INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1992, p. 69-73 Vol. 42, No. I 0020-7713/92/010069-05$02.00/0 Copyright 0 1992, International Union of Microbiological Societies

Listeria ivanovii subsp. londoniensis subsp nova PATRICK BOERLIN,l JOCELYNE ROCOURT,* FRANCINE GRIMONT,3 PATRICK A. D. GRIMONT,3 CHRISTINE JACQUET,* AND JEAN-CLAUDE PIFFARETTIl" Istituto Cantonale Batteriologico, Via Ospedale 6, 6904 Lugano, Switzerland, and Unit&d'Ecologie Bacte'rienne, Centre National de Re'firence pour la Lysotypie et le Typage Mole'culaire de and WHO Collaborating Center for Foodborne Listeriosis2 and Unite' des Ente'robacte'ries, Institut National de la Sante' et de la Recherche Me'dicale, Unit&INSERM 199,3 Institut Pasteur, 75724 Paris Cedex 15, France

An analysis of 23 Listeria ivunovii strains in which we used multilocus enzyme electrophoresis at 18 enzyme loci showed that this bacterial species could be divided into two main genomic groups. The results of DNA-DNA hybridizations and rRNA gene restriction patterns confirmed this finding. The DNA homology data suggested that the two genomic groups represent two subspecies, L. ivunovii subsp. ivanovii and L. ivanovii subsp. londoniensis subsp. nov. The two subspecies can be distinguished biochemically on the basis of the ability to ferment ribose and N-acetyl-P-D-mannosamine.The type strain of L. ivanovii subsp. londoniensis is strain CLIP 12229 (=CIP 103466).

Of the seven recognized Listeria species, only Listeria MATERIALS AND METHODS monocytogenes and Listeria ivanovii are pathogenic (18). Both of these organisms have been isolated from patients In this study we used 3 L. monocytogenes strains, 2 with clinical symptoms, healthy carriers, and the environ- Listeria innocua strains, 2 strains, 2 Lis- ment, but L. ivanovii (formerly called L. monocytogenes teria welshimeri strains, 2 Listeria grayi strains, 2 Listeria serovar 5) has been isolated less frequently than L. mono- murrayi strains, and 23 L. ivanovii strains (Table 1). All of cytogenes (22, 26, 29). L. ivanovii causes mainly abortion in these strains were registered in the Listeria Collection of the sheep (4, 9, 10, 11, 15); more rarely, it causes diseases in Pasteur Institute (CLIP), Paris, France, or in the Special bovines or in humans (21, 26). All L. ivanovii strains belong Listeria Culture Collection (SLCC), Wiirzburg, Germany. to serovar 5, and reciprocally, all serovar 5 strains are The methods which we used for species identification have members of L. ivanovii. These organisms are members of the been described elsewhere (21). When needed, serotyping of only species in the genus Listeria which gives a positive the strains was kindly performed by workers at the Swiss CAMP reaction with and a negative National Listeria Reference Center, Lausanne, Switzerland, CAMP reaction with (24). They who used the reference method (23). produce a particularly wide zone of on sheep MEE. Lysate preparation, electrophoresis, and enzyme blood agar and produce acid from xylose but not from selective staining were done as described by Selander et al. D-mannitol, L-rhamnose, and a-methyl-D-mannoside (25). (27). Electrophoresis preparations for aconitase, alanine Because of these characteristics, L. ivanovii strains can be dehydrogenase, glutamic-oxalacetic transaminase, nucleo- easily distinguished from strains of the other Listeria spe- side phosphorylase, L-phenylalanyl-L-leucine peptidase, and cies. 6-phosphogluconate dehydrogenase were run in buffer sys- From a taxonomic point of view, Ivanov suggested that L. tem A (Tris citrate, pH 8.0). Electrophoresis preparations monocytogenes serovar 5 should be separated as a distinct for NADP-dependent glutamate dehydrogenase, glucose-6- species from L. rnonocytogenes (11). In 1982, Seeliger et al. phosphate dehydrogenase, lactate dehydrogenase, phospho- also recommended that serovar 5 should be considered a glucose isomerase, and mannose phosphate isomerase were taxon that is distinct from L. monocytogenes (26). On the run in buffer system B (Tris citrate, pH 6.7). Electrophoresis basis of its phenotypic characteristics and the results of a preparations for acid phosphatase, adenylate kinase, cata- DNA homology study (20), the species L. ivanovii was lase, fumarase, NAD-dependent glyceraldehyde-3-phos- officially recognized in 1984 (25). phate dehydrogenase, indophenol oxidase, and phosphoglu- In the last few years, multilocus enzyme electrophoresis comutase were run in buffer system F (Tris maleate, pH 8.2). (MEE) has been used successfully with to analyze Specific staining for catalase was performed as described by various epidemiologic and taxonomic problems (17,27). This Harris and Hopkinson (8). The statistical analysis of the data method allows not only differentiation of strains, but also was done with a computer program designed by T. S. estimation of the genomic relatedness of strains, and their Whittam and R. K. Selander as described elsewhere (27). affiliation with species or subspecies. We recently found that DNA-DNA hybridization. DNA-DNA hybridization was a strain identified as L. ivanovii by using conventional performed at 60°C by using the S1 nuclease-trichloroacetic biochemical markers clearly belonged to a genomic group acid method described by Grimont et al. (7) and Rocourt et that has not been described previously when it was exam- al. (21). Bacteria were lysed as described below. Cells were ined by MEE. In this study, we analyzed more L. ivanovii grown for 48 h at 37°C in six Roux flasks containing 150 ml strains by using MEE and identified other members of this of Columbia agar, harvested, and washed in 20 ml of 0.1X group, which we characterized further by using their rRNA SSC (IxSSC is 0.15 M NaCl plus 0.015 M sodium citrate). gene restriction patterns and the results of DNA-DNA They were then incubated for 1 h at 37°C in 6 ml of a hybridization experiments. lysozyme solution (10 mM sodium phosphate-20% sucrose [pH 7.01 containing 0.2% lysozyme [Appligene, Illkirch, France]), lysed by adding 48 ml of a proteinase K solution * Corresponding author. (10 mM Tris-HC1 [pH 8.01, 1 mM EDTA, 1.25% sodium

69 70 BOERLIN ET AL. INT.J. SYST.BACTERIOL.

TABLE 1. Analysis of L. ivanovii strains: with MEE, DNA-DNA hybridization, and rRNA gene restriction patterns

96 DNA Restriction Acid production with labeled DNA patterns from: from' : SDecies SerovaP Strain ET~ Origin' Strain N-acety l-P-D- SLCC Str~~~~lPEcoRI HindIII Ribose mannosamined 3769 L. ivanovii 5 CLIP 8457 1 (I) EIVl HIVl + France, sheep (abortion) L. ivanovii 5 CLIP 12547 1 (I) + France, sheep (abortion) L. ivanovii 5 CLIP 8459 1 (I) t France, sheep (abortion) L. ivanovii 5 CLIP 9441 1 (I) + Germany L. ivanovii 5 CLIP 2300 2 (I) EIVl HIVl + Belgium, human L. ivanovii 5 SLCC 2098 3 (1) 100 (0.3) 61 (3.6) EIV2 HIVl + Australia, sheep (liver) L. ivanovii 5 CLIP 12590 4 (I) + France, cheese L. ivanovii 5 SLCC 3769 4 (I) 100 (0.0) 70 (5.0) EIVl HIVl + Germany, environment L. ivanovii 5 CLIP 8181 5 (I) EIVl HIVl + Italy L. ivanovii 5 SLCC 4723 6 (I) EIVl HIV1 + Germany, bovine (nose) L. ivanovii 5 SLCC 4728 6 (I) + Germany, bovine (nose) L. ivanovii 5 SLCC 4729 7 (I) 103 (1.1) 65 (4.8) EIVl HIV2 + Germany L. ivanovii 5 CLIP 257 8 (I) + France, human (feces) L. ivanovii 5 CLIP 12510T 9 (I) 98 (0.6) 66 (5.6) EIVl HIVl + SLCC 2739T, ATCC 19119T L. ivanovii 5 SLCC 3887 10 (I) 106 (1.5) 68 (5.9) EIV 1 HIVl + Germany, environment L. ivanovii 5 SLCC 4054 11 (I) 99 (0.6) 81 (6.2) EIVl HIVl + Germany, cow (feces) L. ivanovii 5 SLCC 4306 12 (I) +_ Bulgaria L. ivanovii 5 SLCC 3765 13 (11) 60 (4.6) 107 (0.0) EN3 HIVl - Germany, corn leaves L. ivanovii 5 CLIP 2737 14 (11) 58 (6.4) 91 (0.6) HIVl - Czechoslovakia L. ivanovii 5 CLIP 12065 15 (11) 58 (5.3) 94 (0.2) EIV3 HIVl - Belgium, goat L. ivanovii 5 CLIP 1347 16 (11) 64 (4.9) 106 (0.0) EIV3 HIVl - France, dormouse L. ivanovii 5 CLIP 12229 17 (11) 62 (3.1) 100 (0.0) - France, food L. ivanovii 5 CLIP 6645 18 (11) 64 (4.7) 107 (0.6) EIV4 HIV3 - Switzerland L. monocytogenes 1/2 CLIP 14531T 16 ATCC 15313T L. monocytogenes 1l2a CLIP 12498 16 ATCC 35152 L. monocytogenes 4b CLIP 12505 16 ATCC 19115 L. innocua 6a CLIP 12511T 16 ATCC 33O9OT L. innocua 6b CLIP 12512 18 ATCC 33091 L. seeligeri 1/2b CLIP 12513T 42 (7.3) ATCC 35967T L. seeligeri us SLCC 3990 37 L. welshimeri 6b CLIP 12514T 17 ATCC 35897T L. welshimeri 6a SLCC 5332 19 L. grayi NA CLIP 125MT 4 ATCC 19120T L. grayi NA CLIP 640 3 L. murrayi NA CLIP 125MT 8 ATCC 25401T L. murrayi NA CLIP 12516 15 ATCC 25402 US, undesignated serovar; NA, not applicable. The ETs are listed in the same order as in Fig. 1; cluster designations are indicated in parentheses. ' Percentage of relative binding at 60°C. The values in parentheses are AT,,, values (in degrees Celsius). After 18 to 24 h. ' ATCC, American Type Culture Collection, Rockville, Md.

dodecyl sulfate, 20 mg of proteinase K [Appligene]), and solution was filtered to sterility. The methyl red test was incubated overnight at 37°C. The DNA was finally purified performed in MR-VP medium (Difco). by sequential phenol-chloroform extractions. DNAs from strains SLCC 3769 and CLIP 12229T (T = type strain) were RESULTS radioactively labeled by nick translation. rRNA gene restriction patterns. rRNA gene restriction The 23 L. ivanovii strains which we studied by using MEE patterns were determined as described by Jacquet et al. (12) were assigned to 18 electrophoretic types (ETs) (Table 1and by using cloned rDNA (genes coding for rRNA) from Bacil- Fig. 1). A cluster analysis of these ETs showed that they lus subtilis as the probe (3). Restriction enzymes EcoRI and were clearly divided into two main genomic groups (clusters HindIII were used to digest L. ivanovii DNA. I and 11) (Fig. 1).Clusters I and I1 were separated at a genetic For biochemical tests, we used API 50CH galleries (API distance of 0.92, whereas the greatest genetic distances System, Montalieu-Vercieu, France). Additional tests for within clusters I and I1 were 0.34 and 0.17, respectively. fermentation of trehalose, ribose, and N-acetyl-P-D-man- Eight alleles were monomorphic and specific for cluster I nosamine were performed by using a 1% solution of sub- (acid phosphatase, adenylate kinase, alanine dehydrogenase, strate in broth containing (per liter) 10 g of bacteriological glutamic-oxalacetic transaminase , glucose-6-phosphate dehy- peptone (Oxoid), 5 g of Lab-Lemco (Oxoid), 3 g of NaC1, drogenase, mannose phosphate isomerase, nucleoside phos- and 10 ml of a 0.2% bromthymol blue or 0.2% phenol red phorylase, phosphoglucose isomerase) and 12 alleles were solution. The pH was adjusted at 7.0 with NaOH, and the monomorphic and specific for cluster I1 (acid phosphatase, VOL. 42, 1992 LISTERIA IVANOVll SUBSP. LONDONIENSIS SUBSP. NOV. 71

ET

11

1 12

13 II _c: 14 - r- 15 16 II? c2.4

I I a 1 I II I 1.0 a9 0.8 a7 0.6 0.5 0.4 0.3 0.2 0.1 o

GENETIC DISTANCE tl.6 FIG. 1. Genetic relationships among 18 ETs of L. ivanovii. The dendrogram was generated by using the average-linkage method of clustering from a matrix of pairwise coefficients of genetic distances, based on electrophoretically demonstrable allelic variations at 18 FIG. 2. Autoradiogram of Southern blot of EcoRI-digested enzyme loci. ET 3,4, 7,9,10, and 11 (cluster I) strains belong to one whole-cell DNAs obtained from L. ivanovii strains belonging to hybridization group, and ET 13 to 18 (cluster 11) strains belong to genomic groups I and I1 after hybridization with a 16s rDNA probe. another hybridization group (see Table 1). Lane 1, pattern EIVl (strain CLIP 8457); lane 2, pattern EIV2 (strain SLCC 2098); lane 3, pattern EIV3 (strain CLIP 1347); lane 4, pattern EIV4 (strain CLIP 6645). adenylate kinase, alanine dehydrogenase, catalase, NADP- dependent glutamate dehydrogenase, glutamic-oxalacetic When strains SLCC 3769 and CLIP 12229Twere tested as transaminase, NAD-dependent glyceraldehyde-3-phosphate representatives of clusters I and 11, respectively, by using dehydrogenase, glucose-6-phosphatedehydrogenase, nucleo- API 50CH galleries, these strains showed differences only in side phosphorylase, 6-phosphogluconate dehydrogenase, the fermentation of trehalose and ribose. When all of the L. phosphoglucose isomerase, phosphoglucomutase). ivanovii strains were tested for acid production from DNA-DNA hybridization of the DNAs from 12 L. ivanovii trehalose, no correlation was observed between the results strains with labeled DNAs from strains SLCC 3769 and of the test and the genomic groups (data not shown). After 24 CLIP 12229T (representatives of clusters I and 11, respec- h, all of the cluster I strains except strain SLCC 4306 were tively) confirmed the division of the species into two ge- positive for fermentation of ribose (Table 1); strain SLCC nomic groups (Table l). Each DNA hybridization group 4306 produced only a faint acidification of the medium after corresponded to one of the clusters found by using MEE. 48 to 72 h. All of the cluster I1 strains remained clearly The levels of DNA homology between L. ivanovii SLCC negative for this reaction. Conversely, all of the cluster I1 3769 and other strains belonging to cluster I ranged from 98 strains were positive for acid production from N-acetyl-P-D- to 106% (mean, 101%; difference in melting temperature mannosamine, and all of the cluster I strains were negative [AT,] range, 0 to 1.5"C), and the levels of DNA homology for this test after 18 to 24 h (but not after 48 h). The between strain SLCC 3769 and strains belonging to cluster TI differences observed in the methyl red test results did not ranged from 58 to 64% (mean, 61%; AT, range, 3.1 to 6.4"C) correlate with the genetic groups (data not shown). (Table 1). Conversely, strain CLIP 12229T exhibited 61 to 81% DNA homology with strains belonging to cluster I DISCUSSION (mean, 68%; AT, range, 3.6 to 6.2"C) and 91 to 107% homology with strains belonging to cluster I1 (mean, 101%; Listeria species are phenotypically similar (5, 13), and ATm range, 0.0 to 0.6"C). Strain CLIP 12229T exhibited 3 to only a limited number of biochemical tests allow workers to 42% DNA homology with members of other Listeria species differentiate them (21). Most of these organisms can be (Table 1). The AT, for strain CLIP 12229T and the L. separated more distinctly by genetic methods (1, 20); the seeligeri type strain (level of homology, 42%) was 7.3"C. exceptions are L. grayi and L. murrayi, which probably form When the L. ivanovii DNAs were digested with EcoRI, a unique species (19). Until now, no phenotypic subgroups two restriction patterns for rRNA genes (patterns EIVl and have been found within L. ivanovii (5).Differences between EIV2) were associated with cluster I and two other patterns strains belonging to this taxon have been observed for the (patterns EIV3 and EIV4) were associated with cluster I1 following characteristics: acid production from galactose, (Table 1 and Fig. 2 and 3). There was no overlapping of melezitose, sucrose, and trehalose (2,24, 26) and the methyl patterns between the two clusters when preparations were red test (2). In a previous DNA-DNA hybridization study assayed with this restriction enzyme. When Hind111 was (20), nine L. ivanovii strains were shown to be genetically used, we observed three digestion patterns for the rRNA very similar (99 +- 5% DNA homology; AT,, less than genes (Table 1 and Fig. 3 and 4). One of these patterns 1.2"C). (pattern HIV1) was present in both clusters. None of these With MEE, allelic variations at several enzyme loci are patterns was found with other Species of the genus Listeria revealed by differences in the electrophoretic mobilities of (12). the gene products and are used to distinguish genotypes (or 72 BOERLIN ET AL. INT. J. SYST.BACTERIOL.

