INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1988, p. 1-6 Vol. 38, No. 1 OO20-7713/88/01OO01-06$02.W/O Copyright 0 1988, International Union of Microbiological Societies

Serratia entomophila sp. nov. Associated with Amber Disease in the New Zealand Grass Grub zealandica PATRICK A. D. GRIMONT,l* TREVOR A. JACKSON,' ELISABETH AGERON,l AND MICHAEL J. NOONAN3 Unit&des Enttrobacte'ries, Institut National de la Sante' et de la Recherche Me'dicale Unitt 199, Institut Pasteur, F-75724 Paris Ce'dex 15, France'; Agricultural Research Division, Ministry of Agriculture and Fisheries, Lincoln, Canterbury, New Zealand2; and Department of Agricultural Microbiology, Lincoln College, Canterbury, New Zealand3

Serratiu entomophila sp. nov. is a homogeneous deoxyribonucleic acid relatedness group most closely related to ficaria and Serratia marcescens. All 19 strains studied resembled S. marcescens (the phenotypically closest species) by their inability to ferment or utilize L-arabinose, L-rhamnose, D-melibiose, dulcitol, and D-raffinose but differed from S. marcescens by their inability to ferment or utilize sorbitol and their lack of lysine and ornithine decarboxylases. All strains utilized itaconate, a unique characteristic among Serratia species. Two biotypes could be distinguished. Strains of biotype 1 utilized D-arabitol but not L-arabitol or D-xylose. Strains of biotype 2 utilized L-arabitol and D-xylose but not D-arabitol. S. enturnuphila has been isolated from larvae and water. Most strains were isolated in New Zealand from the grass grub infected with amber disease. None of the strains were isolated from infected humans, (other than ), or plants. The type strain is strain AIT (ATCC 43705T).

The grass grub Costelytra zealandica White (Coleoptera; (A2a), 286 (A2b), 253 (A3b), 326 (A4a), 4466 (A4b), 246 (A5), ) is a major pasture pest in New Zealand and 3780 (A6), 268 (A8b), 81, and 3583 (TCT). The type or causes damage estimated at up to $100 million (N.Z. cur- reference strains of other species used for comparison are rency) per year. Larvae of Costelytra zealandica feed on the same as used in earlier works (1, 9, 10). grass, clover, and other plant roots. Typically, their popu- DNA relatedness study. Published procedures to extract, lations grow to a peak in 4 to 6 years after the pasture is sown purify, and shear unlabeled DNAs were used (2). The and then collapse. Grass grub population decline was found procedures used for in vitro labeling of DNA with tritium- to be associated with the .presence of a disease called amber labeled nucleotides and for hybridization experiments (nu- disease (previously referred to as honey disease [22]). The clease S1-trichloroacetic acid method) have been described major bacterial agent of honey disease, a Serratia sp. (pre- previously (12). The denaturation temperature ( Tm), at viously referred to as Hafnia alvei [22] or Serratia marino- which 50% of the reassociated DNA became hydrolyzable rubra [21]), was isolated from naturally infected larvae (22) by S1 nuclease, was determined by the method of Crosa et and shown experimentally to produce the disease when al. (3); AT, is the difference between the T, of the heterol- transmitted to healthy larvae (22). Orally infected larvae stop ogous DNA pair and the T, of the homologous DNA. feeding within a few days, clear the gut, develop an amber G+C content determination. The denaturation tempera- discoloration, and gradually lose weight until death occurs 4 tures of 50-pg/ml DNA solutions in 0.1~SSC buffer (Ix to 6 weeks later (22) (Fig. 1). The colonize the gut SSC is 0.15 M NaCl plus 0.015 M trisodium citrate) were and cause disease symptoms without invading the hemo- measured with a Gilford spectrophotometer (Gilford Instru- coele (a body cavity largely filled with blood). Another ment Laboratories, Oberlin, Ohio). The guanine-plus-cy- bacterium, Serratia proteamaculans (previously referred to tosine (G+C) contents of DNAs were determined from the as Serratia liquefaciens [21]) was also shown to cause amber denaturation temperatures (17). The DNA of Escherichia disease in the grass grub. coli K-12 was used as a standard (G+C content, 51.2 mol%). The purpose of the present work was to determine, by Assimilation tests. Carbon source utilization tests were deoxyribonucleic acid (DNA) relatedness and extensive done by using specially manufactured API strips (API Sys- phenotypic characterization, the taxonomic position of the tem, La Balme les Grottes, France) which contained pure Serratia sp. causing amber disease in the grass grub. The carbon sources and were similar to commercial API CH, outcome of the study is the description of a new species for API AO, and API AA galleries (3,except that the total which the name is proposed. number of tests was 114 carbon sources (listed in the species description below). The minimal medium was supplied by MATERIALS AND METHODS API System. The strips were inoculated as described earlier (l),incubated at 3WC, and examined daily for growth for 4 Bacterial strains. The sources of S. entomophila strains days. are given in Table 1. Grass grub strains were isolated from Conventional biochemical tests. All tests were incubated at the midgut of larvae after dissection under sterile conditions 30°C unless otherwise stated. These tests included produc- (21). The following 13 Serratia $curia strains were repre- tion of gas and H,S in glucose-lactose-iron agar (Diagnostics sentative of the four known serotypes of that species (7): Pasteur, Marne-la-Coquette, France), urea hydrolysis, and 2602, 4024T (T, type strain), 4026, 4028, 4034, 4037, 4038, indole production from tryptophan in urea-indole medium 4039, 4600, 4738, 4739, 4747, and 4750. The following 12 (Diagnostics Pasteur), gelatin hydrolysis (film method) (16), Serratia marcescens strains represented 11 of the known the P-glucuronidase test (14), the y-glutamyltransferase test biotypes (indicated in parentheses): 5 (Ala), 328 (Alb), 504T (6), growth on caprylate-thallous agar (20), growth in tryptic soy broth (Diagnostics Pasteur) at different temperatures, * Corresponding author. growth in peptone water (Bacto-Peptone [Difco Laborato-

1 2, GRIMONT ET AL. INT. J. SYST.BACTERIOL.

bles 2 and 3) since some DNA preparations yielded either high relative binding ratios (up to 72%) or relatively low ATm values (e.g., 5°C). However, high relative binding ratios matched with high AT, values, and low AT, values matched with low relative binding ratios. The average relatedness of S. Jicaria strains to labeled DNA from S. entomophila AIT was 47% at 60°C and 58% at 70°C. The corresponding average ATm values were 6.7"C (hybridization at 60°C) and 7.5"C (hybridization at 70°C). The average relatedness of S. entomophila strains to labeled DNA from S. Jicaria 4024T was 56% at 60°C and 62% at 70°C. The corresponding average AT,,, values were 7°C (hybridization at 60°C) and 6.6"C (hybridization at 70°C). The other Serratia species were 28 to 48% and 14 to 38% related to S. entomophila at 60 and 70"C, respectively. FIG. 1. Second-instar larvae of C. zealandica. Healthy larva The following other members of the Enterobacteriaceae with dark gut on left, translucent larva showing symptoms of amber were 1 to 25% related to S. entomophila A1 at 60°C: disease on right. Budvicia aquatica 20186T, Cedecea davisae 5T, Citrobacter freundii 460-61, Edwardsiella tarda 10396=, Enterobacter aerogenes AIT, Enterobacter agglomerans E20T, Entero- ries, Detroit, Mich.], 10 g; distilled water, 1 liter; pH 7) bacter agglomerans I11 1429-71, Enterobacter agglomerans containing 0, 2, 6, or 8% (wthol) NaC1, and the Voges- VII 6003-71, Enterobacter amnigenus 78-79, Enterobacter Proskauer test, using the modification of Richard (19). All cloacae 1347-71, Enterobacter gergoviae 2-78, Enterobacter other tests were done by using procedures described else- intermedium 33422, Enterobacter sakazaki 4562-70, Erwinia where (9). amylovora EA178, Erwinia carnegieana EC186, Erwinia Production of enterobacterial common antigen. Production carotovora 495, Erwinia chrysanthemi SR32, Erwinia cypri- of enterobacterial common antigen by strain AIT was stud- pedii EC155, Erwinia mallotivora 2851T, Erwinia nigrifuens ied by using a previously described hemagglutination test EN104, Erwinia rhapontici ER106, Erwinia salicis ES102, (15, 18). Escherichia coli K-12, Ewingella americana C24, Hafnia Pathogenicity tests. For each strain tested, 30 grass grub alvei 329-73, Klebsiella pneumoniae 2, Proteus mirabilis larvae were incubated at 20°C for 14 days in vials of 20 g of PR14, Providencia stuartii 2896-6gT, Rhanella aquatilis 77- soil (containing about 1.5 x 10" bacteria) per larva. The lljT, Salmonella typhimurium LT2, Shigella boydii 1610-55, detailed method of the test has been published previously Xenorhabdus luminescens Hb, and Yersinia ruckeri 4535-69. (21). Among these non-Serratia species, the highest percent re- latedness was with E. carotovora (25%), and the lowest (1%) RESULTS was with P. stuartii. DNA-DNA hybridization. Several samples of DNA from S. DNA base composition. DNAs from strains AIT and A14 entomophila AIT and S.Jicaria 4024T were labeled, and the contained 57.6 and 58.0 mol% G+C, respectively (averages S 1 nuclease-resistant core (in the incubated control tubes of three determinations). containing only denatured labeled DNA) was 6 to 10% at Phenotypic characterization. Characteristics that were either 60 or 70°C. The Tm of homoduplexes (in 0.42 M NaC1) common to all strains studied are given below in the species was 95.2 to 99°C (strain Al) or 96.0 to 97.7"C (strain 4024). description. Characteristics that varied among the 19 strains All strains of S. entomophila studied were more than 74% studied are given in Table 4. Although all strains grew on related to strain AIT at both 60 and 70°C (Table 2) with caprylate-thallous agar (a medium containing yeast extract) insignificant divergence (AT, close to OOC). The S. entomo- in 2 to 6 days, only eight could utilize caprylate within 4 days plzila DNA relatedness group was clearly distinct from that in the assimilation strips (Table 4). At least two biotypes of S. marcescens (Table 2). DNA relatedness between S. could be defined: biotype 1 (15 strains), which utilized entomophila and S. Jicaria needed careful examination (Ta- D-arabitol and failed to utilize L-arabitol and D-xylose; and

TABLE 1. Origins of the S. entornophila strains studied Strain Source Site description" 222 Water Spring in Haux, France Al., A2 Grass grub Fairton (Canterbury, New Zealand), mature pasture, 550 grass grubs/m2, 65% diseased A3 Grass grub Ashford forest (Canterbury, New Zealand), mature pasture, 305 grass grubs/m2, 10% diseased A4, A5, A6 Grass grub Carew (Canterbury, New Zealand), mature pasture, 133 grass grubs/m2, 23% diseased A8, A10 Grass grub Ealing (Canterbury, New Zealand), mature pasture, 260 grass grubs/m2, 29% diseased All Grass grub Methven (Canterbury, New Zealand), young pasture, 490 grass grubs/m2, 5% diseased A1.2, A13 Grass grub Wakanui (Canterbury, New Zealand), mature pasture, 100-200 grass grubslm2, 3% diseased A14, A15 Grass grub Montalto (Canterbury, New Zealand), mature pasture, 122 grass grubs/m2, 32% diseased A2:O Grass grub Tihoi (Taupo, New Zealand), mature pasture, 480 grass grubslm2, 2% diseased Mol, Mo7 Grass grub Motueka (Nelson, New Zealand), new pasture, 200400 grass grubs/m2, no apparent disease CI1, C113 Grass grub Chatham Islands (Canterbury, New Zealand), mature pasture, 100-200 grass grubs/m2, 3% diseased

a For grass grub isolates, the following information is provided: place where grass grubs were sampled (with county and country indicated in parentheses), type of pasture, density of grass grub population in pasture, and percentage of grass grubs with honey disease (at the site). VOL. 38, 1988 SERRATIA ENTOMOPHILA SP. NOV. 3

TABLE 3. Reassociation of DNA from S. ficaria 4024T with DNA from S. entomophila TABLE 2. Reassociation of DNA from S. entomophila AIT with DNAs from Serratia species % Reassociation S. entomophila (and AT, values in "C) at % Reassociation (and AT, unlabeled DNA Source of unlabeled DNA values in "C) at: 60°C 70°C ~~ 60°C 70°C Biotype 1 AIT 38 (6.5) S. entomophila 65 (6.5) biotype 1 A2 68 (6.5) 67 (8.0) AIT 100 (0.0) 100 (0.0) a Q A3 - 70 (8.5) A2 - 98 - A4 - 65 (6.0) A3 103 A5 61 (7.0) 62 (5.5) A4 - 108 A20 67 (6.0) 61 (5.0) A5 83 (0.0) 94 Mol 51 55 (6.0) A20 - 95 Mo7 48 51 (5.5) Mol 79 (0.0) 83 222 72 (9.0) 60 (7.5) Mo7 84 86 222 95 (0.0) 101 Biotype 2 A14 - 59 S. entomophila biotype 2 A15 - 69 A14 80 (1.0) 89 CI1 47 60 (7.5) A15 80 (0.5) 100 C113 52 63 (6.5) CI1 74 76 C113 82 (1.0) 88 a -, Not determined. S. ficaria 2602 43 (5.5) 46 (5.5) 2 (4 4024T 49 (6.0) 72 (7.0) biotype strains), which utilized L-arabitol and D-xylose 4026 47 (5.0) 60 (5.0) and failed to utilize D-arabitol. The characteristics allowing 4028 39 (8.0) 60 (12.5) differentiation of S. entornophila from nine other Serratia 4034 51 (7.5) 52 (10.5) species are given in Table 5. 4037 55 (8.5) 33 (7.0) Pathogenicity tests. When grass grub larvae were kept 4038 43 (7.0) 54 (8.0) confined in vials of S. entornophila-treated soil, the survival 4039 62 69 (7.0) or death of the larvae was dependent upon the nature of 4600 46 (6.0) 69 (7.0) bacteria added to the soil. When soil was supplemented with 4738 40 (6.0) 51 (7.0) cells of S. entornophila Al, A2, A3, A4, A5, A6, A8, A10, 4739 50 (5.5) 70 (7.0) All, A12, A13, A14, A15, 79 100% 4747 41 (8.5) 54 (8.0) and to of the tested 4750 47 (7.5) 64 (6.0) larvae were either dead or diseased after 14 days. The mortality and morbidity among larvae was 50% when the S. marcescens cells were from strain CI1. When soil was supplemented 5 36 47 with cells from S. entornophila CI13, Mol, Mo7, 222, and 81 - 44 246 37 53 253 45 55 TABLE 4. Variable characteristics of 19 S. entomophila strains 268 52 (8.0) 51 286 36 51 Characteristic Reaction" Strain with opposite reactionb 326 38 44 328 38 50 Utilization of 504= 46 (8.0) 51 D- Arabitol' +, 15 A14, A15, CI1, CI13 3583 39 - L-Arabitol -? 4 A14, A15, CI1, CI13 3780 53 (7.5) - Benzoate +, 15 Al, A13, Mol, Mo7 4466 46 (7.5) - C a pra t e +, 16 Al, CI1, CI13 Capry late -7 8 A5, A6, A8, A10, All, A12, S. liquefaciens 507 48 38 A13, A20 Glutarate -9 1 A15 S. proteamaculans 3630T 40 32 a-Ketoglutarate +, 12 Al, A4, A5, A15, A20, Mo7, C113 S. grimesii D-Malate -7 2 CI1, C113 503* 30 28 Phenylacetate +, 17 Al, 222 4067 39 27 Propionate +, 15 Al, A4, CI1, C113 Quinate +, 17 CIl, CI13 S. plymuthica 510T 42 34 L-Tyrosine +, 14 A14, 222, Mol, CI1, C113 Xylitol -1 1 222 S. rubidaea 864= 34 32 D-Xylose' -1 4 A14, A15, CI1, C113

