JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/99/$04.0010

Aug. 1999, p. 2399–2401 Vol. 37, No. 8

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Prototypal Diarrheagenic Strains of Hafnia alvei Are ActuallyMembers of the Genus Escherichia

J. MICHAEL JANDA,1* SHARON L. ABBOTT,1 AND M. JOHN ALBERT2

Microbial Diseases Laboratory, Division of Communicable Disease Control, California Department of Health Services,Berkeley, California 94704-1011,1 and International Centre for Diarrhoeal Disease Research, Bangladesh,

Dhaka-1000, Bangladesh2

Received 8 February 1999/Returned for modification 9 April 1999/Accepted 19 April 1999

We analyzed five bacterial strains, designated 19982, 9194, 10457, 10790, and 12502, that were isolated fromstool specimens of individuals with diarrheal illness by the International Centre for Diarrhoeal DiseaseResearch in Dhaka, Bangladesh (M. J. Albert, S. M. Faruque, M. Ansaruzzaman, M. M. Islam, K. Haider, K.Alam, I. Kabir, and R. Robins-Browne, J. Med. Microbiol. 37:310–314, 1992). The strains were initiallyidentified as Hafnia alvei with a commercial identification system and were reported to contain the eae gene ofenteropathogenic Escherichia coli. Results of conventional biochemical analyses, testing of susceptibility tocephalothin, lysis by a Hafnia-specific phage, and amplification of the outer membrane protein gene phoE withspecies-specific primers support the identification of these strains as members of the genus Escherichia ratherthan Hafnia alvei. These strains varied from typical E. coli strains by their inability to produce acid from lactoseor D-sorbitol and failure to elaborate the enzyme b-D-glucuronidase. PCR analysis confirmed previous findingsthat the strains were positive for the eae gene and negative for other virulence markers present amongrecognized categories of diarrheagenic E. coli. Our findings support the hypothesis that these strains are a newcategory of diarrheagenic isolates belonging to the genus Escherichia and illustrate the importance of usingmultiple methodologies when identifying new bacterial agents of diarrheal disease.

Over the past three decades the number of bacterial taxaproven to cause gastrointestinal disease has risen dramatically.In addition to bona fide enteric pathogens, a number of re-puted agents of gastroenteritis which had previously beenthought to be simple commensals of the gastrointestinal tracthave been described (1). One such agent is Hafnia alvei. In1991, John Albert and colleagues at the International Centrefor Diarrhoeal Disease Research, Bangladesh (ICDDRB), re-ported on laboratory investigations concerning a strain of H.alvei (designated 19982) recovered from a 9-month-old girlseen at ICDDRB with vomiting, mild dehydration, fever, ab-dominal distention, and diarrhea of 72 h duration (2). Thisfecal isolate was found to induce diarrhea in 8 of 12 adultrabbits by using the removable intestinal tie-adult rabbit diar-rhea model. Electron microscopic studies of the apical surfaceof the rabbit’s infected gastrointestinal epithelium revealedclassic attachment-effacement lesions traditionally associatedwith enteropathogenic Escherichia coli (EPEC). Subsequentinvestigations by the same investigators at ICDDRB identifiedan additional six strains of H. alvei with similar properties (3).Like EPEC, all seven H. alvei strains were fluorescent actinstaining positive and hybridized with the EPEC attaching-and-effacing (eae) probe (3). These findings led to the hypothesisthat the eae locus is highly conserved among diverse specieswithin the family Enterobacteriaceae (16).

On the basis of these investigations, several case reports andcase-control studies followed, and they documented H. alvei asan enteric pathogen (18–20, 25). However, a 1996 Canadianstudy failed to detect virulence characteristics present in H.alvei 19982 in any of nine H. alvei isolates recovered fromchildren with diarrhea at The Hospital for Sick Children in

Toronto or in two strains previously implicated in outbreaks ofgastroenteritis (12). That study also noted that the outer mem-brane, plasmid, and pulsed-field gel electrophoresis (PFGE)profiles of Canadian isolates differed significantly from thoseof H. alvei 19982. Finally, recent collaborative studies betweenICDDRB and the University of Helsinki found a low level of16S rRNA sequence homology (92%) between eae-positiveand eae-negative H. alvei strains, raising concerns about thecorrect taxonomic position of these strains (20). Here we re-port on the results for five of these strains and provide dataindicating that these isolates are, in fact, unusual biotypes of E.coli or represent a new species in the genus Escherichia.

