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Volume 18, Number 3—March 2012

Dispatch

Clinical Significance of Escherichia albertii

Tadasuke Ooka, Kazuko Seto, Kimiko Kawano, Hideki Kobayashi, Yoshiki Etoh, Sachiko Ichihara, Akiko Kaneko, Junko Isobe, Keiji Yamaguchi, Kazumi Horikawa, Tânia A.T. Gomes, Annick Linden, Marjorie Bardiau, Jacques G. Mainil, Lothar Beutin, Yoshitoshi Ogura, and Tetsuya HayashiComments to Author 
Author affiliations: University of Miyazaki, Miyazaki, Japan (T. Ooka, Y. Ogura, T. Hayashi); Osaka Prefectural Institute of Public Health, Osaka, Japan (K. Seto); Miyazaki Prefectural Institute for Public Health and Environment, Miyazaki (K. Kawano); National Institute of Animal Health, Ibaraki, Japan (H. Kobayashi); Fukuoka Institute of Health and Environmental Sciences, Fukuoka, Japan (Y. Etoh, S. Ichihara, K. Horikawa); Yamagata Prefectural Institute of Public Health, Yamagata, Japan (A. Kaneko); Toyama Institute of Health, Toyama, Japan (J. Isobe); Hokkaido Institute of Public Health, Hokkaido, Japan (K. Yamaguchi); Universidade Federal de São Paulo, São Paulo, Brazil (T.A.T. Gomes); University of Liège, Liège, Belgium (A. Linden, M. Bardiau, J.G. Mainil); Federal Institute for Risk Assessment, Berlin, Germany (L. Beutin)

Suggested citation for this article

Abstract

Discriminating Escherichia albertii from other Enterobacteriaceae is difficult. Systematic analyses showed that E. albertii represents a substantial portion of strains currently identified as eae-positive Escherichia coli and includes Shiga toxin 2f–producing strains. Because E. albertii possesses the eae gene, many strains might have been misidentified as enterohemorrhagic or enteropathogenic E. coli.

Attaching and effacing pathogens possess a locus of enterocyte effacement (LEE)–encoded type III secretion system. They form attaching and effacing lesions on intestinal epithelial cell surfaces by the combined actions of intimin, an eae gene–encoded outer membrane protein, and type III secretion system effectors. Attaching and effacing pathogens include enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) and Citrobacter rodentium (1,2). Escherichia albertii have recently been added to this group (35). However, the clinical significance of E. albertii has yet to be fully elucidated, partly because it is difficult to discriminate E. albertii from other Enterobacteriaceae spp. by using routine bacterial identification systems based on biochemical properties (69). A large number of E. albertii strains might have been misidentified as EPEC or EHEC because they possess the eae gene.

The Study

Figure 1

Thumbnail of Phylogenies of the intimin subtypes and the cdtB genes of 275 eae-positive strains from humans, animals, and the environment that had been originally identified by routine diagnostic protocols as enteropathogenic or enterohemorrhagic Escherichia coli. A) Neighbor-joining tree constructed based on the amino acid sequences of 30 known intimin subtypes and previously undescribed 5 intimin subtypes (N1–N5) that were identified. The sequences of the N1–N5 alleles are substantially diverg

Figure 1. Phylogenies of the intimin subtypes and the cdtB genes of 275 eae-positive strains from humans, animals, and the environment that had been originally identified by routine diagnostic protocols as enteropathogenic or...

We collected 278 eae-positive strains that were originally identified by routine diagnostic protocols as EPEC or EHEC. They were isolated from humans, animals, and the environment in Japan, Belgium, Brazil, and Germany during 1993–2009 (Table 1; Technical Appendix [PDF - 646 KB - 18 pages]). To characterize the strains, we first determined their intimin subtypes by sequencing the eae gene as described (Technical Appendix [PDF - 646 KB - 18 pages]). Of the 275 strains examined, 267 possessed 1 of the 26 known intimin subtypes (4 subtypes—η, ν, τ, and a subtype unique to C. rodentium—were not found). In the remaining 8 strains, we identified 5 new subtypes; each showed <95% nt sequence identity to any known subtype, and they were tentatively named subtypes N1–N5. For subtype N1, 3 variants were identified (N1.1, N1.2, and N1.3, with >95% sequence identity among the 3 variants) (Figure 1, panel A).

Figure 2

Thumbnail of Neighbor-joining tree of 179 eae-positive Escherichia coli and Escherichia albertii strains analyzed by multilocus sequence analysis. The tree was constructed with the concatenated partial nucleotide sequences of 7 housekeeping genes (see Technical Appendix for protocol details). A) The whole image of the 179 strains examined and 10 reference strains (E. coli/Shigella sp., E. fergusonii, and Salmonella enterica serovar Typhi) is shown. B) Enlarged view of the E. albertii lineage and

Figure 2. Neighbor-joining tree of 179 eae-positive Escherichia coli and Escherichia albertii strains analyzed by multilocus sequence analysis. The tree was constructed with the concatenated partial nucleotide sequences of 7 housekeeping genes (see...

