Epidemiological studies of large resistance plasmids in Haemophilus

N. I. Leavesa, I. Dimopouloub, I. Hayesa, S. Kerridgea, T. Fallaa, O. Seckac, R. A. Adegbolac, M. P. E. Slacka,d, T. E. A. Petoe,f and D. W. M. Crooke,f,*

a Public Health Laboratory, John Radcliffe Hospital, Oxford; b Medical School of Alexandroupolis, University of Thrace, Alexandroupolis, Greece; c Medical Research Council Laboratories, Fajara, PO Box 273, Banjul, The Gambia; d Haemophilus Reference Unit, e Interdepartmental Academic Unit of Infectious Diseases and Clinical Microbiology, f Oxford Vaccine Group, John Radcliffe Hospital, Oxford OX3 9DU, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The distribution of large conjugative Haemophilus influenzae plasmids in the nasopharyngeal haemophili of a group of people and in a large collection of 541 H. influenzae type b (Hib) isolates was studied. A newly developed PCR-based assay was used to detect the plasmids. The target sequences were chosen from sequence analysis of part of p1056, a large multiresistance plasmid isolated from a clinical Hib isolate, 1056. Fifty-nine per cent of people were found to carry ß-lactamase-positive (ß-lac+), ampicillin-resistant (ampR) haemophili with detectable plasmid sequences. Of these, 83% were in Haemophilus parainfluenzae and 17% were in H. influenzae. In the collection of 541 Hib, antibiotic resistance [ß-lac+ampR, ß-lac+ampR plus tetracycline resistance (tetR) or tetR] was highly correlated with large plasmids. It was found that 2.3% of the isolates contained large cryptic plasmids (i.e. these isolates were susceptible to antibiotics). The distribution of plasmids between invasive and carried Hib did not differ significantly (25 of 245 and 23 of 276, respectively). Isolates with large plasmids occur at high frequency in the nasopharynx of the normal human population and consist of two populations in Hib, one associated with specific antibiotic resistance traits and the other cryptic. These plasmids do not appear to influence the invasiveness of Hib.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In 1972, the first resistant isolate of Haemophilus influenzae, a ß-lactamase-producing, ampicillin-resistant (ß-lac+ampR) H. influenzae type b (Hib) was identified.1 During the past 35 years, isolates of H. influenzae with ß-lac+ampR, and tetracycline, chloramphenicol and macrolide resistance have become prevalent2 but the exact proportion of the population of H. influenzae with these resistances is unclear. However, in the sub-population of Hib, as many as 80% of isolates from some countries may exhibit ß-lac+ampR.3 The world-wide emergence and rapid spread of resistance in H. influenzae has not been conclusively explained. Molecular studies of resistant H. influenzae have shown that antibiotic resistance of the type referred to here can be encoded by genes located on one of two types of plasmid.4 Small (<10 kb) cytoplasmic plasmids generally code for only a TEM ß-lactamase4 conferring ampicillin resistance, whereas much larger plasmids (usually >30 MDa) often encode multiple antibiotic resistance in addition to TEM ß-lactamase.57 The frequency of large and small plasmids in the colonizing human flora is not known, but may provide an indication of the size of the genetic pool of resistance plasmids circulating in humans and may offer insights into the origin of these plasmids. An indirect measure of plasmid frequency would be obtained by measuring the point prevalence of plasmids in Haemophilus spp. colonizing people living in the community.

Large resistance plasmids have been characterized and have been shown to transfer at low frequency (approximately 10–5) between H. influenzae isolates8 and between H. influenzae and Haemophilus parainfluenzae.9 They are usually undetectable extra-chromosomally in clinical isolates. However, following conjugative transfer they are readily detectable as closed circular plasmids in the recipient.7 The presence of antibiotic resistance genes on these large plasmids led to their detection and gives us a marker by which we can indirectly detect them. However, investigations aimed at detecting chromosomally integrated plasmids that do not possess antibiotic resistance genes (cryptic plasmids) have been limited by the lack of a ready assay. Recently, sequence analysis of plasmid DNA from a 1.2 kb fragment (lodged with GenBank No. U68467), containing the site of plasmid excision from the chromosome, has been used as a target for PCR which can specifically detect the presence of both chromosomally and cytoplasmically located plasmid sequences. By screening a large number of H. influenzae isolates it would be possible to establish the proportion of isolates possessing large cryptic plasmids and the relationship between specific resistance traits and these plasmids.

