Vol. 29, No. 2

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1991, p. 291-296

0095-1137/91/020291-06$02.00/0 Copyright © 1991, American Society for Microbiology

Characterization of Noncapsulate Haemophilus influenzae by Whole-Cell Polypeptide Profiles, Restriction Endonuclease Analysis, and rRNA Gene Restriction Patterns KENNETH D. BRUCE* AND J. ZOE JORDENSt Department of Medical Microbiology, University ofAberdeen Medical School, Foresterhill, Aberdeen AB9 2ZD, United Kingdom Received 16 August 1990/Accepted 20 November 1990

Thirty-four clinical isolates of noncapsulate Haemophilus influenzae representing isolates with either related or dissimilar patterns of whole-cell polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were further characterized by restriction enzyme analysis (REA) and rRNA gene restriction patterns. Total cellular DNA was extracted by a rapid, microcentrifuge-scale method and digested with BamHI, which gave a pattern of about 18 discrete bands. This confirmed the five closely related groupings suggested by SDS-PAGE. Isolates dissimilar by SDS-PAGE were also distinguishable by REA. However, there was no correlation between the degrees of similarity estimated from whole-cell polypeptide profiles and those obtained from REA for the dissimilar isolates. Therefore, inferences of genetic relatedness made on the basis of these data should be interpreted with caution. rRNA gene restriction patterns also confirmed the groupings suggested by the other two techniques. We conclude that the three methods were highly discriminatory and that whole-cell polypeptide patterns or REA with BamHI would be appropriate techniques for epidemiological studies of noncapsulate H. influenzae.

Haemophilus influenzae, although a common commensal of the upper respiratory tract of healthy individuals, is an important human pathogen (28). The species is divided, on the basis of the production of a polysaccharide capsule, into six capsular types and those strains which are noncapsulate. These noncapsulate H. influenzae (NCHi) have been implicated as the cause of diseases such as otitis media, sinusitis, and conjunctivitis in children (28, 29) and can also cause meningitis, pneumonia, and septicemia (4, 25, 27). In addition, NCHi can be responsible for infections in patients with either cystic fibrosis or chronic bronchitis (5, 15). Little is known about the epidemiological behavior of NCHi. It is therefore important to be able to characterize these isolates to aid in such studies. To characterize isolates, a technique is required which ideally should be rapid, reproducible, technically simple, easily interpretable, and highly discriminatory. A variety of techniques have been applied to the characterization of H. influenzae, including biotyping (11), serotyping (16), sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of both outer membrane proteins (17) and whole-cell polypeptides (WCPs) (21), multilocus enzyme electrophoresis (24), and analysis of lipooligosaccharides (8). Profiles of outer membrane proteins and WCPs in particular have been used successfully to characterize clinical isolates of NCHi (1, 21, 26). However, both of these techniques analyze the phenotype rather than the genotype in that they rely on the particular expression of protein. It is therefore preferable to analyze the genome directly, because this type of analysis is not subject to such phenotypic variation. The bacterial genome may be studied either directly, by the staining of fragments resulting from digestion with a restriction endonuclease (direct restriction endonuclease

analysis [REA]), or indirectly, after hybridization with a probe such as rRNA (rRNA probe-REA). Direct REA has been useful in epidemiological studies of other clinically important species (20), and recently rRNA probe-REA was shown to discriminate between isolates of H. influenzae biogroup aegyptius (9). H. influenzae type b consists of subpopulations of organisms which are virtually or absolutely genetically identical, i.e., clonal (18). Previous studies within our laboratory have used WCP profiles to define clones of type b H. influenzae (22). The aim of the present study was to assess the discriminatory value of DNA-based typing techniques (direct REA and rRNA probe-REA) compared with WCP profiles for related (clonal) and dissimilar isolates of NCHi. MATERIALS AND METHODS Isolates. Isolates of NCHi were cultured from clinical specimens and identified by their requirement for X and V factors and by their lack of reaction with commercial antisera against capsular polysaccharides (Wellcome Reagents Ltd., Beckenham, United Kingdom) in the diagnostic bacteriology laboratories at Aberdeen Royal Infirmary or The City Hospital, Aberdeen, Scotland, during the period 1984 to