S. odorifera 4015 40 24 Chitin hydrolyzed +, 17 A2, A8

S. fonticola 4556-71 28 14 Tributyrin hydrolyzed + , 18 A20

a 50 -, " -, Not determined. +, Positive for to 100% of strains; positive for 0 to 49% of strains. Also indicated are numbers of strains positive in 4 days. Opposite to that indicated for the species. Identical results with fermentation tests. 4 GRIMONT ET AL. INT. J. SYST.BACTERIOL.

TABLE 5. Differentiation of S. entomophila and other Serratia species Reaction" Characteristic S. S. S. S. S. liquefaciens S. S. S. entomophila marcescens jicaria plymuthica groupb rubidaea odorifera fonticola lrtilization of" trans-Aconitate Adonitol L-Arabinosed D-Arabitold L-Arabi to1 Benzoate Betaine Dulci told meso-Erythritol Gentisate 3-H ydrox ybenzoate 4-H ydrox ybenzoate I taconate D-Malate Maltitol Melezitosed D-Melibiosed Quinate Rhamnosed Sucrosed D-Tartrate meso-Tartrate Tricarball ytate Trigonelline Xylitol D-Xylosed

Prodigiosine - D

Pyrazinic odor

Glucose, gas L:ysine (Mdller) - + Orinithine (Mdller) - + Airginine (Mdller)

Malonate test - -Tetrathionate reduced D '' +, Positive for 90% or more of strains, D, test used to differentiate biotypes or species (S.liquefaciens group); d, positive for 10 to 89% of strains; -, negative for 90% or more of strains. '' Includes S. liquefaciens sensu stricto, S. proteamaculans, and S. grimesii. (. 4-day reading. '' Same results obtained with fermentation tests.

A20, 10 to 17% of the tested larvae were dead or diseased 60°C. We first ran the hybridization experiments at 60"C, a after 14 days. Control soil (treated with phosphate buffer standard hybridization temperature for the Enterobac- instead of a bacterial suspension in buffer) was associated teriaceae (2, 3). Then the observation of high T,,, of S. with a 24% mortality or morbidity among larvae. entomophila homoduplexes (95.2 to 99.0"C in 0.4 M NaC1) suggested that 60°C was below the optimal temperature for reassociation (normally T,,, - 25°C to T,,, - 30°C) and that DISCUSSION 70°C was a more optimal temperature. This might explain why the percent reassociation values were higher at 70°C The strains of S. entomophila have properties fitting the among those Serratia species known to have high G+C description of the family Enterobacteriaceae (4) and the ratios (S. entomophila, S. Jicaria, and S. marcescens), genus Serratia (8). Among the members of the Enterobac- whereas percent reassociation values were higher at 60°C teriaceae that produce acetoin, the production of extracel- between S. entomophila and those Serratia species known lular enzymes hydrolyzing tributyrin, chitin, gelatin, Tween to have lower G+C ratios (all other Serratia species). 80, and DNA is typical of the genus Serratia. The DNA relatedness experiments showed that the 19 The DNA relatedness study showed some problems. strains of S. entomophila form a single DNA hybridization Percent reassociation values were often higher at 70 than at group closely related to (but separate from) S. Jicaria and VOL. 38, 1988 SERRATIA ENTOMOPHILA SP. NOV. 5