The effect of plasmids on H. influenzae other than encoding resistance is unclear. It is reasonable to postulate that a plasmid could influence the behaviour of its bacterial host. This would probably be most readily detectable in Hib, the most invasive type of H. influenzae, where many of the epidemiological characteristics of the organism are understood. If plasmids influence progression from colonization to invasive disease, it should be measurable as differences in the distribution of plasmids between carried and invasive isolates. The occurrence of more plasmids in carried isolates than in invasive isolates would imply an impediment to invasion by Hib, one possible reason being the genetic burden and fitness cost of plasmids. The probability of such a relationship is supported by inoculation experiments in monkeys where blood and CSF isolates were uniformly ß-lacampS even though the monkeys had been challenged intranasally with ß-lac+ampR isolates.10

We report on a number of studies that investigated, first, the point prevalence of plasmid-containing Haemophilus spp. in people from the community and, second, the relationship between large plasmids and antibiotic resistance, specifically ß-lac+ampR and tetracycline resistance (tetR) in H. influenzae, and on an investigation designed to detect an association between plasmids and invasive disease in Hib.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

The bacterial isolates were collected from a number of sources and were characterized for antibiotic susceptibility and presence of large plasmid sequences as described below. In addition, two well-characterized control isolates, Eagan11 and isolate 1056,7 were used throughout. All isolates were stored long term by freezing at –80°C in 15% glycerol tryptone soy broth.

A collection of isolates was assembled to determine the point prevalence of large plasmids in nasopharyngeal haemophili. As part of a continuing nasopharyngeal carriage study,12 throat swabs were obtained, by a previously described method,13 from 87 (52 children, 35 adults) healthy people living in the community. The swabs were plated on to enriched Columbia agar14 containing ampicillin (2 g/L). Morphologically distinct colonies of Haemophilus spp. growing on this antibiotic selective agar were purified by subculturing to a fresh selective agar plate. The isolates were identified by standard methods including their X and V factor dependence.15 Their antibiotic susceptibility and ß-lactamase status were tested as described below.

To determine the frequency of plasmids in invasive and carried isolates of Hib, simultaneously collected invasive and carried isolates of Hib were obtained from the following three geographically based studies: a regional Oxfordshire survey, a national UK study (based in Bangor, Ipswich, Manchester and Newcastle) and a Gambian study. The carried and invasive isolates were obtained from children with the same age distribution. The Oxfordshire-based studies have been reported previously and were part of a carriage study and a regional surveillance study of invasive disease.12,16 Fifty-six carried Hib (C-Hib) isolates from a longitudinal survey of Hib colonization in a birth cohort during the period of maximal risk from invasive disease were collected for the period December 1991 to May 1993. Fifty-nine consecutive invasive Hib (I-Hib) were selected from the Oxfordshire invasive Hib disease survey for the same period. The I-Hib isolates came from the two regions within Oxfordshire where the carriage studies were being performed. The isolates for the national UK study were obtained from a cross-sectional Hib carriage study of child day-care centres (May–July 1992). The I-Hib isolates were selected from cases identified in a national survey of invasive Hib disease (January–December 1992) and lived in the same cities as the day-care centres. To avoid repeated counting of shared clones of Hib in children from the same day-care centres, the C-Hib were subtyped using ribotyping and OMP-subtyping as previously described.17 Only distinguishable isolates from each day-care centre were used. After this selection, there were 25 C-Hib and 50 I-Hib. One hundred and thirty-six I-Hib Gambian isolates were collected during active surveillance of Hib disease in rural locations between July 1990 and June 1994; 194 C-Hib isolates were collected over the same time period.18,19 All the invasive and carried Gambian isolates were epidemiologically unlinked.

To investigate the relationship between specific resistance traits in H. influenzae and large Haemophilus plasmids, all the H. influenzae isolates collected above were studied. In addition to these, 15 tetR H. influenzae isolates obtained before 1991 were selected for study from the frozen archive of H. influenzae collected during the continuing Oxfordshire Hib invasive diseases survey.

