JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1992, p. 3099-31070095-1137/92/123099-09$02.00/0Copyright © 1992, American Society for Microbiology

Vol. 30, No. 12

Differentiation of Moraxella nonliquefaciens, M. lacunata,and M. bovis by Using Multilocus Enzyme Electrophoresis

and Hybridization with Pilin-Specific DNA ProbesTONE T0NJUM,l* DOMINIQUE A. CAUGANT,2 AND KJELL B0VRE'

Kaptein W. Wilhelmsen og Frues Bakteriologiske Institutt, University of Oslo, Rikshospitalet, N-0027 Oslo,'and Department of Bacteriology, National Institute of Public Health, N-0462 Oslo,2 Norway

Received 26 May 1992/Accepted 27 August 1992

Genetic relationships among strains ofMoraxefla nonliquefaciens, M. lacunata, and M. bovis were studied byusing multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electro-phoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes weregrouped in four major clusters, one representing strains ofM. nonliquefaciens, two representing strains ofM.lacunata, and one comprising strains of M. bovis and the single strain of M. equi analyzed. DNA-DNAhybridization with total genomic probes also revealed four major distinctive entities that corresponded to thoseidentified by multilocus enzyme electrophoresis. The two distinct clusters recognized among the M. lacunatastrains apparently corresponded to the species previously designated M. lacunata and M. liquefaciens.Distinction of the four entities was improved by hybridization with polymerase chain reaction products ofnonconserved parts of pilin genes as DNA probes. With these polymerase chain reaction probes, new isolatesofM. nonliquefaciens, M. lacunata, M. liquefaciens, and M. bovis can be identified easily by hybridization.

The bacterial group called the classical moraxellae or theMoraxella lacunata group consists of M. nonliquefaciens,M. lacunata, and M. bovis, which are particularly closelyrelated (2, 3). M. nonliquefaciens is part of the normal florain the human upper respiratory tract and is frequentlyisolated from the nasal cavity (2, 3). It has also been culturedfrom the blood, eye, cerebrospinal fluid, lower respiratorytract, and other local sites (2, 3, 8). The species is consideredto be of low pathogenicity, depending on reduced hostresistance for invasion and clinical manifestations (3). At theNational Hospital, Oslo, Norway, several of our M. non-liquefaciens strains were isolated from leukemia patientswith septicemic episodes. Certainly, the frequency of M.nonliquefaciens as part of the normal airway flora and itsclinical importance as a potential pathogen are underesti-mated.The species designated M. lacunata unifies the previous

species M. liquefaciens and M. lacunata on the basis ofobservations of particularly close genetic affinities (1, 2, 9,10). M. lacunata is a significant causative agent of humanconjunctivitis and keratitis as well as chronic sinusitis andendocarditis (3, 21) and has been found to infect guinea pigs(3). Local outbreaks of conjunctivitis caused by M. lacunatahave been reported (21).M. bovis is the major etiological agent involved in bovine

infectious keratoconjunctivitis, which is a common oculardisease in cattle (12). M. bovis has also been isolated fromother animals, including horses (2, 3).

All three species display a number of phenotypes asso-ciated with fimbriation, among them spreading and/or cor-roding growth on agar, twitching motility, surface pellicleformation in static broth, and natural competence for trans-formation (2, 3, 22). They possess type 4 pili, which arefound in a wide variety of gram-negative species (6). Themain structural subunits, termed pilins, of these fimbriae or

* Corresponding author.

pili share a highly conserved amino-terminal domain of 25 to30 amino acid residues (11), whereas the C-terminal partscontain variable DNA regions that rearrange by differentforms of recombination (17, 22, 25). The type 4 pilin genes ofstrains of the classical moraxellae have been cloned (18, 22,28).Rapid identification of classical moraxella strains has been

difficult for many laboratories. The distinction betweenstrains of M. nonliquefaciens and M. lacunata is mostrelevant in clinical samples, because the two species colo-nize similar habitats in humans (3). Serum liquefaction andgrowth requirements (1, 15) are traits that may help distin-guish these two species; however, tests of such traits areoften not employed in ordinary routine diagnostic work, andtheir value as specific key tests is not fully exploited.Separation between the two species has previously beendemonstrated by genetic transformation (1-3), but the labo-rious quantitative assay needed for this fine distinction istime-consuming and dependent on competent strains.