significantly related to the other Serratia species. In an days), maltose (1 day), D-mannitol (1 day), D-mannose (1 earlier study (ll),one strain of S. entomophila (strain 222) day), salicin (1 day), sucrose (1 day), and trehalose (1 day). had been found that was closely related to S. ficaria. Acid production from D-arabitol and D-xylose defines Phenotypically, S. entomophila differs by more than eight biotypes. No acid produced from L-arabinose, dulcitol, characteristics from S. Jicaria. In a numerical lactose, melibiose, a-methyl-D-glucoside, D-raffinose, L- study (9), S. entomophila 222 was found to be ambiguously rhamnose, D-sorbitol, and L-sorbose. Esculin hydrolyzed, related to S. marcescens, clustering at the edge of the S. Gluconate test positive. marcescens phenon when the unweighted method of average Growth on Simmons citrate. No growth factor required. was the clustering technique used, but remaining isolated Malonate test negative. The following compounds are uti- when the single linkage method of clustering was used. In lized as sole carbon and energy sources by all strains (in 1 to practice, the lack of lysine and ornithine decarboxylase 4 days): N-acetyl-D-glucosamine, cis-aconitate, trans-aconi- would not allow any confusion between S. entomophila and tate, adonitol, D-alanine, L-alanine, L-aspartate, D-cello- S. marcescens. Furthermore, S. entomophila is the only biose, citrate, p-D-fructose, a-L-fucose, fumarate, D-galac- Serratia species that utilizes itaconate for growth and en- tose, D-galacturonate, P-gentiobiose, D-gluconate, D-glu- ergy. Routine diagnostic laboratories might find S. entomo- cosamine , D-glucose, D-glucuronate, L-glutamate, DL-glyce- phila difficult to identify because the properties given in rate, glycerol, L-histidine, myo-inositol, itaconate, 2-keto-~- Table 5 may not appear when cultures are incubated at 37°C gluconate, 5-keto-~-gluconate, DL-lactate, L-malate, malt- (erratic results were found at this temperature for all Serratia ose, maltotriose, D-mannitol, D-mannose, P-methyl-D- species except S. marcescens). If a commercial strip (e.g., glucoside, L-proline, putrescine, D-ribose, salicin, L-serine, API 20E) is incubated at 30 to 35"C, the identification should succinate, sucrose, and D-trehalose. Utilization of D-ara- be easy, being oriented toward the Klebsiella-Enterobacter- bitol, L-arabitol, and D-xylose define two biotypes. Most Serratia-Erwinia group by positive o-nitropheny-P-D-galac- strains utilize benzoate, caprate, phenylacetate, propionate, topyranoside, citrate, and Voges-Proskauer tests together quinate, and L-tyrosine. The following compounds are not with a negative tryptophan deaminase test. A strong hydro- utilized as sole carbon and energy sources: p-alanine, DL-Y- lysis of gelatin and lack of acid production from rhamnose amino-n-valerate, L-arabinose, betaine, o-coumarate, 2-de- would suggest a Serratia species. In this genus, no other oxy-D-glucose, 2,3-dihydroxybenzoate, 2,4-dihydroxyben- species altogether lack decarboxylases and acid production zoate, 3,5-dihydroxybenzoate, dulcitol, meso-erythritol, from sorbitol, D-melibiose, and L-arabinose. For a reference ethanolamine, ferulate, gentisate, glycine, heptanoate, his- identification of this and other Serratia species, however, tamine, 3-hydroxybenzoate, 4-hydroxybenzoate, DL-P-hy- carbon source utilization tests are invaluable (8-11). droxybutyrate, lactose, lactulose, malonate, maltitol, D- Pathogenicity of S. entomophila for grass grubs was not melezitose, D-melibiose, L-methionine, methyl-a-galactoside, apparently correlated with any biochemical pattern. S. en- methyl-P-galactoside, 3-O-methylglucose, a-methy1-D-gluco- tomophila A1 and A2 