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1 Laboratory of Microbiology, Ghent University, K. L. Ledeganckstr. 35, B-9000 Ghent, Belgium
2 Laboratory of BCCMTM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstr. 35, B-9000 Ghent, Belgium
3 Microbial Diseases Laboratory, Division of Communicable Disease Control, California Department of Health Services, Berkeley, CA 94704-1011, USA
Correspondence
Geert Huys
geert.huys{at}rug.ac.be
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82 % internal DNA relatedness), with the closest affiliation to the type strains of Escherichia coli (55–64 %) and Shigella flexneri (54–60 %). The DNA–DNA hybridization levels were much lower with members of other described Escherichia species (16–45 %) and with the type strain of H. alvei (9–17 %). The G+C content of the ICDDR,B strains ranged from 50·5 to 50·7 mol%. Together with the diagnostic characteristics reported previously, including the presence of the eaeA gene of enteropathogenic E. coli and of the E. coli and Shigella-specific phoE gene, it is concluded that the ICDDR,B strains represent a novel taxon in the genus Escherichia, for which the name Escherichia albertii sp. nov. is proposed. Its type strain is Albert 19982T (=LMG 20976T=CCUG 46494T).
The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of ICDDR,B strain LMG 20976T is AJ508775.
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described a group of five diarrhoeagenic strains that were allocated to the genus Escherichia on the basis of phenotypic and genotypic evidence. These isolates all originated from stool specimens of children with diarrhoeal illness and were recovered by workers at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh (Albert et al., 1991b). Initially, the five ICDDR,B stool isolates were assigned to the species Hafnia alvei by the API 20E system. However, Albert et al. (1992b) later discovered that all of these strains, tentatively identified as H. alvei, possessed the attaching-and-effacing eaeA gene typical of enteropathogenic Escherichia coli. In addition, partial 16S rRNA sequence analysis of eaeA-positive and eaeA-negative H. alvei isolates revealed a relatively low level of similarity (92 %; a partial 16S rDNA sequence of ICDDR,B strain LMG 20973 was submitted to the EMBL database under accession no. Z47360), indicating that the ICDDR,B isolates were probably misidentified as H. alvei (Ridell et al., 1995). Likewise, Ismaili et al. (1996) reported a number of striking phenotypic and genotypic differences between Canadian H. alvei isolates and the ICDDR,B stool isolate 19982 based on eaeA-induced cellular responses and on outer-membrane protein (OMP), PFGE and plasmid profile analysis. More recently, conventional biochemical characterization, determination of susceptibility to the antibiotic cephalothin and to a Hafnia-specific phage, PCR-based detection of the OMP gene phoE, PFGE and partial 16S rDNA sequencing have further supported the notion that the diarrhoeagenic ICDDR,B isolates actually belong to the genus Escherichia (Janda et al., 1999, 2002). It was concluded that these isolates either represent an unusual biotype of E. coli or constitute a novel Escherichia species. In the present study, DNA–DNA hybridizations, additional phenotypic characterization and 16S rDNA sequencing were performed in order to elucidate the taxonomic position of the diarrhoeagenic Escherichia strains.
