[Show abstract][Hide abstract] ABSTRACT: In this study, the complete CoSYPS Path Food workflow including all steps, namely swab sample enrichment, SYBR®Green qPCR detection of Salmonella spp. and Listeria spp., isolation and confirmation of the detected strain, was validated on beef carcass swabs. To perform the validation, the results of the complete workflow were compared, according to the ISO 16140:2003, with the ISO reference methods for detection, isolation and confirmation of Listeria monocytogenes and Salmonella spp. The results showed that the relative level of detection and the limit of detection of the complete workflow and ISO reference methods are in a range from 2 to 16 CFU/swab for both bacteria. The relative specificity, sensitivity and accuracy identified during this validation were all 100% since the results obtained with the complete CoSYPS Path Food workflow and the ISO reference methods were identical (Cohen's kappa index = 1.00). In addition the complete CoSYPS Path Food workflow is able to provide detection results (negative or presumptive positive) in half the time needed as for the ISO reference methods. These results demonstrate that the performance of the complete CoSYPS Path Food workflow is not only comparable to the ISO reference methods but also provides a faster response for the verification of beef carcasses before commercial distribution.
International Journal of Food Microbiology 01/2015; 192:103–110. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacillus cereus, the Gram-positive and spore-forming ubiquitous bacterium, may cause emesis as the result of food intoxication with cereulide, a heat stable emetic toxin. Rapid determination of cereulide-positive B. cereus isolates is of highest importance due to consequences of this intoxication for human health and life. Here we present a one-day pulsed-field gel electrophoresis for emetic B. cereus isolates, which allows rapid and efficient determination of their genomic relatedness and helps determining the source of intoxication in case of outbreaks caused by these bacilli.This article is protected by copyright. All rights reserved
[Show abstract][Hide abstract] ABSTRACT: Bacillus thuringiensis is an entomopathogenic bacterium that has been used worldwide as an efficient biopesticide. Despite that this bacterium is usually described as an insect pathogen, its lifecycle in the environment is still largely unknown. B. thuringiensis belongs to the Bacillus cereus group of bacteria, which has been associated with many mobile genetic elements such as species-specific temperate or virulent bacteriophages (phages). Temperate (lysogenic) phages are able to establish a long-term relationship with their host, providing, in some cases, novel ecological traits to the bacterial lysogens. Therefore, this work focuses on evaluating the potential influence of temperate tectiviruses GIL01 and GIL16 to the development of different life traits of B. thuringiensis. For this purpose, a B. thuringiensis serovar israelensis plasmid-cured (non-lysogenic) strain was used to establish bacterial lysogens for phages GIL01 and GIL16 and, subsequently, the following life traits were compared among the strains: kinetics of growth, metabolic profiles, antibiotics susceptibility, biofilm formation, swarming motility and sporulation. The results revealed that GIL01 and GIL16 lysogeny has a significant influence on bacterial growth, sporulation rate, biofilm formation and swarming motility of B. thuringiensis. No changes in metabolic profiles or antibiotics susceptibilities were detected. These findings provide evidence that tectiviruses can have a putative role in B. thuringiensis lifecycle as adapters of life traits with ecological advantages.
Applied and Environmental Microbiology 09/2014; 80(24):7620-7630. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidate-resistant mutants.
Journal of Microbiological Methods 08/2014; 106:101–103. · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Salting is one of the oldest means of food preservation: adding salt decreases water activity and inhibits microbial development. However, salt is also a source of living bacteria and archaea. The occurrence and diversity of viable archaea in this extreme environment were assessed in 26 food-grade salts from worldwide origin by cultivation on four culture media. Additionally, metagenomic analysis of 16S rRNA gene was performed on nine salts. Viable archaea were observed in 14 salts and colony counts reached more than 10(5)CFU per gram in three salts. All archaeal isolates identified by 16S rRNA gene sequencing belonged to the Halobacteriaceae family and were related to 17 distinct genera among which Haloarcula, Halobacterium and Halorubrum were the most represented. High-throughput sequencing generated extremely different profiles for each salt. Four of them contained a single major genus (Halorubrum, Halonotius or Haloarcula) while the others had three or more genera of similar occurrence. The number of distinct genera per salt ranged from 21 to 27. Halorubrum had a significant contribution to the archaeal diversity in seven salts; this correlates with its frequent occurrence in crystallization ponds. On the contrary, Haloquadratum walsbyi, the halophilic archaea most commonly found in solar salterns, was a minor actor of the food-grade salt diversity. Our results indicate that the occurrence and diversity of viable halophilic archaea in salt can be important, while their fate in the gastrointestinal tract after ingestion remains largely unknown.
International Journal of Food Microbiology 08/2014; 191C:36-44. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Point-of-care (PoC) diagnostics for bacterial detection offer tremendous prospects for public health care improvement. However, such tools require the complex combination of the following performances: rapidity, selectivity, sensitivity, miniaturization and affordability. To meet these specifications, this paper presents a new selectivity method involving lysostaphin together with a CMOS-compatible impedance sensor for genus-specific bacterial detection. The method enables the sample matrix to be directly flown on the polydopamine-covered sensor surface without any pre-treatment, and considerably reduces the background noise. Experimental proof-of-concept, explored by simulations and confirmed through a setup combining simultaneous optical and electrical real-time monitoring, illustrates the selective and capacitive detection of Staphylococcus epidermidis in synthetic urine also containing Enterococcus faecium. While providing capabilities for miniaturization and system integration thanks to CMOS compatibility, the sensors show a detection limit of ca. 108 (CFU/mL).min in a 1.5 μL microfluidic chamber with an additional setup time of 50 min. The potentials, advantages and limitations of the method are also discussed.
[Show abstract][Hide abstract] ABSTRACT: Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here.
[Show abstract][Hide abstract] ABSTRACT: The propidium monoazide (PMA) coupled with PCR (viability PCR) is used in foodborne pathogen detection in order to detect only viable bacteria. Originally presented to fully remove the signal of dead bacteria, the limits of the viability PCR rapidly came out in the literature. In this study, the use of PMA in a viability-qPCR (v-qPCR) was assessed on viable and dead cells of Salmonella enterica subsp. enterica serovar Enteritidis. The PMA treatment protocol was modified (dark incubation duration, concentration of PMA) to evaluate if a complete negative signal of dead Salmonella was possible. However, none of these modifications was found to improve the removal of the remaining qPCR signal observed in the presence of dead bacteria. The present research also underlines that PMA may unexpectedly decrease the qPCR signal observed on living S. Enteritidis at low concentration. Finally, the use of S. Enteritidis cells killed by processes altering, or not, the cell-wall/membrane gives us a clue to answering the question about the non-total extinction of the signal of dead cells sample in the v-qPCR assay. Indeed, the data strongly indicate that the remaining qPCR signal observed in non-culturable cells does not only depend on the cell-wall/membrane integrity of the bacteria. According to these results, the authors suggest that for a rapid and reliable foodborne bacteria detection system, an enrichment followed by a qPCR analysis should be preferred to a v-qPCR.
Journal of Microbiological Methods 06/2014; · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cereulide is a cyclic dodecadepsipeptide ionophore, produced via non-ribosomal peptide synthetases (NRPS), which in rare cases can lead to human death. Early studies had shown that emetic toxin formation belongs to a homogeneous group of Bacillus cereus sensu stricto and the genetic determinants of cereulide (a 24-kb gene cluster of cesHPTABCD) are located on a 270-kb plasmid related to the Bacillus anthracis virulence plasmid pXO1.
[Show abstract][Hide abstract] ABSTRACT: Background & Aim
Inulin-type fructans (ITF) prebiotics promote changes in the composition and activity of the gut microbiota. The aim of this study was to to determine variations on fecal short chain fatty acids (SCFA) concentration in obese women treated with ITF and to explore associations between Bifidobacterium species, SCFA and host biological markers of metabolism.
Samples were obtained in a randomized, double blind, parallel, placebo-controlled trial, with 30 obese women randomly assigned to groups that received either 16 g/day ITF (n=15) or maltodextrin (n=15) for 3 months. The qualitative and quantitative analysis of Bifidobacterium spp. was performed in feces by PCR-DGGE and q-PCR, and SCFA profile was analyzed by gas chromatography. Spearman correlation analysis was performed between the different variables analyzed.
