JOURNAL OF BACTERIOLOGY, Mar. 1992, p. 2002-2013 Vol. 174, No. 6 0021-9193/92/062002-12$02.00/0 Copyright X) 1992, American Society for Microbiology Phylogeny of 54 Representative Strains of Species in the Family as Determined by Comparison of 16S rRNA Sequences FLOYD E. DEWHIRST,1* BRUCE J. PASTER,2 INGAR OLSEN,3 AND GAYLE J. FRASER1 Departments ofPhannacology1 and Microbiology,2 Forsyth Dental Center, Boston, Massachusetts 02115, and Department ofMicrobiology, Dental Faculty, University of Oslo, Oslo, Norway3 Received 3 September 1991/Accepted 14 January 1992 Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family PasteureUlaceae. Of these strains, 15 were Pasteurella, 16 were ActinobaciUlus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacilus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains ofA. seminis and PasteureMa aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains ofPasteureUla multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. galinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragaUlinarum, and A. capsulatus. This cluster also contained PasteureUla species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyti- cus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacilus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the PasteureUlaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving strains (indicated by differing numbers of base changes incorporated into the 16S rRNA sequence relative to outgroup organisms). While the results presented a clear picture of the phylogenetic relationships, the complexity of the branching will make division of the family into genera a difficult and somewhat subjective task. We do not suggest any taxonomic changes at this time.

The family Pasteurellaceae Pohl 1981 (51) is currently nobacillus. Major advances in understanding the phylogeny composed of species in the genera Pasteurella Trevisan 1887 of the members of the family Pasteurellaceae have come (63), Actinobacillus Brumpt 1910 (14), and Haemophilus from the DNA-DNA hybridization studies by the Marburg Winslow et al. 1917 (65). While only 27 species were group as exemplified by the work of Pohl, Mannheim, recognized within this family in Bergey's Manual ofSystem- Mutters, and colleagues (34, 39, 50, 51) and from rRNA- atic Bacteriology (17, 30, 35, 46), over 70 species or taxa DNA hybridization studies by De Ley et al. (21). These from human, mammalian, avian, and reptilian sources have studies have defined species belonging to sensu stricto been described (4-11, 38, 41, 47, 48). Essentially all these definitions of the genera Pasteurella (39), Haemophilus (15), taxa are listed and discussed in a review by Mutters et al. and Actinobacillus (24, 51). These studies have shown that (40). Their review is particularly important in that it desig- the phylogenetic structure of the Pasteurellaceae is complex nates reference strains for each of the described, but not and that more than three genera are required to accommo- formally recognized, taxa. Overviews of research on the date the vast array of species in this group. However, many family Pasteurellaceae can be found in the proceedings of species have not fallen into defined clusters, and the branch- conferences held in Copenhagen in 1980 (31) and Guelph in ing of genus-level clusters remains unclear. To further clarify 1989 (45) and in the monograph "Pasteurella and Pasteurel- the phylogeny of this complex family, we decided to under- losis" (1). take an exhaustive study involving full 16S rRNA sequenc- The of the family Pasteurellaceae as a whole ing of strains the more than and of its component genera has been examined by several representing 70 described methodologies. Major studies based on phenotypic traits species or taxa. Comparison of 16S rRNA sequences has include those on Haemophilus by Kilian (29) and Broom and proved extremely useful for determining phylogenetic rela- Sneath (12) and onActinobacillus and Pasteurella by Sneath tionships among eukaryotic and prokaryotic organisms (66, and Stevens (59). The studies by Sneath suggested an 67). Unlike hybridization studies, it is feasible to use com- overlapping interrelationship between Pasteurella and Acti- plete distance matrices where the similarity of every se- quence to every other sequence is determined (4,900 com- parisons for 70 organisms). Treeing algorithms that correctly account for differing branch lengths are then applied to the * Corresponding author. distance matrix data to produce phylogenetic trees. The 2002 VOL. 174, 1992 PHYLOGENY OF STRAINS OF THE PASTEURELLACEAE FAMILY 2003 present report describes our interim findings for studies tree nodes was also examined by generating 15 trees using 14 performed during the past 3 years in which we obtained full different beta and gamma species as out- 16S rRNA sequence data for over 50 strains. Within the next groups, or no outgroup. The species used as outgroups are year, we hope to obtain 20 to 30 additional sequences for indicated in Table 1. strains representing the remaining described species or taxa within the family Pasteurellaceae. RESULTS AND DISCUSSION MATERLALS