J Med Microbiol 56 (2007), 838-846; DOI: 10.1099/jmm.0.47125-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Diversity of antibiotic resistance integrative and conjugative elements among haemophili
Ioanna D. Dimopoulou1,
Sofia I. Kartali1,
Rosalind M. Harding2,
Tim E. A. Peto3 and
Derrick W. Crook3
1 Department of Microbiology – Infectious Diseases, University Hospital of Alexandroupolis, Democritus University of Thrace, Alexandroupolis 68100, Greece
2 Department of Zoology, University of Oxford, Oxford, UK
3 Infectious Diseases and Clinical Microbiology, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
Correspondence
Derrick W. Crook
derrick.crook{at}ndcls.ox.ac.uk
Received 18 December 2006
Accepted 28 February 2007
The objective of this study was to investigate the sequence diversity in a single country of a family of integrative and conjugative elements (ICEs) that are vectors of antibiotic resistance in Haemophilus influenzae and Haemophilus parainfluenzae, and test the hypothesis that they emerged from a single lineage. Sixty subjects aged 9 months – 13 years were recruited and oropharyngeal samples cultured. Up to 10 morphologically distinct Pasteurellaceae spp. were purified, and then the species were determined and differentiated by partial sequence analysis of 16S rDNA and mdh, respectively. ICEs were detected by PCR directed at five genes distributed evenly across the ICE. These amplicons were sequenced and aligned by the neighbour-joining algorithm. A total of 339 distinguishable isolates were cultured. ICEs with all 5 genes present were found in 9 of 110 (8 %) H. influenzae and 21 of 211 (10 %) H. parainfluenzae, respectively. ICEs were not detected among the other Pasteurellaceae. A total of 20 of 60 (33 %) children carried at least 1 oropharyngeal isolate with an ICE possessing all 5 genes. One of the five genes, integrase, however, consisted of two lineages, one of which was highly associated with H. influenzae. The topology of neighbour-joining trees of the remaining four ICE genes was compared and showed a lack of congruence; though, the genes form a common pool among H. influenzae and H. parainfluenzae. This family of antibiotic resistance ICEs was prevalent among the children studied, was genetically diverse, formed a large gene pool, transferred between H. influenzae and H. parainfluenzae, lacked population structure and possessed features suggestive of panmixia, all indicating it has not recently emerged from a single source.
Abbreviations: ICE, integrative and conjugative element.
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INTRODUCTION
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Antibiotic resistance amongst major human pathogens is becoming an increasing public health problem (Finch, 1998; Williams, 1997). The prevalence of transferable resistance has increased among human bacterial pathogens with the increasing use of antibiotics (Davies, 1997). Despite these major epidemiological changes, a detailed understanding of how conjugative elements have evolved in pathogen populations is incomplete and only gradually emerging. A better understanding of the evolutionary process leading to conjugative resistance elements amongst pathogens will help predict the future spread of resistance.
The emergence of antibiotic resistance among clinical isolates of Haemophilus influenzae dates back to the early 1970s (Gunn et al., 1974; Mathies, 1972). Following the first detection of resistance in 1973 (Mathies, 1972), the prevalence of resistance to ampicillin, and increasingly to other antibiotics, rose rapidly (Syriopoulou et al., 1978). It has recently been shown that the main vector of this antibiotic resistance is not a plasmid, but a large integrative and conjugative element (ICE) (Dimopoulou et al., 2002; Mohd-Zain et al., 2004). These ICEs likely form a related family first regarded as conjugative plasmids in the 1970s (Elwell et al., 1975, 1977; Laufs & Kaulfers, 1977; Laufs et al., 1981; Saunders & Sykes, 1977; Stuy, 1980). Recently, publication of the whole sequence of one of these ICEs, ICEHin1056 (Mohd-Zain et al., 2004), provided the information from which the sequence diversity among these ICEs could be studied and, thereby, make the investigation of the evolution and routes of transmission of these ICEs possible. This approach will allow the hypothesis that they spread from a single point source to be tested by performing a detailed ecological study in a single geographical region, Alexandroupolis, Greece.