The species Bifidobacterium longum, Bifidobacterium pseudocatenulatum and Bifidobacterium adolescentis were significantly increased at the end of the treatment in the prebiotic group (p <0.01) being B. longum negatively correlated with serum lipopolysaccharide (LPS) endotoxin (p< 0.01). Total SCFA, acetate and propionate, that positively correlated BMI, fasting insulinemia and homeostasis model assessment (HOMA) (p< 0.05), were significantly lower in prebiotic than in placebo group after the treatment
ITF consumption selectively modulates Bifidobacterium spp. and decreases fecal SCFA concentration in obese women. ITF could lessen metabolic risk factors associated with higher fecal SCFA concentration in obese individuals.
[Show abstract][Hide abstract] ABSTRACT: Achieving fast, sensitive and selective detection of bacteria in liquid samples is a major challenge for
public healthcare. Unlike conventional methods, CMOS chips with sensing parts provide tremendous
benefits in term of portability, system integration and reduced cost. CMOS-compatible microelectrodes
were shown to selectively detect whole-cell Staphylococci after drying the sensor surface1. Here, we
extend the use of these microelectrodes to bacteria detection in continuous flow for microfluidic
[Show abstract][Hide abstract] ABSTRACT: GIL01/Bam35, GIL16, AP50 and Wip1 are tectiviruses preying on the Bacillus cereus group. Despite the significant contributions of phages in different biological processes, little is known about the dealings taking place between tectiviruses and their Gram-positive hosts. Therefore, this work focuses on characterizing the interactions between tectiviruses and the B. cereus group, by assessing their occurrence, genetic diversity and evaluating their host range. To study the occurrence of tectiviruses in the B. cereus group, 2,000 isolates were evaluated using primers designed on two variable regions detected in previously described elements. PCR and propagation tests revealed that tectiviral elements occurred in less than 3 % of the isolates. Regardless of this limited distribution, several novel tectiviruses were found and partial DNA sequencing indicated that a greater diversity exists within the family Tectiviridae. Analyses of the selected variable regions, along with their host range, showed that tectiviruses in the B. cereus group can be clustered mainly into two different groups: the ones infecting B. anthracis and those isolated from other B. cereus group members. In order to address the host range of some novel tectiviruses, 120 strains were tested for sensitivity. The results showed that all the tested tectiviruses produced lysis in at least one B. cereus sensu lato strain. Moreover, no simple relationship was found between the infection patterns of the tectiviruses and their diversity.
Applied and Environmental Microbiology 05/2014; 80(14):4138-4152. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacillus cereus is an important foodborne pathogen causing diarrhoea, emesis and in, rare cases, lethal poisonings. The emetic syndrome is caused by cereulide, a heat-stable toxin. Originally considered as a rather homogenous group, the emetic strains have since been shown to display some diversity, including the existence of two clusters of mesophilic B. cereus and psychrotolerant Bacillus weihenstephanensis. Using Pulsed-Field Gel Electrophoresis (PFGE) analysis, this research aimed to better understand the diversity and spatio-temporal occurrence of emetic strains originating from environmental or food niches versus those isolated from foodborne cases. The diversity was evaluated using a set of 52 B. cereus and B. weihenstephanensis strains isolated between 2000 and 2011 in ten countries. PFGE analysis could discriminate 17 distinct profiles (pulsotypes). The most striking observations were: i) more than one emetic pulsotype can be observed in a single outbreak, ii) the number of distinct isolates involved in emetic intoxications is limited and these potentially clonal strains frequently occurred in successive and independent food poisoning cases, iii) isolates from different countries displayed identical profiles, and iv) the cereulide-producing psychrotolerant B. weihenstephanensis were, so far, only isolated from environmental niches.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Food microbiology proficiency testing (PT) is a useful tool to assess the analytical performances among laboratories. PT items should be close to routine samples to accurately evaluate the acceptability of the methods. However, most PT providers distribute exclusively artificial samples such as reference materials or irradiated foods. This raises the issue of the suitability of these samples since the equivalence - or "commutability" - between results obtained on artificial vs authentic food samples has not been demonstrated. In the clinical field, the use of non-commutable PT samples has led to erroneous evaluation of the performances when different analytical methods were used. This study aimed to provide a first assessment of the commutability of samples distributed in food microbiology PT.
