INFECTION AND IMMUNITY, JUlY 1986, P. 79-89 Vol. 53, No. 1 0019-9567/86/070079-11$02.00/0 Copyright © 1986, American Society for Microbiology

Difference in Structure between Type b and Nontypable influenzae Populations OSCAR PORRAS,'t DOMINIQUE A. CAUGANT,'t BARRY GRAY,2 TERESA LAGERGARD,3 BRUCE R. LEVIN,4 AND CATHARINA SVANBORG-EDENl* Departments of Clinical Immunologyl and ,3 University of Goteborg, S413 46 Goteborg, Sweden; Department of Pediatrics, University ofAlabama, Birmingham, Alabama 352942; and Department ofZoology, University of Massachusetts, Amherst, Massachusetts 010034 Received 8 August 1985/Accepted 15 January 1986

The extent of chromosomal genetic variability and the genetic structure of Haemophilus influenzae populations was analyzed. A total of 119 isolates from humans in Goteborg, Sweden, and Birmingham, Ala., and 16 strains from a type culture collection were characterized for capsular type, biotype, outer membrane protein profile, and enzyme electrophoretic type (ET). The results of this study indicate that the identified as H. influenzae are a genetically extremely variable array of organisms. For the six enzymes studied, the estimated mean genetic diversity was 0.57 (approximately 20% higher than the corresponding estimate for Escherichia colt). Two lines of evidence indicate that despite its ability to recombine by transformation, H. influenzae maintains a largely clonal population structure. Although there is considerable potential for generating different genotypes, there were only 88 distinct ETs among the 135 strains, and isolates of the same ET and biotype were recovered at frequencies greater than would be anticipated at random. This evidence for a clonal population structure holds for uncapsulated as well as capsulated strains. However, these data also suggest that the stability of H. influenzae clones (clone persistence time) may be less than that of the nontransforming species E. coli. The ET data indicate that there is somewhat less variability among H. influenzae strains that express the same capsular antigens, biotype, and outer membrane proteins than among randomly chosen isolates. Nevertheless, there is substantial genetic variation among isolates within each of these classes and combinations thereof. There is also variation in these typing characteristics among strains of the same ET. These observations and those on genetic variability and population structures have implications for the characterization of H. influenzae isolates in clinical and epidemiological studies.

The clone concept postulates that bacterial populations alleles at an arbitrary locus). Nevertheless, strains with the are arrays of stable lineages (clones) that maintain their same multilocus genotypes are found in very different chromosomal gene composition with little or no rearrange- places, and these genotypes are maintained for considerable ment for extended periods of time (25-27). As a result, periods of time. One of the more dramatic examples of this bacteria of the same (or nearly the same) genotype can be are recent isolates from natural populations that are identical isolated from samples taken from different geographic re- to the E. coli laboratory strain K-12 for all 20 of the enzyme gions or at different times. One practical implication of the loci studied (30). E. coli K-12 was isolated more than 60 clone concept is that general, nonspecific procedures such as years ago. There is also evidence for nonrandom associa- serotyping, biotyping, phage typing, outer membrane pro- tions between typing characters, such as multiple antigen tein (OMP) profiles, restriction fragment patterns, and serotype, enzyme electrophoretic type (ET), and OMP pro- multilocus enzyme electrophoresis can be used to identify file, and the determinants of pathogenic intestinal and extra- bacteria for clinical and epidemiological studies. Chromo- intestinal E. coli infections (1, 5, 18, 26, 32, 34). somal (and possibly plasmid-borne) genes determining the In theory, the persistence of multilocus genotypes could characters that motivated the study, usually virulence prop- be explained by selection for specific gene combinations. erties, are likely to remain associated with those coding for However, this doesn't seem likely, as it requires that natural the typing characters. selection be able to distinguish among different combinations The most extensive evidence for a clonal population of functional variants of the same enzymes. A more parsi- structure has been found for . Initially this monious hypothesis, and one which is currently favored (7, interpretation was based on the results of serotype studies 16, 19), is that the rate of recombination is low and (25, 26), but more recently evidence for the clonality of E. multilocus genotypes are not broken up by gene shuffling. coli populations has come from investigations done with With low rates of recombination and random extinction of multilocus enzyme electrophoresis and OMP profiles (1, 6, 7, local populations (19) or periodic selection (16), a clonal 30). There is considerable genetic variability in this species, population structure can be maintained even without direct a genetic diversity on the order of 0.5 (roughly the probabil- selection for specific combinations of genes. ity of a randomly chosen pair of bacteria having different For E. coli, recombination requires plasmid or phage vectors. Although relatively high rates of recombination may occur, this seems to be an artifact of laboratory strains. Even * Corresponding author. in the rare cases in which chromosomal gene recombination t Present address: Hospital Nacional de Ninios, Apartado 1654, 1000 San Jose, Costa Rica. does occur when naturally occurring E. coli strains are used t Present address: Department of Biology, University of Roches- as donors, the rate is very low (see for example reference 24 ter, Rochester, NY 14627. and the arguments in references 16, 17, and 30). A very 79 80 PORRAS ET AL. INFECT. IMMUN.