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METHODS
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Study subjects, sampling and culture.
Sixty subjects aged 9 months – 13 years were recruited. They were selected from children admitted without infection to the Department of Paediatrics, University General Hospital of Alexandroupolis, during the periods October 2001 – December 2001 and April 2002 – July 2002. Children on or recently receiving antibiotic treatment (that is within a week) were excluded. After consenting, throat and dental swabs were collected. The swabs were placed in haemophilus transport medium (HTM) as previously described (Barbour et al., 1993a). After bacteria were dispersed by vigorous vortexing, 50 µl HTM was cultured and the remainder of the medium was stored at –80 °C for later culture.
Two sets of children were studied. The first set of 20 children was to determine differences between the floras of the throat versus the dental margin. From these 20 children throat and dental swabs were immediately subbed onto HTM (without antiserum) (Barbour et al., 1993b) containing bacitracin (10 units ml–1) alone or plus ampicillin (2 µg ml–1) or tetracycline (4 µg ml–1). The bacitracin alone plate allowed selection of Haemophilus spp. without selection against ampicillin or tetracycline susceptible isolates. Up to ten morphologically different colonies from the antibiotic containing media were purified onto the same media with the above concentrations of antibiotics, and tested for the presence of ß-lactamase using intralactam strips (Mast Diagnostics). For the 40 remaining subjects, only throat swab samples from the frozen stock were cultured on the selective plates. Four, three and two different morphotypes from the bacitracin, ampicillin and tetracycline containing media, respectively, were purified.
Speciation and differentiation of isolates.
In addition to standard phenotypic identification using X, V and X+V utilization, partial sequence of one of two regions of DNA encoding 16S rRNA was used for speciation. Differentiation of haemophili within an individual was based on sequence comparison of partial sequence of mdh (malate dehydrogenase), a gene used for H. influenzae multilocus sequence typing. Sequence difference in mdh of strains from the same individual defined distinguishable isolates. To design primers targeting sequence shared by different haemophili, the primers, MdhFP, MdhRP, MdhFS and MdhRS were designed by alignment of mdh from the whole sequences of H. influenzae KW20 (GenBank accession no. L42023) (Fleischmann et al., 1995), Haemophilus ducreyi (GenBank accession no. NC_002940) and ‘Haemophilus somnus’ (GenBank accession no. NC_008309), and choosing regions with conserved sequence. For differentiating the few H. influenzae and Haemophilus parainfluenzae isolates that failed to produce sequence from these primers, further internal primers were designed from alignment of the mdh sequences generated above, and further nested primer sets were designed targeting conserved sequences to optimize amplification and sequencing. This allowed differentiation of the few remaining strains.
The PCR amplicons were sequenced and read on an ABI PRISM 3700 DNA analyser (Applied Biosystems). The primers used for sequencing are shown in Table 1
. Sequence data were assembled using the STADEN suite of computer programs. 16S rDNA sequence species identification was determined by DNA sequence alignment using the BLASTN algorithm available through the National Center for Biotechnology Information (NCBI). For comparison, mdh sequences from strains isolated from within an individual were aligned using both NRDB (written by Warren Gish, University of Washington, St Louis, MO, USA and available through http://pubmlst.org/) and ClustalW (Thompson et al., 1994).
ICE detection.
For ICE detection, five primer sets were designed from equally spaced core genes (these genes have been described by Mohd-Zain et al., 2004) of ICEHin1056 (GenBank accession no. AJ627386): two targeting a putative replicative region, namely orf1 and orf11 being homologues of parA and topB; two targeting a recently described putative conjugative region consisting of a novel type IV secretion system (Juhas et al., 2007), namely orf43 and orf51, genes tfc14 and tfc22, respectively, with homology to traB and pilT, respectively; and lastly, int, referred to as int1, a homologue of xerC/D, a tyrosine integrase, at the 5' end of ICEHin1056. Because many ICEs in the study contained a divergent xerC/D homologue, a different primer set, int2, was designed from sequence of the ‘cryptic’ ICE present in the antibiotic susceptible H. influenzae 86-028NP, GenBank accession no. CP000057 (Harrison et al., 2005) to detect and sequence this lineage of integrase. This lineage of int, int2, was typical of that found in the previously described H. influenzae type b isolate, 299, from Athens, Greece (Dimopoulou et al., 1997). In addition, PCR products were sequenced, in preference, using nested sequencing primers where suitably available (Table 1
).