To determine the phylogenetic relationships of the strains, we performed multilocus sequencing analysis of 179 strains that were selected from our collection on the basis of intimin subtype and serotype (see Technical Appendix [PDF - 646 KB - 18 pages] for selection criteria and analysis protocol). Among the 179 strains, 26 belonged to the E. albertii lineage (Figure 2). The 26 E. albertii strains were from 14 humans (13 from symptomatic patients), 11 birds, and 1 cat. All of the 5 new intimin subtypes were found in the E. albertii strains. Intimin subtypes found in other E. albertii strains were also rare subtypes found in E. coli (10). This finding suggests that more previously unknown intimin subtypes may exist in the E. albertii population.

We next analyzed the pheV, selC, and pheU loci of the 26 E. albertii strains for the presence of LEE elements as described (Technical Appendix [PDF - 646 KB - 18 pages]). These 3 genomic loci are the known LEE integration sites in E. coli. By this analysis, all E. albertii strains except 1 (EC05–44) contained the LEE in the pheU locus (the integration site in EC05–44 was not identified). This finding indicates that despite the remarkable diversity of intimin subtypes, the LEE elements are preferentially integrated into the pheU tRNA gene in E. albertii.

Because all E. albertii strains isolated so far contained the cdtB gene encoding the cytolethal distending toxin B subunit (8,9), we examined the presence and subtype of the cdtB gene as described (Technical Appendix [PDF - 646 KB - 18 pages]). This analysis revealed that all E. albertii strains except 1 (CB10113) possessed the cdtB gene belonging to the II/III/V subtype group (Figure 1, panel B); this finding is consistent with published findings (9). In addition, 2 strains (E2675 and HIPH08472) each of which was subtype I , possessed a second cdtB gene, (Figure 1, panel B).

We used PCR to further investigate the presence of Shiga toxin genes (stx) and their variants (Technical Appendix [PDF - 646 KB - 18 pages]) and found that 2 E. albertii strains possessed the stx2f gene (Figure 2, panel B). Stx2 production by these strains was confirmed by using a reverse-passive latex agglutination kit (Technical Appendix [PDF - 646 KB - 18 pages]). The 2 stx2f-positive strains were those containing the subtype I cdtB gene in addition to the II/III/V subtype group gene: 1 (HIPH08472) was isolated from a patient with diarrhea and the other (E2675) was from a healthy Corvus sp. bird (Figure 2).

Last, we examined the phenotypic and biochemical properties of the 26 E. albertii strains and compared the results with those obtained in a previous study (9) and with those of E. albertii type strain LMG20976 (Table 2). To identify features that could discriminate E. albertii from E. coli, the results were further compared with those of E. coli (11). Consistent with findings in previous reports (57,9), the lack of motility and the inability to ferment xylose and lactose and to produce β-D-glucuronidase were common biochemical properties of E. albertii that could be used to discriminate E. albertii from E. coli, although 1 E. albertii strain was positive for lactose fermentation. The inability of E. albertii to ferment sucrose has been described as a common feature (9); however, a positive reaction to this test was found for 5 (19.2%) E. albertii strains. Moreover, approximately half of E. coli strains are positive for sucrose fermentation. Thus, the inability to ferment sucrose is not informative. Rather, the inability to ferment dulcitol (all E. albertii strains were negative, 60% of E. coli strains are positive) may be a useful biochemical property for differentiation.

Conclusion

In the current clinical laboratory setting, a substantial number of E. albertii strains are misidentified as EPEC or EHEC. Because 13 of the isolates were from patients with signs and symptoms of gastrointestinal infection, E. albertii is probably a major enteric human pathogen. In addition, E. albertii should be regarded as a potential Stx2f-producing bacterial species, although the clinical significance of Stx2f-producing strains is unknown.

Notable genetic, phenotypic, and biochemical properties of E. albertii, which were identified by analyzing the confirmed E. albertii strains, are 1) possession of intimin subtypes rarely or previously undescribed in E. coli, 2) possession of the II/III/V subtype group cdtB gene, 3) LEE integration into the pheU tRNA gene, 4) nonmotility, and 5) inability to ferment xylose, lactose, and dulcitol (but not sucrose) and to produce β-D-glucuronidase. These properties could be useful for facilitating identification of E. albertii strains in clinical laboratories, which would in turn improve understanding of the clinical significance and the natural host and niche of this newly recognized pathogen. In this regard, however, current knowledge of the genetic and biological properties of E. albertii might be biased toward a certain group of E. albertii strains because, even with this study, only a limited number of strains have been analyzed. To more precisely understand the properties of E. albertii as a species, further analysis of more strains from various sources is necessary.

Dr Ooka is an assistant professor at the Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki. His research interests include the genomics and pathogenicity of pathogenic bacteria, especially attaching and effacing pathogens.

Acknowledgment

This work was supported by the following KAKENHI (Grants-in-Aid for Scientific Research) grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan: Applied Genomics, 17019058, to T.H.; Kiban-B, 20310116, to T.H.; and Wakate-B, 23790480, to T.O.

References

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Figures

Tables

Technical Appendix

Suggested citation for this article: Ooka T, Seto K, Kawano K, Kobayashi H, Etoh Y, Ichihara S, et al. Clinical significance of Escherichia albertii. Emerg Infect Dis [serial on the Internet]. 2012 Mar [date cited]. http://dx.doi.org/10.3201/eid1803.111401

DOI: 10.3201/eid1803.111401

Table of Contents – Volume 18, Number 3—March 2012

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Tetsuya Hayashi, Division of Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan

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