Odile Tresse

Antimicrobial Activities of Isothiocyanates Against Campylobacter jejuni Isolates

Food-borne human infection with Campylobacter jejuni is a medical concern in both industrialized and developing countries. Efficient eradication of C. jejuni reservoirs within live animals and processed foods is limited by the development of antimicrobial resistances and by practical problems related to the use of conventional antibiotics in food processes. We have investigated the bacteriostatic and bactericidal activities of two phytochemicals, allyl-isothiocyanate (AITC), and benzyl isothiocyanate (BITC), against 24 C. jejuni isolates from chicken feces, human infections, and contaminated foods, as well as two reference strains NCTC11168 and 81-176. AITC and BITC displayed a potent antibacterial activity against C. jejuni. BITC showed a higher overall antibacterial effect (MIC of 1.25–5u2009μgu2009mL−1) compared to AITC (MIC of 50–200u2009μgu2009mL−1). Both compounds are bactericidal rather than bacteriostatic. The sensitivity levels of C. jejuni isolates against isothiocyanates were neither correlated with the presence of a GGT (γ-Glutamyl Transpeptidase) encoding gene in the genome, with antibiotic resistance nor with the origin of the biological sample. However the ggt mutant of C. jejuni 81-176 displayed a decreased survival rate compared to wild-type when exposed to ITC. This work determined the MIC of two ITC against a panel of C. jejuni isolates, showed that both compounds are bactericidal rather than bacteriostatic, and highlighted the role of GGT enzyme in the survival rate of C. jejuni exposed to ITC.

Polynucleotide phosphorylase has an impact on cell biology of Campylobacter jejuni.

Polynucleotide phosphorylase (PNPase), encoded by the pnp gene, is known to degrade mRNA, mediating post-transcriptional regulation and may affect cellular functions. The role of PNPase is pleiotropic. As orthologs of the two major ribonucleases (RNase E and RNase II) of Escherichia coli are missing in the Campylobacter jejuni genome, in the current study the focus has been on the C. jejuni ortholog of PNPase. The effect of PNPase mutation on C. jejuni phenotypes and proteome was investigated. The inactivation of the pnp gene reduced significantly the ability of C. jejuni to adhere and to invade Ht-29 cells. Moreover, the pnp mutant strain exhibited a decrease in C. jejuni swimming ability and chick colonization. To explain effects of PNPase on C. jejuni 81-176 phenotype, the proteome of the pnp mutant and parental strains were compared. Overall, little variation in protein production was observed. Despite the predicted role of PNPase in mRNA regulation, the pnp mutation did not induce profound proteomic changes suggesting that other ribonucleases in C. jejuni might ensure this biological function in the absence of PNPase. Nevertheless, synthesis of proteins which are involved in virulence (LuxS, PEB3), motility (N-acetylneuraminic acid synthetase), stress-response (KatA, DnaK, Hsp90), and translation system (EF-Tu, EF-G) were modified in the pnp mutant strain suggesting a more specific role of PNPase in C. jejuni. In conclusion, PNPase deficiency induces limited but important consequences on C. jejuni biology that could explain swimming limitation, chick colonization delay, and the decrease of cell adhesion/invasion ability.

Biofilm spatial organization by the emerging pathogen Campylobacter jejuni: comparison between NCTC 11168 and 81-176 strains under microaerobic and oxygen-enriched conditions

During the last years, Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries. Described as an obligate microaerophile, Campylobacter has puzzled scientists by surviving a wide range of environmental oxidative stresses on foods farm to retail, and thereafter intestinal transit and oxidative damage from macrophages to cause human infection. In this study, confocal laser scanning microscopy (CLSM) was used to explore the biofilm development of two well-described Campylobacter jejuni strains (NCTC 11168 and 81-176) prior to or during cultivation under oxygen-enriched conditions. Quantitative and qualitative appraisal indicated that C. jejuni formed finger-like biofilm structures with an open ultrastructure for 81-176 and a multilayer-like structure for NCTC 11168 under microaerobic conditions (MAC). The presence of motile cells within the biofilm confirmed the maturation of the C. jejuni 81-176 biofilm. Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process. Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts. Overexpression of cosR in the poorer biofilm-forming strain, NCTC 11168, enhanced biofilm development dramatically by promoting an open ultrastructure similar to that observed for 81-176. Consequently, the regulator CosR is likely to be a key protein in the maturation of C. jejuni biofilm, although it is not linked to oxygen stimulation. These unexpected data advocate challenging studies by reconsidering the paradigm of fastidious requirements for C. jejuni growth when various subpopulations (from quiescent to motile cells) coexist in biofilms. These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

Characterization of Campylobacter spp. transferred from naturally contaminated chicken legs to cooked chicken slices via a cutting board

