Biofilms structural diversity of 100 strains of the food-borne pathogen Listeria monocytogenes

International audienc

Assessment of the roles of LuxS, Autoinducer 2 and its precursor S-Ribosyl Homocysteine, in cell attachment during biofilm formation by Listeria monocytogenes EGD-e

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Assessment of the Roles of LuxS, S-Ribosyl Homocysteine, and Autoinducer 2 in Cell Attachment during Biofilm Formation by Listeria monocytogenes EGD-e

LuxS is responsible for the production of autoinducer 2 (AI-2), which is involved in the quorum-sensing response of Vibrio harveyi. AI-2 is found in several other gram-negative and gram-positive bacteria and is therefore considered a good candidate for an interspecies communication signal molecule. In order to determine if this system is functional in the gastrointestinal pathogen Listeria monocytogenes EGD-e, an AI-2 bioassay was performed with culture supernatants. The results indicated that this bacterium produces AI-2 like molecules. A potential ortholog of V. harveyi luxS, lmo1288, was found by performing sequence similarity searches and complementation experiments with Escherichia coli DH5α, a luxS null strain. lmo1288 was found to be a functional luxS ortholog involved in AI-2 synthesis. Indeed, interruption of lmo1288 resulted in loss of the AI-2 signal. Although no significant differences were observed between Lux1 and EGD-e with regard to planktonic growth (at 10°C, 15°C, 25°C, and 42°C), swimming motility, and phospholipase and hemolytic activity, biofilm culture experiments showed that under batch conditions between 25% and 58% more Lux1 cells than EGD-e cells were attached to the surface depending on the incubation time. During biofilm growth in continuous conditions after 48 h of culture, Lux1 biofilms were 17 times denser than EGD-e biofilms. Finally, our results showed that Lux1 accumulates more S-adenosyl homocysteine (SAH) and S-ribosyl homocysteine (SRH) in culture supernatant than the parental strain accumulates and that SRH, but not SAH or AI-2, is able to modify the number of attached cells

Exploring the diversity of listeria monocytogenes biofilm architecture by high-throughput confocal laser scanning microscopy and the predominance of the honeycomb-like morphotype

BADIALListeria monocytogenes is involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation by L. monocytogenes isolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96 L. monocytogenes strains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improve L. monocytogenes biofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA)

Approche de l'éradication ponctuelle d'un insecte invasif par illumination électromagnétique micro-onde circulaire

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Techniques d'études de la formation des biofilms. Retour d'expérience issu du domaine "agro-alimentaire" et du domaine "environnemental"

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The ZKIR assay, a novel real-time PCR method for the detection of Klebsiella pneumoniae and closely related species in environmental Samples

International audienceKlebsiella pneumoniae (Kp) is of growing public health concern due to the emergence of strains that are multidrug-resistant, virulent, or both. Taxonomically, Kp includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here we analysed 1,001 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR assay, was developed and used to detect Kp in 96 environmental samples. Results were compared to a culture-based method using Simmons citrate agar with 1% inositol (SCAI) medium coupled to MALDI-TOF mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed.The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10-1 CFU g-1 after enrichment for 24 h in LB supplemented with ampicillin, and 1.5 × 103 to 1.5 × 104 CFU g-1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 MLST sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific and sensitive novel method to detect the presence of Kp in complex matrices, and indicates that Kp isolates from environmental samples differ from clinical isolates.IMPORTANCE The Klebsiella pneumoniae species complex (Kp) includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR, which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content, from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources

The diagnostic performance of a simulated "short" gadoxetic acid-enhanced MRI protocol is similar to that of a conventional protocol for the detection of colorectal liver metastases

To compare the performance of standard and simulated short gadoxetic acid-enhanced MRI protocols for the detection of colorectal liver metastases (CRLM)

Plants as a realized niche for Listeria monocytogenes

International audienceListeria monocytogenes is a human pathogen. It is the causative agent of listeriosis, the leading cause of bacterial-linked foodborne mortality in Europe and elsewhere. Outbreaks of listeriosis have been associated with the consumption of fresh produce including vegetables and fruits. In this review we summarize current data providing direct or indirect evidence that plants can serve as habitat for L. monocytogenes, enabling this human pathogen to survive and grow. The current knowledge of the mechanisms involved in the interaction of this bacterium with plants is addressed, and whether this foodborne pathogen elicits an immune response in plants is discussed

High Prevalence of Klebsiella pneumoniae in European Food Products: a Multicentric Study Comparing Culture and Molecular Detection Methods

International audienceThe Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices and to characterize food isolates phenotypically and genotypically. Chicken meat (n = 160) and salad (n = 145) samples were collected in five European countries and screened for the presence of KpSC using culture-based and zur-khe intergenic region (ZKIR) quantitative PCR (qPCR) methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44°C for 48 h) was defined as the most suitable selective culture method for KpSC recovery. A high prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and the culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). A total of 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples-same food type and same country (15 cases), different food types and same country (1), and same food type and two countries (1)-suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans. IMPORTANCE Bacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous, and K. pneumoniae is a leading cause of antibiotic-resistant infections in humans. Despite the urgent public health threat represented by K. pneumoniae, there is a lack of knowledge of the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing the presence of KpSC in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies