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Journal of Clinical Microbiology, May 2005, p. 2307-2314, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2307-2314.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid and Accurate Identification of Human Isolates of Pasteurella and Related Species by Sequencing the sodA Gene

Anne-Lise Gautier,1 Damien Dubois,1 Françoise Escande,2 Jean-Loup Avril,1 Patrick Trieu-Cuot,3 and Olivier Gaillot1*

Laboratoire de Bactériologie-Virologie, Faculté de Médecine de Rennes, Rennes,1 Centre National des Pasteurella,2 Unité de Biologie des Bactéries Pathogènes à Gram-positif, CNRS URA 2172, Institut Pasteur, Paris, France3

Received 10 October 2004/ Returned for modification 7 January 2005/ Accepted 18 January 2005


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ABSTRACT
 
The identification of Pasteurella and related bacteria remains a challenge. Here, a 449- to 473-bp fragment (sodAint) internal to the sodA gene, encoding the manganese-dependent superoxide dismutase, was amplified and sequenced with a single pair of degenerate primers from the type strains of Pasteurella (18 strains), Gallibacterium (1 strain), and Mannheimia (5 strains) species. The sodAint-based phylogenetic tree was in general agreement with that inferred from the analysis of the corresponding 16S rRNA gene sequences, with members of the Pasteurella sensu stricto cluster (Pasteurella multocida, Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis) forming a monophyletic group and Gallibacterium and Mannheimia being independent monophyletic genera. However, the sodAint sequences showed a markedly higher divergence than the corresponding 16S rRNA genes, confirming that sodA is a potent target to differentiate related species. Thirty-three independent human clinical isolates phenotypically assigned to 13 Pasteurella species by a reference laboratory were successfully identified by comparing their sodAint sequences to those of the type species. In the course of this work, we identified the first Gallibacterium anatis isolate ever reported from a human clinical specimen. The sodAint sequences of the clinical isolates displayed less than 2.5% divergence from those of the corresponding type strains, except for the Pasteurella pneumotropica isolates, which were closely related to each other (>98% sodAint sequence identity) but shared only 92% sodAint identity with the type strain. The method described here provides a rapid and accurate tool for species identification of Pasteurella isolates when access to a sequencing facility is available.


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INTRODUCTION
 
Pasteurella species are small, gram-negative bacilli that colonize mucous membranes of wild and domestic animals but are usually absent from the normal flora in humans (3, 42). Most species can act as primary or opportunistic pathogens in their hosts and are responsible for significant losses to livestock and poultry (33). Human infections are chiefly associated with some form of animal contact and occur predominantly following cat- and dog-inflicted injuries (17, 40, 42). The most common clinical presentation resulting from direct inoculation is cellulitis and lymphangitis, which can be complicated by abscess, tenosynovitis, septic arthritis, and osteomyelitis (15, 17, 40). Otitis media and conjunctivitis are less frequently encountered localized infections (43). Inhalation of bacteria may result in respiratory tract infection in colonized patients with underlying pulmonary disease (20, 43). In this setting, the most common condition is bronchitis, but sinusitis, epiglottitis, pneumonia, empyema, and lung abscess may develop (9, 36). Less frequently, Pasteurella can be involved in systemic infections such as bacteremia, meningitis, brain abscess, endocarditis, and peritonitis, often associated with cirrhosis and immunosuppression (16, 17, 43). Cases of meningitis in infancy have been reported repeatedly and were usually related to nontraumatic contact with pet animals (41). Genitourinary infections are rare but may cause peritonitis and perinatal sepsis (34, 35, 41).

