistant vancomycin-intermediate Staphylococcus aureus strains (hVISA) is even more challenging, as no routine standardized laboratory methods are available. A total of 124 S. aureus isolates recovered from inpatients attended in hospitals of Santa Catarina State, Southern Brazil, were evaluated. The MIC of vancomycin, teicoplanin, and daptomycin was determined by Etest and prediffusion tests using NeoSensitabs® tablets. All isolates were susceptible to vancomycin (MICs: 0.5-3 µg/mL) by Etest. However, according to prediffusion test, 17 isolates presented reduced susceptibility to vancomycin, and of these, 12 were confirmed as hVISA using populational analysis. Considering daptomycin, prediffusion results were in agreement with susceptibility data (MICs), as all isolates were susceptible. Considering that characterizing hVISA is challenging and that MIC determination is not adequate to characterize this phenotype, prediffusion test was a viable alternative to screening hVISA and reduced susceptibility to vancomycin. It was simple and low cost, with accuracy comparable to other well-established methods.

In vitro antibacterial activity of tigecycIine against clinical isolates of linezolid-Intermediate and linezolid-resistant enterococci by time-kill assay

Introduction: Enterococci have become the third leading cause of nosocomial bacteraemia, an infection which is significantly associated with the risk of developing infective endocarditis. Linezolid provides high rates of clinical cure and microbiologic success in complicated infections due to Enterococcus spp. However, several instances of emergence of resistance during linezolid treatment have been reported. The aim of this study was evaluate the activity of tigecycline against linezolidintermediate (LIE) and linezolid-resistant enterococcus faecalis(LRE) by the timekill assay. Methods: Five isolates of LRE and two isolates of LIE were used in this study. Minimum inhibitory concentration (MIC) was determined by broth dilution following the guidelines from the Clinical and Laboratory Standards Institute (CLSI). Time-kill assay was employed to access the in vitro response profile of tigecycline. Results: All seven isolates presented MIC of 0.125 ìg/mL. Tigecycline activity was individually evaluated according to CLSI criteria. This antibiotic showed bactericidal activity against three of the five isolates of LRE and bacteriostatic activity against the other isolates. Conclusions: Tigecycline presented both bacteriostatic and bactericidal activity against tested isolates, which is an important data that must be considered for new studies.

Rifampicin fails to eradicate mature biofilm formed by methicillin-resistant Staphylococcus aureus.

INTRODUCTION: Antimicrobial activity on biofilms depends on their molecular size, positive charges, permeability coefficient, and bactericidal activity. Vancomycin is the primary choice for methicillin-resistant Staphylococcus aureus (MRSA) infection treatment; rifampicin has interesting antibiofilm properties, but its effectivity remains poorly defined. METHODS: Rifampicin activity alone and in combination with vancomycin against biofilm-forming MRSA was investigated, using a twofold serial broth microtiter method, biofilm challenge, and bacterial count recovery. RESULTS: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration for vancomycin and rifampicin ranged from 0.5 to 1mg/l and 0.008 to 4mg/l, and from 1 to 4mg/l and 0.06 to 32mg/l, respectively. Mature biofilms were submitted to rifampicin and vancomycin exposure, and minimum biofilm eradication concentration ranged from 64 to 32,000 folds and from 32 to 512 folds higher than those for planktonic cells, respectively. Vancomycin (15mg/l) in combination with rifampicin at 6 dilutions higher each isolate MIC did not reach in vitro biofilm eradication but showed biofilm inhibitory capacity (1.43 and 0.56log10 CFU/ml reduction for weak and strong biofilm producers, respectively; p<0.05). CONCLUSIONS: In our setting, rifampicin alone failed to effectively kill biofilm-forming MRSA, demonstrating stronger inability to eradicate mature biofilm compared with vancomycin.

