Jpn. J. Infect. Dis., 67, 204-208, 2014

Short Communication Shiga Toxin 2f-Producing albertii from a Symptomatic Human

Koichi Murakami1*, Yoshiki Etoh1, Eri Tanaka2, Sachiko Ichihara1, Kazumi Horikawa1, Kimiko Kawano3,TadasukeOoka4, Yoshiaki Kawamura2, and Kenitiro Ito5 1Fukuoka Institute of Health and Environmental Sciences, Fukuoka 818-0135; 2Department of Microbiology, School of Pharmacy, Aichi Gakuin University, Aichi 464-8650; 3Miyazaki Prefectural Institute for Public Health and Environment, Miyazaki 889-2155; 4Division of Microbiology, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692; and 5National Institute of Infectious Diseases, Tokyo 162-8640, Japan (Received August 15, 2013. Accepted November 18, 2013)

SUMMARY: The previously identified Shiga toxin (Stx) 2f-producing O115:HNM strain F08/101-31, isolated from a symptomatic human, was confirmed to be E. albertii in the present study by whole genome DNA–DNA hybridizations, by sequencing (cpn60, dnaJ, and 16S rRNA genes), and by multi-locus sequence typing. The F08/101-31 strain was originally identified as E. coli rather than the relatively new bacterial species E. albertii, which was first described in 2003, because it did not display any of the biochemical characteristics of E. albertii. This new classification will impact public health management strategies in Japan because the present study showed that some E. albertii strains, which are often misidentified as E. coli, produce Stx and likely cause diarrhea in humans. Therefore, further guidelines for the management and identification of Stx-producing E. albertii are required in Japan.

The discovery of Shiga toxin (Stx)-producing Es- nome DNA–DNA hybridization and DNA sequencing cherichia albertii strains was significant from a public techniques. The purpose of this study was to describe health perspective in Japan (1). E. albertii was first de- the characteristics of the E. albertii strain for the benefit scribed by Huys et al. in 2003 (2). This newly described of public health. enteropathogen is often misidentified as E. coli (3) or For genetic analysis, species-specific PCR analysis, other members of the family be- whole-genome DNA–DNA hybridization, sequence cause of its poorly defined biochemical characteristics analysis (16S rRNA genes, cpn60 (groEL), and dnaJ), and general obscurity. In addition, E. albertii closely and multi-locus sequence typing (MLST) were per- resembles E. coli in its phenotypic characteristics. formed. The F08/101-31 strain was examined by Retention of the major virulence factor intimin in E. al- species-specific PCR, as described previously, for the bertii is well established (4,5), whereas little is known presence of E. albertii-specific sequences within lysP about the incidence of Stx-producing E. albertii (1). Stxs and mdh (4) and for E. coli, Shigella,andE. albertii/S. are the most significant virulence factors of Stx-produc- boydii lineage-detecting alleles of clpX (4,8). Whole-ge- ing E. coli (STEC) in human infections. However, nome DNA–DNA hybridizations were performed be- STEC strains that produce intimin, a host-cell adhesin, tween the F08/101-31 strain and the E. albertii type often cause more critical symptoms in patients than strain LMG 20976T as well as other strains of closely STEC strains that do not produce intimin (6). There- related species (E. coli JCM 1649T and Hafnia alvei fore, the misidentification of Stx-producing E. albertii JCM 1666T). DNA extraction and purification were per- strains that also produce intimin as E. coli may compli- formed as described by Marmur (9) and Boom et al. cate public health monitoring strategies in many coun- (10), respectively. A membrane-filter DNA–DNA tries. hybridization method was performed as described by The F08/101-31 strain, which was previously reported Tourova and Antonov (11). Photobiotin-labeled DNA as Stx2f-producing E. coli O115:HNM harboring eae was hybridized with filter-bound DNA in 6× standard (7), was recently inferred to be an Stx2f-producing E. al- saline citrate and 50z formamide at 359C (optimal con- bertii strain on the basis of species-specific polymerase dition) and 459C (stringent condition). The amount of chain reaction (PCR) analysis (4,8). Therefore, in the re-associated DNA was measured using Image Quant present study, we attempted to definitively determine LAS4000 (GE Healthcare Bioscience, Tokyo, Japan), whether the strain is an E. albertii isolate by whole-ge- followed by the addition of alkaline phosphatase-conju- gated streptavidin and chemiluminescence substrate *Corresponding author: Mailing address: Fukuoka Institute (CDP-star; Roche Applied Science, Tokyo, Japan) to of Health and Environmental Sciences, 39 Mukaizano, the filters. The amplification and sequencing of 16S Dazaifu, Fukuoka 818-0135, Japan. Tel: ＋81-92-921- rRNA genes, cpn60 (groEL), and dnaJ were performed 9944, Fax: ＋81-92-928-1203, E-mail: murakami＠fihes. as follows: the 16S rRNA genes (1483 bp) were se- pref.fukuoka.jp quenced using previously described primers (12), and