Distinction between M. lacunata and M. bovis can usuallybe obtained easily by the expression of hemolysis of M.bovis (3). However, this test cannot be used with nonhemo-lytic strains of M. bovis, including the nonhemolytic entityM. equi, found in horses (3).Many fastidious gram-negative bacterial species can be

separated and thereby identified by using DNA-DNA hy-bridization with total genomic probes (26). The classicalmoraxellae are exceptions to this finding (26). To determinethe overall genetic relationships among strains of thesespecies, we analyzed electrophoretically demonstrable al-lelic variation at 12 genes encoding metabolic enzymes andperformed hybridization of total chromosomal DNA withfour reference and type strain probes. For diagnostic pur-poses, we investigated the use of hybridization by usingpolymerase chain reaction (PCR) products of selected partsof pilin genes as DNA probes on 84 strains of these and othergram-negative species.

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MATERIALS AND METHODS

Bacterial strains and growth conditions. The strains inves-tigated are listed in Tables 1 and 2. Species assignment wasbased on data from previous studies by genetic transforma-tion in our laboratory, supplemented with recent experi-ments (1-3; unpublished data), and results from Juni et al.(14, 15). M. lacunata E2371, E3808, E7992, E9650, andF4037 and M. bovis EPP63, Tifton-1, Fla64, S009-6,GRE8-5, BET9-18, 0009:09-7, NCSU-1, NCSU-9, 144LE,IBH64(406), and BEJ8-3 were generously supplied by E.Juni, University of Michigan. Strains were grown on 5%human blood agar plates (Difco) and chocolate agar plates at33°C with 5% CO2. Serum liquefaction was investigated byreaction with Loeffler slants of 75% inspissated bovineserum (1).

Multilocus enzyme electrophoresis. Protein extracts wereprepared as described previously for Neisseria meningitidis(4). The methods used for starch gel electrophoresis andselective enzyme staining were similar to those described bySelander et al. (23). Buffer system A was used for allenzymes. The 12 enzymes assayed are listed in Table 1.

Mobility variants (allozymes) of each enzyme, numberedin order of decreasing anodal mobility, were equated withalleles at the corresponding structural gene loci. Each isolatewas characterized by its combination of alleles at the 12enzyme loci, and distinctive multilocus genotypes weredesignated as electrophoretic types (ETs). ETs were num-bered sequentially according to the positions in the dendro-gram (Fig. 1). Genetic diversity and genetic distance werecalculated as described previously (20, 23, 24).DNA-DNA hybridization. DNA-DNA hybridization was

performed essentially as previously described (27). DNAwas extracted by a modification (27) of the method ofMarmur (16) or by the method of Hull et al. (13). Thedifference between relative binding ratios (RBRs) was lessthan 6% when DNAs from the same strains extracted byboth methods were investigated.

(i) Dot blot filters. Eight parallel dots of single-strandedgenomic DNA and a Tris-EDTA buffer control were blottedon nitrocellulose paper for each strain. Filters were storeddry.

(ii) PCR probes. Synthetic oligonucleotides FF14 throughTF19 (Genetic Designs Inc., Houston, Tex.), used as PCRprimers, were made from areas of the pilin genes displayingthe least DNA homology among the three species (18, 22, 28)(Table 3). PCR reactions were performed with a Stoffelfragment of Taq polymerase and DNA thermal cycler (Per-kin Elmer Cetus, Norwalk, Conn.) according to the direc-tions of the manufacturer, except that an annealing temper-ature of 60°C was used. Oligonucleotides TT16 and TT17were also used as degenerate primers with M. lacunata50139 as a template to achieve the PCR product termed La(Table 3). The PCR products were prepared for labeling byelectroelution from SeaKem GTG agarose (FMC, Rockland,Maine).

(iii) Labeling of probes. Mechanically fragmented genomicDNA probes and PCR probes were labeled with [3 P]dcGrpto a specific activity of 108 cpm with a random priminglabeling kit (Amersham) as recommended by the manufac-turer.

(iv) Hybridization. Hybridization was performed as de-scribed previously (27). The sodium salt concentrations ofthe prehybridization and hybridization fluids were 0.1 and 1M for total genomic and PCR probes, respectively. Other-wise, all hybridizations were performed under standard

conditions at 65°C (27). The hybridization results of the dotblot were obtained by overnight autoradiography (HyperfilmMP; Amersham) and scintillation counting (Packard Instru-ments) of pieces of the nitrocellulose filter cut to a standardsize with a vacuum cutting device designed in our labora-tory.

