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
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Abstract |
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Introduction |
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Large resistance plasmids have been characterized and have been shown to transfer at low frequency (approximately 105) 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.
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Materials and methods |
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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 (MayJuly 1992). The I-Hib isolates were selected from cases identified in a national survey of invasive Hib disease (JanuaryDecember 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 I, Figure
) targeting plasmids (derived from the plasmid sequence lodged with GenBank; U468467) and the right junction fragment, attR (Figure
). 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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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, BoehringerMannheim (now Roche Diagnostics), Lewes, UK). After hybridization, banding patterns were detected by chemiluminescence as described previously.17
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Results |
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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 II). Of the 58 isolates, 10 (17%) were H. influenzae and 48 (83%) were H. parainfluenzae.
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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 III). 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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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 (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.
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Discussion |
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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.
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Acknowledgments |
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Notes |
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References |
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Received 10 March 1999; returned 15 September 1999; revised 18 October 1999; accepted 16 January 2000