MATERIALS AND METHODS

H. alvei 19982, 9194, 10457, 10790, and 12502 were provided by M. John Albert(Dhaka, Bangladesh). The enterovirulence characteristics of these strains havebeen described previously (2, 3). Isolates were kept as working cultures onmotility deeps, with permanent stock cultures maintained at 270°C. Conven-tional biochemical tests were performed as described previously (13), and sus-ceptibility to cephalothin was determined by the AB Biodisk E test (Remel,Lexena, Kans.) according to the manufacturer’s instructions.

Testing of individual bacterial strains for Hafnia-specific phage susceptibility(9) and for E. coli-specific genes and virulence characteristics were determinedby multiplex PCR through the courtesy of the Centers for Communicable Dis-ease Control by previously described methods (5, 7, 8, 11, 14, 17, 22–24). ThePFGE profiles of selected strains were determined with a CHEF II system(Bio-Rad, Hercules, Calif.) according to the manufacturer’s instruction with therestriction endonuclease XbaI.

RESULTS AND DISCUSSION

All five ICDDRB strains were oxidase and indole negative,nonmotile, lysine and ornithine decarboxylase positive, o-ni-trophenyl-b-D-galactopyranoside positive, and methyl red pos-itive. Key differential tests useful in the separation of hafniaefrom E. coli are listed in Table 1. Several reactions, includingfailure to produce aceytlmethycarbinol (Voges-Proskauer),lack of growth in KCN broth, and failure to utilize acetate,suggested that the ICDDRB strains were not H. alvei. Among

* Corresponding author. Mailing address: Microbial Diseases Lab-oratory, 2151 Berkeley Way, Berkeley, CA 94704-1011. Phone: (510)540-2242. Fax: (510) 540-2374. E-mail: JJANDA@DHS.CA.GOV.

2399

on March 16, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

the common sugars, only D-glucose, D-mannitol, L-arabinose,trehalose, and maltose were fermented. The inability to pro-duce acid from lactose and D-sorbitol and the failure to elab-orate the enzyme b-D-glucuronidase (MUG) did not, however,fit the typical pattern exhibited by most E. coli strains.

By PCR, all five of the ICDDRB strains were positive for theeaeA gene, as reported previously (14), but were negative bymultiplex PCR (5, 7, 8, 11, 17, 22, 23) for the following viru-lence characteristics: enterohemolysin (18), bundle-forming pi-lus A (bfpA) (20, 25), EAF plasmid (21), Shiga toxins stx1 andstx2 (22, 24), E. coli invasion plasmid antigen H (ipaH) (23),and enterotoxigenic E. coli heat-labile and heat-stable entero-toxins (22).

Evidence indicating that these strains were in fact membersof the genus Escherichia comes from several lines of evidence.Almost all H. alvei strains are resistant to cephalothin (6), butall of these strains were partially or completely susceptible tocephalosporin by the E test. A genus-specific Hafnia phage,originally described by Guinee and Valkenburg in 1968 (9),failed to lyse any of these strains. Furthermore, a PCR probeencoding the phoE outer membrane protein gene, which isfound only in E. coli and Shigella species, hybridized to all fivestrains but not to true H. alvei isolates (24). Finally, when allfive of the ICDDRB strains were typed by PFGE with XbaIthey generated a typical series of 10 to 15 large DNA frag-ments since only a few restriction sites are available (Fig. 1).However, when H. alvei ATCC 13337 and ATCC 29927 weretyped by PFGE they produced a large series of small DNAbands, indicating that many XbaI restriction sites were presentin true hafniae.

Cumulative results from the present investigation indicatethat the diarrheagenic H. alvei isolates originally described byAlbert and associates (2, 3) either are unusual biotypes of E.coli or represent a new species in the genus Escherichia. Thisconclusion is based upon biochemical and antimicrobial sus-ceptibility differences between true hafniae and the ICDDRB“H. alvei-like” strains and the failure of the latter group to belysed by Hafnia-specific phage and homology between thephoE genes of the latter group and that of E. coli. Recentsequence data on a 353-bp fragment from the 59 end of the 16SrRNA sequences of 10 eae-positive H. alvei strains indicateshomology closest to an EPEC strain designated E2348/69 (20),further supporting the identification of these isolates as E. coli.

When these strains were initially characterized as H. alveithey were identified on the basis of API 20E (bioMerieux

Vitek, Hazelwood, Mo.) reactions. Although the likelihood ofa correct identification was not stated (e.g., excellent identifi-cation, acceptable identification), several reactions, notably anegative Voges-Proskauer reaction and the inability to fermentL-rhamnose, could have suggested the possibility that thesestrains were not true hafniae. Because of these initial misiden-tifications, the association of “H. alvei” with bacterial gastro-enteritis was subsequently reinforced and was disseminated ina series of case reports, clinical studies, and a review (10, 18,19, 21, 25).