AND METHODS 16S rRNA sequences. Virtually complete 16S rRNA se- quences, except for 50 bases at the 3' end, were determined Bacterial strains, sources, and sequence accession numbers. for 52 of the 54 strains. Within the sequenced region, 98% of The strains sequenced in this study, and reference strains the bases, about 1,450 bases, were unambiguously deter- used for comparison in constructing phylogenetic trees, are mined. For two strains, partial sequences were obtained. described in Table 1. Included in this table are the strain ForA. suis CCM 5586T, 1,155 bases were determined. There numbers, the source of the strains, the GenBank accession was a single base difference, G for A at position 257, number for the 16S rRNA sequences, and the literature compared with A. suis ATCC 15557. For [H.] haemoglobin- sources for reference sequences. In Table 1, and throughout ophilus NCTC 8540, 335 bases were determined. There was this report, square brackets are used to indicate that a a single base difference, U for C at position 176, compared species does not belong to the sensu stricto definition of its with [H.] haemoglobinophilus NCTC 1659T. The sequences given genus (brackets around genus) or that the organism is of 14 representative species, aligned with and numbered misnamed (brackets around whole name). Our use of square relative to Escherichia coli (13), are shown in Fig. 1. The brackets differs from that of De Ley et al. (21). Throughout sequences for the 52 fully sequenced strains are available for the text, names refer to type strains unless a strain number is electronic retrieval from GenBank under the accession num- given. bers listed in Table 1. Bacterial culture conditions. Strains of Actinobacillus and Comparisons with previously published Pasteurella se- Pasteurella were cultured aerobically at 37°C for 24 h in quences. Chuba et al. (19) published short partial 16S rRNA brain heart infusion broth (Difco). Haemophilus strains were sequences (445 bases) for seven members of the family cultured in brain heart infusion broth supplemented with X Pasteurellaceae. We found the following number of discrep- and V factors. H. paragallinarum was grown on GC agar ancies between sequences for the organisms examined in (BBL) at 37°C anaerobically for 24 h. H. paraphrohaemolyti- both studies: A. actinomycetemcomitans, 4; A. equuli, 5; A. cus was grown on GC agar at 37°C aerobically for 24 h. lignieresii, 6; H. aphrophilus, 6; H. influenzae, 8; and P. Haemophilus taxon C, Haemophilus minor group strain 202, multocida, 20. We believe that our sequences are accurate and H. parasuis Nagasaki were cultured aerobically for 6 to based on having rechecked our sequencing gels at the 7 days in PPLO broth (Difco) supplemented with 10% yeast discrepant positions, compared conservation of secondary extract (Difco), 5% rabbit serum (Biologos), 0.1% dextrose, structure, and examined consistency with the 54 Pasteurel- and 0.025% NAD (Sigma). laceae sequences in our data base. Each of the Chuba et al. Isolation and purification of rRNA. RNA was isolated and (19) sequences contains four errors in a region conserved in partially purified by a modification of the procedure of Pace all members of the Pasteurellaceae (406 to 436): a gap versus et al. (43) as previously described (44). G at 419, and gaps versus GUA at 428 to 430. While this 16S rRNA sequencing. rRNA was sequenced by a modified report was in preparation, the sequence for H. ducreyi CIP Sanger dideoxy-chain termination technique in which prim- 542' was determined by polymerase chain reaction amplifi- ers complementary to conserved regions were elongated cation of the 16S rRNA gene (56). There are eight discrep- with avian myeloblastosis virus reverse transcriptase (33). ancies between this sequence and ours as follows: A versus The primers used in this study are given in Table 2. For most G at 81, gap versus G at 82, G versus A at 539, A versus C strains, primers 2 or 3 and 4 to 9 were used. Some strains at 629, G versus A at 747, gap versus U at 751, A versus G required the use of primer 1 to complete the sequence of the at 1137, and gap versus G at 1144. Reexamination of our 5'-terminal 100 bases. The details of our sequencing protocol sequencing gels for three strains of [H.] ducreyi substanti- have been described previously (22, 44). ates our sequence (GenBank M75078, M75079, and M75084) Data analysis. A program set for data entry, editing, at all positions except 629. sequence alignment, secondary structure comparison, simi- Sequence signatures and DNA probes. Single-base signa- larity matrix generation, and dendrogram construction for tures for the family Pasteurellaceae are presented in Table 3. 