Comparison of DNA extraction methods for 16S rRNA gene sequencing in the analysis of the human gut microbiome.

The gut microbiome is widely analyzed using high-throughput sequencing, such as 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing (SMS). DNA extraction is known to have a large impact on the metagenomic analyses. The aim of this study was to compare DNA extraction protocols for 16S sequencing. In that context, four commonly used DNA extraction methods were compared for the analysis of the gut microbiota. Commercial versions were evaluated against modified protocols using a stool preprocessing device (SPD, bioMérieux) upstream DNA extraction. Stool samples from nine healthy volunteers and nine patients with a Clostridium difficile infection were extracted with all protocols and 16S sequenced. Protocols were ranked using wet- and dry-lab criteria, including quality controls of the extracted genomic DNA, alpha-diversity, accuracy using a mock community of known composition and repeatability across technical replicates. SPD improved overall efficiency of three of the four tested protocols compared with their commercial version, in terms of DNA extraction yield, sample alpha-diversity, and recovery of Gram-positive bacteria. The best overall performance was obtained for the S-DQ protocol, SPD combined with the DNeasy PowerLyser PowerSoil protocol from QIAGEN. Based on this evaluation, we strongly believe that the use of such stool preprocessing device improves both the standardization and the quality of the DNA extraction in the human gut microbiome studies.

Contribution of Clinical Metagenomics to the Diagnosis of Bone and Joint Infections.

Bone and joint infections (BJIs) are complex infections that require precise microbiological documentation to optimize antibiotic therapy. Currently, diagnosis is based on microbiological culture, sometimes complemented by amplification and sequencing of the 16S rDNA gene. Clinical metagenomics (CMg), that is, the sequencing of the entire nucleic acids in a sample, was previously shown to identify bacteria not detected by conventional methods, but its actual contribution to the diagnosis remains to be assessed, especially with regard to 16S rDNA sequencing. In the present study, we tested the performance of CMg in 34 patients (94 samples) with suspected BJIs, as compared to culture and 16S rDNA sequencing. A total of 94 samples from 34 patients with suspicion of BJIs, recruited from two sites, were analyzed by (i) conventional culture, (ii) 16S rDNA sequencing (Sanger method), and (iii) CMg (Illumina Technology). Two negative controls were also sequenced by CMg for contamination assessment. Based on the sequencing results of negative controls, 414 out of 539 (76.7%) bacterial species detected by CMg were considered as contaminants and 125 (23.2%) as truly present. For monomicrobial infections (13 patients), the sensitivity of CMg was 83.3% as compared to culture, and 100% as compared to 16S rDNA. For polymicrobial infections (13 patients), the sensitivity of CMg was 50% compared to culture, and 100% compared to 16S rDNA. For samples negative in culture (8 patients, 21 samples), CMg detected 11 bacteria in 10 samples from 5 different patients. In 5/34 patients, CMg brought a microbiological diagnosis where conventional methods failed, and in 16/34 patients, CMg provided additional information. Finally, 99 antibiotic resistance genes were detected in 24 patients (56 samples). Provided sufficient genome coverage (87.5%), a correct inference of antibiotic susceptibility was achieved in 8/8 bacteria (100%). In conclusion, our study demonstrated that the CMg provides complementary and potentially valuable data to conventional methods of BJIs diagnosis.

Metabolomic Investigation of Staphylococcus aureus Antibiotic Susceptibility by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Staphylococcus aureus is a major human pathogen that can readily acquire antibiotic resistance. For instance, methicillin-resistant S. aureus represents a major cause of hospital- and community-acquired bacterial infections. In this chapter, we first provide a detailed protocol for obtaining unbiased and reproducible S. aureus metabolic profiles. The resulting intracellular metabolome is then analyzed in an untargeted manner by using both hydrophilic interaction liquid chromatography and pentafluorophenyl-propyl columns coupled to high-resolution mass spectrometry. Such analyses are done in conjunction with our in-house spectral database to identify with high confidence as many meaningful S. aureus metabolites as possible. Under these conditions, we can routinely monitor more than 200 annotated S. aureus metabolites. We also indicate how this protocol can be used to investigate the metabolic differences between methicillin-resistant and susceptible strains.

