Pathogenic Properties of Edwardsiella Species

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1991, p /91/ $02.00/0 Copyright 1991, American Society for Microbiology Vol. 29, No. 9 Pathogenic Properties of Edwardsiella Species J. MICHAEL JANDA,1* SHARON L. ABBOTT,' SUSAN KROSKE-BYSTROM,1 WENDY K. W. CHEUNG,' CATHERINE POWERS,' ROBERT P. KOKKA,1 AND K. TAMURA2 Microbial Diseases Laboratory, California Department of Health Services, Berkeley, California , and National Institute of Health, 10-35, Kamiosaki, Shinagawa-ku Tokyo 141, Japan2 Received 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 mannoseresistant hemagglutination against guinea pig erythrocytes. Edwardsiella hoshinae strains bound Congo red and were variable in their invasive and hemolytic capabilities while Edwardsiella ictaluri strains did not produce either factor; neither E. hoshinae nor E. ictaluri expressed mannose-resistant hemagglutination nor elaborated siderophores under the tested conditions. Selected strains of each species tested for mouse lethality indicated strain variability in pathogenic potential, with E. tarda strains being the most virulent; 50% lethal doses in individual strains did not correlate with plasmid content, chemotactic motility, serum resistance, or expression of selected enzyme activities. The results suggest some potential important differences in pathogenic properties that may help explain their environmental distribution and ability to cause disease in humans. One of the less frequently encountered pathogenic genera in the family Enterobacteriaceae is the genus Edwardsiella (8). Although the genus originally consisted of only a single member (Edwardsiella tarda), at least three species are now known to exist. These species often inhabit freshwater sources and can also be recovered from cold-blooded vertebrates. Edwardsiellae additionally produce a wide range of infections in animals and are recognized as pathogens for eels, catfish, and high-order vertebrates. Edwardsiella ictaluri primarily causes enteric septicemia in channel catfish (10, 11, 27), while E. tarda has been implicated in the same animals as the causative agent of emphysematous putrefactive disease, a foul-smelling wound infection with abscess formation (18); other animal diseases caused by E. tarda include "red disease" in eels (31) and enteritis in penguins (4). In humans, E. tarda is the only recognized pathogenic species primarily associated with sporadic cases of gastroenteritis (3, 13); in rare instances, E. tarda has also been reported to cause extraintestinal disease, most commonly involving cases of septicemia or bacteremia (34). In the case of Edwardsiella hoshinae, although this species has been recovered from humans (in feces), it has been most often isolated from lizards and birds (8, 9). A definite association between this species and its isolation as a bona fide pathogen has not been established to date. Very little information is currently available concerning what factors regulate pathogenicity in the three Edwardsiella species. Several recent studies have identified a number of factors potentially associated with the pathogenicity of edwardsiellae. These factors include cell-associated or extracellular enzymes, hemagglutinins, invasins, and extrachromosomal elements (12, 15, 16, 32, 35). Presently, it is unknown whether certain virulence-associated factors are species specific or whether differential expression within a species is defined by site of isolation. How these factors play a role in the disease process is also unclear. To begin to address some of these issues, we surveyed the pathogenic properties of 35 Edwardsiella strains recovered from distinct * Corresponding author ecologic settings to see how expression of these factors might relate to virulence. MATERIALS AND METHODS Bacterial strains. Thirty-five Edwardsiella strains (E. tarda, n = 24; E. hoshinae, n = 6; E. ictaluri, n = 5) were investigated in this study. The E. tarda strains tested were selected for analysis on the bases of their source of isolation and disease presentation; these strains were wild-type isolates (with the exception of strain F63) which failed to produce acid from D-mannitol, sucrose, and L-arabinose and produced H2S on TSI Agar. Of the 35 strains, 21 have been described previously (12, 35); of the remaining 14 strains, 5 environmental isolates were received from J. Lindquist (Madison, Wis.), 6 additional isolates were from the Microbial Diseases Laboratory collection, and 2 cultures (ATCC and ATCC 33379) were obtained from the American Type Culture Collection (Rockville, Md.). The serotype of each E. tarda strain was determined according to the recently revised international typing scheme (30). For all in