INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1981, p. 72-76 Vol. 31, No. 1 0020-7713/81/010072~5$02.00/0 Alcaligenes faecalis subsp. homari subsp. nov., a New Group of Bacteria Isolated from Moribund Lobsters B. AUSTIN,’ C. J. RODGERS,’ J. M. FORNS? AND R. R. COLWELL3 Ministry ofAgriculture, Fisheries and Food, Fish Diseases Laboratory, The Nothe, Weymouth, Dorset, DT4 8UB, England’; Applied Marine Ecology Laboratory, Falmouth, Massachusetts OZ5402; and Department of Microbiology, University of Maryland, College Park, Maryland 207423 Eight strains isolated from the hemolymph of moribund lobsters were classified in a new subspecies of Alcaligenes faecalis on the basis of a study of their phenotypic characteristics. The name Alcaligenes faecalis subsp. homari is proposed for this new subspecies, of which the type strain is L1 (= NCMB 2116 = ATCC 33127). Bacterial diseases of lobsters include gaffke- 8 weeks. The strains were compared with nine marker mia, shell disease, and larval asphyxiation, which strains, including Acinetobacter calcoaceticus ATCC are caused by “Aerococcusviridans subsp. hom- 15308, Aeromonas hydrophila ATCC 9071, Aero- ari“ ( 19), unidentified gram-negative, chitinoly- monas salmonicida ATCC 14174, Alcaligenes fae- tic bacteria and Leucothrix mucor (18)) calk NCTC 655 (= FP/63/78, a laboratory strain), (E), Enterobacter aerogenes NCTC 8172, Escherichia coli respectively. However, bacterial isolates distinct NCTC 8136, Vibrio anguillarum NCMB 1875, and from these organisms were recovered in pure Vibrio parahaemolyticus NCTC 10441. culture from the hemolymph of moribund lob- Characterization of the strains. The strains were sters (Homarus americanus) during 1978. They examined by 107 tests described previously for use in were phenotypically dissimilar to all of the rec- numerical taxonomy studies (2) and by 17 antibiotic ognized fish and crustacean pathogens (15, 18- susceptibility tests detailed below. Unless otherwise 20), and from a detailed taxonomic study, it was stated, marine agar 22163 was used as the basal me- shown that the isolates were members of Alca- dium, and inoculated media were incubated at 19OC Zigenes faecalis. In fact, it is proposed that the for 14 days before the results were recorded. Antibiotic susceptibility. Susceptibilities to am- organisms constitute a new subspecies, Alcali- picillin (10 pg), chloramphenicol (10 pg), chlortetra- genes faecalis subsp. homari. cycline (10 pg), cloxacillin (5 pg), colistin sulfate (10 pg), erythromycin (10 pg), furazolidone (50 pg), gen- MATERIALS AND METHODS tamicin (10 pg), kanamycin (30 pg), neomycin (10 pg), Lobsters. Lobsters (H.americanus) were reared nitrofurantoin (200 pg), novobiocin (5 pg), penicillin G at Martha’s Vineyard, Mass., individually in tanks (1.5 U), streptomycin (10 pg), sulfamethoxazole (25 containing seawater at a salinity of ca. 30 to 33%. The pg), sulfafurazole (500 pg), and tetracycline (50 pg) temperatures of the water in the tanks varied from were determined by the method of Allen et al. (1).In -1°C in winter to 23°C in summer. Feeding, with addition to marine agar 2216E, tryptone soya agar frozen brine shrimp (Artemia salina),was undertaken (Oxoid) was used in determining antibiotic suscepti- twice daily. bility. Zones of clearing around the antibiotic disks For 3 days in August 1977, the water temperature were measured as soon as growth could be detected, exceeded 26”C, and shortly afterwards, after the onset i.e., 24 h at 19°C. of molting, 18% of the 1-year-old lobsters died. The Determination of the deoxyribonucleic acid only external symptoms included the presence of base composition. The quanine-plus-cytosine con- softened shells and occasional opaque areas approxi- tent of purified deoxyribonucleicacid, prepared by the mately 5 mm in diameter on the carapace. Postmortem method of Marmur (ll),was calculated from the ther- examinations were carried out on moribund and dead mal denaturation temperature, T, (12), according to animals. the equation of De Ley (5). Bacterial strains. Eight bacterial strains, desig- Electron microscopy. Strain L1, grown for 24 h nated L1 through L8, were isolated from the hemo- at 19°C on marine agar 22163, was suspended in 0.9% lymph of eight moribund lobsters. Drops of hemo- (wt/vol) saline to ca. log cells per ml, negatively lymph, collected aseptically, were inoculated onto stained with 2% (wt/vol) uranyl acetate, and examined plates of Marine Agar 22163 (Difco Laboratories), and with a JEOL 100 CX transmission electron micro- after incubation at 19°C for 7 days, single-culture scope. growth was observed. The isolates were streaked onto Pathogenicity. Suspensions of L1, NCTC 655, and plates of 22163 agar, with three serial streakings of FP/63/78, washed three times