Among the 17 Pasteurella species in the List of Bacterial Names with Standing in Nomenclature (http://www.bacterio.cict.fr/index.html), Pasteurella multocida subsp. multocida, P. multocida subsp. septica, and Pasteurella canis account for the great majority of human infections because of their association with the pharyngeal flora of domestic cats and dogs (12, 17, 40). The growth and identification of these species are easily achieved by routine methods in clinical laboratories. Conversely, other Pasteurella species may have special growth requirements and are not properly identified by most conventional commercial systems. Therefore, identification relies increasingly on the use of genetic tools derived from those used to establish the phylogenetic structure of the family Pasteurellaceae (4, 5, 17, 18, 24).

The first molecular classification of the genus Pasteurella Trevisan 1887 was provided by Mutters et al., on the basis of whole-genome annealing (25). In that work, the species Pasteurella multocida (subdivided into P. multocida subsp. multocida, P. multocida subsp. septica, and P. multocida subsp. gallicida), Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella gallinarum, Pasteurella anatis, Pasteurella langaa (now P. langaaensis), Pasteurella avium, and Pasteurella volantium were delineated. In 1992, phylogenetic relationships were assessed by comparisons of 16S rRNA sequences (11). Since then, the taxonomy of the Pasteurella genus has been under constant revision. Pasteurella ureae was transferred to the genus Actinobacillus as Actinobacillus ureae (26), while Pasteurella haemolytica, Pasteurella granulomatis, and Pasteurella anatis were, respectively, assigned to the new genera Mannheimia (Mannheimia haemolytica and Mannheimia granulomatis) (1), and Gallibacterium (Gallibacterium anatis) (6). The phylogeny of the entire family Pasteurellaceae was recently updated by thorough analysis of 16S rRNA gene sequences (7), confirming that new genera are probably necessary in order to accommodate species such as Pasteurella aerogenes, Pasteurella bettyae, Pasteurella caballi, Pasteurella mairii, Pasteurella pneumotropica, Pasteurella testudinis, Pasteurella trehalosi, and the newly described Pasteurella skyensis (2). Alternatively, other monocopy target sequences which show a higher divergence than those of the 16 rRNA gene have been evaluated as phylogenetic tools. Partial sequencing of the housekeeping genes atpD, encoding the beta subunit of ATP synthase; infB, encoding the translation initiation factor 2; and rpoB, encoding the RNA polymerase beta subunit have recently provided new insights to delineate the phylogeny of the family Pasteurellaceae (8, 21). However, none of those alternative targets was evaluated for identification purposes with human clinical isolates.

Sequence analysis of the Mn-dependent superoxide dismutase (Mn-SOD) gene sodA has been used successfully to improve diagnostics for the Streptococcus (30), Enterococcus (31), and Staphylococcus (32) genera, and it displayed a higher discriminatory power than the 16S rRNA gene sequence-based method in this setting. Here we describe the construction of a sodA library of the 24 species of the Pasteurella, Gallibacterium, and Mannheimia genera and demonstrate its usefulness for rapid sequence-based identification of human clinical isolates.

(A preliminary report of this work was presented at the 104th General Meeting of the American Society for Microbiology, New Orleans, LA, 23-27 May 2004 [abstract R-009].)


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions. The main characteristics of the 60 isolates used in this work are listed in Tables 1 and 2. The 27 type strains were obtained from the Collection de l'Institut Pasteur (Paris, France). Actinobacillus ureae, Haemophilus influenzae, and Haemophilus parainfluenzae type strains were chosen as representatives of the other main genera of the family Pasteurellaceae. Pasteurella lymphangitidis and Pasteurella piscicida were not included in our investigations because of their relatedness to the families Enterobacteriaceae and Vibrionaceae, respectively (10).


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  		TABLE 1. Type strains of Pasteurella and related species used in this study


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  		TABLE 2. Identification of human isolates of Pasteurella by sequencing the sodAint fragment

The 33 epidemiologically unrelated human isolates were obtained from clinically relevant specimens and were selected to encompass a variety of infections. Among them, only Pasteurella caballi isolate CNP 1019 had been previously reported (13). Throughout this paper, names refer to type strains unless an isolate number is mentioned. All strains were grown aerobically at 37°C on chocolate agar supplemented with Polyvitex (AES Laboratories, Combourg, France), except for Pasteurella skyensis, which was grown anaerobically at 20°C on 10% sheep blood agar. In selected cases, complementary study of acid production from carbohydrates was performed by using Taxo disks and CTA medium (Becton Dickinson, Le Pont-De-Claix, France), and p-nitrophenyl-ß-D-glucoside tests were performed in API 20 NE strips (bioMérieux, Marcy-l'Étoile, France).

DNA manipulations. Based on the published sodA sequence of P. multocida strain Pm70 (23), we assumed that degenerate primers d1 (5'-CCITAYICITAYGAYGCIYTIGARCC-3') and d2 (5'-ARRTARTAIGCRTGYTCCCAIACRTC-3'), designed by Poyart et al. (29), could be used for sodA amplification from Pasteurella and related species. An internal fragment (sodAint) representing approximately 80% of the sodA gene was amplified by colony PCR with d1 and d2. PCR conditions were as described by Poyart et al. for gram-positive cocci (30), and the bacterial lysis was carried out during the initial denaturing step. Briefly, reactions were performed in a final volume of 50 µl containing ca. 103 bacteria from a single colony, 0.5 µM (each) primer (Proligo, Paris, France), 200 µM (each) deoxynucleoside triphosphate (Invitrogen, Oxon, United Kingdom), and 1 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Courtabeuf, France) in a 1x amplification buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2). The PCR mixtures were denatured (7 min at 95°C) and then subjected to 30 cycles of amplification (60 s of annealing at 37°C, 45 s of elongation at 72°C, and 30 s of denaturation at 95°C) and to a final elongation cycle of 72°C for 10 min. The PCR products were resolved by electrophoresis on a 1% agarose gel stained with ethidium bromide. Amplicons were purified on S-400 Sephadex columns (Pharmacia Biotech, Orsay, France).

Cloning and sequencing. Purified amplicons obtained from the type strains were cloned into the pUC18-SmaI dephosphorylated vector by using the Sure-clone ligation kit (Pharmacia Biotech). Recombinant plasmids were analyzed by colony PCR on 12 randomly chosen clones with the universal –21 (5'-GTAAAACGACGGCCAGT-3') and reverse (5'-AACAGCTATGACCATG-3') primers in a final volume of 50 µl containing 103 bacteria, 0.1 µM (each) primer, 200 µM (each) deoxynucleoside triphosphate, and 1 U of AmpliTaq Gold DNA polymerase in a 1x amplification buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2). The PCR mixtures were denatured (10 min at 95°C) and were then subjected to 30 cycles of amplification (90 s of annealing at 45°C, 1 min of elongation at 72°C, and 1 min of denaturation at 95°C). PCR products were directly sequenced after purification on a Sephadex S-400 column. The entire nucleotide sequences of both strands of two cloned amplicons obtained from independent PCRs were determined with the BigDye terminator cycle sequencing kit (version 3.1; Applied Biosystems) per the manufacturer's instructions. Sequencing products were resolved with an ABI 3100 automated sequencer (Applied Biosystems). Purified sodAint amplicons obtained from the clinical isolates were directly sequenced on both strands with the degenerate primers d1 and d2 by using the BigDye terminator kit and the ABI 3100 sequencer.