16S rRNA data was written in Microsoft QuickBASIC for Signatures are positions within the sequence where the base use on IBM PC-AT and compatible computers. RNA se- present differs from that found in other . Included in quences were entered and aligned as previously described Table 3 are the position number, the base found in all (44). Our sequence data base contains approximately 300 Pasteurellaceae strains, the base(s) found in other taxa, and sequences comprising those determined in our laboratory, a list of exceptions: those taxa which share the Pasteurel- published sequences, and unpublished sequences provided laceae signature. The large number of signatures makes to us by other scientists. Similarity matrices were con- design of DNA probes specific for the family Pasteurel- structed from the aligned sequences by using only those laceae relatively easy. Sequencing primer 2 was designed as sequence positions for which all strains had data. The a Pasteurellaceae-specific DNA probe. It recognizes all similarity matrices were corrected for multiple base changes Pasteurellaceae strains except those of [A.] actinomycetem- by the method of Jukes and Cantor (28). Phylogenetic trees comitans and P. anatis, which have single-base mismatches. were constructed by the Neighbor-Joining method (58, 61). Validation of this probe will be reported elsewhere. The reproducibility of tree nodes was analyzed by using the Position of the PasteureUlaceae within the Proteobacteria. To Neighbor-Joining bootstrapping programs PSFIND and place the Pasteurellaceae within the class Proteobacteria, NJBOOT written by T. S. Whittam. One hundred bootstrap we calculated a similarity matrix for 6 representative Pas- trees were generated and examined. The reproducibility of teurellaceae species and 17 reference beta and gamma 2004 DEWHIRST ET AL. J. BACTIERIOL.

TABLE 1. Sources and accession numbers of strains studied Organisma Strainb Source' GenBankd Referencee Actinobacilli [A.] actinomycetemcomitans FDC Y4 FDC M75035f [A.] actinomycetemcomitans ATCC 29522 ATCC M75036 [A.] actinomycetemcomitans ATCC 29524 ATCC M75037 [A.] actinomycetemcomitans ATCC 29523 ATCC M75038 [A.] actinomycetemcomitans ATCC 33384T ATCC M75039 [A. capsulatus] NCTC 11408T NCTC M75062 [A. capsulatus] CCUG 12396T CCUG M75069 A. equuli NCTC 8529T NCTC M75072 A. lignieresii ATCC 19393 ATCC M35017 A. lignieresii NCTC 4189T NCTC M75068f A. pleuropneumoniae (H. pleuropneumoniae) ATCC 27088T ATCC M75074 [A.] seminis ATCC 15768T ATCC M75047f A. suis (H. suis) ATCC 15557 ATCC M75071f A. suis (H. suis) CCM 5586T CCM A. ureae (P. ureae) Henriksen 3520/59T KWBI M75075 Actinobacillus species (A. capsulatus) CCUG 19799 CCUG M75067 Bisgaard taxon 11 Haemophili H. aegyptius NCTC 8502T NCTC M75044 H. aphrophilus ATCC 33389T ATCC M75041f H. aphrophilus (H. parainfluenzae) ATCC 7901 ATCC M75040 [H.] ducreyi ATCC 27722 Albritton (KC57) M75084 [H.] ducreyi HD 35000 Albritton (KC61) M75079 [H.] ducreyi CIP 542T Albritton (KC1) M75078f [H.] haemoglobinophilus NCTC 1659T NCTC M75054 [H.] haemoglobinophilus NCTC 8540 NCTC H. haemolyticus NCTC 10659T NCTC M75045 H. influenzae ATCC 33391T ATCC M35019f [H.] paracuniculus ATCC 29986T ATCC M75061f [H.] paragallinarum NCTC 11296T NCTC M75057 [H.] parahaemolyticus NCTC 8479T NCTC M75073 [H.] parainfluenzae ATCC 33392r ATCC M75081f [H.] parainfluenzae (H. paraphrophilus) ATCC 29242 ATCC M75082 [H.] paraphrohaemolyticus NCTC 10670T NCTC M75076 H. paraphrophilus ATCC 29241T ATCC M75042 [H.] parasuis NCTC 4557T NCTC M75065f [H.] parasuis type 5 Nagasaki Rapp-Gabrielson M75066 H. segnis ATCC 33393T ATCC M75043 [H.] somnus OVCG 43826 Little M75046 [Haemophilus] minor group 202 Rapp-Gabrielson M75077 [Haemophilus] taxon C CAPM 5111 Rapp-Gabrielson M75056 Pasteurellae [P.] aerogenes ATCC 27883T ATCC M75048 P. anatis ATCC 43329T ATCC M75054 P. avium (H. avium) NCTC 11297T NCTC M75058f P. canis ATCC 43326T ATCC M75049 P. dagmatis ATCC 43325T ATCC M75051 P. gallinarum NCTC 11188T NCTC M75059 [P.] haemolytica NCTC 9380T NCTC M75080 P. trehalosi (P. haemolytica biotype T) NCTC 10624 NCTC M75063 P. langaa ATCC 43328T ATCC M75053 P. multocida NCTC 10322T NCTC M35018f [P.] pneumotropica NCTC 8141T NCTC M75083f P. stomatis ATCC 43327r ATCC M75050 P. volantium (H. parainfluenzae) NCTC 4101 NCTC M75060 P. volantium NCTC 3438T NCTC M75070 Pasteurella species A CCUG 18782 CCUG M75055f Pasteurella species B CCUG 19794 CCUG M75052 Reference strains Gamma proteobacteria Escherichia colig rrB cistron J01695f 13 Citrobacterffreundiig ATCC 29935 M59291 A Serratia marcescensg ATCC 13880T M59160 A Proteus vulgarisg Monteil J01874 16 Vibrio parahaemolyticusg ATCC 17802T M59161 A Aeromonas hydrophilag ATCC 7966T M59148 A Ruminobacter amylophilusg DSM 1361T NA 36 Continued on following page VOL. 174, 1992 PHYLOGENY OF STRAINS OF THE PASTEURELLACEAE FAMILY 2005

