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

IgE reactivity of recombinant Der f 36 and Der p 36.

<p>Recombinant non-glycosylated Der f 36 and Der p 36 were produced in <i>P</i>. <i>pastoris</i>. IgE reactivity was assessed by western blot using a pool of sera from HDM-sensitized individuals. Culture supernatant from a mock strain was used as a negative control.</p

Functional analysis of proteomes from HDM bodies and feces.

<p>Proteins identified by LC-MS/MS in fractionated extracts from either body (left panel) or feces (right panel) were classified into functional categories using the KAAS server (details of protein identification are provided in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185830#pone.0185830.s005" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185830#pone.0185830.s006" target="_blank">S3</a> Tables). The histograms denote the numbers of occurrences of KEGG Orthology (KO) annotations for each of <i>D</i>. <i>farinae</i> (A, B) and <i>D</i>. <i>pteronyssinus</i> (C, D) species, assembled in functional categories.</p

A combined transcriptome and proteome analysis extends the allergome of house dust mite <i>Dermatophagoides</i> species

<div><p>Background</p><p>House dust mites (HDMs) such as <i>Dermatophagoides farinae</i> and <i>D</i>. <i>pteronyssinus</i> represent major causes of perennial allergy. HDM proteomes are currently poorly characterized, with information mostly restricted to allergens. As of today, 33 distinct allergen groups have been identified for these 2 mite species, with groups 1 and 2 established as major allergens. Given the multiplicity of IgE-reactive mite proteins, potential additional allergens have likely been overlooked.</p><p>Objective</p><p>To perform a comprehensive characterization of the transcriptomes, proteomes and allergomes of <i>D</i>. <i>farinae</i> and <i>D</i>. <i>pteronyssinus</i> in order to identify novel allergens.</p><p>Methods</p><p>Transcriptomes were analyzed by RNA sequencing and <i>de novo</i> assembly. Comprehensive mass spectrometry-based analyses proteomes were combined with two-dimensional IgE reactivity profiling.</p><p>Results</p><p>Transcripts from <i>D</i>. <i>farinae</i> and <i>D</i>. <i>pteronyssinus</i> were assembled, translated into protein sequences and used to populate derived sequence databases in order to inform immunoproteomic analyses. A total of 527 and 157 proteins were identified by bottom-up MS analyses in aqueous extracts from purified HDM bodies and fecal pellets, respectively. Based on high sequence similarities (>71% identity), we also identified 2 partial and 11 complete putative sequences of currently undisclosed <i>D</i>. <i>pteronyssinus</i> counterparts of <i>D</i>. <i>farinae</i> registered allergens. Immunoprofiling on 2D-gels revealed the presence of unknown 23 kDa IgE reactive proteins in both species. Following expression of non-glycosylated recombinant forms of these molecules, we confirm that these new allergens react with serum IgEs from 42% (8/19) of HDM-allergic individuals.</p><p>Conclusions</p><p>Using combined transcriptome and immunoproteome approaches, we provide a comprehensive characterization of <i>D</i>. <i>farinae</i> and <i>D</i>. <i>pteronyssinus</i> allergomes. We expanded the known allergen repertoire for <i>D</i>. <i>pteronyssinus</i> and identified two novel HDM allergens, now officially referred by the International Union of Immunological Societies (IUIS) Nomenclature Subcommittee as Der f 36 and Der p 36.</p></div

Experimental workflow of HDM transcriptome, proteome and allergome analyses.

<p>Messenger RNAs from <i>D</i>. <i>farinae</i> and <i>D</i>. <i>pteronyssinus</i> were sequenced using next generation sequencing. Following <i>de novo</i> assembly, translated sequence databases were derived after coding sequences (CDS) prediction and used as references to identify proteins in aqueous extracts from mite bodies and feces by LC-MS/MS analysis. In parallel, extracts from whole cultures were submitted to 2D-gel electrophoresis to establish reference 2D maps of species-specific proteomes and assign IgE reactivity.</p

Proteome and IgE reactivity maps of <i>Dermatophagoides</i> species.

<p>Water-soluble HDM proteins were separated by 2D-gel electrophoresis and stained with Sypro Ruby or probed with a pool of serum IgEs from HDM-sensitized donors to establish 2D proteome (left panel) and IgE reactivity (right panel) maps for <i>D</i>. <i>farinae</i> (A, B) and <i>D</i>. <i>pteronyssinus</i> (C, D) species. Protein spots were picked and analyzed by LC-MS/MS after trypsin digestion, using the transcriptome-derived protein sequence collection supplemented with allergen sequences registered in the WHO/IUIS allergen database in order to facilitate identification. Spots corresponding to novel allergens (<i>i</i>. <i>e</i>. Der f 36 and Der p 36) are circled.</p

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 × 10⁵, 5 × 10⁵, and 1 × 10⁶ 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