The five ICDDR,B strains [LMG 20972 (=Albert 9194), LMG 20973 (=Albert 10457), LMG 20974 (=Albert 10790), LMG 20975 (=Albert 12502) and LMG 20976T (=Albert 19982T)] all originated from faeces of diarrhoeal children (<5 years old) and were isolated in 1990–1991 in Dhaka, Bangladesh (Albert et al., 1991b; Ridell et al., 1995; Janda et al., 1999, 2002). Type strains of Escherichia species, H. alvei and Shigella flexneri were obtained from the BCCMTM/LMG Bacteria Collection, Ghent University, Belgium (http://www.belspo.be/bccm/). All strains were cultured aerobically on trypticase soy agar (TSA) containing 3 % (w/v) trypticase soy broth (BBL) and 1·5 % (w/v) bacteriological agar no. 1 (Oxoid) at 37 °C for 24 h. In addition to previous phenotypic studies (Ridell et al., 1995; Janda et al., 1999, 2002), the five ICDDR,B isolates were further characterized biochemically with the API 50 CH system (bioMérieux) according to the manufacturer's instructions. Antimicrobial susceptibilities to penicillin (10 µg), ampicillin (25 µg), tetracycline (30 µg), kanamycin (30 µg), streptomycin (25 µg) and chloramphenicol (30 µg) were determined by the disc diffusion method using Oxoid discs according to the conventional Kirby–Bauer method (Bauer et al., 1966). Isolates were classified as resistant, intermediate or susceptible on the basis of the quantitative interpretation criteria recommended by the NCCLS (1993). For determination of the G+C content, DNA was enzymically degraded into nucleosides according to the protocol of Mesbah et al. (1989). The mixture was then separated by HPLC using a Waters SymmetryShield C8 column thermostatted at 37 °C. The solvent used was 0·02 M NH4H2PO4 (pH 4·0) with 1·5 % acetonitrile. Non-methylated lambda phage DNA (Sigma) was used as the calibration reference. For the purpose of DNA–DNA hybridizations, genomic DNA was prepared using a combination of the protocols of Marmur (1961) and Pitcher et al. (1989) as described by Goris et al. (1998). Hybridizations were performed using the fluorimetric microplate method (Ezaki et al., 1989) with modifications by Goris et al. (1998) at an optimal renaturation temperature of 40 °C in the presence of 50 % formamide. The complete 16S rDNA sequence of strain LMG 20976T was determined as described previously (Huys et al., 2001) using an ABI PRISM 3100 Genetic Analyzer.
As a result of the taxonomic data reported by Ridell et al. (1995) and Janda et al. (1999), the initial placement of the eaeA-positive diarrhoeagenic ICDDR,B isolates in the genus Hafnia (Albert et al., 1992b) was called seriously into question. Although a great deal of phenotypic and genotypic evidence has been put forward in favour of the inclusion of these strains in the genus Escherichia (Janda et al., 1999, 2002), a final conclusion on their taxonomic status was hampered by the lack of a decisive DNA–DNA hybridization study. As determined with the fluorimetric microplate method, the five eaeA-positive diarrhoeagenic ICDDR,B isolates included in the present study were found to constitute a highly homogeneous group, exhibiting internal relatedness between 82 and 100 % (Table 1). In addition, the five isolates under study displayed a very narrow range of G+C content, 50·5–50·7 mol%. These findings clearly demonstrate the genotypic homogeneity among the ICDDR,B isolates as reflected previously by their highly similar RAPD-PCR profiles and identical 353-bp fragments of the 16S rRNA genes (Ridell et al., 1995) and typical PFGE profiles (Janda et al., 1999).
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). A comparable range of genomic relatedness (54–60 %) was also found with the type strain of S. flexneri (LMG 10472T=ATCC 12022T =CDC 3591-52T), which is reflective of the fact that representatives of the genus Shigella cannot be differentiated from E. coli on the basis of DNA–DNA hybridization (Brenner et al., 1973; Watanabe & Okamura, 1992). The DNA relatedness of the two ICDDR,B strains to the type strains of other Escherichia species was much lower, ranging from 14 to 45 % (Table 1). The inclusion of eaeA-positive ICDDR,B isolates in H. alvei, as initially suggested (Albert et al., 1991b, 1992b), was definitely ruled out by the finding that the type strain of the latter taxon, strain LMG 10392T (=ATCC 13337T), was only 9–17 % related to ICDDR,B strains LMG 20974 and LMG 20976T (Table 1). The DNA–DNA hybridization values obtained in this study clearly indicate that the eaeA-positive diarrhoeagenic ICDDR,B isolates constitute a homogeneous taxon in the genus Escherichia that is most closely affiliated with, but still well below 70 % related (concordant with the recommendations of Wayne et al., 1987) to, E. coli and S. flexneri. This conclusion is further supported by previously reported genotypic diagnostic markers, including the presence of the eaeA gene of enteropathogenic E. coli and of the E. coli and Shigella-specific phoE gene (Albert et al., 1992b; Janda et al., 1999; Ridell et al., 1995). Pairwise comparison of the 16S rDNA sequence of ICDDR,B strain LMG 20976T with those of the neighbouring taxa retrieved from the EMBL database (http://srs.ebi.ac.uk) revealed the following similarity values: 99·3 % with Shigella sonnei LMG 10473T (=ATCC 25931T) (accession no. X96964) and S. flexneri ATCC 29903T (X96963), 98·3 % with E. coli LMG 2092T (=ATCC 11775T) (X80725), 97·8 % with E. vulneris LMG 7868T (=ATCC 33821T) (X80734) and 93·5 % with H. alvei LMG 10392T (=ATCC 13337T) (M59155). Taken together, these results reinforce the conclusion that the ICDDR,B group does not belong to H. alvei and confirm its close phylogenetic affiliation to the genera Escherichia and Shigella.