(v) Quantitation of the dot blot hybridization reaction. Themean of the counts per minute for the eight parallels minusthe control counts per minute of salmon sperm DNA foreach of the strains was determined. The mean counts perminute value of the autologous strain was defined to repre-sent a DNA homology ratio or an RBR of 100%. The meanRBR of each strain was calculated by dividing the meancounts per minute of each strain by the mean counts perminute of the autologous reaction and then multiplying by100. The 95% confidence interval of the sample mean of theRBRs was estimated (data not shown). For comparisonbetween total genomic and PCR probes, a two-tailed Wilcox-on's paired test was employed; the level of statistical signif-icance was set at P < 0.05.

Genetic transformation. The media and reagents weredescribed previously (27). Mutants were selected for strep-tomycin resistance, DNA was extracted (27), and the DNAconcentration was adjusted to 200 ,ug/ml. The quantitativetransformation assay was performed by exposing 0.5 ml ofthe recipient suspension to 0.05 ml of each DNA for 20 min.After DNase treatment, 0.1-ml aliquots of each final suspen-sion and appropriate dilutions were inoculated on blood agarplates. These plates were preincubated for 7 h and thenplaced on top of an agar layer giving a final concentration of50 ,ug of streptomycin per ml after diffusion. The plates werefurther incubated for 3 to 5 days; then the colonies werecounted, and the interstrain/intrastrain (autologous) trans-formation ratios were calculated.

RESULTS

Biochemistry and growth. None of the strains of M.nonliquefaciens liquefied bovine serum, whereas all strainsof M. bovis and M. lacunata were positive in this confirma-tory test. The strains of M. lacunata exhibited two distinctgrowth patterns. One group, designated group I, grew withrelatively large, opaque-bluish colonies, some of them withcross-feeding (small colonies when separate); the others,designated group II, had smaller, clearer colonies through-out (strains listed in Tables 1 and 2). The type strain of M.lacunata, ATCC 17967, was the slowest growing of thegroup I strains and also displayed the highest degree ofcross-feeding.

Multilocus enzyme electrophoresis. In the collection of 74isolates analyzed, all 12 enzyme loci were polymorphic for 2to 11 alleles (mean, 7.0 alleles per locus).

Fifty-nine distinctive multilocus genotypes were identified(Table 1), among which the mean genetic diversity per locuswas 0.650.The genetic relationships among the 59 ETs are shown in

Fig. 1. The dendrogram consists of four major clusters,designated A through D. Cluster A includes the 37 ETs of all39 isolates assigned toM. nonliquefaciens; The 14 ETs of the18 isolates of M. lacunata are in clusters B and C, andcluster D includes the 7 ETs of the 16 M. bovis and thegenotype (ET 57) of the single M. equi isolate tested.There was no sharing of ETs among the Moraxella species

examined, but indistinguishable allozymes between M. non-liquefaciens and M. lacunata, M. nonliquefaciens and M.

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DIFFERENTIATION OF CLASSICAL MORAXELLAE 3101

TABLE 1. Allelic profiles of 74 isolates of Moraxella spp.

Allele no. at the indicated enzyme locusaET Species and isolate

ADK MDH MAE PE2 PE3 IDH GDl GD2 FUJM GOT EST IPO

M. nonliquefaciens1 270/602 672/583 ATCC 19975

NCTC 77844 8a5 13385/626 3179/667 B5539/908 0252749 44810 119a11 31a12 VT32/6913 P2581/8814 13015/6215 877516 135a17 178/6218 4378/6219 B2042/9020 2916/6621 468/9122 836/6123 149IV24 118a

4863/6225 B5270/9026 139IV27 37a28 1541V29 ll5a30 75a31 3067/6632 162IV33 45H40/9034 826/6135 182V36 858/8637 28a

M. lacunata group I38 NCTC 13035939 ATCC 1795640 E237141 E380842 ATCC 1796743 NCTC 798544 NCTC 791145 CDC 983346 E7992

3 6 6 3 13 6 5 3 13 4 6 5 1

3 4 6 5 13 4 6 4 13 4 6 5 23 4 6 6 23 4 6 5 23 4 6 4 20 6 6 6 23 6 6 6 23 6 6 6 13 6 6 6 23 4 4 5 23 4 6 5 23 4 6 5 33 6 6 0 13 6 6 5 13 6 6 5 13 6 6 6 13 6 6 5 13 6 6 5 13 4 6 5 13 6 6 5 1

0 6 6 5 13 6 6 5 13 6 6 6 10 6 6 5 13 6 6 5 13 6 6 5 33 6 6 5 33 6 6 5 13 6 6 3 13 6 6 5 21 4 7 4 23 4 7 3 23 8 6 6 2

2 5 3 5 02 5 1 7 04 3 2 5 15 3 2 5 12 3 3 5 12 3 3 5 12 3 3 7 12 3 2 1 32 3 3 5 3

4 2 2 2 7 8 14 2 2 2 7 8 14 2 2 3 4 8 1

4 4 2 3 4 8 14 2 2 3 5 8 14 2 2 1 5 8 14 2 2 1 5 8 14 2 2 3 5 8 14 3 2 1 5 8 14 2 2 3 5 0 14 2 2 3 5 8 14 2 2 3 5 8 14 2 1 2 5 8 14 2 2 2 5 7 14 4 2 2 0 7 14 2 2 1 6 7 15 4 2 3 4 7 15 4 2 3 4 7 14 4 2 3 4 7 14 4 0 3 4 7 14 3 2 1 5 8 14 3 2 1 5 7 14 2 2 1 5 7 14 2 2 1 5 7 1

4 2 2 3 5 7 14 2 2 2 4 7 14 2 2 2 8 7 14 4 2 2 5 7 14 4 2 2 5 7 14 2 2 2 5 7 15 2 2 1 5 7 15 2 0 1 5 7 15 2 2 3 5 7 14 2 3 1 5 7 15 2 2 2 5 7 16 4 2 2 5 7 13 3 2 2 5 7 1

2 0 2 0 4 3 12 7 2 0 5 5 11 0 3 4 1 1 11 8 3 0 1 1 11 0 1 0 3 3 12 0 1 4 3 3 12 1 1 4 3 3 21 7 0 0 3 3 11 4 3 0 3 3 1

M. lacunata group II47 50139

501415014228419/84

48 50136A947

49 ATCC 1174850 E965051 F4037

4 7 2 8 2 2 5 3 4 3 2 1

4 7 2 10 2 2 5 3 4 3 2 1

5 7 2 9 26 7 2 9 24 7 2 9 1

2 4 3 4 4 3 12 4 3 0 4 3 12 4 3 4 1 4 1

M. bovis52 NCTC 9425 7 1 2 3 2 1 4 4 4 2 0 1

Continued on followingpage

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TABLE 1-Continued

Allele no. at the indicated enzyme locusaET Species and isolate

ADK MDH MAE PE2 PE3 IDH GD1 GD2 FUM GOT EST IPO

53 NCTC 8561 7 1 2 3 2 1 4 4 4 2 5 1NCTC 9426

54 BET9-18 7 1 2 3 2 1 4 4 4 1 0 1EPP63GRE8-5Tifton-1

55 1BH64 0 1 2 2 2 1 4 4 4 1 0 156 ATCC 10900 7 1 2 2 2 1 4 4 4 1 6 1

F1a6458 3 8 1 2 3 2 2 4 4 4 1 5 1

59181P3

59 5009-6 7 2 2 3 2 2 4 4 0 1 0 1

57 M. equi NCTC 11012 7 1 2 3 2 2 6 4 4 1 5 1a Enzyme abbreviations: ADK, adenylate kinase; MDH, malate dehydrogenase; MAE, malic enzyme; PE2, phenylalanyl-leucine peptidase; PE3, leucyl-

glycyl-glycine peptidase; IDH, isocitrate dehydrogenase; GD1 and GD2, two glutamate dehydrogenases; FUM, fumarase; GOT, glutamic oxaloacetictransaminase; EST, naphthyl propionate esterase; IPO, indophenol oxidase.

bovis, and M. lacunata andM. bovis were found for 6, 4, and8, respectively, of the 12 enzymes assayed.

Genetic diversity within the three species is shown inTable 4. There was significant heterogeneity in allele fre-quencies at 11 of the 12 enzyme loci among ETs of the threespecies, as measured by the coefficient of genetic differenti-ation (F,,) between the species.

