Vol. 29, No. 9JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1991, p. 1997-20010095-1137/91/091997-05$02.00/0Copyright © 1991, American Society for Microbiology

Pathogenic Properties of Edwardsiella SpeciesJ. MICHAEL JANDA,1* SHARON L. ABBOTT,' SUSAN KROSKE-BYSTROM,1 WENDY K. W. CHEUNG,'

CATHERINE POWERS,' ROBERT P. KOKKA,1 AND K. TAMURA2Microbial Diseases Laboratory, California Department of Health Services, Berkeley, California 94704-1011,

and National Institute of Health, 10-35, Kamiosaki, Shinagawa-ku Tokyo 141, Japan2Received 12 April 1991/Accepted 21 June 1991

The pathogenic characteristics of 35 Edwardsiella strains from clinical and environmental sources were

investigated. Overall, most Edwardsiella tarda strains were invasive in HEp-2 cell monolayers, produced a

cell-associated hemolysin and siderophores, and bound Congo red; many strains also expressed mannose-

resistant hemagglutination against guinea pig erythrocytes. Edwardsiella hoshinae strains bound Congo red andwere variable in their invasive and hemolytic capabilities while Edwardsiella ictaluri strains did not produceeither factor; neither E. hoshinae nor E. ictaluri expressed mannose-resistant hemagglutination nor elaboratedsiderophores under the tested conditions. Selected strains of each species tested for mouse lethality indicatedstrain variability in pathogenic potential, with E. tarda strains being the most virulent; 50% lethal doses inindividual strains did not correlate with plasmid content, chemotactic motility, serum resistance, or expressionof selected enzyme activities. The results suggest some potential important differences in pathogenic propertiesthat may help explain their environmental distribution and ability to cause disease in humans.

One of the less frequently encountered pathogenic generain the family Enterobacteriaceae is the genus Edwardsiella(8). Although the genus originally consisted of only a singlemember (Edwardsiella tarda), at least three species are nowknown to exist. These species often inhabit freshwatersources and can also be recovered from cold-blooded verte-brates. Edwardsiellae additionally produce a wide range ofinfections in animals and are recognized as pathogens foreels, catfish, and high-order vertebrates. Edwardsiella icta-luri primarily causes enteric septicemia in channel catfish(10, 11, 27), while E. tarda has been implicated in the sameanimals as the causative agent of emphysematous putrefac-tive disease, a foul-smelling wound infection with abscessformation (18); other animal diseases caused by E. tardainclude "red disease" in eels (31) and enteritis in penguins(4). In humans, E. tarda is the only recognized pathogenicspecies primarily associated with sporadic cases of gastro-enteritis (3, 13); in rare instances, E. tarda has also beenreported to cause extraintestinal disease, most commonlyinvolving cases of septicemia or bacteremia (34). In the caseof Edwardsiella hoshinae, although this species has beenrecovered from humans (in feces), it has been most oftenisolated from lizards and birds (8, 9). A definite associationbetween this species and its isolation as a bona fide pathogenhas not been established to date.Very little information is currently available concerning

what factors regulate pathogenicity in the three Edwardsiellaspecies. Several recent studies have identified a number offactors potentially associated with the pathogenicity of ed-wardsiellae. These factors include cell-associated or extra-cellular enzymes, hemagglutinins, invasins, and extrachro-mosomal elements (12, 15, 16, 32, 35). Presently, it isunknown whether certain virulence-associated factors arespecies specific or whether differential expression within aspecies is defined by site of isolation. How these factors playa role in the disease process is also unclear. To begin toaddress some of these issues, we surveyed the pathogenicproperties of 35 Edwardsiella strains recovered from distinct

* Corresponding author.

ecologic settings to see how expression of these factorsmight relate to virulence.