Although many new potential enteric pathogens have beendescribed over the past decade, current data do not stronglysupport a role for hafniae in infectious gastroenteritis. Fur-thermore, the present report points out the critical importanceof bacterial taxonomy and the complete biochemical charac-terization of isolates in this process (4, 15). Without suchanalysis the impact of molecular characterization may be lostor provide incorrect information if the isolate is not initiallyplaced in the correct taxa. Therefore, infrequently isolatedgroups, particularly those with aberrant properties, should re-ceive close systematic scrutiny prior to the reporting of newdisease associations or virulence properties linked to suchgroups.

ACKNOWLEDGMENTS

We thank Nancy Strockbine, Evan Sowers, and Caroline O’Hara forfruitful discussions and for assistance in the genotyping of our strains.

REFERENCES

1. Abbott, S. L., and J. M. Janda. 1992. Bacterial gastroenteritis. I. Incidenceand etiologic agents. Clin. Microbiol. Newsl. 14:17–21.

2. Albert, M. J., K. Alam, M. Islam, J. Montanaro, A. S. M. H. Rahman, K.Haider, M. A. Hossain, A. K. M. G. Kibriya, and S. Tzipori. 1991. Hafniaalvei, a probable cause of diarrhea in humans. Infect. Immun. 59:1507–1513.

3. Albert, M. J., S. M. Faruque, M. Ansaruzzaman, M. M. Islam, K. Haider, K.Alam, I. Kabir, and R. Robins-Browne. 1992. Sharing of virulence-associatedproperties at the phenotypic and genetic levels between enteropathogenicEscherichia coli and Hafnia alvei. J. Med. Microbiol. 37:310–314.

FIG. 1. PFGE profile (XbaI) of Albert’s strains and reference strains of H.alvei. Lanes 1 and 9, bacteriophage lambda markers; lanes 2, Albert’s strain12502; lane 3, Albert’s strain 10790; lane 4, Albert’s strain 10457; lane 5, Albert’sstrain 9194; lane 6, Albert’s strain 19982; lane 7, H. alvei ATCC 13337; and lane8, H. alvei ATCC 29927.

TABLE 1. Comparison of properties of Albert’s diarrheal isolatesto characteristic properties of H. alvei and E. coli strains

Test

Result for the following strainsa:

E. colib H. alveibICDDRB strains

19982 9194 10457 10790 12502

Voges-Proskauer 2 1 2 2 2 2 2KCN 2 1 2 2 2 2 2Acetate 1 2 1 1 1 1 1Acid from:

Lactose 1 2 2 2 2 2 2D-Sorbitol 1 2 2 2 2 2 2

MUG 1 2 2 2 2 2 2Cephalothin S R I I S I SHafnia phage 1672 2 1 2 2 2 2 2phoE gene probe 1 2 1 1 1 1 1

a Abbreviations: 1, $85% positive; 2, #15% positive; S, susceptible; I, inter-mediate; R, resistant.

b Typical profiles of each species (active E. coli, H. alvei) are from Farmer (6).

2400 JANDA ET AL. J. CLIN. MICROBIOL.

on March 16, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

4. Brenner, D. J. 1988. Phylogenetic classification of bacteria and recent de-velopments in bacterial classification and nomenclature: impact on clinicalmicrobiology. Clin. Microbiol. Newsl. 10:153–156.

5. Cebula, T. A., W. L. Payne, and P. Feng. 1995. Simultaneous identification ofstrains of Escherichia coli serotype O157:H7 and their Shiga-like toxin typeby mismatch amplification mutation assay-multiplex PCR. J. Clin. Microbiol.32:248–250.

6. Farmer, J. J., III. 1995. Enterobacteriaceae: introduction and identification,p. 438–449. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, andR. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. AmericanSociety for Microbiology, Washington, D.C.

7. Franke, J., S. Franke, H. Schmidt, A. Schwarzkopf, L. H. Wieler, G. Balijer,L. Beutin, and H. Karch. 1994. Nucleotide sequence analysis of enteropatho-genic Escherichia coli (EPEC) adherence factor probe and development ofPCR for rapid detection of EPEC harboring virulence plasmids. J. Clin.Microbiol. 32:2460–2463.