Sequence analysis. The nucleotide sequences were analyzed with Applied Biosystems software (Sequencing Analysis, SeqScape). The sequences corresponding to the oligonucleotides d1 and d2 were excluded for subsequent analysis because of their degeneracy. Multiple alignment was carried out with the CLUSTAL X program (19). Alignment gaps were not taken into consideration for calculations. The construction of the sodAint and 16S rRNA gene phylogenetic trees was performed by the neighbor-joining method (37). The topology of the phylogenetic tree was evaluated by bootstrap analysis to give the degree of confidence intervals for each node on the phylogenetic tree. The reproducibility of tree nodes was evaluated by generating 1,000 bootstraps trees. The sodA and 16S rRNA gene sequences of Escherichia coli K-12 (complete genome, accession no. U00096) were used as outgroups. The 16S rRNA gene phylogenetic tree was based on a 1,342-bp partial sequence corresponding to nucleotide positions 49 to 1391 (E. coli numbering) which was obtained from GenBank for each of the type strains (accession numbers are as follows: P. aerogenes, U66491; P. avium, AY362916; P. bettyae AY362917; P. caballi, AY362918; P. canis, AY362919; P. dagmatis, AY362920; P. gallinarum, AY362921; P. langaaensis, AY362922; P. mairii, AY362923; P. multocida subsp. multocida, AF294410; P. multocida subsp. gallicida, AF294412; P. multocida subsp. septica, AF294411; P. pneumotropica, AY362924; P. stomatis, AY362925; P. skyensis, AJ243202; P. testudinis, AY362926; P. trehalosi, AY362927; P. volantium, AY362928; M. glucosida, AY362912; M. granulomatis, AY362913; M. haemolytica, AF060699; M. ruminalis, AY362912; M. varigena, AY362912; A. ureae, AY362900; G. anatis, AF228001; H. influenzae, M35019; and H. parainfluenzae, AY362908). Ambiguities or gaps present in some of those sequences were solved by partial 16S rRNA gene sequencing of the corresponding type strain before performing alignments.

Nucleotide sequence accession numbers. The 60 sodAint sequences determined in this work were submitted to GenBank and assigned the accession numbers listed in Tables 1 and 2.


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RESULTS AND DISCUSSION
 
A sodA gene is present in all Pasteurella and related species. The degenerate primers d1 and d2 were designed on the basis of a reverse translation of two conserved amino acid domains that are characteristic of the sodA gene products of low-GC gram-positive eubacteria (29). Therefore, they made it possible to amplify a DNA fragment internal to the sodA gene (designated sodAint) of enterococcal, streptococcal, and staphylococcal type strains and clinical isolates (30, 31). Sequence analysis of the genome of P. multocida strain Pm70 revealed the presence of sodA and sodC genes, which are thought to encode Mn-SOD and Cu/Zn-SOD, respectively (23). Interestingly, a computer-assisted analysis of the Pm70 genome indicated that the 642-bp sodA gene was the sole target for primers d1 (no mismatches) and d2 (two mismatches) and that a 506-bp sodAint fragment should be amplified by PCR if d2 misprimability is tolerated. Consistently, by using primers d1 and d2 in a colony PCR assay, we amplified a single DNA fragment of the expected size with all 18 type strains of Pasteurella used in this study. A similar result was obtained with Gallibacterium and Mannheimia species and with the type strains of A. ureae, H. influenzae, and H. parainfluenzae (Fig. 1). The nucleotide sequences of the sodAint fragments from these type strains were determined following cloning into pUC18. Analysis of the deduced amino acid sequences (data not shown) revealed that they all shared one aspartyl residue and three histidyl residues that are thought to be involved in Fe2+ or Mn2+ binding by SOD enzymes and four other residues that are characteristic of Mn-SODs (28). We therefore concluded that the PCR products cloned and sequenced were actual internal fragments of sodA genes. In the family Pasteurellaceae, an Mn-SOD activity has been demonstrated so far only in H. influenzae (22) and Haemophilus ducreyi (38), while a sodA gene is also present in the genomes of four species, Actinobacillus pleuropneumoniae (27), P. multocida (23), M. haemolytica (unpublished, accession no. L47537), and Histophilus (Haemophilus) somnus (unpublished genome shotgun sequence, accession no. NZAACJ01000012). Here we demonstrate that all the species of the Pasteurella, Gallibacterium, and Mannheimia genera also possess a sodA gene.