MATERIALS AND METHODS

Bacterial strains. Thirty-five Edwardsiella strains (E.tarda, n = 24; E. hoshinae, n = 6; E. ictaluri, n = 5) wereinvestigated in this study. The E. tarda strains tested wereselected for analysis on the bases of their source of isolationand disease presentation; these strains were wild-type iso-lates (with the exception of strain F63) which failed toproduce acid from D-mannitol, sucrose, and L-arabinose andproduced H2S on TSI Agar. Of the 35 strains, 21 have beendescribed previously (12, 35); of the remaining 14 strains, 5environmental isolates were received from J. Lindquist(Madison, Wis.), 6 additional isolates were from the Micro-bial Diseases Laboratory collection, and 2 cultures (ATCC35052 and ATCC 33379) were obtained from the AmericanType Culture Collection (Rockville, Md.). The serotype ofeach E. tarda strain was determined according to the re-cently revised international typing scheme (30). For all invitro assays, E. tarda and E. hoshinae strains were culturedand tested at 35°C, while E. ictaluri was grown and assayedat 25°C unless otherwise specified.

Motility. The ability of edwardsiellae to migrate chemo-tactically in motility medium was determined according tothe method of Craven and Montie (5). Standardized suspen-sions (ca. 109 CFU) were point inoculated into motility agarwhich consisted of 1% tryptone, 0.3% yeast extract, 0.5%NaCl, and 0.3% agar. Migration of bacteria in such media isdependent on the motility of individual strains and theirresponsiveness to chemotactic gradients generated by deple-tion of metabolites surrounding growth. The distance ofmigration from the point source inoculum (diameter is ex-pressed in millimeters) for each strain was determined 18 to20 h after initial inoculation. Strains whose zone was .10mm were considered either nonmotile or defective in chemo-tactic mobility.MRHA. Mannose-resistant hemagglutination (MRHA) of

guinea pig erythrocytes (1.5% vol/vol) in the presence of 1%D-mannose was determined according to the protocol of

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Wong et al. (35) by using the rock tile test. Some strains werefurther evaluated for the ability of chemical treatments orspecific analogs to inhibit this MRHA reaction.HEp-2 invasion and CAH. The invasive capabilities of

Edwardsiella species were screened in HEp-2 cells propa-gated in chamber slides in a 5% C02 atmosphere at 35°C (12).Invasive strains were defined as those whose numbers ofgentamicin-resistant progeny at 3 h postinfection were equalto or exceeded 103 CFU; initial infection inocula were ca. 106CFU. Invasive strains were not probed for invasion plasmidantigen-related sequences, as a previous study had indicatedthat E. tarda strains are Sereny test negative (2). Cell-associated hemolysins (CAH) were detected by coincuba-tion of serial dilutions of standardized suspensions of bacte-ria in phosphate-buffered saline with 1% (vol/vol) solutionsof either guinea pig, sheep, or rabbit erythrocytes in micro-titer plates at 35°C for 1 h. Strains producing a CAH weredefined as those whose CAH titer was .4 hemagglutinatingunits (hemolytic units, 100% lysis) against one or more of theerythrocytes tested (12).

STs. Production of heat-stable (ST) enterotoxinlike activ-ity for all 35 Edwardsiella strains was determined in sucklingmice according to the method of Dean et al. (6) as recentlymodified (1); 25 of these strains were additionally screenedfor homologous sequences to the ST of Escherichia coli (20)by using the biotinylated SNAP enterotoxigenic E. coli STprobe (Molecular Biosystems, Inc., San Diego, Calif.).

Phenotypic markers. The ability of edwardsiellae to bindCongo red was determined on tryptic soy agar containing0.006% Congo red. Plates were incubated at the appropriatetemperature for 72 h before final readings were recorded.Siderophore production was determined under identical con-ditions by using Chrome Azurol S agar (1). Chondroitinaseactivity (28) was assessed on brain heart infusion agarcontaining 400 ,ug of chondroitin sulfate per ml and 1%bovine serum albumin (fraction V). For selected strains, theability of 65% pooled human serum to induce complement-

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FIG. 1. Invasion of HEp-2 cells by edwardsiellae.