TABLE 1. Composition of the H. influenzae isolates in Birmingham, Ala. (0. Porras, H. Dillon, B. Capsular type Biotype Gray, and C. Svanborg-Eden, Pediatr. Infect. Dis., in press). The Goteborg isolates were obtained from Origin b NT 1 2 3 separate hosts, Other and the Birmingham isolates were obtained sequentially Goteborg, Sweden 28 47 31 31 12 1 from 26 individuals. Identical strains from the same host Blood 10 3 9 4 0 0 were eliminated from the collection. Cerebrospinal fluid 12 0 12 0 0 0 These bacteria were confirmed as H. influenzae by their Middle ear fluid 0 9 1 6 1 1 for both Nasopharynx 3 32 4 20 11 0 growth requirement NAD and hematin. The isolates Other 3 3 5 1 0 0 were kept frozen. For testing, bacteria were thawed, trans- ferred to hematin plates, and grown overnight at 37°C in 5% Birmingham, Ala. 9 34 25 12 3 3 CO2. Oropharynx 7 28 21 10 2 3 Capsular type. At isolation each strain was designated Middle ear fluid 2 5 4 2 1 0 type b or NT by its reactivity with anticapsular antisera (Phadebact, Pharmacia, Sweden, or Hyland Lab, Costa Mesa, Calif.). different situation may obtain for bacteria in which recom- Biotype. The biotype was determined by the method of bination occurs by the sequestering of free DNA, i.e., Kilian et al. (12) by testing the ability to produce urease, transformation. There is evidence that in seminatural condi- indole, and ornithine decarboxylase. tions recombination through transformation plays a role in Multilocus enzyme electrophoresis. The conditions for the adaptation of Bacillus subtilis (8). starch gel electrophoresis of H. influenzae protein extracts H. influenzae is a gram-negative bacterium, with strains have been described in Porras et al. (29). The protein that are competent for transformation (32). It is a constituent extracts were prepared from bacteria grown overnight at of the normal respiratory tract flora as well as a frequent 37°C with shaking in 100 ml of antigen-free medium. The cause of local and systemic infections (35). Isolates of H. cultures were centrifuged at 8,670 x g (J2-21 rotor JA 10; influenzae may be identified by several typing procedures: Beckman Instruments, Palo Alto, Calif.) for 10 min at 5°C. capsular type, biotype, OMP and profiles The pellet was suspended in 2 ml of 0.01 M Tris-0.001 M (1-3, 11, 28), and, most recently, enzyme electrophoresis EDTA (pH 6.8), sonicated (100-W Ultrasonic Disintegrator; (22, 29). There is an association between some typing MSE, London) for 1 min with cooling and centrifuged at characters, such as the b capsule type and virulence (22). In 2,830 x g for 30 min at 5°C. The supernatants were trans- fact, a recent study presented evidence that type b strains of ferred to 10-ml tubes and stored at -70°C until used. H. influenzae maintain a clonal population structure (22). The protein extracts were subjected to horizontal gel In this report we present the results of an investigation electrophoresis. The 11.4% starch gels were sliced and employing multilocus enzyme electrophoresis, biotype, and stained for six enzymes: malate dehydrogenase (MDH; EC OMP profiles to estimate genetic variability and ascertain the 1.1.1.37), phenylalanylleucine-peptidase (PE2; EC 3.4.11), population genetic structure of capsulated and uncapsulated 6-phosphogluconate dehydrogenase (6PG; EC 1.1.1.49), ad- strains of H. influenzae. We present evidence that genetic enylate kinase (AK; EC 2.7.4.3), glucose 6-phosphate dehy- variability in H. influenzae exceeds that in E. coli and drogenase (G6P; EC 1.1.1.49), and phosphogluconase provide additional support for the hypothesis of a clonal isomerase (PGI; EC 5.3.1.9). The rationale for selecting population structure for type b H. influenzae. We also these enzymes is described in Porras et al. (29). Two buffer provide evidence that a clonal population structure occurs in systems (BS) were used: Tris-citrate, pH 8.0/Tris-citrate, pH uncapsulated (nontypable [NT]) members of this species as 8.0 (BS1) and Tris-citrate, pH 6.3/Tris-citrate, pH 6.7 (BS2). well as in isolates of other capsule types. The results of our MDH, PE2, 6PG, and AK were electrophoresed at 130 V in analysis, however, suggest that the stability (persistence BS1, G6P was electrophoresed at 250 V in BS1, and PGI was time) of H. influenzae clones may be less than that of E. coli electrophoresed at 150 V in BS2. The histochemical stains clones. have been described previously (29; D. Caugant, Ph.D. (Selected aspects of this study were presented in June thesis, University of Goteborg, Goteborg, Sweden, 1983). E. 1985 at the Federation of European Microbiology Societies coli protein extracts were included as controls. meeting [Molecular biology of microbial pathogenesis: role Electrophoretic type. The relative mobility of the elec- of protein-carbohydrate interactions, S. Normark and D. tromorphs was determined and graded as variants of fast Lark, in press].) (F+ + F+, F), medium (M, M-) or slow (S+, S, S1, S2, S3). The number of electromorphs previously identified was five MATERIALS AND METHODS for MDH, six for PE2, four for 6PG, three for AK, and six each for G6P and PGI. In the present material additional Bacteria. A total of 135 H. influenzae isolates were tested. electromorphs were found for MDH (Si), 6PG (S), AK (F+), The following strains were obtained from the National G6P (F+ +), and PGI (S2). N signifies null, i.e., no detectable Collection of Type Cultures, Public Health Laboratory, electromorph. London (NCTC), and from the culture collection of the Each isolate was assigned an ET, the combination of University of Goteborg (CCUG). Type a: CCUG 6881, electromorphs (allozymes) for the six enzymes tested (6). NCTC 8465, and NCTC 8466; type b: CCUG RAB and The allozyme combinations of the individual ETs are shown 12769; type c: CCUG 4852 and NCTC 8469; type d: CCUG in Table 2. A total of 88 ETs were identified. 6878 and 15117; type e: NCTC 8472, CCUG 15518, OMP preparation. The OMP preparations were obtained Montenegro, CCUG 15522, and NCTC 386/66; type f: NCTC by the method of Zollinger et al. (36). The bacteria were 8473, CCUG 15435, and NCTC 7918. The origin, capsular grown in 30 ml of antigen-free medium (4) for 4 h at 37°C. type, and biotype of the remaining RAB clinical isolates used Each culture was divided equally into two Ehrlenmeyer is shown in Table 1. Seventy-five strains originated from flasks containing 400 ml of broth and incubated for 16 h at Goteborg, Sweden; 44 were obtained from children prone to 37°C with shaking. The cultures were centrifuged twice at VOL. 53, 1986 CLONALITY IN H. INFLUENZAE 81

TABLE 2. Characteristics of H. influenzae isolates Capsular Allozymeb f()MPuv type ETa Biotype No. MDH PE2 6PG AK G6P PGI p)rofile a 1 F M M M F F 1 _ 2 2 S M M M F+ F 1 1 c 3 F+ + S F+ M S Fl 2 2 d 4 S S F+ M S S 4 1 5 S S F+ M S F+ 4 1 e 6 S M- F M S Si 4 2 7 S S F+ M S F 3 2 8 S M F+ M M Si 4 1 f 9 S M F+ M M S+ 2 1 10 M M F F F M 1 1 11 S M F F N M 1 1 62 S M M F F M 1 INV 1 b 12 S M F F F M 1 1 13 S S F+ M M F 1 1 14 S S F+ M M S 1 I 17 1 II 1 1 V 1 1 XII 1 2 I 2 1 4 15 S M F+ M M S 1 I 1 52 S M M M M M 1 I 1 1 I 2 60 S M M M M S 1 I 1 74 M M M M M M 1 I 1 76 S S F S M S1 2 XIIX 1 77 Si M F+ M S S 1 II[I 1 85 M S M F F F 1 I 1 86 M S M M F F 1 II][1 1