Collectively, the results of API 50CH characterization obtained in this study agreed well with the phenotypic data reported previously on eaeA-positive diarrhoeagenic ICDDR,B isolates (Ridell et al., 1995; Janda et al., 1999, 2002) and clearly indicate that this novel group of Escherichia strains are also phenotypically significantly different from their closest enterobacterial relatives. The ICDDR,B strains can be clearly distinguished from E. coli by negative reactions for indole and for fermentation of lactose and D-sorbitol and by the inability to produce 
-D-glucuronidase. In addition, the novel group of Escherichia strains can be separated from the other described Escherichia species by at least two phenotypic characteristics, including fermentation of D-xylose, which is negative for the ICDDR,B group (Table 2). Although biochemical distinction from Shigella isolates may be confusing, the ICDDR,B strains can be distinguished from shigellae by gas production from D-glucose and the ability to produce lysine decarboxylase. By serotyping, the ICDDR,B strains were found to be non-reactive against antisera specific to Shigella boydii serotypes 13 and 14 (J. M. Janda, unpublished data). On a genetic basis, it was previously reported that the ICDDR,B strains do not possess the invasion antigen H (ipaH), which is typically present in shigellae and enteroinvasive E. coli (Janda et al., 1999). Clear phenotypic distinction from H. alvei strains can be made on the basis of acetate assimilation, negative reactions for Voges–Proskauer and growth in KCN broth and resistance to the Hafnia-specific bacteriophage 1672.
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; Albert et al., 1991a, 1992a, 2000)].
This description is based on data reported previously by Ridell et al. (1995) and Janda et al. (1999, 2002) and on API 50CH profiling performed in the course of this study. All five isolates display the following characteristics typical of the genus Escherichia: cells are Gram-negative, medium to long rods, chemo-organotrophic with both oxidative and fermentative metabolism, cytochrome oxidase-negative and catalase-positive and acid and gas are produced from D-glucose. Growth occurs after 24 h at 35–37 °C on TSA medium. Cells are non-motile at 35 °C. Positive for lysine and ornithine decarboxylases, methyl red reaction and nitrate reduction but negative for arginine dihydrolase, indole, DNase, urease, gelatinase and Voges–Proskauer reaction. No growth in KCN broth. Acetate but not citrate or malonate are used as carbon sources. 3-Hydroxybenzoate but not 2-ketogluconate or histidine is assimilated. -D-Glucuronidase is not produced. Acid is produced from L-arabinose and D-mannitol but not from adonitol, amygdalin, D-arabitol, D-cellobiose, dulcitol, erythritol, inositol, lactose, 
-D-melibiose, methyl D-glucoside, D-raffinose, L-rhamnose, salicin, D-sorbitol, D-sucrose or D-xylose. Fermentation of D-maltose and D-trehalose is variable (negative for strains LMG 20972 and LMG 20974). By disc diffusion testing, resistant to tetracycline and penicillin but susceptible to chloramphenicol, kanamycin and ampicillin (except strain LMG 20974). All strains are partially or completely susceptible to cephalothin by the E-test. The G+C content is 50·5–50·7 mol%.