TABLE 1. The in vitro pathogenic properties ofE. tarda strainsa

Strain Source Serotype Mot Inv CAH MRHA Crb CAS

Clinical15947T Feces 033:H1 + + + + + +ET-1 Feces 033:H1 - + + + + +ET-2 Feces 040:H19 + + + + + +ET-7 Feces 033:HNM - + + + + +ET-11 Feces 059:H19 + + + - + +ET-12 Feces 024:H11 + + + + + +ET-13 Abscess 036:H1 + + + - + +ET-14 Feces 06:H19 + + + - + +ET-15 Blood 05:H1 + + + + + (+)ET-16 Feces UK:H8 + + + - + +ET-17 Feces 059:H14 - + + + + +ET-19 Feces 032:H14 + + + - + +ET-20 Spleen 04:UK + + + - + -

Nonhuman10A Lake 024:H14 + + + + + +Fl Lake 030:HUK + + + - + (+)F31 Lake 044:H8 + + + - + +F41 Lake UK:UK + + + + + (+)F53 Lake 059:H19 + + + - + +F63 Lake 045:H37 + + + - (+) +ET-18 Heron 05:H5 + + + - + +SA 8318 Flounder 05:H1 + + - + + +AC 8321 Eel R:H1 + + - + + +TK 8403 Eel 09:H1 - - - + + +3592-64 Unknown R:H4 + - + + + +

a Abbreviations: Mot, motility; Inv, invasion of HEp-2 cells; CAH, cell-associated hemolysin; MRHA, mannose-resistant hemagglutination againstguinea pig erythrocytes; Crb, Congo red binding; CAS, siderophore produc-tion on chrome azurol S agar; UK, unknown type; NM, nonmotile. Paren-theses denote weak reactions.

mediated lysis of bacteria was performed in microcentrifugetubes; end point analysis occurred at 2 h postincubation (22).

Plasmid analysis and mouse pathogenicity. All strains werescreened for the presence of extrachromosomal elements byhorizontal electrophoresis of cell lysates on 0.75% agarosegels (22). Plasmids were subsequently visualized with UVillumination. The pathogenic potentials of some E. tarda, E.hoshinae, and E. ictaluri strains were determined by intra-peritoneal injection of viable bacteria into female SwissWebster mice; 50% lethal doses based upon mortality ratesobserved at various dilutions of bacteria were calculated aspreviously described (22).

RESULTSMost strains of E. tarda bound Congo red (100%), pro-

duced siderophores (96%), were invasive in HEp-2 cellmonolayers (92%; Fig. 1), elaborated a CAH (88%), andwere chemotactically motile (88%; Table 1). These associa-tions were independent of serotype designation or source ofthe strain (human, animal, environment). Approximatelyhalf (54%) of the E. tarda strains tested also producedMRHA against guinea pig erythrocytes; this hemagglutininwas expressed by a number of isolates of diverse serotypes,and a difference in positivity rates between human (54%) andnonhuman strains (55%) was not observed. From earlierstudies (35), we presented evidence that the MRHA of E.tarda was an afimbrial protein whose activity could beblocked by fetuin but was not inhibited by a variety ofsugars, polysaccharides, and glycolipids. Since a recentstudy (24) on the afimbrial hemagglutinin of Shigella speciesindicated that hemagglutination could be specifically blocked

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TABLE 2. Effects of various inhibitors and treatments on MRHAactivity in selected E. tarda strains

Hemagglutination activityb ofConditiona the following:

ET-12 ET-15 ET-17 1OA

D-Mannose (1%) 4+ 4+ 3+ 4+Fetuin (67 mg/ml) 0 0 0 0Asialofetuin (67 mg/ml) 0 0 0 0N-Acetylneuraminic acid (10 mM) 4+ 3+ 3+ 3+NaIPO4 (10 mM) 4+ 3+ 3+ 3+

a Final concentrations in parentheses.b Against guinea pig erythrocytes.

by sialic acid-specific glycoproteins, we tested severalMRHA+ E. tarda strains for similar reactivity. As can beseen in Table 2, all four E. tarda strains exhibited strongMRHA activity against guinea pig erythrocytes in the pres-ence of D-mannose; this reactivity was inhibited by fetuin, aspreviously reported, and by asialofetuin, which lacks sialicacid. Furthermore, sialic acid-containing compounds, suchas N-acetylneuraminic acid, failed to inhibit MRHA activity,as did periodate oxidation of carbohydrates.