8. Fratamico, P. M., S. K. Sackitey, M. Wiedmann, and M. Yi Deng. 1995.Detection of Escherichia coli O157:H7 by multiplex PCR. J. Clin. Microbiol.33:2188–2191.

9. Guinee, P. A. M., and J. J. Valkenburg. 1968. Diagnostic value of a Hafnia-specific bacteriophage. J. Bacteriol. 96:564.

10. Gunthard, H., and A. Pennekamp. 1996. Clinical significance of extraintes-tinal Hafnia alvei isolates from 61 patients and review of the literature. Clin.Infect. Dis. 22:1040–1045.

11. Gunzburg, S. T., N. G. Tornieporth, and L. W. Riley. 1995. Identification ofenteropathogenic Escherichia coli by PCR-based detection of the bundle-forming pilus gene. J. Clin. Microbiol. 33:1375–1377.

12. Ismaili, A., B. Bourke, J. C. S. de Azavedo, S. Ratnam, M. A. Karmali, andP. M. Sherman. 1996. Heterogeneity in phenotypic and genotypic charac-teristics among strains of Hafnia alvei. J. Clin. Microbiol. 34:2973–2979.

13. Janda, J. M., S. L. Abbott, W. K. W. Cheung, and D. F. Hanson. 1994.Biochemical identification of citrobacteria in the clinical laboratory. J. Clin.Microbiol. 32:1850–1854.

14. Louie, M., J. De Azavedo, R. Clarke, A. Borczyk, H. Lior, M. Richter, and J.Brunton. 1994. Sequence heterogeneity of the eae gene and detection of

verotoxin-producing Escherichia coli using serotype-specific primers. Epide-miol. Infect. 112:449–461.

15. Marcon, M. J. 1996. The medical relevance of molecular taxonomy andassociated name changes, or “What’s in a name?” Clin. Microbiol. Newsl.18:123–125.

16. McDaniel, T. K., K. G. Jarvis, M. S. Donnenberg, and J. B. Kaper. 1995. Agenetic locus of enterocyte effacement conserved among diverse enterobac-terial pathogens. Proc. Natl. Acad. Sci. USA 92:1664–1668.

17. Olsvik, O., and N. A. Strockbine. 1993. Polymerase chain reaction detectionof heat-stable, heat-labile, and Shiga-like toxin genes in Escherichia coli, p.271–276. In D. H. Persing, F. C. Tenover, T. F. Smith and T. J. White (ed.),Diagnostic molecular microbiology. American Society for Microbiology,Washington, D.C.

18. Ratnam, S. 1991. Etiologic role of Hafnia alvei in human diarrheal illness.Infect. Immun. 59:4744–4745. (Letter.)

19. Reina, J., J. Hervas, and N. Borrell. 1993. Acute gastroenteritis caused byHafnia alvei in children. Clin. Infect. Dis. 16:443. (Correspondence.)

20. Ridell, J., A. Siitonen, L. Paulin, O. Lindroos, H. Korkeala, and M. J. Albert.1995. Characterization of Hafnia alvei by biochemical tests, random ampli-fied polymorphic DNA PCR, and partial sequencing of 16S rRNA gene.J. Clin. Microbiol. 33:2372–2376.

21. Ridell, J., A. Siitonen, L. Paulin, L. Mattila, H. Korkeala, and M. J. Albert.1994. Hafnia alvei in stool specimens from patients with diarrhea and healthycontrols. J. Clin. Microbiol. 32:2335–2337.

22. Schmidt, H., C. Knop, S. Franke, S. Aleksic, J. Heesemann, and H. Karch.1995. Development of PCR for screening of enteroaggregative Escherichiacoli. J. Clin. Microbiol. 33:701–705.

23. Sethabutr, O., M. Venkatesan, G. S. Murphy, B. Eampokalap, C. W. Hoge,and P. Echeverria. 1992. Detection of shigellae and enteroinvasive Esche-richia coli by amplification of the invasion plasmid antigen H DNA sequencein patients with dysentery. J. Infect. Dis. 167:458–461.

24. Spierings, G., C. Ockhuijsen, H. Hofstra, and J. Tommassen. 1993. Poly-merase chain reaction for the specific detection of Escherichia coli/Shigella.Res. Microbiol. 144:557–564.

25. Westblom, T. U., and T. W. Milligan. 1992. Acute bacterial gastroenteritiscaused by Hafnia alvei. Clin. Infect. Dis. 14:1271–1272. (Correspondence.)

VOL. 37, 1999 ATYPICAL E. COLI 2401

on March 16, 2021 by guest

http://jcm.asm

.org/D

ownloaded from