204 E. albertii with Stx2f from a Symptomatic Human

1352 bp of the resultant sequences were compared with of phenotypic assays using both conventional methods corresponding regions from other bacterial species from and a commercial test strip (Table 1). The strain was ex- the DNA Data Bank of Japan (DDBJ) using a the Basic amined for indole production, Voges–Proskauer reac- Local Alignment Search Tool (BLAST) (http://blast. tion, motility at 359C, presence of lysine decarboxylase ddbj.nig.ac.jp/blastn?lang＝ja). Partial sequences of and ornithine decarboxylase, acetate utilization, and gas cpn60 (590 bp) were amplified as described previously production from D-glucose, as described previously (http://homepage.usask.ca/ Ã jeh369/cpn60 _ universal _ (18). Other phenotypic characteristics of the strain were primers.html, accessed June 2012), along with those of tested using API 20E and API 50 CH test strips (bio- dnaJ (554 bp) (13). The amplified sequences were then Máerieux, Ltd., Marcy-l' ÁEtoile, France), according to compared with corresponding sequences from other the manufacturer's instructions. related species listed in DDBJ using BLAST analysis. Genotyping analysis by species-specific PCR revealed MLST analysis was performed as described previously that the F08/101-31 strain exhibited E. albertii-specific (14) using the housekeeping genes adk, fumC, gyrB, icd, bands for lysP (252 bp) and mdh (115 bp). The DNA– mdh, purA,andrecA. Allele sequences for each strain DNA hybridization values obtained under optimal were then concatenated in the order adk–fumC–gyrB– (359C) and stringent (459C) conditions are shown in Ta- icd–mdh–purA–recA to obtain a final composite length ble 2. The values for the F08/101-31 strain and the E. of 3423 bp and analyzed as described previously (14). albertii control strain LMG 20976T were almost identi- Further genotypic analysis of the F08/101-31 strain cal, indicating that F08/101-31 is an E. albertii strain, was performed as detailed below, along with intimin with a hybridization value of À70z obtained even un- typing. In brief, the genes lpfAO26, sfpA,EHEC-hlyA, der stringent conditions. Sequences comparison of the irp2,andfyuA were amplified using primers described 16S rRNA genes, cpn60,anddnaJ of the F08/101-31 by Sonntag et al. (15), and the cytolethal distending tox- strain with those of other related strains revealed that in gene (cdt) was amplified according to the method de- F08/101-31 was more similar to E. albertii than other scribed by Pickett et al. (16). The intimin gene (eae) species (Fig. 1). Only 8 out of 1352 bp of the 16S rRNA from the F08/101-31 strain was subtyped using gene sequence differed between the F08/101-31 strain heteroduplex mobility analysis, as described previously and E. albertii LMG 20976T (Fig. 1). On the other hand, (17). BLAST analysis revealed a mismatch of 20 bp between The F08/101-31 strain was compared with known E. the 16S rRNA gene sequences of the F08/101-31 strain albertii isolates LMG 20976T and LMG 20973 in a series and E. coli ATCC 1352T, its closest relative among non-

Table 1. Biological characteristics of F08/101-31 and other E. albertii strains tested or cited from the literature

Characteristic described C)

Strain or species 9 -Glucose, gas -Mannitol, acid -Sorbitol, acid -Xylose, acid -Arabinose, acid -Arabitol, acid -Rhamnose, acid Cellobiose, acid Dulcitol, acid Lactose, acid Sucrose, acid Adonitol, acid Raffinose, acid Indole production (24 h) Voges-Proskauer Motility (35 D L L D L D D Lysine decarboxylase Ornithine decarboxylase Acetate use (48 h)

F08/101-31 ＋ －－＋ ＋ ＋ ＋－＋－－－－－＋－－＋－ E. albertii LMG20976T and LMG20973 － －－＋ ＋ ＋ ＋－＋－－－－－＋－－－－ E. albertii (biogroup 1)1) 0z2) 0z 0z 100z 100z 40z 100z 0z 100z 0z 0z 0z 0z 0z 100z 0z 0z 100z 0z E. albertii (biogroup 2)1) 100z 0z 0z 0z 100z 0z 40z 0z 100z 0z 0z 0z 0z 0z 100z 0z 0z 0z 0z

1): Cited from ref. 19. 2):0z, percentage of positive strains.