NT 13 S S F+ M M F 2 2 14 S S F+ M M S 2 1 3 1 15 $ M F+ M M S 2 1 16 F S F M S1 S1 2 1

17 S S F+ M F M 3 1 18 S S F+ M F F 3 1 19 S 51 F+ M S S 2 1 20 M S F+ M M M 2 1 21 S S2 F+ M S S1 2 1

22 F+ S2 F M S1 S 7 1

23 S S F M S F 2 - 1 24 S S F+ M M M 3 - 2 24 2 1 25 S F+ M F S1 2 1 26 M S F+ M F M 2 1

27 F M F+ M S1 Si 1 2 28 F+ S F++ M M M 3 2 29 S 51 F+ M S1 S1 2 1 30 S S2 F+ M S1 S1 2 1 31 F+ S3 F++ S S S 2 1

32 S M F+ M Si F 2 1 33 S M F+ M F S1 2 1 34 F M F M S S1 2 1 35 S S1 F+ M M F 2 4 36 S 51 F+ M M M 2 1

37 S S1 F+ M M S1 2 1 Continued on following page 82 PORRAS ET AL. INFECT. IMMUN.

TABLE 2-Continued Capsular ETa Allozymeb Biotype OMP No. type MDH PE2 6PG AK G6P PGI profile 38 S M- F+ M M F 3 2 39 F+ S F+ M S Si 2 1 40 S S F+ M M S1 2 1 41 M Si F+ M S Si 1 1 42 M S F+ M S M 3 1 43 F+ S F+ M S M 3 2 44 S S F+ M S Si 1 1 45 F M- F M Si Si 2 1 46 M F+ F M S1 1 1 47 M S F M F+ Si 2 1 48 S S3 F+ M M Si 2 1 49 S M F+ S S2 M 3 1 50 F+ M F M S2 S 1 I 1 51 M S F M S M 2 XXIV 1 52 S M M M M M 1 I 1 S XXV 1 53 S S2 M M S S 4 XXII 1 54 S M- M M F M 1 1 55 S M S S S 1 XXI 1 56 S M S M M M 1 XV 1 57 S S M F M S 2 XXIII 1 58 M M F M S2 S 2 XI 1 59 S M M F S M 2 XI 1 1 61 S M M M F S 1 XIII 63 M M S M S Si 1 V 1 1 64 S M M M S Si 1 V 65 S S S F F M 1 XXV 1 66 M M M M F 1 I 1 67 S M S S Si M 2 XXVI 1 68 S S F F+ F+ M 1 I 1 69 M F M F S1 1 V 1 70 S S M F+ S 3 XIV 1 71 F++ S3 F+ S F S 2 XVI 1 72 S M S F F+ S2 1 XVII 1 73 N S S S S S 2 X 1 74 M M M M M M 1 I 1 75 S S M F F+ S 2 XXVII 1 78 S M F+ M F++ S 2 XX 1 79 F+ M- S M M S+ 3 VI 1 80 S M- S M M S+ 2 XII 1 81 S S F S S2 Si 1 VIII 1 82 S S F+ + M S2 Si 1 IX 1 83 S S F F S2 M 1 I 1 84 S M F+ M S2 M 2 VII 1 87 M M M S F F N XXVII 1 88 S S F M F M 3 VI 1

a ETs 1 through 12 were from the type culture collection; ETs 13 through 49 were from Goteborg; and ETs 50 through 88 were from Birmingham. b Grades: fast (F+, F+, F), medium (M, M-), and slow (S+, S, Si, S2, S3).

8,670 x g (J2-21 rotor JA 10; Beckman Instruments) for 30 100,000 x g for 3 h at 5°C, and the pellet obtained was min at 5°C. The pellet was suspended in 20 ml of a buffer suspended in 1 ml of sterile distilled water and centrifuged containing 0.02 M EDTA, 0.05 M sodium phosphate, and twice at 551 x g for 10 min at 5°C. The supernatant 0.15 M NaCl (pH 7.4), incubated for 1 h at 60°C, passed once containing the OMPs was divided into portions and kept through a 19-gauge needle, mixed (Omni-Mixer; Sorvall frozen at -20°C until used for sodium dodecyl sulfate Corp., Norwalk, Conn.) with cooling for 2 min, and centri- (SDS)-polyacrylamide gel electrophoresis. fuged twice at 13,800 x g (J2-21 rotor JA 17; Beckman) for SDS-polyacrylamide gel electrophoresis. SDS-polyacryl- 20 min at 5°C. The supernatant was then ultracentrifuged at amide slab gel electrophoresis was performed by the VOL. 53, 1986 CLONALITY IN H. INFLUENZAE 83 daltons) was used, and the gels were stained with 0.25% A m0*w Coomassie brillant blue. For reading, the gels were destained and dried and then analyzed. Strains appearing to express the same OMPs were run in parallel on the same gel 9.9g iff and were assigned the same OMP profile only if the patterns were indistinguishable. A total of 27 OMP profiles were -M ,. identified. An example of each profile is shown in Fig. 1. Statistical analysis. A diversity measure, H, was calculated for the biotypes and OMP profiles and for each of the six i enzymes: H = 1 - X X.2, where is the relative frequency of a - -tl xi the ith biotype, OMP, or allozyme. H is an estimate of the probability of two randomly chosen isolates having a dif- ferent biotype, OMP, or allozyme for a given enzyme. In addition to this measure of genetic variability, the number of electromorph differences between all pairs of isolates and its mean, D, and' variance, V, was calculated for subsets of these data (6). Since six enzymes were examined, D can range from 0 (identical) to 6 (no identity). Statistical infer- ence for the pairwise difference analysis was by a bootstrap- 10.:2 4. Su. £3 8s 9 iof 12 V 14 ping procedure. The distributions of D and V were calculated for random samples of isolates of different sizes. These random populations were generated by a Monte Carlo rou- tine sampling, without replacement, on the total collection of 135 isolates. The distributions thus generated provide esti- 92.5*w fa mates of the probability of obtaining the observed differ- ences among strains by chance alone, i.e., if the criteria 3nOv V. being considered had no effect. The assignment of ETs and the statistical analysis of these 45.0-ma Om data were accomplished through the use of the CLAN program developed by Neal Bogdanovich of the University of Massachusetts, Amherst. More information about this clone analysis program can be obtained from B. R. Levin.

