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Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH LF82 (expressing FimH with an AIEC-associated mutation) with fimH K12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.

Background & Aims Inducible chitinase 3-like-1 (CHI3L1) is expressed by intestinal epithelial cells (IECs) and adheres to bacteria under conditions of inflammation. We performed a structure–function analysis of the chitin-binding domains (CBDs) encoded by the chiA gene, which mediates the pathogenic effects of adherent invasive Escherichia coli (AIEC). Methods We created AIEC (strain LF82) with deletion of chiA (LF82-ΔchiA) or that expressed chiA with specific mutations. We investigated the effects of infecting different IEC lines with these bacteria, compared with non-pathogenic E coli; chitinase activities were measured using the colloidal chitin-azure method. Colitis was induced in C57/Bl6 mice by administration of dextran sodium sulfate (DSS), and mice were given 108 bacteria for 15 consecutive days by gavage. Stool/tissue samples were collected and analyzed. Results LF82-ΔchiA had significantly less adhesion to IEC lines than LF82. Complementation of LF82-ΔchiA with the LF82 chiA gene, but not chiA from non-pathogenic (K12) E coli, increased adhesion. We identified 5 specific polymorphisms in the CBD of LF82 ChiA (at amino acids 362, 370, 378, 388, and 548) that differ from chiA of K12 and were required for LF82 to interact directly with IECs. This interaction was mediated by an N-glycosylated asparagine in CHI3L1 (amino acid 68) on IECs. Mice infected with LF82, or LF82-ΔchiA complemented with LF82 chiA, developed more severe colitis following administration of DSS than mice infected with LF82-ΔchiA or LF82 that expressed mutant forms of chiA. Conclusion AIEC adhere to an N-glycosylated CHI3L1 on IEC via the CBD of chiA. This mechanism of promotes pathogenic effects of AIEC in mice with colitis.

The survival and transfer of Listeria innocua and Clostridium sporogenes, used as surrogates of the food borne pathogens Listeria monocytogenes and Clostridium botulinum, were quantitatively assessed under field conditions. In the soil, spores of C. sporogenes declined by less than 0.7 log cycles within 16 months and were detected on parsley leaves throughout the experiment. In contrast, L. innocua in the soil declined by 7 log cycles in 90 days and was detected on leaves in low numbers (>0.04 MPN g(-1)) during the first 30 days. Rates of decline in soil were similar in the laboratory at 20 degrees C for two strains of L. innocua and L. monocytogenes ; and in the field for L. innocua over two different years. L. innocua survived better in winter, indicating an important influence of temperature. The major cause of transfer of L. innocua from soil to parsley leaves was splashing due to rain and irrigation. As few as 1 CFU g(-1) Listeria in soil led to contamination of parsley leaves. Internalisation of Listeria through parsley roots was not observed. Under the conditions of soil and climate studied, a delay of 90 days between application of potentially contaminated fertilizer and harvest should be sufficient to eliminate L. monocytogenes.

Aims:  To investigate the presence of viable but non‐culturable Listeria monocytogenes during survival on parsley leaves under low relative humidity (RH) and to evaluate the ability of L. monocytogenes to recover from VBNC to culturable state under satured humidity. Methods and Results:  Under low RH (47–69%) on parsley leaves, the initial number of L. monocytogenes populations counted on non selective media (109 L. monocytogenes per leaf on TSA) was reduced by 6 log10 scales in 15 days, whereas number of viable L. monocytogenes counted under the microscope was reduced by 3–4 log10 scales, indicating the presence of VBNC cells. This was demonstrated on three L. monocytogenes strains (EGDe, Bug 1995 and LmP60). Changing from low to 100% RH permitted an increase of the culturable counts of L. monocytogenes and this growth was observed only when residual culturable cells were present. Moreover, VBNC L. monocytogenes inoculated on parsley leaves did not become culturable after incubation under 100% RH. Conclusions:  Dry conditions induced VBNC L. monocytogenes on parsley leaves but these VBNC were likely unable to recover culturability after transfer to satured humidity. Significance and Impact of Study:  Enumeration on culture media presumably under‐estimates the number of viable L. monocytogenes on fresh produce after exposure to low RH.