1989. Isolates were stored as freeze-dried cultures and subcultured on chocolate agar immediately prior to use. A total of 250 isolates had previously been characterized on the basis of their WCP profiles, as determined by SDS-PAGE using methods described elsewhere (22). From this collection, 17 isolates representing dissimilar WCP profiles and 17 representing five groups each containing 2 to 5 isolates with very similar WCP profiles were chosen for chromosomal DNA analyses. The sources of these 34 isolates are given in Table 1. DNA extraction. Total cellular DNA was extracted by a modification of the method described by Pitcher et al. (23). Briefly, bacterial colonies from an overnight culture on a

Corresponding author. t Present address: Public Health Laboratory, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom. *

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TABLE 1. Source of isolates used in the study Type of WCP profile and isolate

Dissimilar a b c d e f g h i j k I m n o p q

Similar ac bc c d e f g h i j k l m n o p q

Date isolated

Source

(hospital/ward)'

Oct. 1987 July 1987 May 1986 X 1987b Sept. 1988 Oct. 1987 X 1984 Nov. 1988 March 1987 Feb. 1986 Nov. 1986 Dec. 1986 Nov. 1986 Nov. 1986 X 1984 July 1989 March 1986

ARI/47 ARI/47 ARI/02

June 1987 June 1987 Feb. 1985 Nov. 1986 Nov. 1988 Nov. 1985 May 1987 Dec. 1988 Nov. 1988 Oct. 1988 Oct. 1988 Oct. 1988 Feb. 1986 March 1987 Apr. 1987 Feb. 1989 Nov. 1988

ARI/33 ARI/33 ARI/25 ARI/14 ARI/25 RAC/03 RAC/CV CIT/02 ARI/26 ARI/20 CIT/X ARI/20

GLA/Xb CIT/02 ARI/25 ARI/A&E ARI/07 ARI/A&E ARI/37 ARI/37 RAC/04 ARI/46 ARI/32 AMH/FF RAC/03 ARI/03

ARI/50 RAC/04 RAC/06 ARI/08 RAC/06

Specimen

Clone

Sputum Sputum Eye swab Sputum Sputum Sputum Finger swab Sputum Sputum Tracheal aspirate Sputum Nasal swab Sputum Sputum

Sputum Tracheal aspirate Sputum

Sputum Sputum Sputum Sputum Sputum Eye swab Nasal catheter tip Sputum Sputum Sputum Sputum Sputum Sputum Nasal catheter tip Nasal catheter tip Sputum Nasal catheter tip

1 1 2 2 3 3 3 3 3 4 4 4 4 5 5 5 5

AMH, Aberdeen Maternity Hospital; ARI, Aberdeen Royal Infirmary; RAC, Royal Aberdeen Childrens Hospital; CIT, City Hospital, Aberdeen, Scotland; GLA, Glasgow Royal Infirmary; CV, cardiovascular ward; FF, first floor unit; A&E, accident and emergency. b X, Information not available. c Both isolates were from the same individual.

chocolate agar plate incubated at 37°C in an atmosphere containing 5% CO2 were harvested with a dry swab into 0.15 ml of 10 mM Tris (pH 8.0)-i mM EDTA (pH 8.0) (TE buffer). Bacterial cells were lysed by the addition of 0.45 ml of a solution containing 5 M guanidium thiocyanate, 0.1 M EDTA, and 0.5% (vol/vol) Sarkosyl. After lysis (approximately 2 min), samples were placed on ice and 0.25 ml of cold (+4°C) 7.5 M ammonium acetate was added. The tubes were mixed by inversion and kept on ice for 10 min. Chloroform-isoamyl alcohol (24:1) (0.5 ml) was then added, and the samples were mixed thoroughly for 5 min. The aqueous and organic phases were then separated by centrifugation at 12,000 x g for 20 min. A measured volume (usually 0.6 ml) of the upper, aqueous layer was transferred to a fresh tube, and 0.54 volume of cold (-20°C) isopropanol was added. The tubes were inverted gently for 1 min and then placed at -200C for 30 min. The DNA pellet was recovered by centrifugation at 6,000 x g for 2.5 min, dissolved in 0.1 ml of TE buffer, and then reprecipitated by