Campylobacter represents the leading cause of gastroenteritis in Europe. Campylobacteriosis is mainly due to C. jejuni and C. coli. Poultry meat is the main source of contamination, and cross-contaminations in the consumers kitchen appear to be the important route for exposure. The aim of this study was to examine the transfer of Campylobacter from naturally contaminated raw poultry products to a cooked chicken product via the cutting board and to determine the characteristics of the involved isolates. This study showed that transfer occurred in nearly 30% of the assays and that both the C. jejuni and C. coli species were able to transfer. Transfer seems to be linked to specific isolates: some were able to transfer during separate trials while others were not. No correlation was found between transfer and adhesion to inert surfaces, but more than 90% of the isolates presented moderate or high adhesion ability. All tested isolates had the ability to adhere and invade Caco-2 cells, but presented high variability between isolates. Our results highlighted the occurrence of Campylobacter cross-contamination via the cutting board in the kitchen. Moreover, they provided new interesting data to be considered in risk assessment studies.

Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain

BackgroundCampylobacter jejuni is a leading cause of bacterial enteritis worldwide. This microaerophilic bacterium can survive in aerobic environments, suggesting it has protective mechanisms against oxidative stress. The clinical C. jejuni Bf strain is characterized by an increased resistance to oxygen. This study aimed to characterize the behavior of the clinical C. jejuni Bf strain under an aerobic atmosphere and in response to ROS-promoter agents.MethodsGrowth was studied in both aerobic and microaerobic conditions using classic cultivable methods. Electronic microscopy and mreB gene expression were used to evaluate the morphology of this strain under aerobic conditions. The survival under oxidative stress was tested in the presence of different concentrations of hydrogen peroxide (H2O2) and paraquat (PQ).ResultsThe results showed that C. jejuni Bf strain can grow aerobically, unlike other strains of C. jejuni tested. Cells of C. jejuni Bf exposed to oxidative stress presented changes in morphology and the gene mreB, responsible for maintaining the bacillary cell morphology, was down-expressed. In aerobically acclimated conditions, C. jejuni Bf exhibited a higher survival rate of 52xa0% in the presence of H2O2 (1xa0mM) compared to the reference strain NCTC 11168. Concentrations above 1xa0mM PQ were lethal for the reference strain but not for C. jejuni Bf.ConclusionsTaken together, these data highlight the resistance to oxidative stress conditions of C. jejuni Bf, indicating that this microorganism seems more adapted to survival in hostile environmental conditions.

Influence of measurement and control of microaerobic gaseous atmospheres in methods for Campylobacter growth studies

Campylobacter is the leading cause of bacterial enteritis in the world. For this reason, this pathogen is widely studied. As a microaerophilic and capnophilic microorganism, this foodborne pathogen requires an atmosphere with reduced oxygen (O2) and elevated carbon dioxide (CO2) concentrations for its optimal growth inxa0vitro. According to the procedure for Campylobacter spp. isolation and cultivation from food products and environmental samples, European and American standards recommend gas proportions of 5% O2 and 10% CO2, complemented with nitrogen (N2). However, in the literature, the reported proportion of O2 for microaerobic growth conditions of Campylobacter spp. can range from 2.5% to 15% and the reason for this variation is usually not explained. The use of different gas generating systems and media to detect and to grow Campylobacter from foodstuff and the lack of information about gas producing systems are the main sources of the loss of consistancy between data. In this review, the relevance, strengths and weaknesses of these methods and their impact on Campylobacter biology are discussed. In conclusion the minimum information concerning microaerobic gaseous atmospheres are suggested in order to better harmonize data obtained from research studies for a better understanding of Campylobacter features.