TABLE 1-Continued Organisma Strainb Sourcec GenBankd Referencee Oceanospirillum linuig ATCC 11336T M22365 A Pseudomonas aeruginosag ATCC 25330 M34133 A Acinetobacter calcoaceticusg ATCC 33604 M34139 A Cardiobacterium hominig ATCC 15826T M35014 23 Suttonella indologenes ATCC 25869T M35015 23 Dichelobacter nodosus 198AR M35016 23 Beta proteobacteria Neisseria gonorrhoeaeg NCTC 8375T X07714 57 Eikenella corrodensg ATCC 23834T M22512 22 Alcaligenes faecalisg ATCC 8750T M22508 22 [Pseudomonas] cepacia ATCC 25416T M22518 22 a Organisms are listed according to current nomenclature. Square brackets are used to indicate that an organism does not belong in the genus or species indicated. Name of strain in culture collection or previous name is given in parentheses. b Abbreviations used for culture collections: ATCC, American Type Culture Collection, Rockville, Md.; CCUG, Culture Collection, University of Goteborg, Goteborg, Sweden; DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; KWBI, Kaptein W. Wilhelmsen og frues Baktgeriologiske Institutt, University of Oslo, Rikshostpitalet, Oslo, Norway; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom; OVCG, Ontario Veterinary College, Guelph, Ontario, Canada. c Strains were obtained directly from the indicated culture collection (abbreviated as above) or from the following individuals: P. B. Little, Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada; V. J. Rapp-Gabrielson, Department of Microbiology, North Dakota State University, Fargo; or W. L. Albritton, Provincial Laboratory of Public Health for Northem Alberta, University of Alberta, Edmonton, Alberta, Canada. d 16S rRNA sequences for these strains are available for electronic retrieval from GenBank under the following accession numbers. Through cross distribution of data bases, these sequences should also be available from European and Japanese data bases. NA, not available. I Literature references to sequences not obtained in this report. A, unpublished sequences deposited in GenBank by C. Woese. f Indicates that sequence is included in Fig. 1. g Indicates species used in outgroup analysis.

Proteobacteria. This matrix is available upon request from sentative strain was used in the analysis. A 49-by-49 distance the corresponding author. Shown in Fig. 2 is a phylogenetic matrix was computed. This matrix is available upon request tree derived from the similarity data by using the Neighbor- from the corresponding author. Only those positions where Joining method. The family Pasteurellaceae is located in the data were present for all sequences were included in the gamma division of the Proteobacteria. Families closely similarity comparison (1,246 bases). Within this region related to the Pasteurellaceae are the Enterobacteriaceae, among the Pasteurellaceae sequences, there were 1,043 Vibionaceae, and Aeromonadaceae. More distant relatives conserved and 203 variable base positions. Strains within the within the gamma division include the Pseudomonas fluo- Pasteurellaceae differ among themselves on average about rescens complex, the Moraxellaceae, and the Cardiobacte- 6%. Species in the Pasteurellaceae have an average differ- raceae. The branching order of the Enterobactenaceae, ence of about 12% with species in the neighboring family Vibrionaceae, Pasteurellaceae, andAeromonadaceae shown Enterobacteniaceae. A dendrogram illustrating the phylog- in Fig. 2 differs slightly from that reported previously (20, eny of the Pasteurellaceae is shown in Fig. 3. The majority 21). The differences may be due to our use of a treeing of the strains sequenced fall into four major clusters. Three algorithm that accommodates differing branch lengths. Ad- of these clusters contain as subsets the sensu stricto genera ditional sequence data for species in the families Vbrion- Haemophilus, Pasteurella, and Actinobacillus (21). While aceae and Aeromonadaceae should further clarify the exact this work is in general agreement with previous DNA-DNA branching in this part of the gamma division of Proteobac- and RNA-DNA studies, there are some discrepancies which tena. are discussed below. Phylogeny of the PasteureUlaceae. A similarity matrix was (i) Cluster 1, including the genus Haenwphilus sensu stricto. determined for E. coli and 48 representative strains. Where Cluster 1 is composed of three subclusters. Cluster 1C multiple strains had identical sequences, only one repre- includes H. influenzae, H. aegyptius, and H. haemolyticus. This cluster represents the genus Haemophilus sensu stricto (15). We have not sequenced "H. internedius" strains TABLE 2. Sequencing primersa which are included in this cluster by Burbach (15). Cluster No. Position Sequence Specificity 1B includes H. aphrophilus, H. paraphrophilus, H. segnis, and H. aphrophilus ATCC 7901. De Ley et al. (21) exclude 1 109-123 5'-GCATTACTCACCCGT-3' P, M this cluster from the genus Haemophilus. Cluster IA is 2 233-256 5'-ACCAACTACCTAATCCCACTTGGG-3' P composed of strains of [A.] actinomycetemcomitans. Potts 3 344-358 5'-ACTGCTGCCTCCCGT-3' E and coworkers (53, 54) and Tanner et al. (62) previously 4 519-536 5'-GWATTACCGCGGCKGCTG-3' U that this be transferred to the genus 5 786-803 5'-CTACCAGGGTATCTAATC-3' E suggested species 6 907-926 5'-CCGTCAATTCMTTTRAGTTT-3' U Haemophilus based on DNA-DNA homology studies. This 7 1096-1113 5'-GGTTGCGCTCGTTGCGGG-3' E suggestion was rejected because while these studies demon- 8 1392-1406 5'-ACGGGOGGTGTGTRC-3' U strated a relationship between [A.] actinomycetemcomitans 9 1493-1513 5'-TACGGYTACCTTGTTACGACT-3' E and H. aphrophilus, they did not demonstrate a convincing relationship between [A.] actinomycetemcomitans and H. a The sequences are complementary to 16S rRNA at the positions listed (E. the The current coli numbering). Base codes are standard IUB codes for bases and ambiguity. influenzae, the type species of genus (26). Specificity: P, Pasteurellaceae; M, miscellaneous genera; E, most eubacteria; results clearly show that [A.] actinomycetemcomitans is U, universal. related to Haemophilus clusters 1B and 1C. However, Aac .AA MM ac Hap .A.A aUnA Hap Ni nAA GUAA in Ase .aAl GUAA Ase PsA .A.A GUAA PsA Pav . .A( GUAA Pav Pmu . .At GUAA Pau Hpc ..At GUaA Hpc Hps .AAI GUAA Hps Asu ..A GUAn Asu Ali ..Al GUAA Ali Hpi . . .I GtJaA Hpi Hdu ..At GUAA Hdu Pne .AA ,GUAA Pne Eco AAAI Go GUAA Eco 10 20 30 40 50 60 70 80 90 100 110 120 Aac Aac Hap Hap Hin Hin Ase Ase PsA PsA Pav Pav Pmu Pu Hpc Hpc Hps Hps Asu Asu Ali Ali Hpi Hpi Hdu Hdu Pne Pne Eco Eco 130 140 150 160 170 180 190 200 210 220 230 240 Aac Aac Hap Hap Hin Hin Ase Ase PsA PMA Pav Pav Pm,u PM Hpc Hpc Hps Hps Asu Asu Ali Ali Hpi Hpi Hdu Hdu Pne Pne Eco Eco 250 260 270 280 290 300 310 320 330 340 350 360 Aac GGAAAUGCGCAAUGGGGGCAACCa UGACGCAGCCAUGCCGCGUGAAGAGA GGCCUUCGGGUGAA------GAUG CCACAU Aac Hap GGAUWGCGCAAUGGGGGCAACCCUaGCGCAGCCAUGCCGCGtJAL GUGUGZGGGCWGCCGUGCGGAA^GC AACWAAA1l Hap Hin GGAAUALGCGCGAUG CGGAG CAUGCCG UGC CLLGCC^GCCU^tUGCCC Hin Ase GGUUGCGCn^UGGGGGGAACCCUJGACGCAGCCAUJGCCGC GGCCWCGGGWGLY UGGGWAUGAGt)GAGGUUSMXAAW Ase PsA GGAUWGCGCAAUGGGGGGACIGACGACCAUGCCGCGUGAAUGAAAAGC ZG-G- AlOAUA GILAGCUAW U A PMA Pav GGAAUAWGCGC&AUGGGGGGAACCCLUGACGCAGCCAUGCCGCGUGAAUUGAAGAGGCCUUCGGGCLtAAtClCGGJC GWGgA GCUCUdUaAWJI Pav Pmu GGAAUAWGCGCAUWGGGGGGAACCCU GACGCAGCCAUGCCGCAA1GA-GAGGCZRGGCCWCGAXqJCC G G AiCGCA^UACALXMA Pu Hpc GGUWCaCAAUGGGGGGAACCCUgAUGCAGCCAUGCCGCGLJALGAAGCUCGWUACWCULAGGUGAGGAAUGGCGWGGAiGC Hpc Hps GC AGCRCAAUGGGGGGAACCCUGAUGCAGCCAUGCCGCGUGAAUAAGAAGCCUUGUUCG_GGAAGUCLAGUGAnA Gi0I A Al-CGACAUACAWU Hps Asu GGAAUAWGCntnAUtGGGGGACCGAUCGCCWAUGCGC APGCCAGMGCC)CGGaGUUAA 1-WCGGWUtC--AA UI CAGA- IG WA Asu Ali GGAAUAUGCACAA^UGGGGGGAACC CUgAUGCAGCCAUGCCCGUGAAUAAGGCCUUCGGGGGUAAAUUCUUCGGLIAGCGAGAGIA1^-GAL G GUAAt Ali Hpi GGAAUAUGCGCnAUGGGGGCAACCCUGACGCAGCCAUGCCGCGUAUAAGAAGGCCWCGGGWGIAGUGCWJCLGGUnGCGA ZUWA AipASA -AGUA Hpi Hdu GGAAUAUGCaCaAUGGGGGAACCCUGAUGCAGCtGCCGCtGAUGAAGAAGUGCCAJGGGCACGUAGGGGUAWbtUGGLGCglGUC Hdu Pne GGAAUJAGCGCaAUGGGGGGAACCCUGACGCAGCCAJGCCGCGUGAAUGAAGAAGGCCLLCGUUCGWUAAA GIAGGUAJGA GIZACCCCI AA Pne Eco GGAAUAWGCACAAUGGGCGCAAGCCUGAUGCAGCCAUJGCCGCGIGAUGAAUGAAGGCCtUUCGGGUWAAUUACUGCGGGGAGGAAGGGAG CCULAGCtJCAUU Eco 370 380 390 400 410 420 430 440 450 460 470 480 Aac Aac Hap Hap Min Min Ase Ase PSA PsA Pav Pav PMU PU Hpc Npc Hps Hps Asu IGA Asu Ali GGA Ali Hpi Hpi Hdu Hdu Pne Pne Eco Eco 490 500 510 520 530 540 550 560 570 580 590 600 FIG. 1. Aligned sequences of members of the family Pasteurellaceae. The numbering system is that of E. coli (13). The sequences are reported by using the IUB single-letter code for nucleotide bases and ambiguities. Lowercase letters indicate some uncertainty in base identity. Dashes indicate gaps inserted for alignment of sequences, and dots indicate regions that were not sequenced. 2006 VOL. 174, 1992 PHYLOGENY OF STRAINS OF THE PASTEURELLACEAE FAMILY 2007

Aac GJGGAACCGGUACGGnWCLCUCGGGtCGAtAAJAGAGrAAAACAGJAGGtGAUCtAAAGGAGAACAac Hap G)GGAACCGGUACGGn GCUJCGCJGGJCaAtAAAAGAGrA"AIACAGGJACGGAAGUAAAGGAGAACHap Hi n GJGGAGCCGCAACGGr GCUJCGCGGACAGAUAAGAGGAALCCCUUGGJGAUCUGGUtGAGAACHi n AsAsJe GAUCCGGUAC AGsUGAAUAAeGGGICAAUuUUGGGGUrAINCAGJAACGJGAUCLAAAGLG&GAICs PsA GAGJAACCGGWALJGALJCUUAAtgUAUGGAUUGGGGUGAUCCUUGGUAAGGaAAGGAGAACPsA Pay AGGAGCCACAACXGGAAGAWAAIGGXAUGGALAAGAGr)GACAGGACGGAUCUGGUUGGAUC Pav PuuGUUAACCGGUACjGACGALJCGCJGAUUGGnUUgGGGUGAUCAGGACGGAUCUGGUtGAGAIC Pnu Hpc GUJGAGCCGCJACLGGAGAAUAAGGAUUGGJUAJAGAGrAGAACAGGACGGAUCUGGUJGAGAACHpc Hps GU)AACCGGAJALLGGAGA CAAGGAJCAAtALJGGGGrAAACCCUUGGJGAUCUGGUtGAGAACHps Asu GAGGAGCCGCUACJGnUGALJAAUGJACAAUCPUGGGGiAAWCCUtACGGAUCUGGUtGAGAACAsu ALiAGl AACCGGUnCUGaLtGAA)AAUGJACAAU GAGgAAUCAGGACQGAUCUGGUUGGAUC Ati Hpi GUUAACCGGUACtGGAIGAUCUCGGCCAAUCUAGAGrJGACAGGAGGUAAGGAAAGGAGUACHpi Hdu HAGJAACCGGWACGGAUCLACUCGICAUGGAMGGGGIAAUCAGJUGGtGAUCLaAAGJGGAUC du Pne GLGGAACCGGUaCLGGAGAUCGCGGACaGGataGAGnAAUCAGGACGGAUCUGGULGAGAACPne Eco GAGJAACCGGUACLGGAtGACJAAUGAGLJALCCtAAGGGAAL)CG)tACGJAAGGAAACGAGAACEco 610 620 630 640 650 660 670 680 690 700 710 720 Aac GAGGAGACCAGGGUUCGCCGUUCAACUGGGAAAGLAGUCUGUGCCCCJAACGGCAAUGGAGGtJAac Hap GAGCAGCGCCAGGAGJCGCCCUUCAACUGGGAAAGLJGUCCGUG)CCCGAAGtGCAWGGWUCJHap H in HAGCAGCGCCAGGAGJCJAGtCULGGAGGGGACAAAGUAAICCJGALCAGUUACCGJGUUGGUGGtin Ase GAGCAGCGCCGGAAAtGCCJAGGGAGGGGACACGAUGLCCJGALCAGIGAAGULC-A GGUGGUAse PsA GAGCAGCGCCUGGGUCJAGtCULGGAGGJGGGaAA CUGIGCAGL CGC-lUGGAAGGUPsA Pay AGCAGCGCCIJGGGUCJAGLCtGGGAGGGGACACGAXaAACUGAUCCCGAAGUUGLAGGAUGC Pay Pmu GAGGAGACCUGGAGACGCCCUIGrAACLGGACAAGALAACCGGAUCCCGAAGGUrANGGAGGC PMu Hpc AGCAGCGCCUGGAAA HCCtCUpGGAGGcGGACACGAAAALACLGLALCAGCJL&AGULCALAGGAUCGip Hps HAGCAGCGCCUGAAUCJAGUAGJCAACtGGACAAAGUAAACUGALCAGtGAAGUUGUr4GLJGCps Asu GAGGAGACC GGAUUCGAGUAGGnAGGGGGGAAAGLJAAACtGUGCAGtGAAGJGJG WGGAGAUAsu AllGAGGAGACCLJGAULALGCCCAGJCAACLGGACACGAAaAACUGUGJCCCJUACCJ)GLJGGA GAt All Hpi GAGGAGACCWGAUJAJGCCJAAGCAACUGGGAAAGLAGUCCGtAJCCGGUACAGCAAJGGUGr Hpi Hdu GAGCAGCGCCUGAUUCJAGLCULGGAGGJGGGAAAGW%AACUGAUCCCJ CAGJGUAGGMJGCJHdu Pne GAGCAGCGCCAGGAGJCJAGUAGJCAACLGGACACGALAAACUGALCAGtGAAGUU'-IUGGGGGUPne Eco GGGCAGCGCCtGAGAALGCCCGUCAACUGGGAAAGUAAACUGAUCCCGAAGULCAUGAGL GCEco 