Table 2. DNA–DNA hybridization values

DNA hybridization (z) with biotin-labelled DNA from:

Escherichia albertii Escherichia coli T Strain F08/101-31 LMG 20976T JCM1649T Hafnia alvei JCM1666

Optimal1) Stringent2) Optimal Stringent Optimal Stringent Optimal Stringent

F08/101-31 100 ± 0100± 0 79.7 ± 5.9 73.7 ± 2.4 55.6 ± 3.8 57.5 ± 1.4 12.0 ± 4.7 7.2 ± 1.6 Escherichia albertii LMG20976T 82.6 ± 6.8 80.6 ± 3.7 100 ± 0100± 057.9± 3.3 44.1 ± 1.0 4.4 ± 1.6 7.7 ± 3.2 Escherichia coli JCM1649T 52.9 ± 2.7 35.9 ± 4.4 59.0 ± 0.4 50.8 ± 5.0 100 ± 0 100 ± 010.8± 1.4 5.5 ± 2.4 Hafnia alvei JCM1666T 5.0 ± 0.4 8.7 ± 0.3 8.4 ± 5.6 5.3 ± 1.9 8.6 ± 0.4 3.4 ± 0.8 100 ± 0100± 0

1): Under optimal hybridization condition (359C). 2): Under stringent hybridization condition (459C). Each value represents the mean ± sd of three experiments.

205 Fig. 1. Phylogenetic tree showing nucleotide sequence clusters constructed from the 16S rRNA gene (upper), cpn60 (groEL)(middle),anddnaJ (lower) sequence data from F08/101-31 and related strains that were selected using BLAST analysis. The numbers in the tree indicate bootstrap values (z) from 1000 replicates. Salmonella enterica was used as an outgroup. The scale represents nucleotide substitutions per site. Accession numbers for reference sequences are in parentheses.

Fig. 2. Phylogenetic tree showing nucleotide sequence clusters of representative isolates with multi-locus sequence typing data. Allele sequences for each strain were concatenated in the order adk–fumC–gyrB–icd–mdh–purA– recA for a final composite length of 3423 bp. Reference sequences were tested in the present study. Shigella boydii serotype 13 ATCC 12032 has been reclassified in the Escherichia albertii-lineage. The scale bar indicates the num- ber of nucleotide substitutions per site.

E. albertii species. The cpn60 and dnaJ sequences of the also revealed that the F08/101-31 strain belongs to the F08/101-31 strain differed by only 3 bp and 1 bp, re- E. albertii and not the E. coli lineage (Fig. 2). A new spectively, from the corresponding sequences of E. al- 16S rRNA gene sequence for the F08/101-31 strain was bertii LMG 20976T (Fig. 2). Similarly, MLST analysis deposited in DDBJ, with the accession no. AB594160.

