Department ofMicrobiology, The Mount Sinai Hospital, New York, New York 10029. Received 17 ... emergency room at The Mount Sinai Medical Center, New.
OF CLINICAL MICROBIOLOGY, Dec. 1984, p. 1221-1222 0095-1137/84/121221-02$02.00/0 Copyright C 1984, American Society for Microbiology
JOURNAL
Vol. 20, No. 6
Value of Blood Agar for Primary Plating and Clinical Implication of Simultaneous Isolation of Aeromonas hydrophila and Aeromonas caviae from a Patient with Gastroenteritis J. MICHAEL JANDA,* AGNES DIXON, BETH RAUCHER, RICHARD B. CLARK, AND EDWARD Department of Microbiology, The Mount Sinai Hospital, New York, New York 10029
J. BOTTONE
Received 17 May 1984/Accepted 14 September 1984
The simultaneous recovery of Aeromonas hydrophila and Aeromonas caviae from the stool of a 49-year-old with watery diarrhea was facilitated through the use of a blood agar medium which detected the hemolytic capability of A. hydrophila. In vitro phenotypic tests support the conclusion that only the A. hydrophila isolate was clinically significant. woman
Gastroenteritis caused by Aeromonas spp. is an increasingly recognized clinical entity which has been reported to occur on a global basis. Diarrhea produced by this gramnegative bacterium, a member of the family Vibrionaceae, most often occurs in pediatric patients as a mild or chronic forrn or as a more invasive, bloody, dysenteric syndrome mimicking that produced by Salmonella or Shigella spp. (2, 7, 8). In adult patients, gastroenteritis induced by Aeromonas spp. is being recognized more frequently, and when it occurs as a fulminant cholera or dysentery-like syndrome, it usually requires medical attention (1, 4, 6, 11, 13). Epidemiological studies suggest that the incidence of diarrhea caused by Aeromonas spp. peaks during the warm months of the year (2), when maximum concentrations of aeromonads occur in aquatic habitats. Gastroenteritis caused by Aeromonas spp. is usually self-limiting, although antibiotic treatment may be required in children presenting with invasive disease or in immunocompromised patients at risk of developing systemic disease (i.e., bacteremia) subsequent to gastrointestinal tract involvement. Recently three Aeromonas species have been taxonomically defined. On the basis of case-controlled studies (7), enterotoxin production (2, 4, 14), and in vitro phenotypic markers associated with enterotoxigenicity (3, 5, 12), Aeromonas hydrophila and Aeromonas sobria have been implicated as major enteric pathogens. The role of Aeromonas caviae, which is commonly isolated from the human gastrointestinal tract, in diarrheal disease is still unresolved. Additionally, the significance of the concomitance of more than one Aeromonas species in a human diarrheal stool is unknown. We report here the simultaneous isolation of A. hydrophila and A. caviae from the stool of a patient with gastroenteritis and present data relative to the clinical interpretation of such an occurrence. The patient was a 49-year-old woman who presented in the emergency room at The Mount Sinai Medical Center, New York, with a complaint of midepigastic pain, vomiting, and diarrhea of 1 day's duration. The patient had vomited three times and had had six nonbloody, loose bowel movements over the past 24 h. The individual was in apparently good health, was not on any medication, and denied recent travel outside the New York City area. Physical examination was unremarkable except for the rectal examination, which revealed a scant amount of light brown, guaiac-negative stool.
Laboratory examinations revealed a leukocyte count of 21,600, with 64 segmented cells, 28 band forms, 5 lymphocytes, and 3 monocytes. A stool culture was collected by inserting a sterile cotton-tipped swab deep into the rectum. At a follow-up visit 4 days later, all gastrointestinal symptoms had subsided, and the patient felt well. The stool specimen was plated from Stuart transport medium to MacConkey, Hektoen-enteric, Campylobacter, and 5% sheep blood agar media. After overnight incubation, several distinct gram-negative colony types (hemolytic and nonhemolytic) were observed in approximately equal numbers (3 + copredominant organisms) on the blood agar. Hektoen-enteric agar revealed yellow colonies, whereas MacConkey agar showed colorless (lactose-negative) colonies interspersed between colonies of Escherichia coli and Klebsiella pneumoniae. Cytochrome oxidase tests of the colony types on blood agar revealed two of them (hemolytic and nonhemolytic) to be positive. Biochemical characterization (API 20E; Analytab Products, Plainview, N.Y.) of each isolate indicated that both strains were A. hydrophila (the API 20E system does not identify strains to species level within the A. hydrophila complex) with isolates 1 (API profile, 3046127) and 2 (API profile, 7047127) generating distinct phenotypic profiles. Definitive identification of these isolates to species level by the criteria of Popoff and Veron as recently