HRE hypomethylation in CEACAM6 promoter correlates with high expression in IEC. Our findings suggest that abnormal DNA methylation leading to CEACAM6 increased expression and AIEC-mediated gut inflammation can be related to changes in nutritional habits, such as low intake in methyl donor molecules, leading to abnormal epigenetic marks in mouse model mimicking CD susceptibility.

Aims:  To investigate the population dynamics of Listeria monocytogenes and Listeria innocua on the aerial surfaces of parsley. Methods and Results:  Under 100% relative humidity (RH) in laboratory and regardless of the inoculum tested (103–108 CFU per leaf), counts of L. monocytogenes EGDe, LO28, LmP60 and L. innocua CIP 80‐12 tended towards approx. 105 CFU per leaf. Under low RH, Listeria spp. populations declined regardless to the inoculum size (104–108 CFU per leaf). L. innocua CIP 80‐12 survived slightly better than L. monocytogenes in the laboratory and was used in field cultures. Under field cultures, counts of L. innocua decreased more rapidly than in the laboratory, representing a decrease of 9 log10 in 2 days in field conditions compared to a decrease of 4·5 log10 in 8 days in the laboratory. Counts of L. innocua on tunnel parsley cultures were always higher (at least by 100 times) than those on unprotected parsley culture. Conclusions:  Even with a high inoculum and under protected conditions (i.e. plastic tunnels), population of L. monocytogenes on the surface of parsley on the field would decrease by several log10 scales within 2 days. Significance and Impact of the Study:  Direct contamination of aerial surfaces of parsley with L. monocytogenes (i.e. through contaminated irrigation water) will not lead to contaminated produce unless it occurs very shortly before harvest.

A critical step in the life cycle of all organisms is the duplication of the genetic material during cell division. Ribonucleotide reductases (RNRs) are essential enzymes for this step because they control the de novo production of the deoxyribonucleotides required for DNA synthesis and repair. Enterobacteriaceae have three functional classes of RNRs (Ia, Ib, and III), which are transcribed from separate operons and encoded by the genes nrdAB, nrdHIEF, and nrdDG, respectively. Here, we investigated the role of RNRs in the virulence of adherent-invasive Escherichia coli (AIEC) isolated from Crohn's disease (CD) patients. Interestingly, the LF82 strain of AIEC harbors four different RNRs (two class Ia, one class Ib, and one class III). Although the E. coli RNR enzymes have been extensively characterized both biochemically and enzymatically, little is known about their roles during bacterial infection. We found that RNR expression was modified in AIEC LF82 bacteria during cell infection, suggesting that RNRs play an important role in AIEC virulence. Knockout of the nrdR and nrdD genes, which encode a transcriptional regulator of RNRs and class III anaerobic RNR, respectively, decreased AIEC LF82's ability to colonize the gut mucosa of transgenic mice that express human CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6). Microarray experiments demonstrated that NrdR plays an indirect role in AIEC virulence by interfering with bacterial motility and chemotaxis. Thus, the development of drugs targeting RNR classes, in particular NrdR and NrdD, could be a promising new strategy to control gut colonization by AIEC bacteria in CD patients.R ibonucleotide reductase (RNR) is an essential enzyme in all living organisms. It catalyzes the reduction of ribonucleotides (nucleoside triphosphates [NTPs]) to their corresponding 2=-deoxyribonucleotides (deoxynucleoside triphosphates [dNTPs]) and therefore plays an essential role in DNA synthesis and repair. Three RNR classes (classes I, II, and III) exist; these classes exhibit different primary structures, subunit cofactor requirements, and quaternary three-dimensional (3D) structures, but they all are allosterically regulated and share similar catalytic mechanisms (1, 2). Class I RNRs are oxygendependent enzymes that occur in eubacteria, eukaryotes, and some viruses. This class comprises two main subgroups (Ia and Ib). Class Ia RNRs are encoded by an operon containing nrdA and nrdB genes. These genes encode the NrdA subunit, which is catalytically and allosterically regulated, and the NrdB subunit, which possesses radical-generating activity. Class Ib RNRs are encoded by an operon containing the nrdH, nrdI, nrdE, and nrdF genes, which encode the corresponding specific redoxin NrdH, the activating subunit NrdI, the catalytic subunit NrdE, and the radical-generating subunit NrdF. Class III RNRs are present in facultative anaerobic and strict anaerobic microorganisms and use S-adenosylmethionine and iron-sulfur clusters in the NrdG accessory protein to create a stab...