Classification of Lactobacillus divergens, Lactobacillus piscicola, and some catalase-negative, asporogenous, rod-shaped bacteria from poultry in a new genus, Carnobacterium

Abstract

Biochemical and chemical studies were performed on some atypical lactobacilli from chicken meat in an attempt to clarify their taxonomy. The present study showed that the majority of the poultry strains could be allocated to the species Brochothrix thermosphacta, Lactobacillus divergens, and Lactobacillus piscicola. The majority of the remaining poultry strains fell into two distinct groups which were worthy of separate species status. Lactobacillus carnis was found to be a member of the species L. piscicola. On the basis of biochemical, physiological, and chemical criteria, we suggest that L. divergens, L. piscicola, and the two unidentified poultry taxa be classified in a new genus, Carnobacterium, as Carnobacterium divergens comb. nov., Carnobacterium piscicola comb. nov., Carnobacterium gallinarum sp. nov., and Carnobacterium mobile sp. nov. The type strains of C. gallinarum and C. mobile are NCFB 2766T and NCFB 2765T, respectively. ~ Thornley (22) reported the isolation of some gram-positive, catalase-negative, nonsporeforming rods from chicken meat maintained at low temperature. These bacteria resembled lactobacilli in many respects but differed in their inability to grow on acetate media. A subsequent, more comprehensive study indicated that these strains formed three groups, none of which at that time corresponded to any Lactobacillus species or any other recognized taxon (23). A recent numerical phenetic study that included the "atypical lactobacilli" described by Thornley and Sharpe confirmed the presence of three major groups (S. Ferusu and D. Jones, J. Gen. Microbiol., in press). One of these was shown to correspond to Brochothrix thermosphacta, whereas the tax-onomic position of the other two groups remained unre-solved. In recent years there has been an increasing number of reports of unusual lactobacilli from vacuum-packed meat that are also unable to grow on acetate agar (8,12,18). These studies have resulted in the establishment of two new species, Lactobacillus carnis (19) and Lactobacillus divergens (12). Unfortunately, these two species were not included in the numerical study of Ferusu and Jones, and as far as we are aware there has been no comparative study of the poultry isolates and vacuum-packed meat strains. Therefore , in the present study the biochemical and chemical characteristics of the atypical lactobacilli described by Thornley and Sharpe (23) have been determined in an attempt to clarify their relationship, if any, to the species L. carnis and L. divergens. The recently described salmonid fish pathogen Lactobacillus piscicola was also included in the study because this species shares many properties with the atypical lactobacilli from poultry and meat (9). MATERIALS AND METHODS Cultures and cultivation. The test strains used are listed in Table 1. Cells for peptidoglycan and lipid analyses were grown in YGPB broth (5) at 30°C for 3 days. Cultures were checked for purity, harvested by centrifugation, washed with distilled water, and freeze-dried. * Corresponding author. DNA studies. Cells for deoxyribonucleic acid (DNA) studies were obtained by cultivation in 1 liter of YGPB medium at 30°C from a 1% inoculum, using a 16-h culture. On reaching early stationary phase, the cells were chilled in ice water and harvested by centrifugation. Preparation of DNA followed a modification (3) of the method of Garvie (4). DNA base composition was estimated by thermal denaturation in standard saline citrate as described by Garvie (9, using DNA from Leuconostoc mesenteroides NCDO 768 (39.3 mol% guanine plus cytosine [G+C]) and Escherichia coli NCDO 1984 (K-12) (51.5 mol% G+C) as standards. DNA-DNA hybridizations were performed by using the membrane filter technique described previously (6). Fatty acid analyses. Fatty acids were extracted from dry cells by acid methanolysis (13), and purified methyl esters were examined by gas-liquid chromatography, using an Erba Science Fractovap series 4160 flame ionization chromatograph. Analyses were performed on a 50-m fused-silica capillary column with OV-1 as the stationary phase at 200"C, using helium as carrier gas. The identity of individual esters was established by comparison of the retention times with those of standard straight-chain saturated, monounsatu-rated, cyclopropane ring, and methyl-branched esters (BAME Mix [4-54361; Field Analytical). The identity of unsaturated and cyclopropane ring acids was confirmed by catalytic hydrogenation (2). Murein analyses. Purified walls were prepared from ca. 250 mg of dry cells as described by Schleifer and Kandler (15,-16). The amino acid composition of complete hydrolysates was determined by using an automatic amino acid analyser (model 4101; LKB Instruments). The isomers of diaminopi-melic acid were separated by thin-layer chromatography as described by Harper and Davis (7). Biochemical and physiological tests. Biochemical tests were performed by using the API 10E and API 50CH systems (Analytab Products) according to the manufacturer's instructions. YGA medium (nutrient broth no. 2 [Oxoid Ltd.], 25 g; yeast extract [Oxoid], 3 g; glucose, 5 g; agar no. 3 [Oxoid], 15 g; water, 1 liter; pH 6.8) was used for test cultures. These cultures were incubated at 25"C, and API STREP basal medium was used for carbohydrate tests. 310