Antibiotic susceptibility testing

The susceptibility of all isolates to ampicillin (2 µg disc), chloramphenicol (10 µg), tetracycline (10 µg) and erythromycin (5 µg) was determined by the comparative Stokes' disc diffusion method with the sensitive H. influenzae control isolate NCTC 11931.20 In addition, all isolates were tested for the production of TEM-ß-lactamase using Intralactam filter paper strips as recommended by the manufacturer (Mast Diagnostics, Bootle, UK).21

Polymerase chain reaction

All serotype b H. influenzae isolates were capsular genotyped by PCR as previously described.22,23 Isolates were screened for the presence of large plasmids by designing PCR primers (Table IGo, FigureGo) targeting plasmids (derived from the plasmid sequence lodged with GenBank; U468467) and the right junction fragment, attR (FigureGo). The PCR primer Y1 was designed from the Haemophilus genome sequence lodged with GenBank; U400070. To detect plasmids, primer sets F1 with R2 (detected both excised and integrated plasmids) and F2 with R1 (detected only excised plasmids) were used in separate reactions on all isolates. In addition, a third set of primers F2 with Y1 (detected only integrated plasmid) was used only with Hib isolates.


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Table I. PCR primers for detection of plasmids
 


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Figure. Plasmid and chromosomal PCR primer attachment sites. The orientation of the PCR primers F1, R2 and R2, Y1 are indicated at their respective sites on the chromosome, closed circular (excised) plasmid and plasmid recombined (integrated) with the chromosome. attB, chromosomal attachment site; attP, plasmid attachment site; attL, left plasmid chomosomal junction; attR, right plasmid chromosomal junction.

 
Each PCR reaction mixture (25 µL) contained 0.1 U of Taq polymerase (Advanced Biotechnologies, London, UK), 10 mM Tris–HCl (pH 8), 50 mM KCl, 2.5 mM MgCl2, 0.01% (w/v) gelatin, 250 µM of each deoxynucleotide triphosphate (Amersham Pharmacia Biotech, Uppsala, Sweden) and 1 µM of each oligonucleotide primer (R&D Systems, Abingdon, UK), and the mixture was overlaid with mineral oil to prevent evaporation during cycling. To this mixture, 1 µL of target DNA extracted by boiling24 was added. The cycling parameters consisted of 25 cycles of: 1 min at 94°C, 1 min at 60°C and 2 min at 72°C, followed by a final extension period of 10 min at 72°C. All PCRs were performed using a Techne PHC-3 thermal cycler (Techne, Duxford, UK). PCR products were separated by electrophoresis in 1.5% (w/v) agarose and bands were visualized by ethidium bromide staining and UV transillumination. Hib isolate Eagan was used as a negative control for plasmid PCR and as a positive control for PCR capsular genotyping. Isolate 1056 was the positive PCR control for plasmid and other negative PCR controls contained no known DNA template. Isolates were scored positive if two of the primer sets produced product of the expected size.

Further analyses of antibiotic-resistant plasmid PCR negative (PCR ) isolates or antibiotic-susceptible plasmid PCR positive (PCR+) isolates

Isolates of H. influenzae that were antibiotic resistant but were PCR were further examined for small plasmids by a standard alkaline lysis ‘miniprep’ method.7,25 Plasmid preparations were electrophoresed in agarose (0.8% w/v) and plasmid DNA was visualized by UV transillumination after staining with ethidium bromide. In addition, these isolates and all isolates that were antibiotic susceptible and PCR+ were further examined for plasmids by hybridization with whole plasmid from isolate 1056 by a method modified from Dimopoulou et al.7 Briefly, total cellular DNA was extracted, digested with the restriction enzyme PstI and electrophoresed. The restriction fragments were then Southern blotted and the blots hybridized with digoxigenin-labelled p1056 (Digoxigenin DNA labelling kit, Boehringer–Mannheim (now Roche Diagnostics), Lewes, UK). After hybridization, banding patterns were detected by chemiluminescence as described previously.17


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prevalence of large plasmid in colonizing Haemophilus spp.

From the 87 individuals in the community (52 children and 35 adults), 58 ß-lac+ampR Haemophilus spp. with large plasmid-specific sequences detected by PCR were isolated from 52 (59%) individuals, some of whom had more than one isolate (Table IIGo). Of the 58 isolates, 10 (17%) were H. influenzae and 48 (83%) were H. parainfluenzae.


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Table II. Carriage of colonizing Haemophilus spp. with plasmids and antibiotic resistance by individuals in the community
 
The relationship of plasmid to antibiotic resistance in Hib

All ampR and/or tetR isolates were investigated for plasmid sequences. As expected, plasmid sequences were highly associated with antibiotic resistance and vice versa (Table IIIGo). It was found that 2.4% of isolates were susceptible to ampicillin, tetracycline, chloramphenicol or erythromycin and were plasmid PCR+, suggesting the presence of cryptic plasmids. Southern blotting and hybridization with whole plasmid p1056 confirmed the presence of multiple bands of homology, which are typical of large plasmids (data not shown).


