JOURNAL OF CLINICAL MICROBIOLOGY, June 1982, p. 1164-1166 9095-1137/82/061164-03$02.

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Vol. 15, No. 6

Evaluation of Three Differential Media for Detection of Enterobacter agglomerans (Erwinia herbicola)
CANDID BUCHER AND ALEXANDER VON GRAEVENITZ* Department of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland Received 1 December 1981/Accepted 17 February 1982

Dextrin-fuchsine-sulfite medium (DFS), Rimler-Shotts agar (RS), and a new lysine-ornithine-mannitol agar (LOM) were tested for detection of Enterobacter agglomerans. In human stools, LOM and DFS were most sensitive at coliform-toE. agglomerans ratios of -102 and E. agglomerans inocula of >102 per plate. Both LOM and DFS detected one strain in 254 stools, but RS proved to be inhibitory.

Enterobacter agglomerans (Erwinia herbi- For selection of an optimal combination, various cola) is ubiquitous in nature and may also cause concentrations of L-lysine hydrochloride (Difco human infections (1, 6). Since infections are Laboratories, Detroit, Mich.; (5.0 and 10.0 g/ often mixed (6), selective or differential media liter) and L-ornithine hydrochloride (Difco; 6.5 for human specimens may be desirable in special and 13.0 g/liter) were added to a base containing yeast extract (Difco; 3 g/liter), sodium chloride situations. Considering such media, we discounted those (5 g/liter), bromthymol blue (Difco; 0.3 g/liter), previously designed by phytopathologists be- agar (Difco; 13.5 g/liter), and distilled water (to 1 cause either they contain neomycin (5, 8),which liter). After autoclaving (121C for 15 min) and inhibits E. agglomerans (7), or they do not cooling to 50C, various concentrations of filterdifferentiate Escherichia coli and E. agglomer- sterilized d-mannitol (Difco; 3.5, 5.25, and 7.0 g/ ans, which share many biochemical characteris- liter) plus 30 ,ug of vancomycin hydrochloride tics (1, 3, 4; M. N. Schroth, personal communi- (E. Lilly S. A. Suisse, Berne, Switzerland) per cation). Failure to ferment lactose, yellow ml were added. After the pH was adjusted tc pigmentation, and gelatin liquefaction are un- either 6.5 or 7.0, the medium was poured into suitable differential characters because they ap- plates. Thirty-two E. agglomerans strains, whose pear late or only in some of the strains (1). The following three differential media were sources are described elsewhere (C. Bucher and considered. (i) Dextrin-fuchsine-sulfite medium A. von Graevenitz, Med. Microbiol. Immunol., (DFS; Aeromonas Differential Agar, E. Merck, in press), were used for inoculation. They Darmstadt, West Germany) is based on dextrin proved to be resistant to 30 p.g of vancomycin fermentation by Aeromonas and Enterobacter per ml in agar dilution tests. Large, intensely yellow colonies of E. agspp. and lack thereof in E. coli. It was originally designed to select aeromonads (7); we supple- glomerans developed on LOM at concentrations mented it with vancomycin to inhibit gram- of 5 g of lysine per liter plus 6.5 of ornithine per positive organisms. (ii) Rimler-Shotts agar (RS) liter plus 5.25 g of mannitol per liter at a pH of is based on the principle of xylose-lysine agars 6.5 after incubation at 370C for 24 h. Shigella (11). It contains novobiocin and differentiates boydii, Shigella flexneri, A. hydrophila, Provigram-negative organisms that do not decarbox- dencia rettgeri, and the uncommon strains of ylate lysine and ornithine (such as Aeromonas lysine- and ornithine-negative E. coli, Citrohydrophila, for which it was designed, and E. bacterfreundii, and Klebsiella pneumoniae gave agglomerans) from others. (iii) Lysine-ornithine- similar colonies. Mannitol-negative species mannitol agar (LOM), our substantially modified (Pseudomonas spp., Proteus mirabilis, Proteus version of RS, differs from RS in that it lacks vulgaris, Morganella morganii, and Providencia maltose, H2S indicators, ferric ammonium ci- stuartii) yielded colorless colonies which turned trate, sodium deoxycholate, and novobiocin, but greenish-blue after more than 24 h, whereas containing mannitol and vancomycin. It should other members of the Enterobacteriaceae (deyield colorless colonies from mannitol-negative carboxylase-positive E. coli, Enterobacter clostrains, yellow colonies from mannitol-positive, acae, and Enterobacter aerogenes, lysine-posiornithine- and lysine-negative strains, and tive Klebsiella spp., Hafnia alvei, and greenish-blue colonies from mannitol-positive Citrobacter diversus, and ornithine-positive C. strains producing one or both decarboxylases. freundii, Serratia marcescens, and Salmonella
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TABLE 1. Recovery of E. agglomerans on three media in relation to coliform counts and inocula Ratio of coliform Recovery with following inoculum of E. agglome-ants per plate: _102 >102_1 O3 count to E. in >103 Medium _ agglomerans
inoculum 0.01 ml 0.001 ml 0.01 ml 0.001 ml 0.01 ml 0.001 ml