EM IIII I1 I EIV2 I i I I Ill I I lil II I ElV4 lil I I i

HIVi I Ill (b] HIV2 Ill HIV3 I1111 FIG. 3. Schematic representation of EcoRI (a) and HindIII (b) rRNA gene restriction patterns for L. ivunovii strains belonging to genomic groups 1 and 11. Dashed lines indicate faint bands.

ETs) and to estimate the levels of relatedness of strains (27). longing to one cluster with strains belonging to the other Using this method, we divided the 23 L. ivanovii strains cluster led to intermediate DNA homology values and AT,,, which we analyzed into two major clusters (clusters I and values (Table 1).Thus, genomic groups I and I1 (correspond- 11). The same division was revealed by the results of the ing to clusters I and I1 [Fig. 11) should be considered to be DNA-DNA hybridization experiments, which also demon- two distinct subspecies of L. ivanovii. This result is not in strated that L. ivanovii cluster I1 strains do not belong to one agreement with the genetic distance of 0.92 obtained by of the six other Listeria species (Table 1).Strains that exhibit MEE, which is significantly greater than the distance of 0.7 70% or more DNA homology and AT,,, values of less than that is empirically considered to be sufficient to distinguish 5°C are considered to belong to the same bacterial species two species. DNA-DNA hybridization is based on the whole (28). Strains that exhibit less than 50% DNA homology and bacterial genome, whereas only a portion of the genome is AT,,, values of more than 7°C are generally considered considered when MEE is used. This may occasionally lead members of different species. However, pairing strains be- to bias in estimates of genetic relatedness and could explain the overestimated genetic distance between clusters I and I1 obtained by MEE. Analysis of rRNA gene restriction patterns has been proposed as a potential tool for bacterial (6, 16). When we digested L. ivanovii DNAs with EcoRI, we ob- served no overlap between the patterns obtained for the two strain clusters; therefore, these results support the division of this species into two genomic groups. However, the relatively close genetic relatedness determined by DNA- DNA hybridization could explain the fact that the two genomic groups shared a common rRNA gene restriction pattern (pattern HIV1) when the DNAs were digested with HindIII (Table 1). The biochemical assays showed that ribose fermentation is not a constant characteristic of L. ivanovii strains and, consequently, cannot be used as a differential test for the identification of this species as previously proposed (14). However, the ability to ferment ribose and the ability to produce acid from N-acetyl-P-D-mannosamine within 24 h correlate with the placement of the strains in clusters I and I1 (Table 1). Therefore, these biochemical reactions can be used to identify the subspecies of L. ivanovii. Only one isolate (strain SLCC 4306) gave an unclear reaction for FIG. 4. Autoradiogram of Southern blot of HindIII-digested ribose fermentation. This exception among the 17 strains whole-cell DNAs obtained from L. ivanovii strains belonging to belonging to cluster I was probably due to a mutation that led genomic groups I and I1 after hybridization with a 16s rDNA probe. to poor expression of the phenotype. Lane 1, pattern HIVl (strain CLIP 1347); lane 2, pattern HIV2 In conclusion, our results clearly demonstrate that L. (strain SLCC 4729); lane 3, pattern HIV3 (strain CLIP 6645). ivanovii contains two genomic groups of strains at the VOL. 42, 1992 LISTERIA ZVANOVZZ SUBSP. LONDUNZENSZS SUBSP. NOV. 73