vitro assays, E. tarda and E. hoshinae strains were cultured and tested at 35 C, while E. ictaluri was grown and assayed at 25 C unless otherwise specified. Motility. The ability of edwardsiellae to migrate chemotactically in motility medium was determined according to the method of Craven and Montie (5). Standardized suspensions (ca. 109 CFU) were point inoculated into motility agar which consisted of 1% tryptone, 0.3% yeast extract, 0.5% NaCl, and 0.3% agar. Migration of bacteria in such media is dependent on the motility of individual strains and their responsiveness to chemotactic gradients generated by depletion of metabolites surrounding growth. The distance of migration from the point source inoculum (diameter is expressed in millimeters) for each strain was determined 18 to 20 h after initial inoculation. Strains whose zone was.10 mm were considered either nonmotile or defective in chemotactic 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

2 1998 JANDA ET AL. Wong et al. (35) by using the rock tile test. Some strains were further evaluated for the ability of chemical treatments or specific analogs to inhibit this MRHA reaction. HEp-2 invasion and CAH. The invasive capabilities of Edwardsiella species were screened in HEp-2 cells propagated in chamber slides in a 5% C02 atmosphere at 35 C (12). Invasive strains were defined as those whose numbers of gentamicin-resistant progeny at 3 h postinfection were equal to or exceeded 103 CFU; initial infection inocula were ca. 106 CFU. Invasive strains were not probed for invasion plasmid antigen-related sequences, as a previous study had indicated that E. tarda strains are Sereny test negative (2). Cellassociated hemolysins (CAH) were detected by coincubation of serial dilutions of standardized suspensions of bacteria in phosphate-buffered saline with 1% (vol/vol) solutions of either guinea pig, sheep, or rabbit erythrocytes in microtiter plates at 35 C for 1 h. Strains producing a CAH were defined as those whose CAH titer was.4 hemagglutinating units (hemolytic units, 100% lysis) against one or more of the erythrocytes tested (12). STs. Production of heat-stable (ST) enterotoxinlike activity for all 35 Edwardsiella strains was determined in suckling mice according to the method of Dean et al. (6) as recently modified (1); 25 of these strains were additionally screened for homologous sequences to the ST of Escherichia coli (20) by using the biotinylated SNAP enterotoxigenic E. coli ST probe (Molecular Biosystems, Inc., San Diego, Calif.). Phenotypic markers. The ability of edwardsiellae to bind Congo red was determined on tryptic soy agar containing 0.006% Congo red. Plates were incubated at the appropriate temperature for 72 h before final readings were recorded. Siderophore production was determined under identical conditions by using Chrome Azurol S agar (1). Chondroitinase activity (28) was assessed on brain heart infusion agar containing 400,ug of chondroitin sulfate per ml and 1% bovine serum albumin (fraction V). For selected strains, the ability of 65% pooled human serum to induce complement FIG. 1. Invasion of HEp-2 cells by edwardsiellae. OF. TABLE 1. J. CLIN. MICROBIOL. The in vitro pathogenic properties of E. tarda strainsa Strain Source Serotype Mot Inv CAH MRHA Crb CAS Clinical 15947T Feces 033:H ET-1 Feces 033:H ET-2 Feces 040:H ET-7 Feces 033:HNM ET-11 Feces 059:H ET-12 Feces 024:H ET-13 Abscess 036:H ET-14 Feces 06:H ET-15 Blood 05:H (+) ET-16 Feces UK:H ET-17 Feces 059:H ET-19 Feces 032:H ET-20 Spleen 04:UK Nonhuman 10A Lake 024:H Fl Lake 030:HUK (+) F31 Lake 044:H F41 Lake UK:UK (+) F53 Lake 059:H F63 Lake 045:H (+) + ET-18 Heron 05:H SA 8318 Flounder 05:H AC 8321 Eel R:H TK 8403 Eel 09:H Unknown R:H a Abbreviations: Mot, motility; Inv, invasion of HEp-2 cells; CAH, cellassociated hemolysin; MRHA, mannose-resistant hemagglutination against guinea pig erythrocytes; Crb, Congo red binding; CAS, siderophore production on chrome azurol S agar; UK, unknown type; NM, nonmotile. Parentheses denote weak reactions. mediated lysis of bacteria was performed in microcentrifuge tubes; end point analysis occurred at 2 h postincubation (22). Plasmid analysis and mouse pathogenicity. All strains were screened for the presence of extrachromosomal elements by horizontal electrophoresis of cell lysates on 0.75% agarose gels (22). Plasmids were subsequently visualized with UV illumination. The pathogenic potentials of some E. tarda, E. hoshinae, and E. ictaluri strains were determined by intraperitoneal injection of viable bacteria into female Swiss Webster mice; 50% lethal doses based upon mortality rates observed at various dilutions of bacteria were calculated as previously described (22). RESULTS Most strains of E. tarda bound