RESULTS .0- The H. influenzae strains are listed in Table 2 by their ET, capsular type, biotype, and OMP profile. In this collection of 14*40D 135 strains, there were 88 distinct ETs, five capsular types, and 27 OMP profiles. The more common biotypes were 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0- shared among the type culture strains and the Goteborg and FIG. 1. OMP profiles of H. influenzae. (A) Lanes 1 through 14 Birmingham isolates. There was no overlap among the ETs represent OMP profiles I through XIV; (B) lanes 1 through 13 in these different subsets of the collection. represent OMP profiles XXVII through XV. Lane 0 contains the Biotype and OMP diversity. The relative frequencies of low-molecular weight standards lysozyme (14,400), carbonic e s b anhydrase (31,000), ovalbumin (45,000), and phosphorylase'phosphorylase B each specific biotype and OMP pattern and the estimated diversity of these typing characters are presented in Table 3. For both biotype and OMP, the NT strains were more diverse than the type b isolates. Collectively, bacteria ex- method of Laemmli (14, 15) in a discontinuous system. pressing the b capsular antigen(s) were more frequently Samples were diluted 1:4 in 0.0625 M Tris hydrochloride biotype 1-OMP I than strains not expressing these antigens. buffer (pH 6.8) containing 2% SDS, 10% glycerol, 5% This preponderance of biotype 1-OMP I obtained for both 2-mercaptoethanol, and 0.001% bromophenol blue and the Goteborg and Birmingham type b strains. On the other heated at 95°C for 10 min. The stacking gel contained 4% hand, for the NT isolates, the distribution of biotypes acrylamide, 0.2% diallyl tartar diamide, and 0.1% SDS in between Goteborg and Birmingham was quite different. 0.125 M Tris hydrochloride buffer (pH 6.8). The separation Biotype 2 was dominant among the Goteborg NT isolates, gel contained 13% acrylamide, 0.8% diallyl tartar diamide, with biotypes 3 and 1 being second and third, respectively, in and 0.1% SDS in 0.375 M Tris hydrochloride buffer (pH 8.8). relative frequency. Among the Birmingham NT isolates, Ammonium persulfate and tetramethylenediamine were biotype 1 dominated, with biotype 2 second in relative used to polymerize the gels. Electrophoresis was performed frequency and biotype 3 as a distant third. on gels (14 cm wide, 0.75 mm thick, and 12 cm long) at 15 Electromorph diversity. The relative frequencies of each mA per gel in 0.025 M Tris hydrochloride buffer (pH 8.4) allozyme and the (genetic) diversity of each enzyme locus containing 0.192 M glycine and 0.1% SDS for 4 h. A are presented in Table 4 for the total collection and for the low-molecular-size standard (Bio-Rad Laboratories, Rich- major subsets. The failure to demonstrate common ETs in mond, Calif.) containing lysozyme (14,400 daltons), soybean the Birmingham and Goteborg collection (Table 2) and the trypsin inhibitor (21,500 daltons), carbonic anhydrase differences in the relative frequencies of biotypes among the (31,000 daltons), ovalbumin (45,000 daltons), bovine serum NT strains (Table 3) suggest that the Goteborg and Birming- albumin (66,200 daltons), and phosphorylase B (92,500 ham samples have been taken from different H. influenzae 84 PORRAS ET AL. INFECT. IMMUN.

TABLE 3. Biotype and OMP profile frequencies and genetic diversity (H) Frequency' Parameter NT only Type b only All strains All Goteborg Birmingham All Goteborg Birmingham Biotype 1 0.466 0.275 0.106 0.515 0.892 0.929 0.875 2 0.346 0.500 0.617 0.333 0.081 0.071 0.125 3 0.120 0.188 0.255 0.091 0.027 - 4 0.053 0.013 0.030 - 5 0.008 0.013 0.030 7 0.008 0.013 0.021 H 0.645 0.638 0.502 0.613 0.197 0.133 0.219 OMP profile I 0.478 0.206 0.206 0.781 0.870 0.556 II 0.045 0.094 0.087 0.111 III 0.030 0.062 0.222 V 0.045 0.083 0.083 VI 0.030 0.059 - 0.059 XI 0.030 0.059 0.059 -- XII 0.030 0.031 0.044 XXV 0.030 0.059 0.059 - All otherb (no.) 0.284 (19) 0.523 (18) 0.523 - H 0.759 0.925 0.925 0.376 0.234 0.617 a The number of strains/biotypes were as follows. For biotype determinations: all, 133/6; NT only: all, 80/6; Goteborg, 47/4; Birmingham, 33/5; type b only: all, 37/3; Goteborg, 28/2; Birmingham, 9/1. For OMP profile determinations: all, 67/27; NT all, 34/23; type b only: all, 32/5; Goteborg, 23/3; Birmingham, 9/5. b 19 OMPs appeared only once: IV, VII, VIII, IX, X, XIII, XIV, XV, XVI, XVII, XIX, XX, XXI, XXII, XXIII, XXIV, XVI, XVII, and XVIII. These contributed to the calculated diversity. populations. The results (Table 3) provided additional sup- type c. In fact, the four type f strains were of four different port for this interpretation. While for four of the six enzymes ETs, with a pairwise allozyme difference similar in magni- (MDH, PE2, 6PG, and PGI) the genetic diversity was similar tude to that of the uncapsulated strains. for the NT subsets of the Goteborg and Birmingham collec- The pairwise isozyme difference among isolates classified tions, this was not the case for two of the enzymes (AK and by each of the six biotypes was not significantly different G6P). Furthermore, among the NT strains from these two from that of randomly chosen strains. Isolates of the OMP I cities, there were substantial differences in the relative class were more similar to each other than randomly chosen frequency of many of the allozymes. Finally, for all six strains. There were too few isolates with the other OMP enzymes the genetic diversity and allozyme frequencies in profiles to estimate the magnitude of allozyme variation the type b samples were very different for the Goteborg among strains sharing these OMPs or to determine whether and Birmingham isolates. There were only three ETs and the allozyme differences among isolates sharing these OMP correspondingly little genetic diversity among the 28 profiles was less than that of randomly chosen strains. Goteborg type b strains. The nine Birmingham type b However, it was clear that there was some ET variation isolates were of seven different ETs and had substantial among isolates expressing the same OMP pattern. There genetic diversity. were at least two ETs among all OMP profiles that were Electromorph variation among strains classified by capsule represented more than once. type, biotype, and OMP. The number of ETs and average The electromorph variation among isolates classified by allozyme difference among pairs of isolates (D) and its combinations of two and three of these typing criteria is variance (VD) are presented in Table 5 for isolates within shown in Table 6. As would be expected when the associa- each capsular type, biotype, and OMP type. As suggested by tion between different typing characters is not absolute, the lower genetic diversity in the collection as a whole (Table there were fewer isolates in each multiply typed class than 4), there were significantly fewer allozyme differences there were in the corresponding classes of strains that were among isolates of the b capsular type than among strains typed for single characters (Table 5). chosen at random. However, this result could be primarily As would be anticipated, the two- and three-part combi- attributed to the Goteborg type b strains. The average nations of capsular type b, biotype 1, and OMP I (b:1:-, allozyme difference among pairs of Birmingham type b b:-:I, -:1:1, and b:1:I) were significantly less variable than strains, D = 3.306 (with a high of 6), was not significantly in randomly chosen strains. Save for OMP I, the pairwise different from that of a random sample of the same size. The differences among these multiply typed strains were less corresponding value for the Goteborg type b strains was D = than among those typed for single characters. Nevertheless, 0.143 (with a high of 2). It is of interest that the variance in in all these common classes of multiply typed isolates, there D for the type B isolates was significantly greater than that was substantial variation among ETs. On the average, the for randomly chosen strains. There were too few isolates of 253 pairs of b:1:I isolates differed by 1.364 allozymes and the other capsular types to obtain good estimates of the included some pairs that differed in all six. There were too magnitude of within-type diversity or to ascertain whether few other multiply typed strains to estimate the magnitude of there was significantly less variation among isolates with within-type variation or to determine whether the pairwise these capsular antigens. It was, however, apparent that there allozyme difference among them was less than that for was ET variation within all but one of these capsular types, randomly chosen strains. However, it is clear that there is VOL. 53, 1986 CLONALITY IN H. INFLUENZAE 85