REQUASUD and IPH organized 13 food microbiology PTs including 10 to 28 participants. Three types of PT items were used: genuine food samples, sterile food samples and reference materials. The commutability of the artificial samples (reference material or sterile samples) was assessed by plotting the distribution of the results on natural and artificial PT samples. This comparison highlighted matrix-correlated issues when non-food matrices, such as reference materials, were used. Artificially inoculated food samples, on the other hand, raised only isolated commutability issues.
In the organization of a PT-scheme, authentic or artificially inoculated food samples are necessary to accurately evaluate the analytical performances. Reference materials, used as PT items because of their convenience, may present commutability issues leading to inaccurate penalising conclusions for methods which would have provided accurate results on food samples.
For the first time, the commutability of food microbiology PT samples was investigated. The nature of the samples provided by the organizer turned out to be an important factor since matrix effects can impact on the analytical results. This article is protected by copyright. All rights reserved.
Journal of Applied Microbiology 11/2013; · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this work, a three-level Salmonella detection system based on a combination of seven SYBR®Green qPCR was developed. This detection system discriminates Salmonella at the genus, species and subspecies levels using a single 96-well plate. The SYBR®Green qPCR assays target the invA, rpoD, iroB and safC genes, as well as the STM0296 locus, putatively coding for a cytoplasmic protein. This study includes the design of primer pairs, in silico and in situ selectivity, sensitivity, repeatability and reproducibility evaluations of the seven SYBR®Green qPCR assays. Each detection level displayed a selectivity of 100 %. This combinatory SYBR®Green qPCR system was also compared with three commercially available Salmonella qPCR detection kits. This comparison highlighted the importance of using a multi-gene detection system to be able to detect every target strain, even those with deletion or mutation of important genes.
Applied Microbiology and Biotechnology 10/2013; · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell surface proteins of bacteria play essential roles in mediating the attachment of pathogens to host tissues and, therefore, represent key targets for anti-adhesion therapy. In the opportunistic pathogen Staphylococcus epidermidis , the adhesion protein SdrG mediates attachment of bacteria to the blood plasma protein fibrinogen (Fg) through a binding mechanism that is not yet fully understood. We report the direct measurement of the forces driving the adhesion of S. epidermidis to Fg-coated substrates using single-cell force spectroscopy. We found that the S. epidermidis -Fg adhesion force is of ∼150 pN magnitude and that the adhesion strength and adhesion probability strongly increase with the interaction time, suggesting that the adhesion process involves time-dependent conformational changes. Control experiments with mutant bacteria lacking SdrG and substrates coated with the Fg β6-20 peptide, instead of the full Fg protein, demonstrate that these force signatures originate from the rupture of specific bonds between SdrG and its peptide ligand. Collectively, our results are consistent with a dynamic, multi-step ligand-binding mechanism called "dock, lock, and latch".
[Show abstract][Hide abstract] ABSTRACT: pGIAK1 is a 38-kb plasmid originating from the obligate alkaliphilic and halotolerant Bacillaceae strain JMAK1. The strain was originally isolated from the confined environments of the Antarctic Concordia station. Analysis of the pGIAK1 38,362-bp sequence revealed that, in addition to its replication region, this plasmid contains the genetic determinants for cadmium and arsenic resistances, putative methyltransferase, tyrosine recombinase, spore coat protein and potassium transport protein, as well as several hypothetical proteins. Cloning the pGIAK1 cad operon in Bacillus cereus H3081.97 and its ars operon in Bacillus subtilis 1A280 conferred to these hosts cadmium and arsenic resistances, respectively, therefore confirming their bona fide activities. The pGIAK1 replicon region was also shown to be functional in Bacillus thuringiensis, Bacillus subtilis and Staphylococcus aureus, but was only stably maintained in B. subtilis. Finally, using an Escherichia coli - B. thuringiensis shuttle BAC vector, pGIAK1 was shown to display conjugative properties since it was able to transfer the BAC plasmid among B. thuringiensis strains.
PLoS ONE 08/2013; 8(8):e72461. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacillus cereus sensu lato is a species complex that includes the anthrax pathogen Bacillus anthracis and other bacterial species of medical, industrial, and ecological importance. Their phenotypes of interest are typically linked to large plasmids that are closely related to the anthrax plasmids pXO1 and pXO2. Here, we present the draft genome sequences of 94 isolates of B. cereus sensu lato, which were chosen for their plasmid content and environmental origins.