Congo red (100%), produced siderophores (96%), were invasive in HEp-2 cell monolayers (92%; Fig. 1), elaborated a CAH (88%), and were chemotactically motile (88%; Table 1). These associations were independent of serotype designation or source of the strain (human, animal, environment). Approximately half (54%) of the E. tarda strains tested also produced MRHA against guinea pig erythrocytes; this hemagglutinin was expressed by a number of isolates of diverse serotypes, and a difference in positivity rates between human (54%) and nonhuman strains (55%) was not observed. From earlier studies (35), we presented evidence that the MRHA of E. tarda was an afimbrial protein whose activity could be blocked by fetuin but was not inhibited by a variety of sugars, polysaccharides, and glycolipids. Since a recent study (24) on the afimbrial hemagglutinin of Shigella species indicated that hemagglutination could be specifically blocked

3 VOL. 29, 1991 TABLE 2. Effects of various inhibitors and treatments on MRHA activity in selected E. tarda strains Conditiona Hemagglutination activityb of the following: ET-12 ET-15 ET-17 1OA D-Mannose (1%) Fetuin (67 mg/ml) Asialofetuin (67 mg/ml) N-Acetylneuraminic acid (10 mm) NaIPO4 (10 mm) a Final concentrations in parentheses. b Against guinea pig erythrocytes. by sialic acid-specific glycoproteins, we tested several MRHA+ E. tarda strains for similar reactivity. As can be seen in Table 2, all four E. tarda strains exhibited strong MRHA activity against guinea pig erythrocytes in the presence of D-mannose; this reactivity was inhibited by fetuin, as previously reported, and by asialofetuin, which lacks sialic acid. Furthermore, sialic acid-containing compounds, such as N-acetylneuraminic acid, failed to inhibit MRHA activity, as did periodate oxidation of carbohydrates. In contrast to the findings regarding E. tarda, a number of major differences were noted among E. hoshinae and E. ictaluri strains surveyed. Although E. hoshinae strains were motile and bound Congo red, they failed to produce a siderophore or demonstrate MRHA activity (Table 3). Invasive capabilities and expression of CAH were detected at a lower frequency (50 to 67%) in E. hoshinae than in E. tarda. Surprisingly, all five E. ictaluri strains failed to produce any of the above-described factors even though they were assayed at 25 C (except for CAH and invasion assays). In addition to the above assays, all edwardsiellae were screened for the presence of plasmids. Half of the E. tarda and E. hoshinae strains studied harbored extrachomosomal elements as detected by agarose electrophoresis. The molecular masses of these plasmids and numbers varied from strain to strain, ranging from 2 to 120 MDa. All E. ictaluri strains contained two low-molecular-mass plasmids of ca. 2 and 4 MDa that appeared similar or identical to those previously found in this species (15). We also tested all edwardsiellae for ST-like activity in suckling mice since some E. tarda isolates have reputedly been shown to produce such factors (2). All edwardsiellae were found to be ST- in suckling mouse assays with intestinal weight/body weight ratios ranging from to TABLE 3. The in vitro pathogenic properties of E. hoshinae and E. ictaluria Species Strain Source Mot Inv CAH MRHA Crb CAS E. hoshinae EH-1 Unknown Puffin Monitor T Monitor Gecko Puffin E. ictaluri 33202T Catfish Catfish Catfish Catfish Catfish NT a NT, not tested; for other abbreviations, see footnote to Table 1. EDWARDSIELLA VIRULENCE FACTORS The 25 Edwardsiella strains (including all E. ictaluri and two E. hoshinae strains) that were also tested for homologous sequences to the ST of E. coli in the SNAP assay were unreactive. Finally, the relative virulence of a number of E. tarda, E. hoshinae, and E. ictaluri strains in mice was investigated and compared with the qualitative and quantitative expression of selected virulence factors by individual strains (Table 4). Overall, E. tarda was 3- to 400-fold more virulent in mice than either E. hoshinae or E. ictaluri. Differences in pathogenic potential within or between species did not directly correlate with qualitative or quantitative expression of cellassociated or extracellular factors, although E. tarda strains were more hemolytic and motile than E. hoshinae. DISCUSSION Few studies to date have centered on the relative pathogenicity and potential virulence-associated factors of Edwardsiella species; however, previous investigations from our laboratory (12, 35) indicate that a number of ultrastructural and cell-associated differences do exist among these species. In the present study, we extend these former observations by identifying additional factors potentially related to pathogenicity and determine their relative