206 E. albertii with Stx2f from a Symptomatic Human

New cpn60 sequences were also deposited for including F08/101-31, could not be categorized as either F08/101-31, LMG 20972, LMG 20973, and LMG biotype 1 or 2. Therefore, we suggest that it is necessary 20976T under accession numbers AB594161–AB594164. to reorganize the biotypes of E. albertii. Although a few dnaJ sequences for these four strains were also common biochemical properties have been reported, deposited in the DDBJ under accession numbers lack of motility and inability to ferment D-xylose and L- AB594166–AB594169. The sequences generated by rhamnose (1,8,25) may be common characteristics of E. MLST are available from the MLST database (http:/ albertii strains. /mlst.ucc.ie/). Of the other tested genes, only cdt was Genotyping methods remain particularly helpful in detected in the F08/101-31 strain, and the intimin type the identification of E. albertii. Species-specific PCR in the 5? region was the b1-variant. based on MLST (4,8) is particularly useful for confirm- Phenotyping revealed that the F08/101-31 strain pro- ing E. albertii strains. We previously isolated a few E. duced indole, and contrary to previous reports (19), it albertii strains from the feces of wild birds using a cul- was positive for lysine decarboxylase (Table 1). The ture identification method aided by PCR (data not API 20E test strips identified F08/101-31 as E. coli shown). Sequencing of cpn60 and dnaJ also helped in (probability, 81.5z; T value, 0.86) (https://apiweb. the identification of the F08/101-3 strain in the present biomerieux.com/servlet/Authenticate, accessed June study (Fig. 2). We recommend that these genetic tests 2013), with the code 4144502. should be performed for the correct identification of Two major results were obtained from the present non-motile Stx-producing isolates that harbor eae and study. First, the F08/101-31 strain, isolated from a are unable to ferment D-xylose and L-rhamnose to con- symptomatic patient, was identified as an Stx2f-produc- firm whether the isolates are E. coli or E. albertii. ing strain of E. albertii, and not E. coli. Second, the In conclusion, in the present study, we showed that biochemical characteristics of the F08/101-31 strain de- certain E. albertii strains, which are often misidentified termined by the present study were not consistent with as E. coli, produce Stx and likely cause diarrhea in hu- those described for E. albertii by Nataro et al. (19). mans. Therefore, further guidelines for the manage- Other than E. coli, Stx production has been reported in ment and identification of Stx-producing E. albertii S. dysenteriae type 1 (20), S. flexneri (21), Enterobacter strains are required in Japan. cloacae (22), Citrobacter freundii (23), and Aeromonas species (24). Stx production is an unusual phenomenon Acknowledgments This work was supported by a grant from the in all these species, except for S. dysenteriae type 1. JSPS KAKENHI Grant Number 24590846 to Murakami. We are However, E. albertii strains harboring stx2f appear to be grateful to Professor Hayashi, Frontier Science Research Center, Uni- relatively common and have been reported by Ooka et versity of Miyazaki, for invaluable advice. We also thank Dr. Hirata, al. (1); moreover, we have isolated 4 E. albertii strains Dr. Chijiwa, Dr. Sera, Mr. Kimoto, Ms. Uemura, and Dr. Takenaka, Fukuoka Institute of Health and Environmental Sciences, for their harboring stx2f from wild birds (data not shown). Fur- advice. ther studies investigating the observation that Stx2f- producing E. albertii strains cause more severe symp- Conflict of interest None to declare. toms than Stx-negative E. albertii are required. Identification of the F08/101-31 strain as E. albertii REFERENCES using just phenotypic characteristics is difficult. The 1. Ooka T, Seto K, Kawano K, et al. Clinical significance of Es- strain had not been previously identified as E. albertii cherichia albertii. Emerg Infect Dis. 2012;18:488-92. on the basis of routine examinations using biochemical 2. Huys G, Cnockaert M, Janda JM, et al. Escherichia albertii sp. tests because it displayed none of the biochemical nov., a diarrhoeagenic species isolated from stool specimens of characteristics of E. albertii, as defined in a previous Bangladeshi children. Int J Syst Evol Microbiol. 2003;53:807-10. 3. Ooka T, Tokuoka E, Furukawa M, et al. Human gastroenteritis report (19). Hyma grouped a few S. boydii type 13 iso- outbreak associated with Escherichia albertii, Japan. Emerg In- lates into E. albertii lineages (4). In addition, 2 biotypes fect Dis. 2013;19:144-6. of E. albertii have been documented by Nataro et al. 4.HymaKE,LacherDW,NelsonAM,etal.Evolutionarygenetics (19). According to the description of Nataro et al. (19), of a new pathogenic Escherichia species: Escherichia albertii and related Shigella boydii strains. J Bacteriol. 2005;187:619-28. E. albertii biotype 1 is negative for indole production, 5. Lacher DW, Steinsland H. Whittam TS. Allelic subtyping of the positive for lysine decarboxylase, and positive for acid intimin locus (eae) of pathogenic Escherichia coli by fluorescent production from D-sorbitol (hereafter referred to as RFLP. FEMS Microbiol Lett. 2006;261:80-7. ``negative-indole/positive-lysine/positive-D-sorbitol''), 6. Jenkins C, Willshaw GA, Evans J, et al. Subtyping of virulence while biotype 2 is positive-indole/negative-lysine/ genes in verocytotoxin-producing Escherichia coli (VTEC) other than serogroup O157 associated with disease in the United King- negative-D-sorbitol (Table 1). However, the F08/101-31 dom. J Med Microbiol. 2003;52:941-7. strain was positive-indole/positive-lysine/positive-D- 7. Etoh Y, Murakami K, Ichihara S, et al. Isolation of Shiga toxin sorbitol (Table 1). Stock et al. (25) reported that 10z of 2f-producing Escherichia coli (O115:HNM) from an adult symp- E. albertii isolates (2/21) showed a positive-indole/ tomatic patient in Fukuoka Prefecture, Japan. Jpn J Infect Dis. 2009;62:315-7. positive-lysine/negative-D-sorbitol phenotype, while the 8.OaksJL,BesserTE,WalkST,etal.Escherichia albertii in wild remaining 90z showed a negative-indole/positive- and domestic birds. Emerg Infect Dis. 2010;16:638-46. lysine/negative-D-sorbitol phenotype. Ooka et al. (1) 9. Marmur J. A procedure for the isolation of deoxyribonucleic acid reported that 96.2z of E. albertii isolates (25/26) were from micro-organisms. J Mol Biol. 1961;3:208-18. positive-indole/positive-lysine, while Oaks et al. (8) 10. Boom R, Sol CJ, Salimans MM, et al. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. reported that isolates (uncertain number) from birds 1990;28:495-503. were positive-indole/positive-lysine with varying D-sor- 11. Tourova TP, Antonov AS. Identification of microorganisms by bitol fermentation. Subsequently, several E. albertii iso- rapid DNA-DNA hybridization. Methods Microbiol. lates with positive-indole/positive-lysine phenotypes, 1987;19:333-55.