Hybridization with total genomic probes. Mean RBRs ofstrains of M. nonliquefaciens, M. lacunata, M. bovis, andreference strains of other gram-negative species, hybridizedwith total genomic DNA probes of type and reference strainsof the three species (NCTC 7784, ATCC 17956, 50139, andATCC 10900), are shown in Table 2 and Fig. 2. Data for only17 of the 38 M. nonliquefaciens strains included in thehybridization study are presented in Table 2. Total genomicDNA of the M. nonliquefaciens reference strain NCTC 7784showed mean RBRs to other strains assigned to this speciesranging from 80 to 112%. Strain NCTC 7784 had mean RBRsof between 18 and 37% to strains of M. lacunata and M.bovis. Strains of M. lacunata fell into two categories corre-sponding to M. lacunata groups I and II (Table 2; Fig. 2).Strain ATCC 17956 had RBRs ranging from 73 to 113% (95%confidence interval from 66 to 118%), respectively, to thegroup I strains and RBRs of 41 to 86% (confidence interval of38 to 89%) to the group II strains. M. lacunata 50139 hadRBRs of 61 to 86% (95% confidence interval of 56 to 89%)and 83 to 113% (95% confidence interval of 79 to 118%) tostrains of M. lacunata groups I and II, respectively. Totalgenomic DNA of M. bovis ATCC 10900 hybridized to otherM. bovis strains with mean RBRs ranging from 73 to 115%(95% confidence interval from 70 to 121%). Hybridization tothe M. equi reference strain also fell into that range (meanRBR of 92%). In hybridization to strains of M. lacunatagroup I, the total genomic probe from M. bovis had RBRsranging from 29 to 69%, but with M. nonliquefaciens and M.lacunata group II, the probe showed weaker affinity (meanRBRs of 17 to 38%). The M. nonliquefaciens RBRs werequite homogeneous within one cluster, as were the M. bovisRBRs. The group I probe (ATCC 17956) of M. lacunatashowed an affinity to the strains ofM. bovis that was as high

as or higher than that of the M. lacunata group II strains(Table 2; Fig. 2). The group II probe (50139) ofM. lacunataexhibited higher RBRs with group I strains of M. lacunatathan with M. nonliquefaciens and M. bovis.

Hybridization with pilin-specific PCR probes. The pilin-specific PCR probes (No, Li, La, and Bo, respectively) forM. nonliquefaciens, M. lacunata groups I and II, and M.bovis distinguished generally better between the entities ofthe classical moraxellae than the total genomic probes did(Table 2; Fig. 2). The M. nonliquefaciens probe No had anRBR of 32% or less with strains of M. lacunata and M.bovis, whereas RBRs to M. nonliquefaciens itself rangedfrom 89 to 112%. M. lacunata pilin-specific PCR probes alsogave a clearer distinction between the two groups of M.lacunata, without overlapping mean RBRs. PCR probes Liand La had RBRs of 69 to 112% to strains of their group,whereas their RBRs to the other group of M. lacunatastrains were between 37 and 69%. The PCR probe for M.lacunata group I (labeled Li) improved distinction betweenM. lacunata and M. bovis and between M. lacunata and M.nonliquefaciens.The classical moraxellae were also compared with a

variety of other gram-negative bacterial species representinga relevant spectrum of differential diagnoses in this context(Table 2; Fig. 2). These 10 species were all well distinguishedfrom the moraxellae with both total genomic and PCRprobes. The RBRs of these strains were slightly higher withthe PCR probes than with the total genomic probes.

Genetic transformation. Representative recipient and do-nor strains of M. lacunata groups I and II were analyzed byquantitative genetic transformation (Table 5). These experi-ments indicated two closely related clusters of M. lacunatastrains, which were distinguished from each other but moreso from both M. nonliquefaciens and M. bovis.

DISCUSSION

In this study, the genetic relationships of the classicalmoraxellae were investigated by means of multilocus en-zyme electrophoresis and DNA-DNA hybridization with

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VOL. 30, 1992 DIFFkERENTIATION OF CLASSICAL MORAXELLAETABLE 2. RBRs in DNA-DNA hybridizations between Moraxella strainsa

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Mean RBRC with the following probe:

ET Filter DNA sourceb NCTC ATCC 9 ATCC PCR probes

778417956 50( PCR00(Mn) (n) (MIlII M) No Li La BoIII)~~ ~ ~ '~ (Mn) (MiT) (Mlli) (Nib)

M. nonliquefaciensg3 ATCC 19975T3 NCTC 77846 3179/667 B5539/908 025274/9013 P2581/8815 877519 B2042/9021 468/9124 4863/6224 118a25 B5270/9031 3067/6633 45H40/9034 826/6135 182V36 858/86