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Table III. The relationship of plasmid carriage to antibiotic resistance in Hib
 
Invasive Hib versus carried Hib

In the three I-Hib and C-Hib study populations, 48 of the 521 isolates contained plasmid-specific sequences. Of the 48 isolates with plasmid sequences, nine were susceptible to all antibiotics tested. There was no significant difference ({chi}2 = 0.57; P = 0.45) in the distribution of plasmids between C-Hib and I-Hib, 23 of 276 (8%) versus 25 of 245 (10%), respectively.


    Discussion
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 Materials and methods
 Results
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 References
 
Sixty per cent of a normal healthy community-based population was shown to carry ß-lac+ampR Haemophilus spp., the majority (83%) of the isolates being H. parainfluenzae. Sequences homologous to a large H. influenzae conjugative plasmid (p1056) could be detected in all of these isolates. This study provides further direct evidence that ß-lac+ampR H. parainfluenzae contain the same family of large plasmids as H. influenzae. This observed prevalence is likely to be an underestimate, as the selection for plasmid-containing isolates was based only on obtaining ß-lac+ampR isolates. Plasmids occurring in tetR (shown in this study to be highly correlated with plasmid-specific sequences), or chloramphenicol-resistant or -susceptible isolates (cryptic plasmids), would not have been detected by the selection of ß-lac+ampR isolates used in this study. Even so, such high-frequency presence of plasmids both indicates a large genetic pool of plasmids and implies that it is a highly successful and well-adapted genetic element.

Previous carriage studies with a cross-sectional design, one in Vancouver, Canada,26 and another in Toulouse, France,27 demonstrated a prevalence of ß-lac+ampR Haemophilus spp. of 70% and 30%, respectively. The majority of these isolates were H. parainfluenzae. On the basis of both the preponderance of resistant H. parainfluenzae over resistant H. influenzae and the assumption that these isolates contained the same large plasmids, the researchers26,27 speculated that the H. influenzae plasmids with ß-lac+ampR originated in the population of H. parainfluenzae. However, without knowing the relative distribution of plasmids within each of the denominator populations of colonizing H. influenzae and H. parainfluenzae, it may be misleading to conclude that plasmids originated in H. parainfluenzae. For instance, the occurrence of plasmids (both cryptic and resistant) at a higher frequency in the population of H. influenzae compared with H. parainfluenzae would be consistent with H. influenzae rather than H. parainfluenzae being both the preferred host and the source of these large resistance plasmids.

The relationship between large plasmids and H. influenzae resistance genes, and the factors that influence this relationship, have been only partially investigated. This study has shown that ß-lac+ampR, tetR and ß-lac+ampR plus tetR in H. influenzae is highly correlated with the presence of large plasmid sequences. A small proportion (5%) of ß-lac+ampR H. influenzae isolates is associated with the presence of small, approximately 5 kb plasmids. This study, on a large number of Hib isolates from the UK and The Gambia, provides direct confirmation of observations from individual case studies and a large number of restricted population-based observations2831 that large plasmids account for essentially all the ß-lac+ampR, tetR and ß-lac+ampR plus tetR. This investigation also gives the first direct measure of the proportion of H. influenzae isolates with cryptic plasmids. A previous study by Laufs et al., based on a systematic search for extra-chromosomal cryptic plasmids, detected plasmids in one (0.14%) of 699 isolates examined.28 In this study, cryptic plasmids were detected in 10 (2.4%) of 410 susceptible H. influenzae isolates. This higher frequency is probably ascribable to chromosomally integrated plasmids that were previously undetectable.

The present study establishes, as surmised by others, that there are two populations of plasmids in H. influenzae, one (the majority) with resistance genes and one (the minority) without resistance genes.5,28 This lends support to the hypothesis that, before the antibiotic era, core (cryptic) plasmids existed in the population of H. influenzae and under antibiotic pressure acquired enterobacterial resistance transposons such as Tn3 or Tn10.28 The presence of cryptic plasmids in an appreciable proportion of isolates also emphasizes that selective factors other than antibiotic pressure may be important in plasmid survival. Also, the presence of cryptic plasmids, as with many other aspects of this family of large plasmids, has been investigated only in H. influenzae. To gain a full understanding of these large plasmids and their host flora requires an investigation of their relationship with other haemophili and other genera of the nasopharyngeal flora.