730 740 750 760 770 780 790 800 810 820 830 840 Aac -UAGCUGJCCAGUAGGUAUGCGCGGAUCGCCAGAAACCAUAUgCGGCCCCACGJGGAGGLAAJAac Hap -W-GLJGGCGJGUAGGUAUGCGCJGGGJCGCCAGUAAUAAGrJnCGG-CCCACGGACULGUAW Hap H in -AAJCGGACGAGJACtGUAUGCGCGGAUCGCCAGUAAUAUGAAGCGGCGCCAC JGACUtGUAAJHi n Ase -rJ-GUGtGCGACJAGJAAACACCLGGALAGCGAGUAACCAUALJAGGGCGAAgGtGACLGGUUAJAs. PsA -W-GUGUCCUGUAGJAAACACCLGGAtAGCGAGUAACCAU-ANGCGGCCCCACGGACULGUAJW PSA Pay PayGCXGGJCCUGUAGJAUAUGCGC)GGAUCGCCAGUACUAAGAGCGGCCGAAGGtGACtJtGMINIAa Pmu -rJ-GUGtGCGACJAGJAAMGCGCJGGGAGCGAGUA CCAUALJAGGGCGAAGGJGGAGGULM Pau Hpc HUGACUGJCCtAC)AGGUAUGCGCGGALAGCGAGLJAALCAUAWAGGGCGAaGGUGGAGJGLJpc Hps -nGACUGJCCUGUAGGUAUGCGCGGAtAGCGAGLJAAUAAnAAGCGG-cCCACGGACUUGUAUHps Asu -W-GCGUCCAGUAGGUAUGCGCGGAtAGCGAGUaACCAUMuAGGGCGa&GGUGGa)UGUaUAsu Al i -UnAtJGJCCAGIACIGUAUGCGCGGAUCGCCAGUAAUAAGAUAGGGCGAAGGUGGAMGUAM Atl Hpi -AJACGGGCGACACJGUAUGCGCGGGGAGCGaGAJACrm GALJAGGGCGAnGGGAGHpi Hdu HW-GCGCCCAGUAGGUAUGCGCGGAtAGCGAGUAACJAAn&GCGGCCCCACGJGGAGGUUAdu Pne -W-GWGGCGJGUAGJAAACACCUGGGJCGCCAGAACJAAGAUAGGGCGAAGGJGGAJLGUAAUPne Eco CWAGGJGLJCGGUAGGUAtCACCtGGAtAGCGAGUAACJAAGAAGCGGCCCCACMGACUUGXMUEco 850 860 870 880 890 900 910 920 930 940 950 960 Aac UGUcAGGAACUACACCGAACGAAGAUAAAGGJUtGUAGGGUJGGCGUCGAGCGCUACCGGGGAAac Hap UCAGACCAGACXACJCCUAACAGAUUGAAAAGGGGCACGACAGGCGUCGAGCJUGCGUGJUGJAHap H in UCAGACCAGACAACJCCUAACUAAGGtCGGUACUUCUCGACUGGCGJCGAGCGC)ACCJLJUAHin AseUCAGACCAGACJJCUCJUGCUCUGAAtGGGUtG GGCUCGACAGGCGtGLGAGCGCUACCUAGtG As PsA UCAGMGGAACLJCUCJLJAACAAAGAUAAAGM GJCUCGACWAAAGGUCUGLGCUACCUUGJAPsA Pay UCAGACCAGACRACALCAGCUCAGACtGJGGUcgAIUCUCGACUGGCGUCGAJGUUGCGICUUGG av Pmu UGUrCCAGACACAUAGAACtAGAACCGGUACGtGCCGGAUAAAAGGUGAGGtGCGCGtCU GUAPmuu Hpc UGUcAGGAACUACAUUGCUCAGAACCAAAGGUGGCUCGAUAGGCGUCGAGGgCGCGUGGGUAHpc Hps HCAGACCAGACAACAtCUAACUAAGAICGGUAuUUCUcGACUGGCGUCGAGCgCUACCUUGGps Asu UCAGACCAGACACAUUGCUCUGACAGJGGUCAAtGCCGACAGGCGJCJC)GUUGCGUGGGJAAsu Alti UCAGACCAGACUCLAUUGCUCUGACUtAAAAGGGJCWGGACUAAAGGUCUGU)GCGUGJLJJAALl Hpi HCAGACCAGACAACJCCXGCUCGGAACAAAGAAGUCWGUUAAAGGUCUGtGJGCGtC)UGG pi Hdu HCAGaCCAGACXACANCUAACAAAGAUAAAGGLJJCWGGAUU)AAGGUCUGtG)MCGUGGULGdu PneUGUrACCAGAC1ACJCWXGCACAAAGGCiG UWtGCUGGAUWAAAGJCGAGCGCUACCUUMG Pne Eco UCAGACCAGACAACGULJAACAGAGLXCGGUAAUUCUCGACGGGCGIGUCUGUUGCGUGJUG)AEco 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080 Aac AAGUGUAGCCCAGGGACCAACUAGAGCGG-GJGJGGAtCAGAAUCGUAAACGGAGUGGUAGCAtCAac Hap AU GG AAtCCCAGGGACCACUGXGCGG-LJGCGGAtCAGAC)GCGGAAACGGAGUGGUAGCAtCHap H in AAGUGWAUCGACACCACLACUGAGCGG-UGtCGACCAGAAUCAtGUACGAGAGnGAGCUAGCH in Ase AUUGGXAGCCCAGGGACCACUGUGCUG-AGtCGACCAGACGCGUAAaCGGAGUGGUAGCAtCAse PsA AULGGLJAUCGACACCACUACLJGXGCGA-CGJGGCUAAAGCGCGGUACGAGAGGGGUAGJAGCPsA Pavy AGAGGUAUCGACACCACLLACL GAGCGA-ACGGGGAICAGAALGCGGUACJGGAGLGGAGCUAGJ Pay PimuAj XGGAAGCCCAGGGACCUUCUGXGCGG-ACGttGACCAGAAtGCGJAnAUGGAGUGGUAGCA Pmiu Hpc aUUGGrwUCCCAGGGACCJAUCAIGAGCGA-CGUGACCAGAAGCALGUACGGGAGtGGAGCUAGCHpc Hps AUUGGUAUCgACACCAC AUCAGUGCGG-CGCGGAUAAGGtGCAJGUACgGGAGGGGUAGCAtCHps Asu AUUGGtnAUCGACACCAC AUCIJGIGCACGUJGCGGCUAGGGCGCGGUACGAGAGGGGUAGCAGCAsu Al i AlULJGLJAUCGACACCACWUCAUWCACAWGUGGAUAAGGCGCGGUACGAGAGgGAGCtCAUt Hpi AAGAGGUAUCGACACCACUACLXGGCGG-CGCGACCAGAAtGCG)AmCGAGAGGGAGCtCAtCHpi Hdu HAGKGGAnGCCCAGGGaCCXACLJJLJCACUUG)AGGAUAAGGCGCGJArAtGAGAGGGAGCUAG)du PneAU1W GLtaGCCCAGGGACCAACUAGAGCGG UUGJGGAUAAGGCGCrJAAACGGAGUGGUAGCAU Pne Eco AAGAGGXAGCCCAGGGACCACLkGAGCGG-CGCGGAUAAGGCGCGGUACGAGAGJGGUAGJAGCEco 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 FIG. 1-Continued. 2008 DEWHIRST ET AL. J. BACTERIOL.

Aec Aac Hap Hap Hin Hin Ase Ase PsA PsA Pav Pav Pau Psu Hpc Hpc Hps Hps Asu Asu Ali Ali Hpi Hpi Hdu Pr Pne Eco Eco 12 10 1230 1240 I; !60 1270 1280 1290 1300 1310 1320 Aec UUxG Aac Hap UWCAG NGC Hap Nin UWCG GC Hin Ase NGC Ase PsA NGC PM Pav kAUCAG Pav PM PMu Hpc ;UCGUWCAG Hpc Npt Hps Asu UWCG Asu Ali UCG1360 Ali Hpi M4UCAC Hpi Ndu UWCAG Hdu Pre MCG ccw Pne Eco LMG 1GCCW Eco 13330 1340 13!5iO 1360 1370 1380 1390 1400 1410 1420 1430 140

Aac WaCCgAgGGc_GG C CACGQGlJngacu ...... Aac ActinobecitLus actinmycetemcomitans FDC Y4 Hap ACCGUUAGGGGGCQJUUACCACCQIAIJGAUUCAUGAC ...... Hap Heemophilus aphrophilus ATCC 33389 T Hin A UGG...AGGGCQ CACG IAIGAU IUGACUGGGG...... Hinm Haemophilus influenzae ATCC 33391 T Aso c AG-GGGGGcGAACCJAC JAUgUCA ...... Ase Actinobacillus seminis ATCC 15768 T PM ACCGCAAGGGGUU _GIAC_CAC-GWGkIAZCA...... PsA Pasteurella species A CCUG 18782 Pav UACOCaAG ACCGG A ...... Pav Pasteurella aviuL. NCTC 11297 PM ACC.__ GGGGGG UACCACG UCAI...... Pi Pasteurella inltocida ACTC 10322 T Hpc UA_CUCA.A.A.GGGGGGCQAAU UA ...... Hpc Haemophilus paracuniculus ATCC 29986 T Hps IANAAACGGGGG- CACGGUAUGAUUCAu...... Hps Haemophilus parasuis NCTC 4557 T Asu UCcnuCgGGGGGGcG.kUA--ACGGIAUG Ca ...... Asu Actinobacillus suis ATCC 15557 T Ali ACC_CU GGGGGCGLRJACCA C.UG CGGGG...... Ali Actinobacillus lignieresii NCTC 4189 T Hpi WM=UUCG G UIACCA U AJUGaCJGGGG...... Hpi Haemophilus parainfluenzae ATCC 33392 T Ndu AC_OCJCGGGAGGGCQJACCAC.AIJGAIIXAAC ...... Hdu Haemophilus ducreyi CPI 542 T Prle ACUAGGGCC U XAUUCA ...... Pne Pasteurella pneumotropica NCTC 8141 T Eco UACUCGGGAGGGCGCUUACCACUUUUJGALAtAGACUGGGQIGUGAGtUAACA Eco Escherichia coli 1450 1460 1470 1480 1490 1500 FIG. 1-Continued.

