Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H−/Hnt Isolates from Cattle and Clonal Relationship Analysis

Abstract

Since enterohemorrhagic Escherichia coli (EHEC) isolates of serogroup O156 have been obtained from human diarrhea patients and asymptomatic carriers, we studied cattle as a potential reservoir for these bacteria. E. coli isolates serotyped by agglutination as O156:H25/H؊/Hnt strains (n ‫؍‬ 32) were isolated from three cattle farms during a period of 21 months and characterized by rapid microarray-based genotyping. The serotyping by agglutination of the O156 isolates was not confirmed in some cases by the results of DNA-based serotyping as only 25 of the 32 isolates were conclusively identified as O156:H25. In the multilocus sequence typing (MLST) analysis, all EHEC O156:H25 isolates were characterized as sequence type 300 (ST300) and ST688, which differ by a single-nucleotide exchange in the purA gene. Oligonucleotide microarrays allow simultaneous detection of a wider range of EHEC-associated and other E. coli virulence markers than other methods. All O156:H25 isolates showed a wide spectrum of virulence factors typical for EHEC. The stx 1 genes combined with the EHEC hlyA (hlyA EHEC ) gene, the eae gene of the subtype, as well as numerous other virulence markers were present in all EHEC O156:H25 strains. The behavior of eight different cluster groups, including four that were EHEC O156:H25, was monitored in space and time. Variations in the O156 cluster groups were detected. The results of the cluster analysis suggest that some O156:H25 strains had the genetic potential for a long persistence in the host and on the farm, while other strains did not. As judged by their pattern of virulence markers, E. coli O156:H25 isolates of bovine origin may represent a considerable risk for human infection. Our results showed that the miniaturized E. coli oligonucleotide arrays are an excellent tool for the rapid detection of a large number of virulence markers.

Zoonotic Agents in Small Ruminants Kept on City Farms in Southern Germany

“…We found S. aureus at lower prevalences of 29.4% in sheep and 11.5% in goats. These strains all belonged to the same clonal complex (CC133), which has been predominant in isolates from small ruminants in other studies (17,20). They carry the leukocidin lukM-lukF P83, a toxin host specific for ruminants (35).…”

“…The primer elongation and the hybridization, washing, and straining of ArrayStrips were described previously (17). The ArrayStrips were photographed using an ArrayMate instrument (Alere Technologies, Jena, Germany) and automatically analyzed.…”

Development of a Rapid Microarray-Based DNA Subtyping Assay for the Alleles of Shiga Toxins 1 and 2 of Escherichia coli

“…In contrast, a standard commercial kit for DNA isolation takes Ͼ1 h. We observed no significant differences in the signal and background quality of the microarray between the methods, which allows the conclusion that the DNA isolated by heat lysis is sufficiently pure and highly concentrated (0.5 to 1.5 g) in order to be used in the standard protocol for labeling and hybridization. A further advantage of the microarray is that the number of spotted probes on the array can be extended by additional probes, e.g., for resistance genes or markers for further characterization of E. coli or the identification of other Gramnegative bacteria that we have described before (12,(25)(26)(27).…”

Presence of Enterohemorrhagic Escherichia coli ST678/O104:H4 in France Prior to 2011

“…The two French isolates carried none of the resistance genes covered by the array (9). The 2004 isolate was completely susceptible to nalidixic acid, whereas the 2009 isolate was resistant.…”