TABLE 4. Electrophoretic variation: allozyme frequencies and genetic diversity Frequency in strains (no. of isolates/no. of ETs): Allozyme and ET NT only Type b only H values All (135/88) All Goteborg Birmingham All Goteborg Birmingham (81/67) (47/37) (34/30) (38/11)a (28/3) (9/7) MDH F 0.052 0.062 0.106 0.000 0.000 0.000 0.000 S 0.719 0.642 0.617 0.676 0.895 1.000 0.556 F++ 0.022 0.012 0.000 0.029 0.000 0.000 0.000 M 0.126 0.161 0.128 0.206 0.079 0.000 0.333 F+ 0.067 0.111 0.149 0.059 0.000 0.000 0.000 Si 0.007 0.000 0.000 0.000 0.026 0.000 0.111 N 0.007 0.012 0.000 0.029 0.000 0.000 0.000 H 0.460 0.546 0.569 0.495 0.193 0.000 0.568 PE2 M 0.289 0.284 0.149 0.471 0.210 0.036 0.667 S 0.518 0.420 0.447 0.382 0.790 0.964 0.333 M- 0.067 0.086 0.085 0.088 0.000 0.000 0.000 Si 0.067 0.111 0.192 0.000 0.000 0.000 0.000 S2 0.030 0.049 0.064 0.029 0.000 0.000 0.000 S3 0.022 0.037 0.043 0.029 0.000 0.000 0.000 N 0.007 0.012 0.021 0.000 0.000 0.000 0.000 H 0.637 0.719 0.728 0.622 0.332 0.069 0.444 6PG M 0.178 0.161 0.000 0.382 0.184 0.000 0.778 F+ 0.578 0.506 0.809 0.088 0.763 1.000 0.111 F 0.156 0.185 0.128 0.265 0.053 0.000 0.111 F++ 0.030 0.049 0.064 0.029 0.000 0.000 0.000 S 0.059 0.099 0.000 0.235 0.000 0.000 0.000 H 0.606 0.672 0.326 0.720 0.381 0.000 0.370 AK M 0.837 0.803 0.936 0.618 0.921 1.000 0.778 F 0.089 0.086 0.021 0.177 0.053 0.000 0.111 S 0.067 0.099 0.043 0.177 0.026 0.000 0.111 F+ 0.007 0.012 0.000 0.029 0.000 0.000 0.000 H 0.287 0.339 0.121 0.555 0.148 0.000 0.370 G6P F 0.156 0.148 0.106 0.206 0.079 0.000 0.222 F+ 0.044 0.062 0.021 0.118 0.000 0.000 0.000 S 0.185 0.222 0.234 0.206 0.026 0.000 0.111 M 0.481 0.358 0.447 0.235 0.895 1.000 0.667 N 0.007 0.000 0.000 0.000 0.000 0.000 0.000 Si 0.067 0.111 0.170 0.029 0.000 0.000 0.000 S2 0.052 0.086 0.021 0.176 0.000 0.000 0.000 F++ 0.007 0.012 0.000 0.029 0.000 0.000 0.000 H 0.700 0.777 0.704 0.813 0.193 0.000 0.494 PGI F 0.156 0.161 0.234 0.059 0.079 0.036 0.222 S1 0.215 0.284 0.383 0.147 0.026 0.000 0.111 S 0.348 0.210 0.128 0.323 0.763 0.964 0.222 F+ 0.007 0.000 0.000 0.000 0.000 0.000 0.000 S+ 0.022 0.025 0.000 0.059 0.000 0.000 0.000 M 0.244 0.309 0.255 0.382 0.132 0.000 0.444 S2 0.007 0.012 0.000 0.029 0.000 0.000 0.000 H 0.748 0.754 0.717 0.720 0.393 0.069 0.691 Mean H 0.573 0.635 0.532 0.645 0.273 0.023 0.490

a One type b strain was from a type culture collection and was not included with the Goteborg or Birmingham samples. some genetic variation among isolates expressing the same Capsule, biotype, and OMP pattern variation within ETs. combinations of capsule, biotype, and OMP profile. Except Save for the 28 ET14 strains, most of the ETs were repre- for the two isolates each of c:2:-, -:2:1, and b:2:I, there sented by one or at most five isolates (Table 2). Thus, it is were allozyme differences among the strains sharing two or difficult to make a general statement about the extent of more of the other typing combinations. capsule, biotype, and OMP variation within ETs. However, 86 PORRAS ET AL. INFECT. IMMUN.