In contrast to the findings regarding E. tarda, a number ofmajor differences were noted among E. hoshinae and E.ictaluri strains surveyed. Although E. hoshinae strains weremotile and bound Congo red, they failed to produce asiderophore or demonstrate MRHA activity (Table 3). Inva-sive capabilities and expression of CAH were detected at alower frequency (50 to 67%) in E. hoshinae than in E. tarda.Surprisingly, all five E. ictaluri strains failed to produce anyof the above-described factors even though they were as-sayed at 25°C (except for CAH and invasion assays).

In addition to the above assays, all edwardsiellae werescreened for the presence of plasmids. Half of the E. tardaand E. hoshinae strains studied harbored extrachomosomalelements as detected by agarose electrophoresis. The mo-lecular masses of these plasmids and numbers varied fromstrain to strain, ranging from 2 to 120 MDa. All E. ictaluristrains contained two low-molecular-mass plasmids of ca. 2and 4 MDa that appeared similar or identical to thosepreviously found in this species (15).We also tested all edwardsiellae for ST-like activity in

suckling mice since some E. tarda isolates have reputedlybeen shown to produce such factors (2). All edwardsiellaewere found to be ST- in suckling mouse assays withintestinal weight/body weight ratios ranging from 0.051 to

TABLE 3. The in vitro pathogenic properties ofE. hoshinae and E. ictaluria

Species Strain Source Mot Inv CAH MRHA Crb CAS

E. hoshinae EH-1 Unknown + - - - + -1-78 Puffin + - + - + -9-66 Monitor + + + - + -35051T Monitor + + - - + -35052 Gecko + + + - + -33379 Puffin + - + - + -

E. ictaluri 33202T Catfish -

6006 Catfish -

6012 Catfish -

6013 Catfish -

6017 Catfish - - - - - NTa NT, not tested; for other abbreviations, see footnote to Table 1.

0.069. The 25 Edwardsiella strains (including all E. ictaluriand two E. hoshinae strains) that were also tested forhomologous sequences to the ST of E. coli in the SNAPassay were unreactive.

Finally, the relative virulence of a number of E. tarda, E.hoshinae, and E. ictaluri strains in mice was investigated andcompared with the qualitative and quantitative expression ofselected virulence factors by individual strains (Table 4).Overall, E. tarda was 3- to 400-fold more virulent in micethan either E. hoshinae or E. ictaluri. Differences in patho-genic potential within or between species did not directlycorrelate with qualitative or quantitative expression of cell-associated or extracellular factors, although E. tarda strainswere more hemolytic and motile than E. hoshinae.

DISCUSSION

Few studies to date have centered on the relative patho-genicity and potential virulence-associated factors of Ed-wardsiella species; however, previous investigations fromour laboratory (12, 35) indicate that a number of ultrastruc-tural and cell-associated differences do exist among thesespecies. In the present study, we extend these formerobservations by identifying additional factors potentiallyrelated to pathogenicity and determine their relative fre-quency in each of these species as related to source ofisolation. These differences offer possible explanations forobserved variations in overt pathogenicity, disease spec-trum, and host tropisms recognized in each of the threeEdwardsiella species.One of the interesting observations noted in the present