An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS).

Sepsis is the leading cause of death among patients in intensive care units (ICUs) requiring an early diagnosis to introduce efficient therapeutic intervention. Rapid identification (ID) of a causative pathogen is key to guide directed antimicrobial selection and was recently shown to reduce hospitalization length in ICUs. Direct processing of positive blood cultures by MALDI-TOF MS technology is one of the several currently available tools used to generate rapid microbial ID. However, all recently published protocols are still manual and time consuming, requiring dedicated technician availability and specific strategies for batch processing. We present here a new prototype instrument for automated preparation of Vitek®MS slides directly from positive blood culture broth based on an "all-in-one" extraction strip. This bench top instrument was evaluated on 111 and 22 organisms processed using artificially inoculated blood culture bottles in the BacT/ALERT® 3D (SA/SN blood culture bottles) or the BacT/ALERT VirtuoTM system (FA/FN Plus bottles), respectively. Overall, this new preparation station provided reliable and accurate Vitek MS species-level identification of 87% (Gram-negative bacteria = 85%, Gram-positive bacteria = 88%, and yeast = 100%) when used with BacT/ALERT® 3D and of 84% (Gram-negative bacteria = 86%, Gram-positive bacteria = 86%, and yeast = 75%) with Virtuo® instruments, respectively. The prototype was then evaluated in a clinical microbiology laboratory on 102 clinical blood culture bottles and compared to routine laboratory ID procedures. Overall, the correlation of ID on monomicrobial bottles was 83% (Gram-negative bacteria = 89%, Gram-positive bacteria = 79%, and yeast = 78%), demonstrating roughly equivalent performance between manual and automatized extraction methods. This prototype instrument exhibited a high level of performance regardless of bottle type or BacT/ALERT system. Furthermore, blood culture workflow could potentially be improved by converting direct ID of positive blood cultures from a batch-based to real-time and "on-demand" process.

Annotation of the Staphylococcus aureus Metabolome Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry and Application to the Study of Methicillin Resistance.

Staphylococcus aureus can cause a variety of severe disease patterns and can readily acquire antibiotic resistance; however, the mechanisms by which this commensal becomes a pathogen or develops antibiotic resistance are still poorly understood. Here we asked whether metabolomics can be used to distinguish bacterial strains with different antibiotic susceptibilities. Thus, an efficient and robust method was first thoroughly implemented to measure the intracellular metabolites of S. aureus in an unbiased and reproducible manner. We also placed special emphasis on metabolome coverage and annotation and used both hydrophilic interaction liquid chromatography and pentafluorophenyl-propyl columns coupled to high-resolution mass spectrometry in conjunction with our spectral database developed in-house to identify with high confidence as many meaningful S. aureus metabolites as possible. Overall, we were able to characterize up to 210 metabolites in S. aureus, which represents a substantial ∼50% improvement over previously published data. We then preliminarily compared the metabolic profiles of 10 clinically relevant methicillin-resistant and susceptible strains harvested at different time points during the exponential growth phase (without any antibiotic exposure). Interestingly, the resulting data revealed a distinct behavior of "slow-growing" resistant strains, which show modified levels of several precursors of peptidoglycan and capsular polysaccharide biosynthesis.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60-80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

Rapid discrimination of bacteria by paper spray mass spectrometry.

Paper spray mass spectrometry ambient ionization is utilized for rapid discrimination of bacteria without sample preparation. Bacterial colonies were smeared onto filter paper precut to a sharp point, then wetted with solvent and held at a high potential. Charged droplets released by field emission were sucked into the mass spectrometer inlet and mass spectra were recorded. Sixteen different species representing eight different genera from Gram-positive and Gram-negative bacteria were investigated. Phospholipids were the predominant species observed in the mass spectra in both the negative and positive ion modes. Multivariate data analysis based on principal component analysis, followed by linear discriminant analysis, allowed bacterial discrimination. The lipid information in the negative ion mass spectra proved useful for species level differentiation of the investigated Gram-positive bacteria. Gram-negative bacteria were differentiated at the species level by using a numerical data fusion strategy of positive and negative ion mass spectra.