207207 12. Kawanami T, Fukuda K, Yatera K, et al. Severe pneumonia with Salmonella. In: Murray PR, Baron EJ, Jorgensen JH, et al. Leptotrichia sp. detected predominantly in bronchoalveolar editors. Manual of Clinical Microbiology. 9th ed. Washington lavage fluid by use of 16S rRNA gene sequencing analysis. J Clin DC: ASM Press; 2007. p. 670-87. Microbiol. 2009;47:496-8. 20. Thorpe CM, Ritchie JM, Acheson DWK. Enterohemorrhgic and 13. Pham HN, Ohkusu K, Mishima N, et al. Phylogeny and species other Shiga toxin-producing Escherichia coli. In: Donnenberg identification of the family Enterobacteriaceae based on dnaJ se- MS, editor. Escherichia coli: Virulence Mechanisms of a Versatile quences. Diagn. Microbiol Infect Dis. 2007;58:153-61. Pathogen. San Diego: Academic Press; 2002. p. 119-54. 14. Noda T, Murakami K, Asai T, et al. Multi-locus sequence typing 21. Yavzori M, Cohen D, Orr N. Prevalence of the genes for shigella of Salmonella enterica subsp. enterica serovar Enteritidis strains enterotoxins 1 and 2 among clinical isolates of shigella in Israel. in Japan between 1973 and 2004. Acta Vet Scand. 2011;53:38. Epidemiol Infect. 2002;128:533-5. 15. Sonntag AK, Zenner E, Karch H, et al. Pigeons as a possible 22. Paton AW, Paton JC. Enterobacter cloacae producing a Shiga- reservoir of Shiga toxin 2f-producing Escherichia coli pathogenic like toxin II-related cytotoxin associated with a case of hemolytic- to humans. Berl Munch Tierarztl Wochenschr. 2005;118:464-70. uremic syndrome. J Clin Microbiol. 1996;34:463-5. 16. Pickett CL, Pesci EC, Cottle DL, et al. Prevalence of cytolethal 23. Herold S, Karch H, Schmidt H. Shiga toxin-encoding bac- distending toxin production in Campylobacter jejuni and related- teriophages—genomes in motion. Int J Med Microbiol. ness of Campylobacter sp. cdtB gene. Infect Immun. 2004;294:115-21. 1996;64:2070-8. 24. Alperi A, Figueras MJ. Human isolates of Aeromonas possess 17. Ito K, Iida M, Yamazaki M, et al. Intimin types determined by Shiga toxin genes (stx1andstx2) highly similar to the most viru- heteroduplex mobility assay of intimin gene (eae)-positive Es- lent gene variants of Escherichia coli. Clin Microbiol Infect. cherichia coli strains. J Clin Microbiol. 2007;45:1038-41. 2010;16:1563-7. 18. Barrow GI, Feltham RKA. Cowan and Steel's Manual for the 25. Stock I, Rahman M, Sherwood KJ, et al. Natural antimicrobial Identification of Medical . Cambridge: Cambridge Uni- susceptibility patterns and biochemical identification of Es- versity Press; 2003. cherichia albertii and Hafnia alvei strains. Diagn Microbiol Infect 19. Nataro JP, Bopp CA, Fields PI, et al. Escherichia, Shigella,and Dis. 2005;51:151-63.

208208