After 24 h, the buffer was removed, and fresh buffer was added. Agarose blocks were washed three times with 1.3 ml Tris-EDTA (TE) buffer containing 2 mM Pefabloc KPT-330 Sigma SC (Roche, Basel, Switzerland) at 37��C for 30 min. Subsequently, blocks were incubated with 40 U XbaI (Fermentas, Ontario, Canada) and incubated at 37��C under gentle shaking for 20 h. DNA fragments were resolved in 1% peqGOLD universal agarose (Peqlab Biotechnologie GmbH, Erlangen, Germany) in 0.5�� TBE buffer with the Pharmacia Biotech Gene Navigator apparatus (GE Healthcare, Vienna, Austria). Electrophoresis was performed for 24 h at 6��C at a constant voltage of 200 V (6 V/cm) with a linear ramp of pulse times from 1 to 40 s. Gels were stained with ethidium bromide, and restriction patterns were visualized under UV irradiation.

Analysis of restriction fragment patterns was performed with ImageMaster 1D software (Amersham Pharmacia Biotech, Uppsala, Sweden). A control strain, K. pneumoniae ATCC 4352, was run with each gel to assess variation within gels. Matching and cluster analyses were done by using the unweighted-pair group method with average linkages (UPGMA) with the Dice coefficient. RESULTS Cytotoxic properties detected at the early stationary phase of K. oxytoca growth. The capacity of toxin-producing AAHC K. oxytoca isolate 04/1O to impair Hep2 cell viability compared to laboratory control strain ATCC 13182 was monitored over 48 h of bacterial growth. Cytolethal properties were first observed for the conditioned culture medium of the clinical isolate at the end of the logarithmic growth phase (Fig.

(Fig.1B).1B). The capacity to mediate substantial cell killing was maintained throughout an additional 30-h period. At this point, after 44 h of cultivation, no variation in the viability of isolate 04/1O was observed (Fig. (Fig.1A),1A), but the culture medium no longer exhibited cytotoxic effects on Hep2 cells. Cytotoxin production by isolate 04/1O was not observed under similar conditions in M9 minimal medium (not shown). FIG. 1. Cytotoxin production during growth of two K. oxytoca strains. (A) Growth curves of laboratory strain ATCC 13182 and clinical K. oxytoca isolate 04/1O, originating from an AAHC patient, were determined by colony counts on agar plates. (B) Effect of culture … Clinical source of Klebsiella isolates.

The clinical diagnoses for the patients and the sources of isolation of Klebsiella strains are shown in Table Table11 Cilengitide and Table S1 in the supplemental material. Fifteen K. oxytoca isolates from 13 patients with AAHC (group I) were isolated during the active phase of colitis. AAHC was diagnosed based on typical clinical and endoscopic and/or radiological features (9, 10). All AAHC patients tested negative for C. difficile and other intestinal pathogenic bacteria. K. oxytoca strains of group II were obtained from patients with C.