TABLE 3. Sequence signatures for the family Pasteurellaceae because A. actinomycetemcomitans differs phenotypically and has a long evolutionary branch, it can be argued that this Positiona rellaceae"Pasteu- Eubacteria' Exceptions' species should be placed in its own genus. What is unargu- able is that [A.] actinomycetemcomitans is not a member of 237 A C, U, G Acinetobacter the genus Actinobacillus (cluster 4A). [A.] actinomycetem- 248 G C, a Bacteroides, Rickettsia comitans ATCC 29522, FDC Y4, and ATCC 29524 are 2761 C G, u serotype b, ATCC 29523 is serotype a, and ATCC 33384T is 562 A, g U, C, g Ruminobacter serotype c (68). The 16S rRNA sequences for serotypes a, 599 G C, u, a None b, 639] C G,a,u and c differ from one another by more than many species 665 G A Treponema, Rickettsia elsewhere on the tree. Change of serotype to subspecies may 667 A G None be warranted following further analysis. 739 U C Strains obtained from culture collections as H. parainflu- 723 A U Serpulina enzae and H. paraphrophilus fell into three different areas of 831 G U, a, c None the phylogenetic tree, as previously reported by Pohl (51). 8551 C G, A, u The type strain of H. paraphrophilus and [H. parainfluen- 868 U C Capnocytophaga, spirochetes 1298 A U, C None zae] ATCC 7901 (actually H. aphrophilus) fall in cluster 1B, 1 the type strain of H. parainfluenzae and [H. paraphrophilus] 1439 A G, U, C Aeromonas, Legionella ATCC 29242 (actually H. parainfluenzae) fall in cluster 4B, 14621 U C, A, G and [H. parainfluenzae] NCITC 4101 (reclassified as P. Position using E. coli numbering. Positions connected by brackets repre- volantium) falls in cluster 3A. It is recognized that these sent paired bases. b Bases found in Pasteurellaceae species. Bases are abbreviated as in Fig. 1. species are genetically heterogeneous and contain many ' Bases found in other eubacteria. Lowercase letters denote bases found in misidentified strains (15, 29, 50, 51, 62). less than one-fourth of the sequences. (ii) Cluster 2. Cluster 2 is composed of three species d Genera with species possessing the same base as Pasteurellaceae. VOL. 174, 1992 PHYLOGENY OF STRAINS OF THE PASTEURELLACEAE FAMILY 2009

Scale (Percent) Family or Taxa Escherichia coli Citrobacter freundii I En terobac teri aceae Serratia marcescens Proteus vulgaris Vibrio parahaemolyticus - Vibrionaceae Haemophi lus aphrophilus Haemophi 1us in fluenzae Pasteurella canis r I Pasteurellaceae Pasteurella multocida Actinobacillus suis Actinobacillus lignJeresii Aeromonas hydrophi la - Aeromonadaceae Ruminobacter amylophi lus Proteobacteria Oceanospiri llum linum Pseudomonas aeruginosa - Pseudomonas fluorescens Complex gamma - Acinetobacter calcoaceticus - Moraxellaceae Cardiobacterium hominis - Suttonella indologenes Cardiobacteriaceae Dichelobacter nodosus Neisseria gonorrhoeae ] Neisseriaceae Eikenella corrodens beta - Alcaligenes faecalis -Alcaligenaceae [Pseudomonas] cepaci a -Pseudomonas solanacerum Complex FIG. 2. Phylogenetic tree for the beta and gamma divisions of Proteobacteria. The scale bar represents a 10% difference in nucleotide sequence as determined by measuring the lengths of horizontal lines connecting two species. previously cast out of their given genera: [H.] somnus, [A.] isms have avian hosts, while cluster 3B organisms have seminis, and [P.] aerogenes. These species are less closely mammalian hosts. related to one another than are members of other clusters. Cluster 3C is an odd assortment of species that have not This is in part due to the long branch of [H.] somnus, which previously been related to one another or to other members has accumulated many changes in its 16S rRNA sequence of cluster 3. This group includes [H.] paracuniculus, [A. relative to other species. Previous investigators found anti- capsulatus] NCTC 11408T and CCUG 12396T, P. trehalosi genic cross-reactivity between [H.] somnus and [A.] seminis NCTC 10624, [H.] haemoglobinophilus, [H.] parasuis, and (55, 60) but failed to find a convincing relationship by [H.] parasuis type 5 Nagasaki. [H.] haemoglobinophilus DNA-DNA (49, 64) and DNA-RNA (21) hybridization stud- NCTC 8540 fell with the type strain (355 bases). [H.] ies. The stability of this cluster to outgroup switching and parasuis type 5, which was previously shown by DNA-DNA bootstrapping (see below) convinces us that it is a legitimate hybridization (37) to differ from other H. parasuis strains (61 cluster. to 72% identity), represents a separate species based on 16S (iii) Cluster 3, including the genus PasteureUa sensu stricto. rRNA distance. Previous investigators found that A. capsu- Clusters 3A and 3B contain the species generally recognized latus strains fell in cluster 4A with the true Actinobacillus as Pasteurella sensu stricto (39). Included in cluster 3A are (11, 24, 40, 42). Therefore, our initial finding that strain P. anatis, P. avium, P. gallinarum, P. langaa, Pasteurella NCTC 11408T did not fall in the expected phylogenetic species A, P. volantium, [Haemophilus] taxon C CAMP cluster prompted us to sequence the 16S rRNA of three 5111, and [H.] paragallinarum. The inclusion of [H.] additional strains. We obtained strain 11408T a second time paragallinarum in cluster 3A is consistent with DNA-DNA from National Collection of Type Cultures (NCTC) and the hybridization data, which showed [H.] paragallinarum to same strain from Culture Collection, University of Goteborg share 35% identity with Pasteurella species A, P. volantium, (CCUG), strain 12396T. The partial sequence (340 bases) of and P. gallinarum, about the same level of similarity as was the reacquired NCTC strain was identical to that previously found between Pasteurella species A and the species P. obtained. Similarly, the full 16S rRNA sequence for strain avium and P. volantium (41, 48). Cluster 3B contains P. CCUG 12396T was identical to that of NCTC 11408T. We canis, P. dagmatis, P. multocida, P. stomatis, and Pas- then obtained what we thought was a different strain of A. teurella species B CCUG 19794. We find a very clear capsulatus, CCUG 19799. The 16S rRNA sequence of this delineation between clusters 3A and 3B which is not always strain was markedly different from the previously sequenced apparent in DNA-DNA studies (39). The deeper members of strains and fell in the expected position in cluster 4A. clusters 3A and 3B have many base signatures which clearly Unfortunately, strain CCUG 19799 = Carman 8-11272 = SSI differentiate these groups. For example, all cluster 3A or- P585 is not a strain of A. capsulatus but is an A. suis-like ganisms except P. langaa contain the sequence 5'-GAAAC strain which has been placed in Bisgaard's taxon 11 biovar 2 GAUGGCUAAUACCGCAUAG-3' at positions 159 to 182, (11). Thus, we are left with an unanswered question as to whereas cluster 3B organisms contain the sequence 5'- whether currently available strains NCTC 11408T and GAAACUCICAGCUAAUACCGC-iUAK-3' (mismatch posi- CCUG 12396T are the same as SSI strain p243T and related tions are underlined, K = G or U). A DNA probe, 5'- to strains P244, P564, P572, and CIP 704.80 as shown by CTATGCGGTATTAGCCATCGT'ITC-3', should recognize Escande et al. (24). The structure of cluster 3 poses inter- cluster 3A organisms (except P. langaa) and have five to six esting taxomomic questions. Should Pasteurella sensu mismatches with cluster 3B organisms. All members of stricto be limited to cluster 3B species, or if it also includes cluster 3B are indole positive (or indole variable), while cluster 3A species, how can it exclude species from cluster 3C? cluster 3A organisms are indole negative. Cluster 3A organ- (iv) Cluster 4, includingActinobacillus sensu stricto. Cluster 2010 DEWHIRST ET AL. J. BACTERIOL.