in 0.9% (wt/vol) saline each culture made to ensure purity. After purification, and suspended to ca. lo7cells per ml, were injected (1- cultures were inoculated onto 22163 agar slants and ml volumes) into the heart of each of three 1-kg maintained at 4°C. Subculturing was done every 6 to European lobsters (H. vulgaris). Additional lobsters, 72 VOL. 31,1981 A. FAECALIS SUBSP. HOMARI SUBSP. NOV. 73 serving as controls, were each injected with 1 ml of with the two marker strains of A. faecalis at the 0.9% (wt/vol) saline. The lobsters were kept in sepa- 85% similarity level. rate 50-liter fiber glass tanks which received a constant Characteristics of the lobster isolates. flow of oxygenated seawater (20 liters/h) maintained The eight strains isolated from lobsters each at 9°C. Observations were made daily, and moribund consisted of gram-negative, straight or curved, or dead lobsters were removed for postmortem exam- ination. Hemolymph and tissue from the heart, spleen, peritrichous rods (Fig. 2), ca. 1.5 pm in length, liver, and abdominal muscle were collected aseptically with rounded ends and bipolar staining reac- and were subjected to bacteriological and histopatho- tions; they produced off-white, translucent, logical examination. raised, smooth colonies, 2 to 3 mm in diameter, Computer analyses. The 17 strains were exam- on marine agar 22163 within 24 h at 37°C. The ined for 124 unit characters coded in binary format by results of the remaining tests performed on these scoring positive, negative, and noncompatible results isolates are shown in Table 1. as 1, 0, and 9, respectively. Similarities between strains The isolates possess the characteristics of the were calculated by using the simple matching (SSM; genus AZcaligenes insofar as they are gram-neg- 17) and Jaccard ( SJ;16) coefficients, and sorted simi- larity matrices and dendrograms were obtained by use ative, motile, peritrichous (three to eight fla- of the unweighted average-linkage algorithm (17). gella), oxidase-positive, coccal rods which pro- Identification of lobster isolates. The diagnostic duce an alkaline reaction in glucose (5-7, 14). criteria of Hendrie et al. (6) and Holding and Shewan They matched the general description of A. fae- (7) were consulted in identifying the isolates. caZis (5-7), viz., utilization of carbon compounds, including sodium acetate, as the sole source of RESULTS carbon for energy and growth at 37°C (6, 7). Examination of results of computer anal- However, a number of characteristics differen- yses. The numerical classifications obtained tiated them from A. faecalis, and these are with the SSM and SJ coefficients were almost summarized in Table 2. identical, and only the one based on the SJ Pathogenicity. Lobsters injected with a sus- coefficient is considered further. One cluster and pension of L1 died within 7 days. After bacteri- seven single-member clusters were defined at ological and histopathological examinations, the the 85% similarity level (Fig. 1).The eight bac- hernolymph and spleen were found to be packed terial isolates from moribund lobsters clustered with bacterial cells which possessed the charac- together in a single, well-defined group at the teristics of L1. In contrast, lobsters inoculated 90% similarity level (Fig. 1).This cluster joined with suspensions of A. faecalis NCTC 655 (= No of Percentage similarity strains Identity I source 60 70 ao 90 100 R I obster isolates 1 Alcaligcxnrs faecalis (XCTC’ 65.3 1 Xlcaligents faecalis (FP/63/78) 1 Aeromonas hvdrophiln (ATCC 90711 1 Aeromonas salmonicida (ATCC‘ 14174) 1 Vibrio parahaemol).ticus (NCTC 10441) 1 Viljrio anguillaruni (NChII3 187.7) 1 Escherichia c& (SCTC 81.76) 1 Entcrohacter aerogenes (NCTC H173 1 Acinetolmcter ralcoaceticus (ATCC 1 .,DOH) FIG. 1. Simplified dendrogram based on the SJ coefficient and unweighted average-linkage clustering algorithm. FIG. 2. Negatively stained preparation of Ll showing peritrichous cell with seven flagella. Bar, 1 p. TABLE1. Phenotypic characteristics of A. faecalis subsp. homari subsp. nov. and its type strain Ll (= NCMB 2116 = ATCC 33127) Characteristic Reaction 11 Characteristic Reaction Peritrichous .......................... + Blood .............................. - Strictly aerobic ....................... + Casein ............................. - Nitrate reduction ...................... + Chitin .............................. - Growth at 4-42°C ..................... + Gelatin ............................. - Growth in 1-10% (wt/vol) NaCl . , . , , . + Tweens 5, 20,40,60, and 80 .......... - Susceptible to: Tyrosine .......................... - Ampicillin .......................... + Xanthine ........................... - Chloramphenicol .................... + Utilization of: Furazolidone .......................