Salicin fermentation by Shigella sonnei.

Shigella species are generally considered to be unable to ferment salicin (Breed, Murray, and Smith, Bergey's Manual of Determinative Bacteriology, The Williams & Wilkins Co., Baltimore, 1957; Kauffmann, Enterobacteriaceae, E. Munksgaard, Copenhagen, 1950; Edwards and Ewing, Identification of Enterobacteriaceae, Burgess Publishing Co., Minneapolis, 1955). Recently, however, Ser6ny (Acta Microbiol. Hung. 6:217, 1959) reported that, of 69 strains of S. sonnei tested, 8 fermented salicin slowly; the fermentation required 4 to 26 days. He suggested that salicinpositive strains should not be excluded from the genus Shigella. In view of the importance of these findings, I decided to reinvestigate this phenomenon with cultures of S. sonnei currently being maintained in this laboratory. Twenty-one strains of S. sonnei, two strains of S. flexneri, two strains of S. boydii, two strains of S. dysenteriae, and one strain of the alkalescensdispar group (Kauffmann, Acta Pathol. Microbiol. Scand. 26:879, 1949) were examined. These cultures were obtained from various sources, namely, U.S. Naval Medical Center, Bethesda, Md.; Maxwell Finland, Boston City Hospital, Boston, Mass.; W. H. Ewing, Communicable Disease Center, Atlanta, Ga.; and Frank P. Pauls, Southeentral Regional Laboratory, Anchorage, Alaska. The cultures were constantly checked morphologically, biochemically, and serologically for purity. The organisms were maintained and transferred daily in nutrient broth and a chemically defined medium (Erlandson and Mackey, J. Bacteriol. 75:530, 1958). There was no apparent difference in the fermenting ability of the organisms when grown in these two media. The salicin fermentation broth tubes were prepared from the following formula: beef extract (Difco), 3 g; peptone (Difco), 5 g; salicin (Difco), 5 g; bromothymol blue dye solution, 1 ml; and distilled and deionized water, 1,000 ml. The bromothymol blue solution was prepared by dissolving 0.4 g of bromothymol blue (Difco) in 500 ml of 95% ethyl alcohol and making a total volume of 1,000 ml with water. The solution was filtered through Whatman no. 1 filter paper and stored in a dark bottle at 4 C. The salicin fermentation broth was dispensed in screw-capped test tubes, 10 ml per tube. In addition, Phenol Red Broth Base (Difco) containing 0.5% salicin was used. The fermentation reactions were essentially the same in the two fermentation media employed. Ten tubes of fermentation broth were inoculated with each strain; the volume of the inocula was approximately 0.05 ml, obtained by washing 24-hr cultures in buffer and resuspending to yield a population of approximately 107 cells/ml. The tubes were incubated for 40 days at 37 C before being discarded as negative. Of the 21 strains of S. sonnei, 5 fermented salicin slowly. The other organisms did not ferment salicin. Positive reactions developed after 12 to 22 days (mean of 18.5 days). However, it is necessary to point out that, in strains that fermented salicin, all ten tubes did not become acid. Among the salicin-fermenters, a mean of 7.5 tubes became acid. After several months of subcultivation in nutrient broth, the salicin-fermenting S sonnei strains were retested for the ability to 487 NOTES