the addition of 2.5 volumes of cold (-20°C) absolute ethanol followed by 30 min of storage at -20°C and centrifugation as described above. The resulting pellet was dissolved in sufficient TE buffer to saturate the DNA (0.2 to 0.3 ml). The integrity of the DNA was assessed by horizontal gel electrophoresis on a 0.8% agarose gel in 89 mM Tris-89 mM borate-2 mM EDTA (TBE buffer). Yield was quantitated by measuring the A260Restriction enzyme digestion. Five different restriction endonucleases with 6-base recognition sequences (EcoRI, BamHI, BglII, Sall, and HindlIl) were tested under the conditions recommended by the manufacturer (BoehringerMannheim, Lewes, East Sussex, U.K.) for their abilities to digest the DNA preparations. The total reaction volume of the digests was 10 [l, contained 4 to 5 ,ug of DNA, and was incubated for 16 to 18 h at 37°C. The resulting digests were analyzed by gel electrophoresis on a 0.9% agarose gel using TBE buffer. After being stained with ethidium bromide, gels were photographed under UV transillumination with a Polaroid camera. A 1-kb ladder (GIBCO-Bethesda Research Laboratories) was used as a size marker. For Southern blotting and probing with rRNA, Serratia fonticola 3965 digested with HindIII was included as a size marker (kindly provided by F. Grimont, Institut Pasteur, Paris, France). Probe. 16S plus 23S rRNA from Escherichia coli (Boehringer) (10 ,ug) was end labeled with 50 ,uCi of [-y-32P]ATP (Amersham International, U.K.) by using 10 U of T4 polynucleotide kinase in 50 mM Tris hydrochloride (pH 7.6)-10 mM MgCl2-5 mM dithiothreitol-0.1 mM spermidine-0.1 mM EDTA. After 60 min of incubation at 37°C, the reaction was terminated by addition of 10 ,ul of a solution containing 0.2% SDS and 20 mM EDTA, and the probe was used without further purification. Southern blotting and DNA hybridization. RE digests of chromosomal DNA, separated on agarose gels, were depurinated, denatured, and transferred to nylon membranes (Hybond-N; Amersham International) by the method of Southern as recommended by Maniatis et al. (14). The Southern blots were prehybridized for 3 h at 65°C in 2 x SSC (lx SSC is 0.15 M NaCl and 0.015 M sodium citrate)-5x FPG (lx FPG is 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone 350, and 0.02% glycine)-0.5% SDS-100 ,ug of denatured salmon sperm DNA per ml. The probe was added to this solution, and the hybridization mix was incubated at 65°C overnight. Hybridized filters were then washed as previously described (6), dried, and exposed to X-ray film with an intensifying screen overnight or, as required, at -700C. Analysis.The percent similarity of banding patterns obtained by direct REA or SDS-PAGE of WCPs was estimated by the method of Dice (3): % similarity = (number of shared bands/total number of bands) x 100. For Dice analysis of WCPs, all bands present in each track of the gel were used. On average, 40 polypeptide bands were resolved by this technique. Dice analysis of restriction endonuclease digests of chromosomal DNA was performed on fragments in the size range of 1.6 to 8 kb. BamHI gave 15 to 21 bands in this size range (mean, 18 bands).

RESULTS DNA extraction and restriction. The extraction technique yielded about 0.3 mg of high-molecular-weight DNA from the bacterial growth on a single agar plate in 2 h. This DNA was suitable for digestion with restriction endonucleases. For direct REA, enzymes were selected on the basis of their