LOM DFS
RS

1.2-102
>102

5/12a 1/3
11/12 2/3
0/12 0/3

5/5
5/5 1/5

2/5 0/5
4/5 0/5
0/5 0/5

6/7 2/3
6/7 3/3
0/7 0/3

1/5 0/5

5/10 0/5

1.2-102
>102

4/10 0/5 0/10

0/5

1.2-102
>102

0/5

a Number of plates from which E. agglomerans was recovered to number of plates inoculated with E.

agglomerans.
sp.) and Vibrio alginolvticus yielded greenishblue colonies. Other combinations of pH and lysine, ornithine, and mannitol concentrations either produced yellowish-green E. agglomerans colonies or yielded yellow colonies in other gram-negative rods. We then determined whether our media were inhibitory to E. agglomerans. Each strain was grown overnight in try,tic soy broth (Difco). A 0.1-ml amount of 10- and 10-6 dilutions was plated on tryptic soy agar with 5% sheep blood (BA), LOM, DFS, and RS. After 24 h at 37C, BA grew 32 (100%) of the strains, LOM grew 31 (98%) of the strains, DFS grew 26 (82%) of the strains, and grew RS 17 (54%) of the strains. Colonies on DFS were small and dark red; on RS, colonies were medium sized and yellow. Colony counts on LOM and DFS were not significantly different from counts on BA, but RS counts were regularly lower by one log. We tested the detecting ability of our media with human stools (2) since they represent ideal mixed cultures. Ten stools from healthy individuals were each inoculated with three dilutions of different pigmented E. agglomerans strains so that various ratios of coliform (i.e., non-E. agglomerans as determined by serial dilutions after inoculation) to E. agglomerans inocula resulted (Table 1). Ten control stools had no E. agglomerans added. Amounts of 0.01 and 0.001 ml of stool diluted 1:10 were spread evenly on plates. The 120 plates per medium were incubated at 37C for 24 h. Of the 60 control plates, 5 each with LOM and RS and 2 with DFS, grew E. agglomerans-like, but nonpigmented, colonies which were not identified as E. agglomerans by API 20E (API International S.A., Geneva, Switzerland). For the "spiked" stools, RS proved to be insensitive, possibly because of the citrate, novobiocin, or deoxycholate content, and was eliminated from further consideration. DFS and LOM recovered E. agglomerans 35 and 27 times, respectively (P > 0.05). The total recovery on

both media decreased significantly at ratios of >102 (P < 0.01) and at E. agglomerans inocula of _102 in 0.001-ml samples (P < 0.05). The optimal recovery was achieved with ratios of 1.2 to 102 and E. agglomerans inocula of >102 in 0.001-ml samples (sensitivities, five of five and six of seven; i.e., 100 and 92%, respectively). To investigate whether human stools could be a source of E. agglomerans, we checked 254 stools from outpatients with and without diarrhea for E. agglomerans by plating 0.001 ml of stool suspension (approximate coliform count, 108/g; dilution of 1:10 in modified Cary-Blair transport medium [Difco]) on LOM and DFS. One strain of E. agglomerans was isolated on plates inoculated in parallel (coliform-to-E. agglomerans ratio, 10:1). A total of 46 and 26 samples gave false-positive colonies on LOM and DFS, respectively (P < 0.05); i.e., specificities were 82 and 90%, respectively. Since E. agglomerans lacks specific sensitivity or nutritional characters, a liquid or solid selective medium could not be devised. Because LOM and DFS are differential media, their sensitivity is limited by the amount of other gramnegative flora present which could exert a "covering-up" or inhibitory effect on E. agglomerans. Although they are not suitable for plating of undiluted stools, their sensitivity with diluted inocula by far exceeds that of MacConkey Agar for enteric pathogens. The latter are missed by MacConkey Agar if coliforms outnumber them by 20:1 (12). E. agglomerans, if it ever occurs in stools (10), does not seem to attain large numbers, i.e., >106/g. LOM and DFS seem to be even more suitable for detecting E. agglomerans in specimens with a less numerous gram-negative flora, e.g.. in sputa.
LITERATURE CITED 1. Ewing, W. H., and M. A. Fife. 1971. Ente-obaclter- agglomerans. The Herbicola-Lathyri bacteria, part III. p. 127. Centers for Disease Control, Atlanta, Ga.

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