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  		FIG. 1. Amplification of type strains of Pasteurella and related species with the primers d1 and d2 and separation of the sodAint amplicons (arrowheads) by 1% agarose gel electrophoresis. Lanes: 1 and 21, 50-bp DNA ladder (Fermentas, Lithuania); 2, P. avium; 3, P. gallinarum; 4, P. volantium; 5, P. skyensis; 6, P. testudinis; 7, A. ureae; 8, Mannheimia sp.; 9, P. trehalosi; 10, P. canis; 11, P. dagmatis; 12, P. multocida; 13, P. stomatis; 14, P. aerogenes; 15, P. mairii; 16, G. anatis; 17, H. parainfluenzae; 18, P. bettyae; 19, P. langaaensis, 20, P. caballi. The size of the amplicons is either 503 bp (lanes 2 to 9), 506 bp (lanes 10 to 17), 515 bp (lane 18), 518 bp (lane 19), or 527 bp (lane 20).

Comparison of the sodAint sequences of type strains. Depending on the species, the length of most sodAint sequences (primers excluded) was either 449 bp (P. avium, P. gallinarum, P. volantium, P. skyensis, P. testudinis, A. ureae, Mannheimia sp., and P. trehalosi), 452 bp (P. canis, P. dagmatis, P. multocida, P. stomatis, P. aerogenes, P. mairii, G. anatis, and H. parainfluenzae), or 455 bp (P. pneumotropica and H. influenzae). However, P. bettyae, P. langaaensis, and P. caballi sodAint fragments displayed longer sequences of 461, 464, and 473 bp, respectively. The 9, 12, and 21 extra nucleotides correspond to supplemental blocks of three codons (P. bettyae), four codons (P. langaaensis), and four plus three codons (P. caballi) in the deduced amino acid sequences. Interestingly, these blocks are located in a short and otherwise conserved region (Fig. 2) and were not found in other bacterial SodA sequences determined in this work or obtained from protein data banks (data not shown). The authenticity of these additional codons was confirmed by sequencing the sodAint fragments of unrelated clinical isolates of P. caballi (GenBank accession number AY702524) and P. bettyae (GenBank accession numbers AY702521, AY702522, and AY702523).



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  		FIG. 2. Additional amino acids (boxed) in the deduced SodA sequence of Pasteurella bettyae, Pasteurella caballi, and Pasteurella langaaensis are located in the same region. The corresponding codons are absent from sodA sequences of Pasteurella multocida and other bacterial species. The authenticity of these additional codons was confirmed by sequencing the sodAint fragments of independent P. caballi and P. bettyae isolates. Amino acid numbering refers to the SodA sequence of P. multocida Pm70 (accession no. AAK02085).

Multiple alignment of the sodAint DNA sequences was carried out with the CLUSTAL X program. Pairwise comparison of these sequences showed that their mean identity (83.5%) is less than that calculated from a comparison of their 16S rRNA gene sequences (mean identity, 98.2%). The identity matrix of the sodAint sequences of Pasteurella, Gallibacterium, and Mannheimia type strains is shown in Table 3. These data indicate that sodA might be a more discriminative target than the 16S rRNA gene to differentiate closely related species of the Pasteurellaceae family, as was previously demonstrated for differentiation of streptococci, enterococci, and coagulase-negative staphylococci (30-32). The phylogenetic positions of P. testudinis and P. skyensis were the most distant from those of other species (Fig. 3A; Table 3), an observation consistent with the phenotypic characteristics and host specificities of both species (2, 39). The topology of the sodAint phylogenetic tree obtained (Fig. 3A) was in general agreement with that inferred from an analysis of partial 16S rRNA (Fig. 3B) or rpoB (21) gene sequences, although some differences could be observed. Only three species clusters were supported by a significant bootstrap value of >95% (14). The Pasteurella sensu stricto core group (P. canis, P. dagmatis, P. multocida, and P. stomatis), which encompasses the species most frequently associated with human infections (17), constituted a monophyletic clade defined in 98% of the bootstrap trees (Fig. 3A), in agreement with DNA-DNA hybridization studies (25) and 16S rRNA phylogeny (11). However, the P. canis, P. dagmatis, and P. stomatis cluster defined by 16S rRNA gene analysis (bootstrap value, 97%) was not supported by sodAint analysis, which implied only the possible association of P. canis and P. stomatis (bootstrap value, 91%) (Fig. 3).