TABLE 5. Electromorph variation among isolates of the same TABLE 7. Expected and observed frequency of ETsa capsule type, biotype, and OMP profile' Frequency Probability" No. of: ETb Capsule Expected Category D VD Dmax Dmin type Observed Total Local Isolates ETs Total Local Capsule type 1+ a 2 0.0074 0.000027 a 3 2 1.333 1.333 2.000 0.000 3+ c 2 0.030 0.00043 b 38 11 1.684* 3.367t 6.000 0.000 6+ e 2 0.028 0.00039 c 2 1 0.000 0.000 0.000 7 e 2 0.70 - 0.16 d 2 2 1.000 1.000 1.000 13+ NT 2 1.9 3.2 0.57 0.84 e 5 3 2.800 2.400 4.000 0.000 14 b/NT 28 4.0 8.9 6.0 x 10-14 1.2 x 10-8 f 4 4 3.167 1.767 5.000 2.000 15 b/NT 2 2.3 1.5 0.67 0.47 NT 81 70 3.853 1.479 6.000 0.000 24+ NT 2 2.8 3.2 0.78 0.84 27+ NT 2 0.014 0.012 0.000099 0.000072 Biotype 28+ NT 2 0.014 0.018 0.000099 0.00016 1 62 33 3.243 2.989t 6.000 0.000 35+ NT 4 0.23 0.60 0.000093 0.0031 2 46 39 3.586 1.839 6.000 0.000 38+ NT 2 0.23 0.27 0.023 0.030 3 16 13 2.925 1.398 6.000 0.000 43+ NT 2 0.10 0.090 0.0049 0.0037 4 7 6 2.952 1.548 4.000 0.000 52 b/NT 5 0.48 0.60 0.00014 0.00029 NT 2 2 5.000 5.000 5.000 74+ b/NT 2 0.086 0.21 0.0035 0.018 OMP profile a The expected number of ETs is calculated as the product of the number of 68 49 3.326 1.763 6.000 0.000 isolates, N, and the anticipated frequency of each ET, assuming that the I 32 11 2.248* 3.666t 6.000 0.000 allozymes are randomly distributed, i.e., the product of the allozyme frequen- 2 2.000 3.000 3.000 0.000 cies for the different enzymes. The total number of isolates (135) and the II 3 allozyme frequencies in that total were used for "Total." The number of XII 2 2 3.000 3.000 3.000 isolates and allozyme frequencies used for "Local" were those for the XI 2 2 6.000 6.000 6.000 Goteborg and Birmingham subsets. The Goteborg ETs were 13 through 49 (75 V 3 3 3.000 0.000 3.000 3.000 isolates) and the Birmingham ETs were 50 through 88 (44 isolates). ETs 1 XXV 2 2 3.000 3.000 3.000 through 12 came from the type culture collection. III 2 2 5.000 5.000 5.000 b +, ETs that had the same biotype. VI 2 2 5.000 5.000 5.000 The probability of obtaining at least the observed number of each ET was calculated from the binomial formula: *, 0.002); t, significantly higher than that anticipated by chance (P < 0.05). Nobs-1 N!

x = oX! (N-x)! where N is the total number of isolates, Nob. is the observed number of the particular ET, and p is the anticipated frequency of that ET. This was calculated separately for the total and local populations. TABLE 6. Electromorph variation among isolates classified by two and three categoriesa No. of: Categories D VD Dmax Dmin it is possible to conclude that there is some variation in these Isolates ETs characters among strains of the same six-enzyme ET. This Capsule:biotype can be seen from Table 2. Some isolates with multiply a:1 3 2 1.333 1.333 2.000 0.000 occurring ETs were capsulated, while others are not. Five b:1 34 9 1.563* 3,382t 6.000 0.000 ETs had representatives among the type b and NT isolates b:2 3 2 2.000 3.000 3.000 0.000 (13-15, 52, 74). Even capsulated strains with the same ET c:2 2 1 0.000 - 0.000 0.000 had different OMP profiles and biotypes. The type b strains d:4 2 2 1.000 - 1.000 1.000 of ET14 include some that expressed OMP I, II, V, and XII e:4 4 3 2.833 2.167 4.000 0.000 and some that were of biotype 2 as well as 1. f:1 3 3 2.000 0.000 2.000 2.000 Capsule:OMP DISCUSSION b:I 25 6 1.270* 2.960 6.000 0.000 b:II 3 2 2.000 3.000 3.000 0.000 The results of the present investigation extend the obser-