The finding that plasmids were distributed equally in carried and invasive isolates of Hib suggests that plasmids do not have a large effect on either fitness or invasive traits. This study lacked the power to detect significant differences in plasmid frequency of <=6%. The prospect that these large resistance plasmids are well adapted to Haemophilus spp. and have little or no cost to fitness would predict that the population of resistant haemophili would persist even if antibiotic usage was curtailed. Investigation of the relative fitness of antibiotic-resistant isolates has proven difficult in nature. Given the accessibility of the nasopharyngeal flora to culture, the relationship between plasmids, resistance and antibiotic usage in haemophili and other nasopharyngeal bacteria could be readily investigated. This would provide valuable insights into the transmission of antibiotic resistance between bacterial cells in the nasopharynx and more generally the transmission of antibiotic resistance in the community.


    Acknowledgments
 
The authors would like to thank Mrs E. Coles for her editorial assistance in the preparation of this manuscript.


    Notes
 
* Correspondence address. Interdepartmental academic unit of Infectious Diseases and Clinical Microbiology, John Radcliffe Hospital, Oxford OX3 9DU, UK. Tel: +44-1865-221226; Fax: +44-1865-764192; E-mail: dcrook{at}molbiol.ox.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Mathies, A. W. (1972). Penicillins in the treatment of bacterial meningitis. Journal of the Royal College of Physicians of London 6, 139–46.[Medline]

2 . Powell, M. (1988). Antimicrobial resistance in Haemophilus influenzae. Journal of Medical Microbiology 27, 81–7.[ISI][Medline]

3 . Campos, J., Garcia-Tornel, S., Gairi, J. M. & Fabregues, I. (1986). Multiply resistant Haemophilus influenzae type b causing meningitis: comparative clinical and laboratory study. Journal of Pediatrics 108, 897–902.[ISI][Medline]

4 . De Graaff, J., Elwell, L. P. & Falkow, S. (1976). Molecular nature of two ß-lactamase-specifying plasmids isolated from Haemophilus influenzae type b. Journal of Bacteriology 126, 439–46.[ISI][Medline]

5 . Brunton, J., Clare, D. & Meier, M. A. (1986). Molecular epidemiology of antibiotic resistance plasmids of Haemophilus species and Neisseria gonorrhoeae. Reviews of Infectious Diseases 8, 713–24.[ISI][Medline]

6 . Campos, J., Chanyangam, M., deGroot, R., Smith, A. L., Tenover, F. C. & Reig, R. (1989). Genetic relatedness of antibiotic resistance determinants in multiply resistant Haemophilus influenzae. Journal of Infectious Diseases 160, 810–7.[ISI][Medline]

7 . Dimopoulou, I. D., Kraak, W. A., Anderson, E. C., Nichols, W. W., Slack, M. P. & Crook, D. W. (1992). Molecular epidemiology of unrelated clusters of multiresistant strains of Haemophilus influenzae. Journal of Infectious Diseases 165, 1069–75.[ISI][Medline]

8 . Stuy, J. H. (1980). Chromosomally integrated conjugative plasmids are common in antibiotic-resistant Haemophilus influenzae. Journal of Bacteriology 142, 925–30.[ISI][Medline]

9 . Scheifele, D. W., Fussell, S. J. & Roberts, M. C. (1982). Characterization of ampicillin-resistant Haemophilus parainfluenzae. Antimicrobial Agents and Chemotherapy 21, 734–9.[ISI][Medline]

10 . Daum, R. S., Syriopoulou, V. P., Smith, A. L., Scheifele, D. W. & Willard, J. E. (1981). Loss of plasmid DNA coding for ß-lactamase during experimental infection with Haemophilus influenzae type b. Journal of Infectious Diseases 143, 548–53.[ISI][Medline]

11 . Anderson, P., Johnstone, R. B. & Smith, D. H. (1972). Human serum activities against Haemophilus influenzae, type b. Journal of Clinical Investigation 51, 31–8.[ISI][Medline]

12 . Barbour, M. L., Mayon-White, R. T., Coles, C., Crook, D. W. & Moxon, E. R. (1995). The impact of the conjugate vaccine on carriage of Haemophilus influenzae type b. Journal of Infectious Diseases 171, 93–8.[ISI][Medline]

13 . Barbour, M. L., Booy, R., Crook, D. W., Griffiths, H., Chapel, H. M., Moxon, E. R. et al. (1993). Haemophilus influenzae type b carriage and immunity four years after receiving the Haemophilus influenzae oligosaccharide-CRM197 (HbOC) conjugate vaccine. Pediatric Infectious Disease Journal 12, 478–84.[ISI][Medline]

