3 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
Phage Amplification and Immunomagnetic Separation Combined with Targeted Mass Spectrometry for Sensitive Detection of Viable Bacteria in Complex Food Matrices
We
have developed and describe here for the first time a highly
sensitive method for the fast and unambiguous detection of viable <i>Escherichia coli</i> in food matrices. The new approach is based
on using label-free phages (T4), obligate parasites of bacteria, which
are attractive for pathogen detection because of their inherent natural
specificity and ease of use. A specific immunomagnetic separation
was used to capture the progeny phages produced. Subsequently, T4
phage markers were detected by liquid chromatography coupled to targeted
mass spectrometry. Combining the specificity of these three methodologies
is of great interest in developing an alternative to conventional
time-consuming culture-based technologies for the detection of viable
bacteria for industrial applications. First, optimization experiments
with phage T4 spiked in complex matrices (without a phage amplification
event) were performed and demonstrated specific, sensitive, and reproducible
phage capture and detection in complex matrices including Luria–Bertani
broth, orange juice, and skimmed milk. The method developed was then
applied to the detection of <i>E. coli</i> spiked in foodstuffs
(with a phage amplification event). After having evaluated the impact
of infection duration on assay sensitivity, we showed that our assay
specifically detects viable <i>E. coli</i> in milk at an
initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h
infection. This excellent detection limit makes our new approach an
alternative to PCR-based assays for rapid bacterial detection
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