b:III 2 2 5.000 - 5.000 5.000 vations presented in Porras et al. (29). Bacteria identified as H. influenzae are a genetically extremely variable array of Biotype:OMP organisms. Considering just the enzyme electrophoresis 1:1 30 11 2.352* 3.593t 6.000 0.000 results, the typing method most likely to reflect simple allelic 1:11 3 2 2.000 3.000 3.000 0.000 differences in structural genes (5-7, 22, 30); in this collection 1:V 3 3 3.000 0.000 3.000 3.000 of 135 isolates, there were a minimum of 88 genotypes (the 2:1 2 1 0.000 - 0.000 0.000 ETs) and a mean genetic diversity of 0.573. For the same six 2:XI 2 2 6.000 - 6.000 6.000 enzymes examined in this study, the corresponding esti- 3:VI 2 2 5.000 - 5.000 5.000 mates of genetic diversity in two collections of E. coli from Capsule:biotype:OMP natural populations were 0.471 (30) and 0.462 (7). One E. coli b:1:I 23 6 1.364* 3.058t 6.000 0.000 collection (30) included isolates from humans and other b:1:II 3 2 2.000 3.000 3.000 0.000 mammals, and although it was dominated by strains from the b:2:I 2 1 0.000 - 0.000 0.000 United States, isolates from other countries were also in- a Symbols: *, significantly lower than that anticipated by chance (P < cluded. Thus, it seems reasonable to conclude that genetic 0.002); t, significantly greater than that anticipated by chance (P < 0.05). variability in H. influenzae is at least as great and probably VOL. 53, 1986 CLONALITY IN H. INFLUENZAE 87 greater than that in E. coli, a conjecture raised in a recent TABLE 8. Linkage disequilibrium analysisa paper by Musser et al. (22). df or Q It is clear that the genes determining these ETs are not Organism (no. of ETs) Y MDH PE2 6PG AK G6P PGI randomly distributed among isolates but that there is some underlying structure to the population. This can be readily H. influenzae MDH 30 24 18 42 32 seen from the observed and expected frequencies of the ETs (88) PE2 30.3 20 15 35 30 that appeared more than once (Table 7). If these allozymes 6PG 37.1 30.0 12 28 24 were randomly distributed, the majority of these multiply AK 16.9 24.6 21.1* - 21 18 G6P 34.5 28.8 - appearing ETs would not be anticipated to occur in more 30.1 33.2 42 PGI 27.0 32.2 39.8 20.4 45.1 than a single isolate. While the most common single ET, - ET14, was the one expected to occur most frequently, its E. coli MDH 5 9 3 7 5 anticipated frequency (4.0 and 8.9 isolates for the total and (62)b PE2 14.7** 45 15 35 25 local estimates, respectively) was far lower than the 28 6PG 4.0 30.7 27 63 45 isolates found. Indeed, the likelihood of ET14 being repre- AK 1.3 28.5** 26.0 - 21 15 sented in as many as 28 isolates by chance alone is, at best, G6P 0.9 16.4 75.4 6.7 - 35 1.2 x 10-8. In general, the expected and observed frequen- PGI 4.4 58.7*** 20.6 16.6 20.6 - cies of the multiply appearing ETs were closer when the a The integers (upper triangles) are degrees of freedom; the real numbers estimates were calculated from allozyme frequencies esti- (lower triangles) are the Q values (10, 37): mated from the local population. However, this was not m n D2 always the case, and large deviations between the number Q=NE E u expected and that observed still occurred (Table 7). The i j Pi4i biotype data provide additional evidence for a nonrandom where Dij is the observed relative frequency of the individuals with the ij distribution of genes. The members of 11 of the 15 groups of allozyme combination minus the expected relative frequency, piq,. When the ETs had the same disequilibrium is zero, Q is approximately chi-sqaure distributed with (m - 1) multiply appearing biotype. (n - 1) degrees offreedom, where n and m are the number of allozymes for the The nonrandom structure of this bacterial population can two enzymes being compared. Symbols: *, 0.025 < P < 0.05; **, 0.01 < P < be attributed, in part, to the fact that these strains were 0.025; ***, 0.005 < P < 0.01. selected by capsule type. Of the 28 isolates of ET14, 26 were bFrom Selander and Levin (30). capsule type b. This clearly indicates that the gene(s) coding for the b capsule is not randomly distributed among H. influenzae genotypes. While these data suggest that the other there were 62 ETs in the collection of E. coli strains from capsular types are not randomly distributed among geno- humans and animals examined by Selander and Levin (30). types (Table 7), they are not sufficient to provide estimates Applying this disequilibrium analysis to these data, there of the amount of genetic variation within these other were three highly significant disequilibria among the 15 serotypes. comparisons (Table 8). Nonrandom genetic structure is not only an attribute of Second, the failure to observe identical ETs in the capsulated populations of H. influenzae. Of the seven unique Goteborg and Birmingham samples also supports the hypo- NT representatives of the multiply occurring ETs (Table 7), thesis of a shorter clone persistence time for H. influenzae five had a probability of less than 0.03 of occurring as than for E. coli. For the 72 strains in the standard reference frequently as they did, and for four of these five this collection of E. coli (23) and the six enzymes considered probability was less than 0.004 (based on local estimates) here, there were 40 ETs. Included among these are six ETs (Table 7). Furthermore, the members of all seven unique NT that were obtained in both Swedish and U.S. samples, one of groups had the same biotype. which was also obtained in a sample from Tonga (South We interpret the observations that specific ETs appear Pacific). more frequently than anticipated by chance alone and that Finally, the somewhat higher genetic diversity in the H. association of the capsule antigens among ETs is nonrandom influenzae population can also be interpreted as consistent as evidence that the genetic structure of H. influenzae with the hypothesis of a less stable clonal structure for this populations is that of an array of lineages (clones) that bacterial species relative to E. coli. The genetically effective maintain their genetic identity for extended periods of time, size of asexually reproducing populations is directly propor- i.e., the clone concept (25, 26). This hypothesis has been tional to their rate of recombination (16). If allozyme varia- presented by Musser et al. (22) for type b H. influenzae tion is due to recurrent mutation and random genetic drift of strains. We see the present results as extending this conclu- selectively neutral alleles (13), the standing genetic variation sion of a clonal structure to uncapsulated populations of H. would be directly proportional to effective population size. influenzae, the NT strains as well as capsulated types. At this point we present these observations and arguments A clonal population structure would be anticipated for any for lower stability of H. influenzae clones than of E. coli organism that reproduces asexually. However, the term of clones to raise this issue rather than because we consider maintenance of specific multilocus gene combinations, the them definitive. On the other hand, we find them sufficiently clone persistence time, may vary among species and even compelling to postulate that on the average, the rate of among clones of the same species. Three lines of evidence chromosomal gene recombination in natural populations of suggest that collectively the persistence time of H. influ- H. influenzae is greater than that in natural populations of E. enzae clones is less than that of E. coli clones. First, the coli. distribution of allozyme pairs appears to be closer to random One of the most striking observations in this data set is the for H. influenzae than it is for E. coli. Applying the multiple difference between the Goteborg and Birmingham type b allele linkage disequilibrium analysis (10, 37) for the 88 isolates. While collectively there was less ET variation distinct H. influenzae ETs, there was one barely significant among isolates of this capsular type than in the collection at (0.04 < P < 0.05) pairwise disequilibrium among the 15 large, the Birmingham type b isolates were much more comparisons (Table 8). For the six enzymes considered, variable than those from Goteborg. The nine Birmingham 88 PORRAS ET AL. INFECT. IMMUN. type b isolates examined were of seven different ETs. On the their genetic identity for extended periods. In particular, it is average, pairs of these ETs differed by 3.4 allozymes, with essential that the