Editorial: about the foodborne pathogen Campylobacter

The name “Campylobacter” comes from ancient Greek meaning “curved rod” which describes the shape of this microorganism. Campylobacter was firstly isolated as a Vibrio species from epizootic ovine abortion in 1906 by McFadyean and Stockman (1913), and renamed in 1973 as the neotype strain Campylobacter after showing significant biological differences with Vibrio species (Véron and Chatelain, 1973). Rather than a curved rod, the shape looks more like to a spiral and can develop in to filamentous or coccoid forms under stressful conditions (Tangwatcharin et al., 2006; Ghaffar et al., 2015; Rodrigues et al., 2016). Nowadays, Campylobacter spp. are classified among the ε-proteobacteria in the family of Campylobacteriaceae (Vandamme et al., 1991). Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries, having surpassed Salmonella several years ago, and represents a significant economic burden (EFSA and ECDC, 2016). Although new species of Campylobacter have been recently discovered, human cases of campylobacterosis are dominated by two main species, Campylobacter jejuni and, to a lesser extent, Campylobacter coli. Quantitative epidemiology reports reveal high rates of contamination for broiler chickens and carcasses by Campylobacter (Hue et al., 2010; Lawes et al., 2012; Powell et al., 2012). The presence of Campylobacter was also detected in other farm animals or foodstuffs due to cross contamination (EFSA and ECDC, 2016). Campylobacter in poultry remains a problem with no effective control measures available that can be recommended for microbial food/farm safety guidelines to mitigate the risk of flock colonization. Campylobacter also remains a puzzle as to how an obligate microaerobic bacterium can survive from farm to retail outlets. The underlying molecular mechanisms of persistence, survival and pathogenesis appear to represent a combination peculiar to this pathogen, which are not shared with other foodborne bacterial pathogens such as Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Staphylococcus aureus. This topic includes 18 published articles describing original studies of C. jejuni and C. coli that deal with (1) epidemiology and animal carriage, (2) host interaction, (3) control strategies, (4) metabolism and regulation specificities of these two pathogen species, (5) methodology to improve cultural technique and (6) chicken gut microbiota challenged with Campylobacter.

Protective Effect of Carnobacterium spp. against Listeria monocytogenes during Host Cell Invasion Using In vitro HT29 Model

The pathogenesis of listeriosis results mainly from the ability of Listeria monocytogenes to attach, invade, replicate and survive within various cell types in mammalian tissues. In this work, the effect of two bacteriocin-producing Carnobacterium (C. divergens V41 and C. maltaromaticum V1) and three non-bacteriocinogenic strains: (C. divergens V41C9, C. divergens 2763, and C. maltaromaticum 2762) was investigated on the reduction of L. monocytogenes Scott A plaque-forming during human infection using the HT-29 in vitro model. All Carnobacteria tested resulted in a reduction in the epithelial cell invasion caused by L. monocytogenes Scott A. To understand better the mechanism underlying the level of L. monocytogenes infection inhibition by Carnobacteria, infection assays from various pretreatments of Carnobacteria were assessed. The results revealed the influence of bacteriocin production combined with a passive mechanism of mammalian cell monolayers protection by Carnobacteria. These initial results showing a reduction in L. monocytogenes virulence on epithelial cells by Carnobacteria would be worthwhile analyzing further as a promising probiotic tool for human health.

An adapted in vitro assay to assess Campylobacter jejuni interaction with intestinal epithelial cells: Taking into stimulation with TNFα

Campylobacter jejuni is the most prevalent foodborne bacterial infection agent. This pathogen seems also involved in inflammatory bowel diseases in which pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), play a major role. C. jejuni pathogenicity has been extensively studied using in vitro cell culture methods, and more precisely healthy cells. In fact, no information is available regarding the behavior of C. jejuni in contact with TNFα-stimulated cells. Therefore, this research was designed to investigate the effect of TNFα on C. jejuni interaction with human intestinal epithelial cells (HT29 and HT29-MTX). To ensure IL-8 production induced by TNFα, human rtTNFα was added to HT29 and HT29-MTX before adhesion and invasion assays. About 108 CFU bacteria of C. jejuni strains cells were added to measure their adherence and invasion abilities using TNFα-stimulated cells versus non stimulated cells. Exposure to TNFα results in IL-8 overproduction by intestinal epithelial cells. In addition, the effect of TNFα pre-treatment on C. jejuni adhesion and internalization into eukaryotic cells is strain-dependent. Indeed, the adhesion/invasion process is affected in <50% of the strains tested when TNFα is added to the intestinal cells. Interestingly, TNFα affects more strains in their ability to adhere to and invade the mucus-secreting HT29-MTX cells. Among the 10 strains tested, the aero-tolerant C. jejuni Bf strain is one of the most virulent. These results suggest that the TNFα signalling pathway could participate in the internalization of C. jejuni in human intestinal cells and can help in understanding the pathogenicity of this microorganism in contact with TNFα-stimulated cells.

Proteomics Analyses Applied to the Human Foodborne Bacterial Pathogen Campylobacter spp.

Abstract Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries over recent years. Epidemiological studies reported a constant increase of campylobacteriosis cases representing a significant economic burden worldwide. As an obligate microaerobic bacterium, Campylobacter puzzled scientists as to how it could survive from the farm to retail outlets. In order to propose effective control strategies, a better understanding of its adaptation, virulence, and survival in food products and the food environment is critical. Considering the enigmatic behavior and the idiosyncrasy of Campylobacter jejuni , many of its biological functions could not be inferred only from bacterial genomic comparisons. Consequently structural, functional and expression proteomics analyses performed on Campylobacter have been invaluable in unraveling the biological pathways and responses of Campylobacter . This chapter gives the main discoveries using proteomics tools since 2000 on Campylobacter spp.