M. lacunata group I38 NCTC 1035939 ATCC 1795640 E237141 E380842 ATCC 17967T43 NCTC 798544 NCTC 791145 CDC 983346 E7992

M. lacunata group II47 5013947 5014147 5014247 28419/8448 A94748 5013649 ATCC 1174850 E965051 F4037

525353545454545656NDe58585859

M. bovisNCTC 9425NCTC 9426NCTC 8561EPP63Tifton-1BET9-18GRE8-5ATCC 10900TFI-A 64BEJ8-33595009-6

57 M. equi NCTC 11012T

M. (BranhameUa) catarrhalis Nell(ATCC 25238T)

M. (B.) ovis 199/55 (ATCC 33078T)M. osloensis 5873M. atlantae 1922M. phenylynsvica 752/52Kingella denitificans NCTC 10995TDichelobacter nodosus ATCC 25549Neisseria gonorrhoeae GCN. meningitidis 8152Escherichia coli K-12

89 16 38 17 89 10 15 7100 29 41 28 100 14 32 8103 19 46 29 112 12 24 9110 38 34 27 109 9 24 1189 32 34 31 110 12 28 9109 48 38 26 110 13 23 15105 44 50 25 112 11 21 9112 38 49 29 111 19 29 881 28 34 29 106 16 22 7105 33 29 24 108 8 29 8106 36 40 34 99 15 23 13105 27 44 31 102 13 32 4105 32 32 25 103 14 18 9104 15 32 19 105 15 31 7111 29 48 30 109 15 14 8104 36 35 25 110 12 16 1080 42 39 27 88 13 17 8

33 110 86 31 14 111 67 1834 100 84 45 19 100 62 2331 112 82 69 15 88 72 2235 113 85 53 24 75 63 1935 73 71 43 17 84 69 921 74 73 29 16 93 64 1021 81 61 39 15 72 52 1337 83 78 28 22 85 39 1232 87 71 42 14 96 49 21

28 47 100 23 20 45 100 630 44 109 29 31 42 93 1119 43 107 26 19 46 91 1221 48 111 27 17 47 95 621 41 83 25 25 43 106 1231 64 108 30 22 47 110 1335 86 113 36 32 48 112 822 47 112 21 21 37 73 1020 48 103 24 16 51 69 8

19 59 41 81 16 18 22 9319 58 36 82 14 19 21 9022 68 37 91 13 18 18 9018 42 38 92 9 16 19 10020 54 43 79 12 15 22 10320 64 41 89 15 17 25 8620 69 55 77 13 10 39 8821 64 28 100 15 15 29 10121 64 35 109 20 10 34 9518 43 29 73 14 16 18 7624 67 54 89 17 15 55 8820 66 39 82 16 16 28 8524 68 32 102 12 30 19 8723 69 35 115 19 19 15 84

22 62 57 92 17 17 32 105

16 10 6 9 6 10 16 9

9 8 7 7 5 6 11 75 4 4 5 1 4 8 43 1 1 4 1 1 6 62 1 1 6 2 2 5 86 5 4 6 7 5 12 53 2 1 5 6 4 4 63 3 1 4 6 2 9 54 5 4 5 2 3 7 41 1 0 1 1 6 4 4

a Strains of M. nonliquefaciens (Mn), M. lacunata group I (MIu), M. lacunata group II (MIII), and M. bovis (Mb) were subjected to DNA-DNA hybridizationwith total genomic DNA and pilin gene-specific PCR products with radioactively labeled probes.

b The numbers to the right of shills (/) in some strain designations indicate the year isolated.c Mean of the sample mean of eight parallels in each experiment. The 95% confidence intervals were calculated for all RBRs (data not shown).d Only 17 of the 38 M. nonliquefaciens strains included in the hybridization study are presented in this table.e ND, not determined.

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Genetic Distance0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

L== 59

FIG. 1. Genetic relationships among 59 ETs of isolates of M.nonliquefaciens, M. lacunata groups I and II, and M. bovis. Thedendrogram was generated by the average-linkage method of clus-tering from a matrix of coefficients of genetic distance based on 12enzyme loci. ETs are numbered sequentially from top to bottom.The letters A through D indicate the clusters referred to in the text.

total genomic probes. The information provided was em-ployed to evaluate potential species-specific DNA probes fordiagnostic use for these species, which generally give weakphenotypic expression by conventional diagnostic assays.The electrophoretic analysis of allelic variation at 12

enzyme loci supported previous lines of evidence derivedfrom genetic transformation studies (2, 3, 14, 15) of a cleardistinction between strains assigned to M. nonliquefaciens,M. lacunata, and M. bovis. Although a substantial numberof electromorphs were shared between these species, therewere no overlaps in multilocus enzyme genotypes and

strains of the three species diverged on the average at six ormore of the enzyme loci assayed (a genetic distance ofgreater than 0.55). Compared with other closely relatedspecies, such asActinobacillus actinomycetemcomitans andHaemophilus aphrophilus (5) and oral streptococci (7), in-vestigated by multilocus enzyme electrophoresis, the spe-cies of the classical moraxellae appeared even more closelyrelated to each other.