survey concerns the ability of Edwardsiella species to elab-orate siderophores and bind Congo red. For E. tarda,siderophore (iron chelator) production may facilitate ironacquisition in the host and provide an essential micronutrientrelated to microbial virulence (23). Several underlying con-ditions (33) leading to the hyperferremic state in humans,such as sickle cell hemoglobinopathy, and the neonatal stagehave been associated with a variety of extraintestinal E.tarda infections (21, 25, 26, 34). Under circumstances of irondeprivation in the host, the CAH of E. tarda may provide aniron source for the bacterium by causing the release ofhemoglobin from lysed erythrocytes (23). In the case ofCongo red binding, both E. tarda and E. hoshinae werefound to absorb this dye, while all E. ictaluri strains werenegative for this trait. Congo red is structurally and confor-mationally similar to protoporphyrin IX, and binding of thisdye could be related to the initial stages of iron acquisition.The commercial dye, however, is also highly hydrophobicand could simply react with the colonial surface of thebacterium through hydrophobic-hydrophobic interactions.For E. hoshinae, a species that apparently does not producesiderophores yet binds this commercial dye and has previ-ously been found to be autoagglutination positive and mod-erately hydrophobic (35), the latter possibility is more likely.Almost all serotypes of E. tarda were also found to be

invasive and produce a CAH whether of clinical or nonhu-man origin; the only major exception to this rule noticedwere for three CAH- fish isolates. By far the predominantsyndrome associated with E. tarda infections in humansinvolves cases of gastroenteritis (8). E. tarda-associatedgastroenteritis spans the full panorama of intestinal symp-toms, and cases of colitis or dysenterylike disease (14, 17,19) are compatible with an enteroinvasive mechanism andsupport the present and previous observations (12) regardingthe invasive nature of most E. tarda strains. The CAH

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TABLE 4. Pathogenicity of selected Edwardsiella strains related to phenotype

Organism Strain Chemotaltic CAHa Plasmidb resistancec Chnd pathogencitye

E. tarda 15947T 80 32 120, 3, 2 + (0.4) - 1.0 X 108ET-11 80 128 - + (0.5) - 6.3 x 107ET-12 80 64 - + (0.4) + 3.1 x 1063592-64 18 4 120 + (0.1) - 4.4 x 108

E. hoshinae 3505 T 25 2 - + (1.0) + 1.0 x 1099-66 35 2 - NDf + 4.8 x 108

E. ictaluri 33202T 2 0 4, 2 + (0.2) + 1.3 x 1096006 1 0 4,2 +(0.1) + 3.2x 108

a Hemolytic titer against sheep erythrocytes.b In MDa.cResistance (+) or susceptibility to 65% pooled human serum at 120 min.d Chondroitinase activity.e 50% lethal dose in outbred Swiss-Webster mice.f ND, not determined.9 Zone of migration (spreading in soft agar, in millimeters).

cytolysin may additionally play a role in infection via epi-thelial cell destruction leading to an inflammatory infiltrate inthe intestinal mucosa or alternatively could destroy villuscells or disturb intestinal motor function resulting in diarrhea(7). The lack of such factors in E. ictaluri (coupled withgrowth temperature restrictions) and the lower frequency ofinvasive strains and CAH activity in E. hoshinae maypreclude a similar role for these organisms in human cases ofgastroenteritis.

It is clear that a number of major differences potentiallyimportant in the pathogenesis of E. tarda infections help toseparate this species from both E. ictaluri and E. hoshinae.Such differences include an MRHA that may play a role incolonization, an active CAH, siderophores, strong chemo-tactic motility, and invasive characteristics. The uniformlynegative results obtained with E. ictaluri support the previ-ous hypothesis regarding the clonality of this species on thebasis of isoenzyme analysis, plasmid content, and the lack ofbiochemical variability (15, 27, 29). Recent studies supportprevious sporadic observations that the recovery of E. tardafrom human feces is not invariably associated with diarrhealdisease. Whether disease status in humans is linked tostrains possessing the required virulence factors remains tobe determined. It is apparent for edwardsiellae listed inTable 4 that strains of E. tarda appear to be more virulent inmice than either E. hoshinae or E. ictaluri. This result is notsurprising since E. ictaluri is a fish pathogen that grows bestat 25°C. However, E. hoshinae, which grows at 35°C, had50% lethal doses similar to those of E. ictaluri, presumablyindicating that critical determinants in addition to metabolicactivity at elevated temperatures are required for pathoge-nicity. Future studies identifying what factors are critical inthis process seem warranted.

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