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Bacterial invasion of six different human epithelial ceUl lines showed that some strains of the intestinal pathogen Campylobacterjejuni invaded intestinal cell lines at a level 102-104 times higher than reported previously for other Campylobacter strains. Separately, urinary tract isolates of Citrobacter freundii triggered a high-efficiency invasion of bladder cells. Use of multiple inhibitors with known effects on eukaryotic cell structures/processes allowed us to define in these genetically distinct bacterial genera unusual bacterial invasion mechanisms that uniquely require microtubules but not microfilaments. Campylobacterjejuni strain 81-176 uptake into 407 intestinal ceUls and Citrobacter entry into T24 bladder ceUls was blocked by microtubule depolymerization and inhibitors of coated-pit formation but not by microrflament depolymerization. Inhibitors of endosome acidification had no significant impact on intracellular survival of Campylobacterjejuni or Citrobacterfreundii, but monensin markedly reduced Citrobacter uptake. Epithelial cel invasion by both of these bacterial genera was dependent upon de novo bacterial protein synthesis but not upon de novo eukaryotic cel protein synthesis. In contrast to the T24 cel line-specific, strict microtubule-dependent uptake, Citrobacter entry into other cell lines was inhibited by both microtubule-and microfilament-depolymerization, suggesting that these bacteria encode two separate pathways for uptake (i, microtubule-dependent; ii, microfflament-dependent) that are cel line-specific and are recognized perhaps depending on the presence and abundance of appropriate eukaryotic receptors.

A genomic island encoding the biosynthesis and secretion pathway of putative hybrid nonribosomal peptidepolyketide colibactin has been recently described in Escherichia coli. Colibactin acts as a cyclomodulin and blocks the eukaryotic cell cycle. The origin and prevalence of the colibactin island among enterobacteria are unknown. We therefore screened 1,565 isolates of different genera and species related to the Enterobacteriaceae by PCR for the presence of this DNA element. The island was detected not only in E. coli but also in Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter koseri isolates. It was highly conserved among these species and was always associated with the yersiniabactin determinant. Structural variations between individual strains were only observed in an intergenic region containing variable numbers of tandem repeats. In E. coli, the colibactin island was usually restricted to isolates of phylogenetic group B2 and inserted at the asnW tRNA locus. Interestingly, in K. pneumoniae, E. aerogenes, C. koseri, and three E. coli strains of phylogenetic group B1, the functional colibactin determinant was associated with a genetic element similar to the integrative and conjugative elements ICEEc1 and ICEKp1 and to several enterobacterial plasmids. Different asn tRNA genes served as chromosomal insertion sites of the ICE-associated colibactin determinant: asnU in the three E. coli strains of ECOR group B1, and different asn tRNA loci in K. pneumoniae. The detection of the colibactin genes associated with an ICE-like element in several enterobacteria provides new insights into the spread of this gene cluster and its putative mode of transfer. Our results shed light on the mechanisms of genetic exchange between members of the family Enterobacteriaceae.

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