Detection and characterization of the fimbrial sfp cluster in enterohemorrhagic Escherichia coli O165:H25/NM isolates from humans and cattle

Abstract

The sfp cluster, encoding Sfp fimbriae and located in the large plasmid of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157 (pSFO157), has been considered a unique characteristic of this organism. We discovered and then characterized the sfp cluster in EHEC O165:H25/NM (nonmotile) isolates of human and bovine origin. All seven strains investigated harbored a complete sfp cluster (carrying sfpA, sfpH, sfpC, sfpD, sfpJ, sfpF, and sfpG) of 6,838 bp with >99% nucleotide sequence homology to the sfp cluster of SF EHEC O157:NM. The sfp cluster in EHEC O165:H25/NM strains was located in an approximately 80-kb (six strains) or approximately 120-kb (one strain) plasmid which differed in structure, virulence genes, and sfp flanks from pSFO157. All O165:H25/NM strains belonged to the same multilocus sequence type (ST119) and were only distantly phylogenetically related to SF EHEC O157:NM (ST11). The highly conserved sfp cluster in different clonal backgrounds suggests that this segment was acquired independently by EHEC O165:H25 and SF EHEC O157:NM. Its presence in an additional EHEC serotype extends the diagnostic utility of PCR targeting sfpA as an easy and efficient approach to seek EHEC in patients' stools. The reasons for the convergence of pathogenic EHEC strains on a suite of virulence loci remain unknown.

FIG. 1.

Physical map of the sfp cluster and its flanks in SF EHEC O157:NM strain 3072/96 and PCR strategy used for analysis of the sfp cluster in EHEC O165:H25/NM. The large arrows indicate sfp genes (F, sfpF) and genes adjacent to the sfp cluster in strain 3072/96. A transposon-like element is dotted, and insertion sequences are horizontally striped. The dashed line below repA depicts the RepFIB origin of replication of pSFO157. The small arrows indicate the positions of PCR primers (details for PCRs I to XIX are given in Table 2). In all but four PCRs, amplicons of identical sizes were obtained from each of seven EHEC O165 strains and strain 3072/96. In the remaining PCRs, no amplicons (*) or amplicons of different sizes (**) than those from strain 3072/96 (∼600 bp instead of 1,343 bp) were elicited from EHEC O165 strains. The double arrow below the graph depicts the 8,219-bp plasmid region which was sequenced from EHEC O165:H25 strains 820/08 and 04-07734 and which comprises the sfp cluster (6,838 bp) and its upstream (828 bp) and downstream (553 bp) flanks. The numbers above this arrow indicate nucleotide sequence homologies of the corresponding regions between EHEC O165:H25 strains (which were 100% identical) and strain 3072/96. NS, no significant homology was found using BLAST algorithms (http://www.ncbi.nlm.nih.gov/BLAST). The gray regions (−) adjacent on both sides to the sequenced stretch were not detected in EHEC O165 by the PCR strategy used.

FIG. 2.

Plasmid profiles (A) and plasmid hybridization with the sfpA probe (B) of EHEC O165 and control strains. In lanes 1 and 4 to 8, the following EHEC O165:H25/NM strains are shown: lanes 1, 820/08; lanes 4, 04-07734; lanes 5, 02-11228; lanes 6, 00-09087; lanes 7, 99-02258; and lanes 8, 98-08419-1. In lanes 2 and 3, SF EHEC O157:NM strain 3072/96 and EHEC O157:H7 strain EDL933, respectively, are shown for comparison. For strains which contained two plasmids, the plasmid which hybridized with the sfpA probe is marked by an arrow (A); the sizes of the sfpA-hybridizing plasmids are indicated in panel B. M, molecular size marker (DNAs of plasmids R27 [180 kb] and R100 [94 kb]) (; http://www.biotech.bham.ac.uk/Plasmids/default.htm).

FIG. 3.

Minimum spanning tree based on MLST of the sfp-positive EHEC O165:H25/NM strains (ST119; depicted by arrow) in comparison to the HUSEC collection (33) and Shigella dysenteriae strain M1354 as an outgroup (ST243) (data are from http://web.mpiib-berlin.mpg.de/mlst/dbs/Ecoli). Each circle represents a unique ST (with the designation given in the circle). The numbers on the connecting lines display the numbers of differing alleles. Clonal complexes (CC) of the major EHEC serogroups (33) are highlighted in gray.