genes determining the characters used for one set of pairs differing by all six. Of the 28 Goteborg type typing maintain a relatively stable association with those b isolates, 26 were of the same ET (ET14), and the remaining coding for the characters that motivate the epidemiological two differed from ET14 by only one allozyme. study, such as virulence. For this reason, it is important to The reason for the greater genetic diversity in the Birming- ask whether the clone persistence time of H. influenzae is ham type b strains relative to those from Goteborg is sufficiently long to justify extensive typing programs to something of a puzzle. From the way these samples were identify and monitor particular pathogenic strains. collected, one would have anticipated greater variability in The present results provide an equivocal answer to this the Swedish collection. The Goteborg type b bacteria were question. The limited diversity of the ET14 Goteborg type b isolated over the course of approximately 10 years from strains certainly suggests that clones of this bacterial species individuals of different ages with different types of clinical could maintain their genetic identity for sufficient time to be infections. The Birmingham type b isolates were obtained recognized in independent samples taken over an extended during the course of a single year from young persons (1.7 to period. The results of Musser et al. (22) provide even more 11.7 years old) with ear infections. While one may argue that compelling evidence for the long-term clonal stability of the greater variability of the Birmingham type b strains is an some type b strains of H. influenzae. In their collection, artifact of the small sample size considered here, this doesn't isolates with the same 16-enzyme ET, same OMP profile, seem likely, as there seems to be considerable genetic and same biotype were obtained in Australia, Holland, and variability in the U.S. type b H. influenzae populations. For various places in the United States. On the other hand, it the same six enzymes considered here, Musser et al. (22) remains possible that not all H. influenzae clones have the estimate a mean genetic diversity of 0.423 in their study of same persistence time. The present results with the NT 177 type b H. influenzae isolates (of which 173 were from the strains and the Birmingham type b isolates were consistent United States). The mean genetic diversity for the nine with that proposition. It is certainly possible that the fre- Birmingham type b isolates considered here was 0.490 quency of transformational competence varies among H. (Table 3). One could speculate that the difference in variabil- influenzae clones. Indeed, it may well be that the rate of ity in the Goteborg and Birmingham type b isolates is a recombination in noncapsulated H. influenzae strains is consequence of the Swedish host population being more greater than that in capsulated strains. homogeneous (racially, economically, etc.), but it seems While it was not the primary goal, the present investiga- prudent to postpone speculation until more populations have tion does provide information about the relative merits of been studied. four different methods for typing H. influenzae: multilocus The observation that the same ETs were represented in enzyme electrophoresis, capsule antigens, biochemical char- capsulated and uncapsulated forms is not surprising. It acteristics (biotyping), and OMP profile. It is clear from the seems reasonable to assume that mutation could lead to the present results that none of these procedures by itself would loss of function of genes coding for capsular antigens. These allow unambiguous identification of clones. There was vari- uncapsulated forms could increase in frequency by chance ation among strains identified by each of these procedures. alone or possibly be favored by selection in particular Individually, capsule type, biotype, and OMP profile are environments. More interesting is the observation that ge- not very discriminating. A number of genetically very dif- netically very different H. influenzae strains express the ferent genotypes may express the same capsule, biotype, same capsular antigens. Among the pairs of type b isolates, and OMP. Even collectively, they seem to provide much less there were some that differed by five and even six allozymes. discriminatory power than the three-antigen serotypes of E. Among the four type f ETs there were pairs that differed by coli (6). Multilocus enzyme electrophoresis was the most five allozymes. discriminating of the methods. Nevertheless, variation in We see two plausible (and testable) hypotheses that can biotype, OMP, and capsules can be anticipated within iso- account for the expression of the same capsule antigens (or lates of the same ET. This was the case even when the other polymorphic characters) by genetically different lin- discriminating power of the procedure was augmented by eages: convergent evolution and infectious transmission of using more enzymes than the six employed here. In their capsule-determining genes. If different lineages separately study, Musser et al. (22) used 16 enzymes and still picked up evolved the capacity to produce capsules that are immuno- biotype and OMP variations within ETs of type b. logically indistinguishable, then it is likely (if not absolutely The limited discriminatory power of specific typing meth- necessary) that the genes coding for these capsules and even ods does not mean they have no utility. For clinical appli- the molecular structure of these antigens are different. In the cations, bacterial typing is not a quest for an arbitrary strain, case of infectious transmission, one would anticipate greater but an attempt to ascertain whether a particular, already homology among the capsule-determining genes than the identified, bacterial strain occurs in more than one sample; rest of the for different genotypes that express the the concern is whether a particular clone is responsible for same antigen. One would also expect to be able to infec- continued or recurrent infections in single patients or for an tiously transfer the capsule under realistic experimental outbreak. Thus, for general applications, any reliable typing conditions (mammalian hosts). The classical pneumococcus method or combination of methods could be useful, even if transformation experiments of Griffith (9) certainly provide a they have only fair discriminating power. Greater efficiency precedent for the infectious transfer of capsule-determining would be obtained if the method used for typing were genes in bacteria as well as evidence for those genes being directed at the characters that motivated the study, e.g., favored by selection when they are associated with viru- virulence determinants. lence. The clone persistence time of bacteria is a subject of some ACKNOWLEDGMENTS interest from the clinical as well as the academic perspec- This study was supported by grants from the Swedish Medical tive. If typing is to be of use in epidemiological studies, the Research Council (no. 215), the Medical Faculty, University of bacteria identified by the typing procedures have to maintain Goteborg, the Ellen, Walter and Lennart Hesselmann Foundation VOL. 53, 1986 CLONALITY IN H. INFLUENZAE 89 for Scientific Research, the Swedish Board for Technical Develop- Lancet ii:1312-1314. ment, SAREC, and the U.S. Public Health Service (grant GM-33728 19. Maruyama, T., and M. Kimura. 1980. Genetic variability and to B.R.L.) from the National Institutes of Health. Funds for effective population size when local extinctions and recoloniza- computer time were provided by the University of Massachusetts tions of subpopulations are frequent. Proc. Natl. Acad. Sci. Computing Center. O.P. is the recipient of a training grant from the USA 77:1024-1026. Costa Rica Social Security System and the National Council for 20. Moxon, E. R., R. A. Deich, and C. Connelly. 1984. Cloning of Scientific and Technological Research. chromosomal DNA from Haemophilus influenzae. Its use for The excellent technical assistance of Ingela Delgado Annelie studying the expression of type b capsule and virulence. J. Clin. Lundell and Carol Laursen and the typing by Britta Andersson and Invest. 731:298-306. Ann-Charlotte Malmefeldt are greatly appreciated. A special debt of 21. Murphy, T. F., K. C. Dudas, J. M. Mylotte, and M. A. 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