All three species, especially M. lacunata, whose strainsclustered into a group of relatively closely related ETs(cluster C) and markedly distinct strains (cluster B) (Fig. 1),were quite heterogeneous. This distinction corresponded tothe different growth patterns, colony morphology, and nu-tritional requirements of the two M. lacunata groups (15,19). On the basis of genetic variation at the 12 loci, the twogroups of M. lacunata strains were as distinct from oneanother as they were from the two other species. The singleisolate of M. equi fell into cluster D with the M. bovisstrains, confirming earlier suggestions from genetic transfor-mation experiments that M. equi strains should in fact bereassigned to M. bovis as nonhemolytic variants of thisspecies found in horses (3).

Strains of M. nonliquefaciens were recovered from sys-temic infections, local infections, and carrier states. Therewas no association between ETs of these strains and theirclinical sources.The clustering of strains of M. lacunata into two distinct

groups was also apparent from total genomic hybridization,although the clusters overlapped slightly. Some of thisoverlap may be due to variations in this method of hybrid-ization. Very stringent conditions are used to obtain optimalseparation, and this facilitates variation. Some variability ofthe DNA quality and steric conditions may also affect theRBRs found.The two groups of M. lacunata corresponded to the two

cell wall fatty acid patterns reported for M. lacunata byMoss et al. (19) as well as to the two auxotrophic donortransformation activities toward M. bovis found by Juni etal. (15). Quantitative transformation analysis of strains of thetwo groups as both recipients and streptomycin-resistantdonors, as compared with earlier observations with otherstrain combinations, improved the separation of the twogroups (1, 3, 10). The M. lacunata groups I and II probablyrepresented strains of the species previously referred to asM. liquefaciens and M. lacunata, respectively.

Although the classical moraxellae are particularly closelyrelated, they appeared as four separate and distinct unitswith regard to enzyme genotypes, RBRs, and transformationratios. The two phenotypic groups of M. lacunata wereseparable as unique genetic entities. Retention of the previ-ous designations ofM. lacunata and M. liquefaciens (3, 4, 9)would be convenient for purposes of identification and

TABLE 3. Nucleotide sequences of the PCR primers

Species or probe Oligonucleotide Sequence product (bp)

M. nonliquefaciens TT14 5'-GATACAGCTGCAGATGGCOG-3'No TT15 5'-CTAGCACTAGCTTACCTGC-3' 195M. lacunata TT16 5'-GGTTTTGGATGCTGGCAAC-3'Li and La' TT17 5'-GTGCCTTGCTGTACGTGAC-3' 254M. bovis TT18 5'-CCAAACTAGGTAAAGCTGC-3'Bo TT19 5'-GGCAGAACCGTTAATGGTG-3' 257

a The PCR product with 1T716 and 1T17 as degenerate primers migrated slightly slower than Li did on gel electrophoresis.

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DIF'FERENTIATION OF CLASSICAL MORAXELLAE 3105

TABLE 4. Genetic diversity at 12 enzyme loci in Moraxella spp.

Genetic diversity in ETs of: Variance componenthEnzyme locus4

M. nonliquenfaciens M. lacunata M. bovis h5 h, Ft

ADK 0.203 0.692 0.524 0.360 0.654 0.450MDH 0.503 0.648 0.286 0.512 0.771 0.336PE2 0.629 0.791 0.476 0.650 0.744 0.126IDH 0.368 0.495 0.476 0.412 0.685 0.398ME 0.206 0.582 0.000 0.272 0.610 0.554GD1 0.523 0.846 0.000 0.538 0.710 0.242GD2 0.206 0.648 0.000 0.288 0.600 0.520PE3 0.563 0.747 0.000 0.540 0.614 0.121FUM 0.683 0.538 0.286 0.600 0.808 0.257GOT 0.517 0.703 0.476 0.557 0.745 0.252EST 0.503 0.670 0.667 0.563 0.774 0.273IPO 0.000 0.143 0.000 0.034 0.034 0.000

Mean 0.409 0.625 0.266 0.444 0.646 0.313

a See footnote a of Table 1.b h5 is the within-species component of genetic variance, which estimates the probability of a mismatch of alleles at an enzyme locus for two ETs chosen

randomly from the same species; ht, the total variance, is the same estimate for two ETs drawn from the three species pooled. F3, = (h, - h,)/h, is a measureof the relative magnitude of genetic differentiation at an enzyme locus among species.