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abilities to restrict the DNA into a discernible number of fragments (about 20) which could be resolved in the minimum period of time on an agarose gel. For probing with rRNA, enzymes were chosen on the basis of their ability to restrict most DNA. Direct REA. Initial studies on 6 NCHi isolates showed that SalT did not digest any of the six DNA samples and that HindlIl digested only three of the six into fragments of less then 12-kb. BglII and EcoRI both gave large numbers of fragments. BamHI gave the most suitable pattern for direct REA, as it produced a discernible number of discrete bands (between 15 and 21, with an average of 18) in the 1.6- to 8-kb range; therefore, these patterns were used for Dice analysis. Isolates with highly similar WCP profiles, which were therefore regarded as clones (Dice coefficients of similarity of 92 to 100%), gave identical groupings with direct REA (Dice coefficients of similarity all 100%) (Fig. 1B and 2B). The banding patterns of isolates with dissimilar profiles of WCPs were all distinguishable by REA (Fig. 1A and 2A). However, Dice coefficients of similarity for these direct REA patterns were more variable (56 to 86% similarity;

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variance, 41) than the corresponding values from WCP profiles (75 to 90% similarity; variance, 9) for these dissimilar isolates. There was no correlation between Dice coefficients obtained with WCP profiles and those obtained from direct REA. This is illustrated by the scatter diagram of WCP and REA results for all pairwise combinations of dissimilar isolates (Fig. 3). Dendrograms constructed from these Dice coefficients to illustrate relationships between isolates (13) suggested very different phylogenies (Fig. 4). rRNA probe-REA. Banding patterns resulting from the hybridization of EcoRI digests with the rRNA probe are shown in Fig. 5. This technique confirmed both the clonal groupings and the differences found with WCPs and direct REA with one exception: isolates e and i were indistinguishable by this technique (Fig. SA). DISCUSSION Infections caused by NCHi are often considered to be of endogenous origin, but spread of a multiresistant NCHi strain within a pulmonary rehabilitation center has been B M

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recently reported (26). In order to monitor and control such spread, a highly discriminatory method of bacterial characterization is needed. Established methods have various disadvantages which preclude them from being suitable for epidemiological studies. Biotyping, though one of the original typing systems, provides insufficient discrimination for epidemiological studies. Multilocus enzyme electrophoresis can be used to differentiate between isolates, but the cost and relative labor intensity involved probably limit this technique to applications such as the investigation of the

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population structure of the organism (24). Antisera raised against outer membrane proteins from NCHi have been shown to subtype isolates (16), but the reliance of this technique on a reagent in limited supply makes interlaboratory reproducibility difficult. Analyses of outer membrane proteins (2, 30) or WCPs (21) have also been applied to the study of NCHi. Of the above techniques, WCP profiles provide the most rapid results because no extraction procedure is required. Any protein-based typing system can be subject to phenotypic variation; therefore, techniques which examine the genome directly are preferable. REA of the genome has beena used successfully for a number of bacterial species (for review, see reference 20) and has been shown to be more consistent than protein profiles. This was recently demonstrated for NCHi; the majority of isolates from patients with chronic obstructive pulmonary disease had indistinguishable direct REA patterns, although they expressed different outer membrane proteins (7). Similarly, the present study showed that isolates with indistinguishable direct REA profiles could used by vary slightly in WCP patterns. Direct REA was also from the Loos et al. (12) to show that pairs of NCHi isolates nasopharynx and middle ear of the same patient gave identical profiles. However, most previous methods of REA have required both large volumes of bacterial culture and considerable time to extract the DNA and have used restriction enzymes which generated large numbers of overlapping fragments. The use of such enzymes makes comparisoniso-of patterns difficult between anything other than identical lates in adjacent tracks. Further, any attempt at quantitative analysis is virtually impossible because of the large numberto and poor resolution of the bands. It is therefore desirable combine a rapid, microcentrifuge-scale DNA extraction procedure with digestion by a restriction endonuclease that produces a pattern of discrete bands which can bea easily using such techinterpreted. Jordens and Hall (10), by between methicillinnique, were able to readily differentiate resistant Staphylococcus aureus strains. The choice of restriction endonuclease for a particular species is therefore crucial.

