Article pubs.acs.org/ac
Phage Amplification and Immunomagnetic Separation Combined with Targeted Mass Spectrometry for Sensitive Detection of Viable Bacteria in Complex Food Matrices Armelle Martelet,†,‡ Guillaume L’Hostis,†,‡ Marie-Claire Nevers,§ Hervé Volland,§ Christophe Junot,‡ François Becher,*,‡ and Bruno H. Muller*,†,‡ †
bioMérieux S.A., chemin de l’orme, 69280 Marcy-l’Etoile, France CEA, iBiTec-S, SPI, Laboratoire d’Etude du Métabolisme des Médicaments (LEMM), 91191 Gif-sur-Yvette, France § CEA, iBiTec-S, SPI, Laboratoire d’Etudes et de Recherches en Immunoanalyse (LERI), 91191 Gif-sur-Yvette, France ‡
S Supporting Information *
ABSTRACT: We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli 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 E. coli 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 E. coli 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.
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in harsh environmental conditions.5 As an illustration, Hsu et al.6 used a real-time PCR assay for the sensitive detection of Escherichia coli O157:H7 in milk and in apple juice samples. After a 4-h enrichment phase, the sensitivity for the detection of E. coli O157 in milk samples (devoid of inhibitors) was 1 cfu/ mL, whereas a 10-h enrichment period was required to obtain the same sensitivity in apple juice samples (containing inhibitors). Another major drawback of PCR-based methods is that they do not discriminate between dead and living microorganisms, thus potentially increasing the number of false positive results.7,8 On the other hand, immunoassays are based on the highly specific interaction between antibody and antigen. The targeted antigen may be a component of the pathogen, such as a cell or flagellar antigen, or a product of the bacteria (e.g., virulence determinants).9 For instance, enzyme linked immunosorbent assays (ELISAs) have been successfully used to detect pathogens in less than 2 h such as 8 × 103 cfu/mL
n the United States, foodborne diseases have been estimated to cause 76 million illnesses and up to 9000 deaths each year.1 Global incidence of foodborne illness is increasing, and the food industry is the main party concerned with the presence of pathogenic microorganisms, where failure to detect a pathogen may lead to a dreadful effect.2 Standardized methods (e.g., ISO methods) are acknowledged as the reference analytical methods for official control. Among these standardized methods, culture-based methods are considered to be the “gold standard” for detection of foodborne pathogens, as they are able to detect a single target cell in the sample. The general approach consists of the time-consuming steps of enrichment culture (1−4 days), confirmation, and strain typing (up to a week).3 A new generation of innovative methods developed in recent decades significantly reduces the time to result in the context of rapid screening and associated batch release of negative samples.4 For instance, the enrichment culture has been replaced by more rapid immunological or nucleic acid amplification-based assays.3 Although powerful, the latter are prone to false-negative results because of enzymatic inhibitors © XXXX American Chemical Society
Received: December 4, 2014 Accepted: May 1, 2015
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DOI: 10.1021/ac504508a Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry Salmonella enterica serovar Typhimurium10 and 104−105 cfu/ mL Escherichia coli O157:H7 among others.11 The potential of recombinant binding proteins from bacteriophages (phages) as a replacement for antibodies has also been demonstrated12 and commercialized (i.e., the VIDAS UP E. coli test).13 Phages are bacterial viruses highly specific to their target hosts, which is particularly interesting in the case where foodborne pathogens are mostly present in very low numbers (