Moraxella nonliquefaciens Moraxella lacunata Group I

7784 No

Probes

nr-9 n-s9 n22Gr. MIIcunata Gr 11| M. bovis

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M. nonlkqubfaclns Gr. Mblacunata G II M. bovis Olher M. nonliquefac.ns| Gr. I Miacunats Gr II M. bovis Others

FIG. 2. Graphic presentation of the mean of the RBRs and standard deviations of M. nonliquefaciens, M. lacunata groups I and II, andM. bovis with a set of total genomic and PCR probes for each entity. RBRs were obtained by using DNA-DNA hybridization with radioactivelabeling. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

,100

0

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0

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_100

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3106 T0NJUM ET AL.

TABLE 5. Affinities of strains of M. lacunata by quantitativestreptomycin resistance genetic transformation

Frequency ratioa in the followingDonor DNA strain recipient:(M. lacunata group)

A947 NCITC 10359

A947 (II) 1 1.4 x 10-150136 (II) 6.2 x 10- 1.2 x 10-lATCC 17956 (I) 4.4 x 10-2 1.2NCTC 10359 (I) 6.3 x 10-2 1E 2371 (I) 9.9 x 10-2 1ATCC 199751b 2.3 x 10-2 5.1 x 10-2ATCC 109001c 3.3 x 10-3 6.8 x 10-2a Ratio of interstrain transformant frequency to intrastrain transformant

frequency.b M. nonliquefaciens.c M. bovis.

epidemiology as well as for pathogenicity studies. However,the final designations in this matter need additional studieson DNA reassociation in solution.

In this study, four major clusters of classical moraxellastrains were distinguished by multilocus enzyme electropho-resis and DNA-DNA hybridization. The stronger geneticaffinity of M. lacunata group I and M. bovis found byDNA-DNA hybridization with total genomic and pilin-spe-cific probes was not reflected by multilocus enzyme electro-phoresis. The range of enzymes that could be used formultilocus enzyme electrophoresis in these moraxellae wasparticularly limited, and distinction reflected by multilocusenzyme electrophoresis may therefore be at a level otherthan that shown by DNA hybridization. The dendrogram inFig. 1 shows relatedness of M. lacunata group II to M.bovis, but this type of analysis is rather inaccurate whenmore distantly related strains are clustered.The pilin genes of only one strain each of M. nonlique-

faciens, M. lacunata, and M. bovis have been sequenced(18, 22, 28), and knowledge about the variability of thesepilin genes is still quite limited (22). The probes No, Li, La,and Bo are located in the nonconserved parts of the pilingenes, where the genes of the type and reference strains ofeach species displayed minimal mutual DNA homology (22,28). The areas of the genome surrounding the pilin geneshave higher mutual DNA homology than the genes them-selves do (22). The relatively high heterologous RBRs foundwith the pilin-specific probes could be due partly to satura-tion of homologous target DNA, which would reduce thescale for distinction, although there in this case were several(partial) gene targets for each bacterial genome (28).

Pilin-specific PCR probes No and Bo gave good separationofM. nonliquefaciens andM. bovis, respectively, from otherspecies. The PCR probes Li and La provided improveddistinction between the two M. lacunata groups, althoughthe discrimination was not as clear as that usually seenbetween separate species. The distinctive property of the Laprobe would most probably have been more clear ifwe couldhave avoided the use of degenerate primers. Still, bothpilin-specific PCR probes Li and La failed to hybridizestrongly to DNA of strains of the other M. lacunata group,demonstrating that if one still regarded them as one species,one would have to use a combined pilin-specific DNA probeto identify strains of the whole entity.The four pilin-specific PCR probes described herein rep-

resent tools for a simple and inexpensive hybridization assay

for identification of new isolates of M. nonliquefaciens, M.lacunata, M. liquefaciens, and M. bovis.

ACKNOWLEDGMENTS

The excellent technical assistance of Eva L0vstad and PiaStavnes is greatly acknowledged. We thank M. Rostrup for assis-tance in statistical analysis of the hybridization results.

This work was supported by grants from the Norwegian Councilfor Science and Humanities and Anders Jahres Foundation to T.T.

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