have produced high levels of disease in side, methyl-a-D-mannoside, a-methyl-D-xyloside, mucate, field trials for the biological control of grass grub (13). palatinose, 3-phenylpropionate, protocatechuate, D-raffinose, Description of Serratia entomophila sp. nov. Serratia ento- L-rhamnose, saccharate, sarcosine, D-sorbitol, L-sorbose, D- mophila (en.to.mo'phi.la. Gr. noun entomon, insect; Gr. vb. tagatose, D-tartrate, L-tartrate, meso-tartrate, tricarballylate, phylein, love; L. fem. adj. entomophila, insect loving). Cells trigonelline, tryptamine, D-tryptophan, D-turanose, and van& are gram-negative, nonsporeforming, nonencapsulated , late. Most strains failed to utilize glutarate, D-malate, and straight rods with peritrichous flagella. The species conforms xylitol. to the definition of the family Enterobacteriaceae (4) and The G+C content of the DNA is 58 mol%. produces the enterobacterial common antigen. Isolated from larvae of C. zealundica (grass grub) with All strains grow on nutrient agar, producing colonies of amber disease, and from the environment. No strain has about 2.5 to 3 mm in diameter after 24 h at 30 or 37°C. The been identified as being involved in a human, (other colonies are whitish and opaque and have smooth, entire than insect), or plant disease. edges. Facultatively anaerobic. All strains grow well in 1 day The type strain is strain AIT (ATCC 43705T). Strain AIT at 10, 20, 30, and 37°C and weakly at 4°C (in 4 days) or 41°C has all the characteristics given above for the species. In (in 1 day) in tryptic soy broth. However, 37°C is not optimal addition, this strain failed to utilize benzoate, caprate, a- for biochemical characterization, and incubation at 30°C is ketoglutarate, phenylacetate, or propionate as the sole preferred. No growth occurs at or above 42°C. Growth source of carbon and energy. Experimentally (per 0s) patho- occurs on caprylate-thallous agar at 30°C in 2 to 6 days. genic for C. zealandica larvae. Catalase produced. Oxidase not produced. Nitrates re- duced to nitrites. Tetrathionate not reduced. H,S not pro- ACKNOWLEDGMENTS duced in glucose-lactose-iron agar or triple sugar iron agar. We thank Joan Pearson and Sharon Ashworth for assistance with Urea not hydrolyzed in urea-indole medium. o-Nitrophenyl- the bioassays. p-D-galactopyranoside hydrolyzed. y-Glutamyltransferase test positive; p-glucuronidase and P-xylosidase tests nega- LITERATURE CITED tive. Indole not produced in peptone water or urea-indole 1. Bouvet, 0. M. M., P. A. D. Grimont, C. Richard, E. Aldova, 0. medium. Tryptophan and phenylalanine not deaminated. Hausner, and M. Gabrhelova. 1985. Budvicia aquatica gen. Lysine, arginine, and ornithine not decarboxylated (Mgller nov., sp. nov.: a hydrogen sulfide-producing member of the medium). Tributyrin and chitin hydrolyzed by most strains; Enterobacteriaceue. Int. J. Syst. Bacteriol. 3560-64. 60, 2. Brenner, D. J., A. C. McWhorter, J. K. Leete Knudson, and DNA, gelatin, Tween 20, Tween 40, Tween and Tween A. G. Steigerwalt. 1982. Escherichia vulneris: a new species of 80 hydrolyzed. Starch not hydrolyzed in 4 days. Glucose Enterobacteriaceae associated with human wounds. J. Clin. fermented without gas production. Voges-Proskauer test Microbiol. 151133-1140. positive. Methyl red usually negative. Acid produced from 3. Crosa, J. H., D. J. Brenner, and S. Falkow. 1973. Use of a adonitol (2 to 4 days), D-cellobiose (3 to 4 days), galacturo- single-strand-specific nuclease for analysis of bacterial and nate (1 to 4 days), glycerol (1 to 4 days), myo-inositol (1 to 4 plasmid deoxyribonucleic acid homo- and heteroduplexes. J . 6 GRIMONT ET AL. INT. J. SYST.BACTERIOL.

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