14 . Barbour, M. L., Crook, D. W. & Mayon-White, R. T. (1993). An improved antiserum agar method for detecting carriage of Haemophilus influenzae type b. European Journal of Clinical Microbiology and Infectious Diseases 12, 215–7.[ISI][Medline]

15 . Kilian, M. (1976). A taxonomic study of the genus Haemophilus with the proposal of a new species. Journal of General Microbiology 93, 9–62.[ISI][Medline]

16 . Tudor-Williams, G., Frankland, J., Isaacs, D., Mayon-White, R. T., MacFarlane, J. A., Slack, M. P. et al. (1989). Haemophilus influenzae type b disease in the Oxford region. Archives of Disease in Childhood 64, 517–9.[Abstract]

17 . Leaves, N. I. & Jordens, J. Z. (1994). Development of a ribotyping scheme for Haemophilus influenzae type b. European Journal of Clinical Microbiology and Infectious Diseases 13, 1038–45.[ISI][Medline]

18 . Adegbola, R. A., Mulholland, E. K., Falade, A. G., Secka, O., Sarge-Njai, R., Corrah, T. et al. (1996). Haemophilus influenzae type b disease in the western region of The Gambia: background surveillance for a vaccine efficacy trial. Annals of Tropical Paediatrics 16, 103–11.[ISI][Medline]

19 . Adegbola, R. A., Mulholland, E. K., Secka, O., Jaffar, S. & Greenwood, B. M. (1998). Vaccination with a Haemophilus influenzae type b conjugate vaccine reduces oropharyngeal carriage of Haemophilus influenzae type b among Gambian children. Journal of Infectious Diseases 177, 1758–61.[ISI][Medline]

20 . Report of the Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50.[ISI][Medline]

21 . Slack, M. P., Wheldon, D. B. & Turk, D. C. (1977). A rapid test for ß-lactamase production by Haemophilus influenzae. Lancet ii, 906.

22 . Falla, T. J., Crook, D. W., Brophy, L. N., Maskell, D., Kroll, J. S. & Moxon, E. R. (1994). PCR for capsular typing of Haemophilus influenzae. Journal of Clinical Microbiology 32, 2382–6.[Abstract]

23 . Leaves, N. I., Falla, T. J. & Crook, D. W. (1995). The elucidation of novel genotypes of Haemophilus influenzae capsular type b with the polymerase chain reaction. Journal of Medical Microbiology 43, 120–4.[Abstract]

24 . Jordens, J. Z., Leaves, N. I., Anderson, E. C. & Slack, M. P. (1993). Polymerase chain reaction-based strain characterization of noncapsulate Haemophilus influenzae. Journal of Clinical Microbiology 31, 2981–7.[Abstract]

25 . Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7, 1513–23.[Abstract]

26 . Scheifele, D. W. & Fussell, S. J. (1981). Frequency of ampicillin- resistant Haemophilus parainfluenzae in children. Journal of Infectious Diseases 3, 495–8.

27 . Dabernat, H., Bauriaud, R., Delmas, C. & Lareng, M. B. (1983). Colonisation de l'enfant par Haemophilus; sérotypes, biotypes et sensibilité aux antibiotiques. Pathologie–Biologie 31, 103–6.

28 . Laufs, R., Riess, F. C., Jahn, G., Fock, R. & Kaulfers, P. M. (1981). Origin of Haemophilus influenzae R factors. Journal of Bacteriology 147, 563–8.[ISI][Medline]

29 . Dimopoulou, I. D., Jordens, J. Z., Legakis, N. J. & Crook, D. W. (1997). A molecular analysis of Greek and UK Haemophilus influenzae conjugative resistance plasmids. Journal of Antimicrobial Chemotherapy 39, 303–7.[Abstract]

30 . Mendelman, P. M., Syriopoulou, V. P., Gandy, S. L., Ward, J. I. & Smith, A. L. (1985). Molecular epidemiology of plasmid-mediated ampicillin resistance in Haemophilus influenzae type b isolates from Alaska. Journal of Infectious Diseases 151, 1061–71.[ISI][Medline]

31 . Elwell, L. P., Saunders, J. R., Richmond, M. H. & Falkow, S. (1977). Relationships among some R plasmids found in Haemophilus influenzae. Journal of Bacteriology 131, 356–62.[ISI][Medline]

Received 10 March 1999; returned 15 September 1999; revised 18 October 1999; accepted 16 January 2000