subspecies level, which can be distinguished by the ability to 12. Jacquet, C., S. Aubert, N. El Sohl, and J. Rocourt. Use of degrade ribose and N-acetyl-P-D-mannosamine. Therefore, subtifis 16s ribosomal RNA genes as a probe to identify we propose that genomic group I should be named Listeria Listeria species. Syst. Appl. Microbiol., in press. ivanovii subsp. ivanovii and genomic group I1 should be 13. Jones, D. 1989. Taxonomic position of Listeria strains. Acta named Listeria ivanovii subsp. londoniensis subsp. nov. Microbiol. Hung. 36:113-118. 14. MacGowan, A. P., R. J. Marshall, and D. S. Reeves. 1989. Description of Listeria ivanovii subsp. londoniensis subsp. Evaluation of API20 STREP system for identifying Listeria nov. Listeria ivanovii subsp. londoniensis (1on.don.i.en’sis. species. J. Clin. Pathol. 42548-550. N.L. fern. adj. londoniensis, from London, Ontario, Canada, 15. MacLeod, N. S. M., J. A. Watt, and J. C. Harris. 1974. Listeria where E. G. D. Murray and R. G. E. Murray worked on rnonocytogenes as a cause of abortion in sheep. Vet. Rec. Listeria). Most of the characteristics are similar to those of 95 :365-367. L. ivanovii (25), except that L. ivanovii subsp. londoniensis 16. Owen, R. J. 1989. Chromosomal DNA fingerprinting-a new does not produce acid from ribose but produces acid from method of species and strain identification applicable to micro- N-acetyl-P-D-mannosamine after 18 to 24 h of incubation at bial . J. Med. Microbiol. 30:89-99. 37°C. The type strain is strain CIP 103466 (=CLIP 12229). 17. Piffaretti, J. C., H. Kressebuch, M. Aeschbacher, J. Bille, E. Bannerman, J. M. Musser, R. K. Selander, and J. Rocourt. 1989. Genetic characterization of the bacterium Listeria monocyto- ACKNOWLEDGMENTS genes causing epidemic disease. Proc. Natl. Acad. Sci. USA 86:38 18-3822. We thank N. El Sohl for supplying the rDNA probe and E. 18. Rocourt, J., J. M. Alonso, and H. P. R. Seeliger. 1983. Virulence Ageron and B. Catimel for technical help in the DNA-DNA hybrid- comparke des cinq groupes de (sensu ization experiments and DNA extraction, respectively. lato). Ann. Inst. Pasteur Microbiol. 134A:359-364. This research was supported by a grant from the Swiss Federal 19. Rocourt, J., P. Boerlin, F. Grimont, C. Jacquet, and J. C. Veterinary Office and by grant 31-9396.88 from the Swiss National Piffaretti. Submitted for publication. Science Foundation. 20. Rocourt, J., F. Grimont, P. A. D. Grimont, and H. P. R. Seeliger. 1982. DNA relatedness among serovars of Listeria REFERENCES monocytogenes sensu fato. Curr. Microbiol. 7:383-388. 1. Boerlin, P., J. Rocourt, and J. C. Piffaretti. 1991. Taxonomy of 21 * Rocourt, J., A. Schrettenbrunner, and H. P. R. Seeliger. 1983. the genus Listeria by using multilocus enzyme electrophoresis. Differentiation biochimique des groupes gdnomiques de Listeria Int. J. Syst. Bacteriol. 4159-64. monocytogenes (sensu lato). Ann. Inst. Pasteur Microbiol. 2. Cooper, R. 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