The type strain, strain Albert 19982T (=LMG 20976T =CCUG 46494T), was isolated from the stool specimen of a diarrhoeal child in Bangladesh. The G+C content of this strain is 50·6 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Albert, M. J., Alam, K., Ansaruzzaman, M., Montanaro, J., Islam, M., Faruque, S. M., Haider, K., Bettelheim, K. & Tzipori, S. (1991a). Localized adherence and attaching-effacing properties of nonenteropathogenic serotypes of Escherichia coli. Infect Immun 59, 1864–1868.
Albert, M. J., Alam, K., Islam, M., Montanaro, J., Hamidur Rahman, A. S. M., Haider, K., Hossain, M. A., Kibriya, A. K. M. G. & Tzipori, S. (1991b). Hafnia alvei, a probable cause of diarrhea in humans. Infect Immun 59, 1507–1513.
Albert, M. J., Alam, K., Ansaruzzaman, M. & 7 other authors (1992a). Pathogenesis of Providencia alcalifaciens-induced diarrhea. Infect Immun 60, 5017–5024.
Albert, M. J., Faruque, S. M., Ansaruzzaman, M., Islam, M. M., Haider, K., Alam, K., Kabir, I. & Robins-Browne, R. (1992b). Sharing of virulence-associated properties at the phenotypic and genetic levels between enteropathogenic Escherichia coli and Hafnia alvei. J Med Microbiol 37, 310–314.[Abstract]
Albert, M. J., Ansaruzzaman, M., Talukder, K. A. & 8 other authors (2000). Prevalence of enterotoxin genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, and the environment. J Clin Microbiol 38, 3785–3790.
Bauer, A. W., Kirby, W. M. M., Sherris, J. C. & Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45, 493–496.[Medline]
Brenner, D. J., Fanning, G. R., Miklos, G. V. & Steigerwalt, A. G. (1973). Polynucleotide sequence relatedness among Shigella species. Int J Syst Bacteriol 23, 1–7.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[CrossRef]
Farmer, J. J., III, Davis, B. R., Hickman-Brenner, F. W. & 12 other authors (1985). Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. J Clin Microbiol 21, 46–76.
Goris, J., Suzuki, K.-I., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA-DNA hybridization method compared with the initial renaturation method. Can J Microbiol 44, 1–7.
Huys, G., Gevers, D., Temmerman, R. & 8 other authors (2001). Comparison of the antimicrobial tolerance of oxytetracycline-resistant heterotrophic bacteria isolated from hospital sewage and freshwater fishfarm water in Belgium. Syst Appl Microbiol 24, 122–130.[CrossRef][Medline]
Ismaili, A., Bourke, B., de Azavedo, J. C. S., Ratnam, S., Karmali, M. A. & Sherman, P. M. (1996). Heterogeneity in phenotypic and genotypic characteristics among strains of Hafnia alvei. J Clin Microbiol 34, 2973–2979.[Abstract]
Janda, J. M., Abbott, S. L. & Albert, M. J. (1999). Prototypal diarrheagenic strains of Hafnia alvei are actually members of the genus Escherichia. J Clin Microbiol 37, 2399–2401.
Janda, J. M., Abbott, S. L., Khashe, S. & Probert, W. (2002). Phenotypic and genotypic properties of the genus Hafnia. J Med Microbiol 51, 575–580.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3, 208–218.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
NCCLS (1993). Performance Standards for Antimicrobial Disk Susceptibility Tests, 5th edn. Approved Standard M2-A5. Villanova, PA: National Committee for Clinical Laboratory Standards.
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151–156.
Ridell, J., Siitonen, A., Paulin, L., Lindroos, O., Korkeala, H. & Albert, M. J. (1995). Characterization of Hafnia alvei by biochemical tests, random amplified polymorphic DNA PCR, and partial sequencing of 16S rRNA gene. J Clin Microbiol 33, 2372–2376.[Abstract]
Watanabe, H. & Okamura, N. (1992). The genus Shigella. In The Prokaryotes. A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, 2nd edn, pp. 2754–2759. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef]
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