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  		TABLE 3. DNA identity matrix based on pairwise comparison of sodAint sequences of type strains of Pasteurella and related species



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  		FIG. 3. Phylogenetic trees showing relationships among sodAint (A) and rrs (16S rRNA gene) (B) sequences from type strains of Pasteurella, Gallibacterium, and Mannheimia species. The trees were established by using the neighbor-joining method from an analysis of the sodAint sequences obtained in this work (listed in Table 1) (A) and rrs sequences obtained from GenBank (B). The sodAint and rrs sequences of A. ureae, H. influenzae, and H. parainfluenzae type strains are also included in order to show the relationships between representatives of the main genera of the family Pasteurellaceae. The sodAint and rrs sequences of E. coli strain K-12 were used as outgroups to root the trees. The value on each branch is the estimated confidence limit (expressed as a percentage) for the position of the branch as determined by bootstrap analysis. Only bootstrap values of greater than 75% are indicated. The scale bar represents 10% differences in nucleotide sequences.

An avian Pasteurella group consisting of P. avium, P. gallinarum, and P. volantium appeared clearly independent from the Pasteurella sensu stricto core group, as it was recovered in 100% of the bootstrap trees (Fig. 3A). Gallibacterium anatis did not cluster with this group, in agreement with infB-based phylogeny (8). This finding is at odds with results from phylogenetic analysis of 16S rRNA gene sequences (Fig. 3B), which indicate that G. anatis is related to the P. avium, P. gallinarum, and P. volantium group (7). Our results confirm the recognition of Gallibacterium as an independent monophyletic genus (6).

The third monophyletic clade encompassed all Mannheimia species, with a branching order identical to that of the 16S rRNA gene sequence-derived tree (Fig. 2), in agreement with the findings of Angen et al. (1).

The sodAint-based phylogenetic positions of P. pneumotropica and A. ureae (formerly Pasteurella ureae) indicated that these species are not related to the aforementioned Pasteurella and Mannheimia clusters (Fig. 2). Moreover, a bootstrap value of 92% suggests that P. pneumotropica might be related to the Haemophilus influenzae group. Preliminary analysis of sodAint sequences of other Haemophilus and Actinobacillus species is in progress in our laboratory and seems to favor that hypothesis (V. Cattoir and O. Gaillot, unpublished data).

Species identification of human clinical isolates. Direct sequencing of the amplicons obtained from the 33 clinical strains yielded electropherograms devoid of overlapping peaks, confirming that the strains contained a single type of sodA gene. Both primers d1 and d2 produced unambiguous sequences of the whole length of the sodAint amplicons. In most cases, the sodAint sequences displayed less than 2.5% divergence with those of the corresponding type strains (Table 2). The only major discrepancy between initial species assignment and sodAint-based identification concerned isolate CNP 531, tentatively identified as "Pasteurella haemolytica." Its sodAint sequence was found to be 74.8% and 99.1% identical to those of M. haemolytica and G. anatis, respectively. Acid production from D-(+)-mannose and p-nitrophenyl-ß-D-glucoside hydrolysis, which allow for differentiation of G. anatis from M. haemolytica (6), subsequently confirmed our molecular identification of G. anatis. To our knowledge, this hemolytic isolate, from a patient with chronic bronchitis, constitutes the first G. anatis human strain ever reported. Conversely, the sodAint sequence of the other "Pasteurella haemolytica" isolate used in this work was 99.8% identical to that of M. haemolytica.

