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.

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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 0.56log 10 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.

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.

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

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.

Background. Urinary Tract Infections (UTIs) are among most common infections in humans. The vast majority are caused by Escherichia coli, occasionally responsible for severe clinical manifestations. Although the species frequently adheres and colonizes the bladder mucosa, its reservoir is the host gastrointestinal tract. Therefore, the study was designed to evaluate genomic features for niche adaptation of urinary and gastrointestinal strains of E. coli by data mining approach. Results. In the E. coli strains, the repertoire of genes was higher than those found in previous studies, and the majority of genes associated to primary metabolism did not depend of bacteria niche, with exception of cell cycle-division, cell motility and secondary metabolite metabolism. Urinary tract isolates of E. coli had great density of virulence and resistance genes carried by prophages. Conclusion. The urinary and gastrointestinal strains of E. coli evaluated in the study presented an open pan-genome, with groups of functional annotation genes associated to specific niches. In addition, gastrointestinal isolates of E. coli were demonstrated as important reservoir of resistance genes.

Background. Vancomycin-resistant enterococci (VRE) are common in some hospital settings and their clonal spread has been described in different regions of the world. We determined the antimicrobial susceptibility profile and the clonal relationship of VRE isolates recovered from inpatients at three general hospitals of Porto Alegre, Brazil. Results. Ninety-four VRE were characterized as Enterococcus faecium and exhibited resistance to teicoplanin, ampicillin, ciprofloxacin, and susceptibility to linezolid, quinupristin-dalfopristin and daptomycin. High level resistance to gentamicin was detected in 13.8% of them. All VREfm harbored vanA gene, while 85.1% and 94.7% harbored respectively esp and acm virulence genes. PFGE profile analysis revealed 23 clonal types including 79 isolates, while 15 isolates exhibited unique pattern type, showing a polyclonal distribution of VREfm in Southern Brazil. Conclusion. These findings contribute to the local understanding regarding the characteristics of the circulating VREs in the region.