SummaryEscherichia coli strain 1404, isolated from a septicaemic calf, carries a transferable plasmid called pVir which codes for the cytotoxic necrotizing factor type 2 (CNF2). A 4 h interaction between strain 1404 and HeLa cells induced the formation of giant mononucleated cells blocked in G2/M phase. Mating experiments between strain 1404 and a non-pathogenic recipient strain demonstrated that the factor(s) encoded by pVir mediated the cell-cycle arrest. A 3.3 kb DNA fragment isolated from a DNA bank of pVir was shown to code for the factor(s) causing the cell-cycle arrest. Nucleotide sequence analysis revealed the presence of three genes encoding proteins sharing significant amino acid homology with the cytolethal distending toxins (CDTs) previously isolated from E. coli, Campylobacter jejuni and Shigella dysenteriae. Southern hybridization experiments demonstrated that the pVir of other CNF2-producing E. coli strains contained sequences related to cdt. Although the amino acid sequences amongst CDT diverged significantly, the two other CDTs previously isolated from E. coli were also able to block the HeLa cell cycle. In conclusion, this study demonstrates the mode of action of CDT and will help us to elucidate the role of this emerging toxin family in microbial pathogenesis.

Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of Salmonella responsible for disease outbreaks is crucial to minimize infections and eliminate ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP) typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated Salmonella strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in Salmonella. In this study, the prophage sequence diversity in different Salmonella serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of S. enterica representing 151 Salmonella serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in S. enterica genomes. Prophage sequences were highly variable among S. enterica serovars with a median ± interquartile range (IQR) of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of S. Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating S. enterica subtypes during foodborne outbreaks.

Salmonella enterica serovar Typhi (S. typhi) is a human‐restricted pathogen which causes typhoid fever. Relatively little is known about S. typhi host interaction as animal models of this disease are severely limited by the lack of virulence of S. typhi in other hosts. The virulence of other Salmonella serovars in animal models is dependent on the abilities of these bacteria to survive within host macrophages. We have used selective capture of transcribed sequences (SCOTS) to identify S. typhi genes expressed during growth in human macrophages. This positive cDNA selection technique identified 28 distinct clones representing previously identified as well as novel, uncharacterized and hypothetical gene sequences that are expressed intracellularly. Transcripts for the Vi capsular antigen and genes whose products are involved in stress responses and nutrient acquisition were obtained from intracellular bacteria using SCOTS. Most of these clones are present in the S. typhimurium genome and are also expressed in murine macrophages. Nineteen of these gene sequences were disrupted insertionally in S. typhi, and most of the resulting mutants exhibited a lower level of survival within macrophages compared with the wild‐type parent strain. Mutant strains, transformed with a copy of a wild‐type gene, exhibited a macrophage survival level similar to that of the parental strain.

The genetic determinant coding for F1651 fimbriae was cloned from the chromosome of the porcine Escherichia coliwild-type strain 4787 (0115: K-: H51: F165). The fimbrial determinant was further subcloned into the BmHI site of pACYC184 and a restriction map was established. On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated. The cloned F1651 fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa. Strains expressing F1651 fimbriae were detected using an F165-specilic polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads. Antiserum against the cloned F16$4 fimbriae recognized a 18.5 kDa band in the parent strain 4787.

SummaryThe PhoPQ two-component system of the intracellular pathogen Salmonella enterica senses and controls resistance to a-helical antimicrobial peptides (AMPs) by regulating covalent modifications of lipid A. A homologue of the phoPQ operon was found in the genome of the murine enteric extracellular pathogen, Citrobacter rodentium. Here we report that C. rodentium PhoPQ was apparently unable to mediate activation of target genes in the presence of a-helical AMPs. However, these AMPs activated C. rodentium PhoPQ expressed in a S. enterica DphoPQ mutant. Analysis of the outer membrane (OM) fractions of the C. rodentium wild-type and DphoPQ strains led to the identification of an omptin family protease (CroP) that was absent in DphoPQ. Deletion of croP in C. rodentium resulted in higher susceptibility to a-helical AMPs, indicating a direct role of CroP in AMP resistance. CroP greatly contributed to the protection of the OM from AMP damage by actively degrading a-helical AMPs before they reach the periplasmic space. Accordingly, transcriptional activation of PhoP-regulated genes by a-helical AMPs was restored in the DcroP mutant. This study shows that resistance to a-helical AMPs by the extracellular pathogen C. rodentium relies primarily on the CroP OM protease.