All but one of the P. multocida isolates from various clinical origins were unambiguously identified to the subspecies level, including P. multocida subsp. gallicida CNP 982, which displayed 100% sodAint identity with the corresponding type strain. Only P. multocida subsp. multocida isolate CNP 978 (Table 2) was identified as P. multocida subsp. septica based on sodAint sequence identity (100% and 96% identity with P. multocida subsp. septica and P. multocida subsp. multocida, respectively), although it fermented sorbitol, a key characteristic of P. multocida subsp. multocida which is absent in P. multocida subsp. septica (25). This result confirms that the differentiation of the two dulcitol-negative P. multocida subspecies based solely on sorbitol fermentation might not be reliable and that sorbitol-positive P. multocida subsp. septica might exist, as previously suggested (18). Other members of the Pasteurella stricto sensu core group were unambiguously identified, as were isolates of P. bettyae, P. aerogenes, P. caballi, and P. trehalosi (Table 2). Some heterogeneity is seen among the five P. canis isolates, with 97.6% to 99.1% sodAint identity with the type strain, although they formed a lineage clearly distinct from that of P. stomatis, P. dagmatis, and P. multocida.

Lastly, we compared the sodAint sequences of four unrelated clinical isolates of P. pneumotropica and found out that they were distributed in a single cluster with 98.1 to 99.2% identity but shared only 91% identity with the sodAint sequence of the type strain (Table 2). As type strain CIP 66.16 (ATCC 35149) is a murine isolate not associated with human infection, the possibility arises that P. pneumotropica human clinical isolates may belong to a different lineage than murine colonizing isolates, although they share identical phenotypic features (biotype Jawetz). However, clinical isolates CNP 728 and RSP 877 are most likely of rodent origin, since they were isolated from rat and guinea pig bite wounds, respectively. The analysis of the genetic relatedness of these and other isolates of various animal or human origins is in progress in our laboratory.

Conclusion. Conventional identification of Pasteurella and related bacteria remains a challenge to many laboratories. Most commercial identification systems are likely to overlook species other than P. multocida, and further phenotypic characterization is long, fastidious, and sometimes inconclusive or misleading. In spite of its cost, sequence-based identification is a convenient alternative which should be recommended when dealing with unusual or atypical isolates and when performing epidemiological studies. The method described here provides a rapid and accurate tool for species identification when access to a sequencing facility is available. Heat lysis was adequate for extracting DNA for amplification, and although sodAint fragments were sequenced on both strands in this work, single-strand sequencing with either primer d1 or d2 was accurate enough for routine identification. The high discriminative power related to the sodA gene variability is particularly helpful in recognizing closely related species or subspecies, which cannot be achieved with the same confidence through 16S rRNA gene analysis. The database generated from the type strains allowed unambiguous identification of all human isolates tested, including those belonging to the unusually encountered species G. anatis, P. trehalosi, and P. caballi. The sequencing of sodA in Haemophilus and Actinobacillus species is in progress in our facility and should provide new insights on the phylogeny of the family Pasteurellaceae. This convenient genetic approach might help to investigate the distribution of Pasteurellaceae species in human infections, a prerequisite for the study of their pathogenesis.


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ACKNOWLEDGMENTS
 
We thank Nicolas Fortineau for the gift of strains and Olivier Lemenand and Vincent Cattoir for their help in bacterial identifications.

This work was supported by a grant from the Conseil Scientifique de la Faculté de Médecine de Rennes.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Bactériologie-Virologie, Faculté de Médecine, Université de Rennes 1, 35034 Rennes, France. Phone: (33) 223234759. Fax: (33) 299284159. E-mail: Olivier.Gaillot{at}univ-rennes1.fr. Back


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Journal of Clinical Microbiology, May 2005, p. 2307-2314, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2307-2314.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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