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4A contains those species recognized as true actinobacilli: node occur together. The number below the nodes is the A. lignieresii, Actinobacillus species strain CCUG 19799, A. percentage of outgroup switching trials in which the taxo- equuli, A. pleuropneumoniae, A. suis, andA. ureae (39, 52). nomic units to the right of the node occur together. Exami- [H.] parahaemolyticus is also a member of this cluster. This nation of the tree shows that the stability of nodes is directly latter finding is surprising based on DNA-DNA hybridization proportional to the length of the branch from the node to studies (15, 21), and we will therefore sequence rRNA from outgroup sequences (branch to left of node). The stability of additional strains of this species. Not actually forming a 8 of 10 of the subclusters is 100% to outgroup switching. The separate cluster, but branching ever more deeply, are cluster stability of cluster 3A is 100% below P. anatis but only 40% 4B organisms [H.] paraphrohaemolyticus, [H.] ducreyi overall, due to P. langaa and P. anatis switching to deeper (three strains), [P.] haemolytica, [H.] parainfluenzae, [H.] positions in the tree. Bootstrapping values are lower but parainfluenzae ATCC 29242, and [Haemophilus] "minor support the existence of most subclusters. The stability of group" strain 202 (32). [H.] ducreyi was previously shown to clusters 1, 2, and 4 is 85 to 100% by outgroup swapping. be unrelated to true haemophili in cluster 1 (2, 3, 18). Clusters 1 and 2 are present in the majority of trees by However, suspicions that it was not a member of the bootstrapping. The major changes seen in the overall tree Pasteurellaceae were unfounded as shown by DNA-RNA when using different outgroups are the frequent movement hybridization (21). While this report was being prepared, of the root of the tree to a position between P. langaa and P. Rossau et al. (56) reported the 16S rRNA sequence for the anatis and movement of P. pneumotropica to cluster 1. The type strain and also concluded that it is a legitimate member tree in Fig. 3 is correct in most of its details but probably has of the Pasteurellaceae. Pohl (50, 51) had previously demon- some discrepancies with a true phylogenetic tree. We be- strated that [H. paraphrophilus] ATCC 29242 is a misnamed lieve that this tree is close to the true tree and that it H. parainfluenzae. Cluster 4 is interesting taxonomically, as represents a major step forward in understanding the phylo- it is a cluster in which each species branches slightly deeper genetic relationships among the members of the family than the one above, giving no logical point at which to end Pasteurellaceae. the genus Actinobacillus sensu stricto. While we think that taxonomy should reflect phylogeny, (v) Cluster 5, [P.] pneumotropica. [P.]pneumotropica is the we do not propose any taxonomic reordering of the family most deeply branching organism of the family Pasteurel- Pasteurellaceae in this report. There are several reasons for laceae. Its 16S rRNA lacks five base changes that differen- deferring taxonomic realignment. First, we plan to sequence tiate Pasteurellaceae from other gamma proteobacteria. 20 to 30 additional strains belonging to the family Pasteurel- Yet, at the same time, it has some of the signatures found in laceae, including a majority of the unnamed Bisgaard taxa Pasteurella sensu stricto organisms (cluster 3). Using differ- (40). These additional data will allow 16S rRNA sequence ent organisms for an outgroup (or no outgroup), P. pneumo- comparison analysis for essentially all of more than 70 tropica branched as a deep member of cluster 1 73% of the described taxa within the Pasteurellaceae. Second, it seems time. In none of our analyses did [P.] pneumotropica fall prudent to allow time to resolve discrepancies between 16S with cluster 3. The phylogenetic position of this species rRNA analyses and other methods where they exist. Finally, deserves further examination. the reordering of a large taxonomic group should reflect the Reliability of trees. For many years, phylogenetic trees thinking of microbiologists in more than one laboratory; were reported with no indication of their reliability. Those therefore, we suggest that the implications of this and other who generated trees were aware that changing outgroups, molecular studies should be addressed by the Committee on including more or fewer bases in the analysis, or using Systematic Bacteriology Subcommittee on Pasteurellaceae different treeing algorithms often changed the tree obtained. and Related Organisms and that this body should have a role Sometimes changes are minor, such as the exchanging of in creating new genera and renaming species. Because a two branches or slight changes in branch lengths. However, solid 16S rRNA sequence data base is now available for over even the exchanging of branches can cause great scientific 50 taxa in the family Pasteurellaceae, and because we and debate, as, for example, in the relationship between chim- many other investigators around the world are willing to panzee, human, gorilla, and orangutan (27). Analysis of the collaborate in determining 16S rRNA sequences, we would stability of trees generated from sequence data is a new field, hope that any future descriptions of new species within the and methods such as the bootstrap and jackknife have only family Pasteurellaceae would include full 16S rRNA se- recently been developed (25). A bootstrap program for use quence information. with the Neighbor-Joining treeing algorithm has recently been written by T. S. Whittam. In our analysis, hypervari- ACKNOWLEDGMENTS able regions were not excluded so that differences between closely related species could be seen; however, this does We thank V. J. Rapp-Gabrielson, Department of Microbiology, decrease the bootstrap values obtained. Therefore, the boot- North Dakota State University, Fargo, for providing cultures of H. are a conservative estimate of the parasuis Nagasaki, Haemophilus taxon "minor group," and Hae- strap values reported mophilus taxon C strain CAMP 5111. We thank W. L. Albritton, reliability of the tree. An alternative way of examining the University of Alberta, Edmonton, Alberta, Canada, for cell pellets reliability of the tree is to use different outgroups. In Fig. 3, of H. ducreyi ATCC 27722, HD 35000, and CIP 542. We thank P. B. there are numbers above and below each node in the tree. Little, Ontario Veterinary College, University of Guelph, Guelph, The number above the nodes is the percentage of bootstrap- Ontario, Canada, for providing a cell pellet of H. somnus 43826. We ping trials in which the taxonomic units to the right of the thank M. Nei and T. S. Whittam, Department of Biology, Institute

FIG. 3. Phylogenetic tree for the family Pasteurellaceae. The scale bar represents a 5% difference in nucleotide sequence as determined by measuring the lengths of horizontal lines connecting two species. The numbers above each node are the percentage of times that the strains to the right of the node occur together by bootstrapping. Numbers below each node are the percentage of times that the strains to the right of the node occur together using different outgroups. *, sequence for strain given in Fig. 1. 2012 DEWHIRST ET AL. J. BACTERIOL. of Molecular Evolution and Genetics, Pennsylvania State Univer- of systematic bacteriology, vol. 1. The Williams & Wilkins Co., sity, University Park, for providing Neighbor-Joining and bootstrap- Baltimore. ping computer programs, respectively. 18. Casin, I., F. Girmont, P. A. D. Girmont, and M.-J. Sanson-Le This work was supported by Public Health Service grants DE- Pors. 1985. Lack of deoxyribonucleic acid relatedness between 04881 and DE-08303 from the National Institute of Dental Research Haemophilus ducreyi and other Haemophilus species. Int. J. and grants from the Dental Faculty, University of Oslo. Syst. Bacteriol. 35:23-25. 19. Chuba, P. J., R. Bock, G. Graph, T. Adam, and U. Gobel. 1988. REFERENCES Comparison of 16S rRNA sequences from the family Pasteurel- 1. Adlam, C., and J. M. Rutler. 1989. Pasteurella and pasteurel- laceae: phylogenetic relatedness by cluster analysis. J. Gen. losis. Academic Press, Inc. (London), Ltd., London. Microbiol. 134:1923-1930. 2. Albritton, W. L. 1989. Biology of Haemophilus ducreyi. Micro- 20. Colwell, R. R., M. T. MacDonell, and J. De Ley. 1986. Proposal biol. Rev. 53:377-389. to recognize the familyAeromonadaceae fam. nov. 1986. Int. J. 3. Albritton, W. L., J. K. Setlow, M. L. Thomas, and F. 0. Sottnek. Syst. Bacteriol. 36:473-477. 1986. Relatedness within the family Pasteurellaceae as deter- 21. De Ley, J., W. 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