6 research outputs found
Annotation of the <i>Staphylococcus aureus</i> Metabolome Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry and Application to the Study of Methicillin Resistance
<i>Staphylococcus aureus</i> 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 <i>S. aureus</i> 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<i> S. aureus</i> metabolites
as possible. Overall, we were able to characterize up to 210 metabolites
in <i>S. aureus</i>, 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 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. <i>E. coli</i> at 1 × 10<sup>5</sup>, 5 × 10<sup>5</sup>, and 1 × 10<sup>6</sup> 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
Data_Sheet_1_An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS).DOC
<p>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<sup>®</sup>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<sup>®</sup> 3D (SA/SN blood culture bottles) or the BacT/ALERT Virtuo<sup>TM</sup> 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<sup>®</sup> 3D and of 84% (Gram-negative bacteria = 86%, Gram-positive bacteria = 86%, and yeast = 75%) with Virtuo<sup>®</sup> 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.</p
Data_Sheet_2_An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS).DOC
<p>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<sup>®</sup>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<sup>®</sup> 3D (SA/SN blood culture bottles) or the BacT/ALERT Virtuo<sup>TM</sup> 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<sup>®</sup> 3D and of 84% (Gram-negative bacteria = 86%, Gram-positive bacteria = 86%, and yeast = 75%) with Virtuo<sup>®</sup> 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.</p
Presentation_1_An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS).ppt
<p>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<sup>®</sup>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<sup>®</sup> 3D (SA/SN blood culture bottles) or the BacT/ALERT Virtuo<sup>TM</sup> 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<sup>®</sup> 3D and of 84% (Gram-negative bacteria = 86%, Gram-positive bacteria = 86%, and yeast = 75%) with Virtuo<sup>®</sup> 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.</p