Rifampicin fails to eradicate mature biofilm formed by methicillin-resistant Staphylococcus aureus / Rifampicina falha na erradicação de biofilmes maduros formados por Staphylococcus aureus resistentes à meticilina

INTRODUCTION: Antimicrobial activity on biofilms depends on their molecular size, positive charges, permeability coefficient, and bactericidal activity. Vancomycin is the primary choice for methicillin-resistant Staphylococcus aureus (MRSA) infection treatment; rifampicin has interesting antibiofilm properties, but its effectivity remains poorly defined. METHODS: Rifampicin activity alone and in combination with vancomycin against biofilm-forming MRSA was investigated, using a twofold serial broth microtiter method, biofilm challenge, and bacterial count recovery. RESULTS: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration for vancomycin and rifampicin ranged from 0.5 to 1mg/l and 0.008 to 4mg/l, and from 1 to 4mg/l and 0.06 to 32mg/l, respectively. Mature biofilms were submitted to rifampicin and vancomycin exposure, and minimum biofilm eradication concentration ranged from 64 to 32,000 folds and from 32 to 512 folds higher than those for planktonic cells, respectively. Vancomycin (15mg/l) in combination with rifampicin at 6 dilutions higher each isolate MIC did not reach in vitro biofilm eradication but showed biofilm inhibitory capacity (1.43 and 0.56log10 CFU/ml reduction for weak and strong biofilm producers, respectively; p<0.05). CONCLUSIONS: In our setting, rifampicin alone failed to effectively kill biofilm-forming MRSA, demonstrating stronger inability to eradicate mature biofilm compared with vancomycin.

INTRODUÇÃO: A atividade dos antimicrobianos em biofilmes depende do seu peso molecular, de cargas positivas, coeficiente de permeabilidade e atividade bactericida. Vancomicina é a escolha primária para o tratamento de infecções causadas por Staphylococcus aureus resistentes à meticilina (MRSA) e rifampicina possui interessante propriedade antibiofilme, apesar da sua efetividade ainda ser fracamente definida. MÉTODOS: Foi investigada a atividade da rifampicina sozinha e em combinação com vancomicina frente à MRSA formadores de biofilme, utilizando o método das microplacas com diluição seriada e recuperação bacteriana em biofilme após exposição antimicrobiana. RESULTADOS: Concentração inibitória minima (MIC) e concentração bactericida mínima (MBC) para vancomicina e rifampicina foi de 0,5-1mg/l e 0,008-4mg/l; 1-4mg/l e 0,06-32mg/l, respectivamente. Biofilmes maduros foram expostos à vancomicina e rifampicina, e a concentração mínima para erradicar o biofilme (MBEC) foi 64-32.000 e 32-512 vezes maior do que para células planctônicas, respectivamente. A combinação de vancomicina (15mg/l) com rifampicina (6-diluições maior do que o MIC de cada isolado) não atingiu erradicação do biofilme in vitro, porém apresentou capacidade inibitória do biofilme formado (redução de 1,43 e 0,56log10 UFC/ml para produtores fracos e fortes, respectivamente; p<0,05). CONCLUSÕES: Rifampicina sozinha falhou em efetivamente matar MRSA formadores de biofilme, demonstrando fraca habilidade para erradicação de biofilmes maduros comparado com vancomicina.

Enhancement of antistaphylococcal activities of six antimicrobials against sasG-negative methicillin-susceptible Staphylococcus aureus: an in vitro biofilm model.

This study was designed to evaluate antimicrobial activities against methicillin-susceptible Staphylococcus aureus in both sessile and planktonic forms and to detect genes associated with this biofilm phenotype. Minimal biofilm inhibition and eradication concentrations (MBIC and MBEC, respectively) were determined by an in vitro biofilm model, and icaA, atlA, and sasG genes were detected by polymerase chain reaction. Vancomycin and tigecycline presented better biofilm inhibitory activity (MBIC range: 4-8 µg/mL) (P ≤ 0.05) and lower MBEC/MIC ratios (P ≤ 0.001) than other antimicrobials. All isolates harbored icaA and atlA, whereas sasG was present only in strong biofilm formers (P ≤ 0.05). Interestingly, antimicrobial activities against sasG- weak biofilm formers were significantly higher than those against sasG+ strong biofilm formers (P ≤ 0.05), demonstrating that number of cells in a biofilm matrix affected the antimicrobial activity, which was also variable, and might be associated with specific genetic determinants. To our knowledge, this was the first study reporting the presence of sasG in clinical isolates of S. aureus in South America.