modified in our laboratory (9) revealed one to be A. caviae and the other to be A. hydrophila (Table 1). The simultaneous recovery of A. hydrophila and A. caviae from clinical specimens is significant for at least two reasons. First, such an occurrence is apparently rare, although A. sobria and A. hydrophila were recovered from a wound infection of a diver who sustained a deep puncture to the left leg while diving in polluted waters (10). Second, only the A. hydrophila isolate produced a cytotoxin and cell-free hemolysin and possessed the in vitro phenotypic markers (lysine decarboxylase and Voges-Proskauer) that have previously been found to be associated with enterotoxigenic strains (3, 5, 8, 12, 14). These data suggest that only the A. hydrophila isolate was toxigenic and hence was the putative etiological agent of the patient's gastrointestinal disturbance. This is particularly noteworthy, since both strains were lactose negative and sucrose positive, and one or both of these species could easily have been overlooked if blood agar were not utilized and only MacConkey and Hektoen-enteric agars were inoculated. In such an instance, if the A. caviae isolate had been selected for virulence-associated phenotypic markers, the true etiology of this patient's diarrhea would not
* Corresponding author. 1221
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NOTES
TABLE 1. Biochemical properties of Aeromonas species recovered from a patient with gastroenteritis Test' Isolate 1 Isolate 2 Esculin hydrolysis + + Growth in KCN broth + + L-Arabinose fermentation + Salicin fermentation + + Glucose (gas) + Voges-Proskauer + H2S + Elastase + Species designation hydrophila caviae + Oxidase + Indole + + ONPG + + Arginine dihydrolase + + Lysine decarboxylase + Growth on TCBS Growth on 6.5% NaCl Sensitivity to compound 0/129 Acid from: D-Mannose + Dextrin + + D-Xylose Lactose Myo-inositol + Sucrose + CHO cell cytotoxicity + Hemolytic activity (HAU/ml) 0 58 a Abbreviations: ONPG, o-Nitrophenyl-,3-D-galactopyranoside, TCBS, thiosulfate citrate-bile salts agar (Eiken Chemical Co.. Tokyo, Japan); CHO, Chinese hamster ovary cells; HAU, hemagglutination units.
have been established. The use of blood agar may greatly accelerate the recovery of enterotoxigenic aeromonads (e.g., A. hydrophila, A. sobria) and also facilitate the recovery of hemolytic vibrio species, such as V. fluvialis, which are being incriminated as agents of bacterial gastroenteritis. LITERATURE CITED 1. Baman, S. I. 1980. Aerornonas hydrophila as the etiologic agent in severe gastroenteritis: report of a case. Am. J. Med. Technol.
J. CLIN. MICROBIOL. 46:179-181. 2. Burke, V., M. Gracey, J. Robinson, D. Peck, J. Beaman, and C. Bundell. 1983. The microbiology of childhood gastroenteritis: Aeromonas species and other infective agents. J. Infect. Dis. 148:68-74. 3. Burke, V., J. Robinson, H. M. Atkinson, and M. Gracey. 1982. Biochemical characteristics of enterotoxigenic Aeromonas spp. J. Clin. Microbiol. 15:48-52. 4. Champsaur, H., A. Andremont, D. Mathieu, E. Rottman, and P. Auzepy. 1982. Cholera-like illness due to Aeromonas sobria. J. Infect. Dis. 145:248-254. 5. Cumberbatch, N., M. J. Gurwith, C. Langston, R. B. Sack, and J. L. Brunton. 1979. Cytotoxic enterotoxin produced by Aeromonas hydrophila: relationship of toxigenic isolates to diarrheal disease. Infect. Immun. 23:829-837. 6. Dhalla, S., and J. T. Flynn. 1980. Aeromonas hydrophilainduced fulminant diarrhea. N.Y. State J. Med. 80:1965-1966. 7. Gracey, M., V. Burke, and J. Robinson. 1982. Aeromonasassociated gastroenteritis. Lancet ii:1304-1306. 8. Janda, J. M., E. J. Bottone, C. V. Skinner, and D. Calcaterra. 1983. Phenotypic markers associated with gastrointestinal Aeromonas hydrophila isolates from symptomatic children. J. Clin. Microbiol. 17:588-591. 9. Janda, J. M., M. Reitano, and E. J. Bottone. 1984. Biotyping of Aeromonas isolates as a correlate to delineating a speciesassociated disease spectrum. J. Clin. Microbiol. 19:44-47. 10. Joseph, S. W., 0. P. Daily, W. S. Hunt, R. J. Seidler, D. A. Allen, and R. R. Colwell. 1979. Aeromonas primary wound infection of a diver in polluted waters. J. Clin. Microbiol. 10:4649. 11. Palfreeman, S. J., L. K. Waters, and M. Norris. 1983. Aeromonas hydrophila gastroenteritis. Aust. N. Z. J. Med. 13:524525. 12. Pitarangsi, C., P. Echeverria, R. Whitmire, C. Tirapat, S. Formal, G. J. Dammin, and M. Tingtalapong. 1982. Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides: prevalence among individuals with and without diarrhea in Thailand. Infect. Immun. 35:666-673. 13. Srahman, A. F. M., and J. M. T. Willoughby. 1980. Dysenterylike syndrome associated with Aeromonas hydrophila. Lancet ii:976. 14. Turnbull, P. C. B., J. V. Lee, M. D. Miliotis, S. Van de Walle, H. J. Koornhof, L. Jeffery, and T. N. Bryant. 1984. Enterotoxin production in relation to taxonomic grouping and source of isolation of Aeromonas species. J. Clin. Microbiol. 19:175-180.