Bacterial detection using unlabeled phage amplification and mass spectrometry through structural and nonstructural phage markers.

According to the World Health Organization, food safety is an essential public health priority. In this context, we report a relevant proof of feasibility for the indirect specific detection of bacteria in food samples using unlabeled phage amplification coupled to ESI mass spectrometry analysis and illustrated with the model phage systems T4 and SPP1. High-resolving power mass spectrometry analysis (including bottom-up and top-down protein analysis) was used for the discovery of specific markers of phage infection. Structural components of the viral particle and nonstructural proteins encoded by the phage genome were identified. Then, targeted detection of these markers was performed on a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. E. coli at 1 × 10(5), 5 × 10(5), and 1 × 10(6) CFU/mL concentrations was successfully detected after only a 2 h infection time by monitoring phage T4 structural markers in Luria-Bertani broth, orange juice, and French bean stew ("cassoulet") matrices. Reproducible detection of nonstructural markers was also demonstrated, particularly when a high titer of input phages was required to achieve successful amplification. This strategy provides a highly time-effective and sensitive assay for bacterial detection.

Nonstructural protein NS1 immunodominant epitope detected specifically in dengue virus infected material by a SELDI-TOF/MS based assay.

Dengue virus (DV) infection is the most common mosquito-born viral disease of public health significance. Though most patients only suffer from flu-like symptoms, a small group of patients experiences more severe forms of the disease. The viral nonstructural protein 1 (NS1), a secreted protein correlating with viremia, is a key element used for dengue diagnosis with potential implications in severe dengue prognosis. Capture-ELISAs for the early detection of the NS1 protein in the sera during the acute febrile stage are commonly used in routine by diagnostic laboratories. In this study, the detection of NS1 protein in DV-infected material was assessed by an alternative method combining a single NS1-directed monoclonal antibody and the SELDI-TOF/MS technology. According to the epitope mapping, the antibodies used are mainly directed against an immuno-dominant peptide located on the C-terminal part of the protein. The NS1 SELDI-TOF assay is specific, has a sensitivity level close to capture-ELISAs and is potentially useful for a coupled serotyping/detection assay or for the detection of subtle post-translational modifications on the protein.

Antigenicity of recombinant proteins after regioselective immobilization onto polyanhydride-based copolymers.

We previously demonstrated that the introduction of a tag consisting of several contiguous lysines at the N- or C-terminus of a recombinant protein greatly improved the covalent grafting of the protein onto negatively charged maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer, under many different experimental conditions (Ladavière, C., et al. (1998) Bioconjugate Chem. 9, 655; Allard, L., et al. (2002) Biotechnol. Bioeng. 80, 341). The grafting efficiency was dependent on the charge and amine density of the tag, characteristics which were determined by the tag composition. The six lysine tag (Lys6) was found to be the most efficient (Allard, L., et al. (2001) Bioconjugate Chem. 12, 972). In the present work, the biological activity of Lys6-proteins covalently bound to polymer was investigated. N- or C-terminal Lys6-tagged HIV-1 p24 recombinant proteins (RK24H and RH24K) were grafted onto MAMVE, and the antigenicity each of the bioconjugates was evaluated using six monoclonal antibodies that recognized different epitopes distributed along the protein. We demonstrate that the position of the tag and the hydrolysis rate of the anhydride moieties of the polymer are the two main parameters involved in the conservation of the biological activity of the immobilized protein. We thus present a process which allows an efficient oriented immobilization of proteins onto copolymers with optimal biological activity that is suitable for the controlled production of active bioconjugates.

Erratum to "characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris".

The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexa-histidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni2+-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellman's reagent allowed the measurement, respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with, respectively, a diameter of 34 and 28 nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect antibodies to HBcAg (anti-HBc) in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.

Characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris.

The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexahistidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni(2+)-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellman's reagent allowed the measurement respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with respectively, a diameter of 34 and 28-nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect anti-HBc antibodies in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.