frequency in each of these species as related to source of isolation. These differences offer possible explanations for observed variations in overt pathogenicity, disease spectrum, and host tropisms recognized in each of the three Edwardsiella species. One of the interesting observations noted in the present survey concerns the ability of Edwardsiella species to elaborate siderophores and bind Congo red. For E. tarda, siderophore (iron chelator) production may facilitate iron acquisition in the host and provide an essential micronutrient related to microbial virulence (23). Several underlying conditions (33) leading to the hyperferremic state in humans, such as sickle cell hemoglobinopathy, and the neonatal stage have been associated with a variety of extraintestinal E. tarda infections (21, 25, 26, 34). Under circumstances of iron deprivation in the host, the CAH of E. tarda may provide an iron source for the bacterium by causing the release of hemoglobin from lysed erythrocytes (23). In the case of Congo red binding, both E. tarda and E. hoshinae were found to absorb this dye, while all E. ictaluri strains were negative for this trait. Congo red is structurally and conformationally similar to protoporphyrin IX, and binding of this dye could be related to the initial stages of iron acquisition. The commercial dye, however, is also highly hydrophobic and could simply react with the colonial surface of the bacterium through hydrophobic-hydrophobic interactions. For E. hoshinae, a species that apparently does not produce siderophores yet binds this commercial dye and has previously been found to be autoagglutination positive and moderately 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 nonhuman origin; the only major exception to this rule noticed were for three CAH- fish isolates. By far the predominant syndrome associated with E. tarda infections in humans involves cases of gastroenteritis (8). E. tarda-associated gastroenteritis spans the full panorama of intestinal symptoms, and cases of colitis or dysenterylike disease (14, 17, 19) are compatible with an enteroinvasive mechanism and support the present and previous observations (12) regarding the invasive nature of most E. tarda strains. The CAH

4 2000 JANDA ET AL. J. CLIN. MICROBIOL. TABLE 4. Pathogenicity of selected Edwardsiella strains related to phenotype Organism Strain Chemotaltic CAHa Plasmidb resistancec Chnd pathogencitye E. tarda 15947T , 3, 2 + (0.4) X 108 ET (0.5) x 107 ET (0.4) x (0.1) x 108 E. hoshinae 3505 T (1.0) x NDf x 108 E. ictaluri 33202T 2 0 4, 2 + (0.2) x ,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 epithelial cell destruction leading to an inflammatory infiltrate in the intestinal mucosa or alternatively could destroy villus cells or disturb intestinal motor function resulting in diarrhea (7). The lack of such factors in E. ictaluri (coupled with growth temperature restrictions) and the lower frequency of invasive strains and CAH activity in E. hoshinae may preclude a similar role for these organisms in human cases of gastroenteritis. It is clear that a number of major differences potentially important in the pathogenesis of E. tarda infections help to separate this species from both E. ictaluri and E. hoshinae. Such differences include an MRHA that may play a role in colonization, an active CAH, siderophores, strong chemotactic motility, and invasive characteristics. The uniformly negative results obtained with E. ictaluri support the previous hypothesis regarding the clonality of this species on the basis of isoenzyme analysis, plasmid content, and the lack of biochemical variability (15, 27, 29). Recent studies support previous sporadic observations that the recovery of E. tarda from human feces is not invariably associated with diarrheal disease. Whether disease status in humans is linked to strains possessing the required virulence factors remains to be determined. It is apparent for edwardsiellae listed in Table 4 that strains of E. tarda appear to be more virulent in mice than either E. hoshinae or E. ictaluri. This result is not surprising since E. ictaluri is a fish pathogen that grows best at 25 C. However, E. hoshinae, which grows at 35 C, had 50% lethal doses similar to those of E. ictaluri, presumably indicating that critical determinants in addition to metabolic activity at elevated temperatures are required for pathogenicity. Future studies identifying what factors are critical in this process seem warranted. REFERENCES 1. Abbott, S. L., R. P. Kokka, and J. M. Janda Laboratory investigations on the low pathogenic potential of Plesiomonas shigelloides. J. Clin. Microbiol. 29: Bockemuhl, J., V. Aleksic, R. Wokatsch, and S. 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