Comparisons of the results from WCP analysis and REA b --~~~~~~~~~~~~~~~n showed exact correlations between the clonal groups formed on the basis of SDS-PAGE profiles and both direct REA and I~~~~~~~~~~ rRNA probe-REA. This supports the definition of these isolates as related (or clonal) organisms and confirms that the on the cutoff ~~~~~~~p point chosen for the initial formation of groupsqualita1 Although da aq WCP was basis of appropriate. 4 wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwd^~ profiles I mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmh tively the same overall patterns of similarity and dissimilarity were observed, no quantitative correlation was observed between the Dice coefficients of similarity for SDS-PAGE WCP analysis and the Dice coefficients of similarity for direct REA. It is possible that the lack of correlation DICE SIMILARITY COEFFICIENT between SDS-PAGE of WCPs and direct REA is due to the 100 95 90 85 80 fact that they both examine relatively small (approximately 3 and 3.5%, respectively) and probably differing sections of the chromosome. Because of the lack of correlation, denof similarity drograms constructed from Dice coefficients suggest very estimated from WCP analysis or direct REA different phylogenies. This implies that dendrograms may therefore not be an accurate indication of relatedness and should be viewed with caution. A similar observation was made by Olyhoek et al. (19), who found that there was only electrophopartial correlation between REA and both the retic type of enzymes and outer membrane protein profiles FIG. 4. Dendrograms showing the relationship between the Dice estimated by direct REA (top) for isolates of Neisseria meningitidis. The use of techniques values for the dissimilar isolates, or WCP analysis (bottom). which examine a larger portion of the genome, e.g., pulsed| 0

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field gel electrophoresis, may provide a more reliable basis from which to estimate relatedness. rRNA gene restriction patterns showed the dissimilar isolates to be extremely diverse. However, two previously unrelated isolates were indistinguishable by this technique, indicating at least some similarity in evolution. This technique has also previously shown H. influenzae biogroup aegyptius to be diverse (9). Combined with the present study, this indicates that H. influenzae consists of a very heterogeneous group of organisms. The discriminatory value of rRNA probe-REA and its implications as an estimator of evolutionary divergence make this a useful technique for taxonomic studies. At present, however, the time and number of procedures required probably make it inapplicable for routine epidemiological studies. In conclusion, there was complete concordance between the results from direct REA and rRNA analysis for those isolates which were regarded as clones on the basis of WCP analysis, therefore supporting the thesis that they were clonally related. Isolates regarded as not being related on the basis of WCP analysis were similarly shown to have dissimilar fragment patterns by direct REA, but no quantitative correlation was found between the two techniques. The techniques of SDS-PAGE of WCP and direct REA, as described above, provide highly discriminatory methods of characterizing isolates of NCHi, and either technique would be applicable to epidemiological studies. ACKNOWLEDGMENTS K.D.B. is the recipient of a University of Aberdeen Medical Endowment Fund studentship, and J.Z.J. is funded by the Medical Research Council. We thank T. Hugh Pennington for his support and encouragement. REFERENCES 1. Barton, L. L., D. M. Granoff, and S. J. Barenkamp. 1983. Nosocomial spread of Haemophilus influenzae type b infection documented by outer membrane protein subtype analysis. J. Pediatr. 102:820-824. 2. Coverdale, C. H., and G. S. Temple. 1989. Outer membrane protein and biotype analysis of non-serotypable strains of Haemophilus influenzae. J. Clin. Pathol. 42:409-413. 3. Dice, L. R. 1945. Measures of the amount of ecologic association between species. Ecology 26:297-302.

4. Everett, E. D., A. E. Rahm, Jr., R. Adaniya, D. L. Stevens, and T. R. McNitt. 1977. Haemophilus influenzae pneumonia in adults. JAMA 238:319-321. 5. Govan, J. R. W., and S. Glass. 1990. The microbiology and therapy of cystic fibrosis lung infections. Rev. Med. Microbiol. 1:19-28. 6. Grimont, F., and P. A. D. Grimont. 1986. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur/Microbiol. (Paris) 137B:165-175. 7. Groeneveld, K., L. van Alphen, P. P. Eijk, H. M. Jansen, and H. C. Zanen. 1988. Changes in outer membrane proteins of nontypable Haemophilus influenzae in patients with chronic obstructive pulmonary disease. J. Infect. Dis. 158:360-365. 8. Inzana, T. J. 1983. Electrophoretic heterogeneity and interstrain

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