The sequences were trimmed and the individual sequences of each gene and/or the concatenated sequences were aligned using the neighbour-joining algorithm available in MEGA, version 3.1 (Kumar et al., 2004). Bootstrap values were used to judge the reliability of clusters. These values on the neighbour-joining tree are percentages of 100 computer-generated trees produced by randomly sampling the sequences and are shown at branch nodes.
Detecting antibiotic resistance genes by PCR.
For all the 41 ICE containing isolates the presence of the following resistance genes were assayed using PCR: TEM-1 and ROB-1 ß-lactamase or tet(B) and the other associated Tn10 genes tetC and tetD. For details of the primers used see Table 1.
Statistical analysis.
Standard statistics were used for frequency analyses.
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RESULTS AND DISCUSSION
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Culture, speciation and differentiation of isolates
Five hundred and eighty strains were isolated and studied further. These isolates consisted mostly of strains picked from the bacitracin plate; a minority of resistant isolates were picked from the bacitracin plus ampicillin or tetracycline plate. Within individuals, only isolates distinguishable by 16S rDNA and/or mdh sequence analysis (i.e. unique sequence types for a child) were included for further analysis. There were 339 such isolates, 266 were isolated from throat swabs and 73 were isolated from dental margin cultures. A wide diversity of Haemophilus spp. isolates (up to eight distinguishable types) were observed for isolates cultured from the throat or dental margin, see Fig. 1. Furthermore, of the 20 people in whom both throat and dental margin cultures were available, 2–12 distinguishable strains were present within an individual (including both sites), 9 shared only 1 strain and 2 shared 2 strains at both swabbing sites. Consequently, most isolates cultured from each geographical site were unique with limited overlap.
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Fig. 1. Charts indicating the number of distinguishable isolates of Haemophilus spp. cultured from either the throat (a) or the dental margin (b) in each child. Grey bars, the number of morphologically distinguishable isolates;  , unique isolates identified by sequence analysis.
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This ecological study indicates a diverse population of haemophili much in line with the work of Kilian & Schiott (1975). The simple culture technique used in this study is inevitably biased away from detecting minority populations of susceptible haemophili. Notwithstanding this limitation, a large number of distinguishable (unique) Haemophilus isolates were recovered per individual with little overlap between the two sampling sites; therefore, this suggests that the diversity is not being exhausted by the sampling. This also suggests there are even more strains present in people’s throats than found in this study. This consequently points to a very large pool of host haemophili per individual that can potentially support the family of Haemophilus ICEs.
ICE detection and characterization
Of the 339 distinguishable isolates, 30 contained complete ICEs (namely, all 5 target genes, i.e. orf1, orf11, orf43, orf51 and int, being present). Five of these ICEs were from dental cultures. A total of 11 incomplete ICEs (i.e. 
4 target genes) were found, of which 4 were from dental cultures. Those elements designated as incomplete are based purely on failing to detect a PCR product following amplification using the sets of primers described in this study. Further characterization of these elements has not been done to determine whether they are in fact genetically coherent using other methods such as Southern blotting or whether they are functional, that is able to conjugate. Further studies need to be done to answer these questions.
The distribution of antibiotic resistance phenotypes and resistance genes was investigated among all the 41 ICE positive (both complete and incomplete) isolates, see Table 2. Of the 339 isolates, 66 expressed ß-lactamase and/or were tetracycline resistant, and 41 of these were found to contain ICE sequences; therefore, ICE sequences are significantly associated with this phenotype (
2=193, P0.001). This confirms in another geographical location the finding that ICE sequences associate with antibiotic resistance, as reported by Leaves et al. (2000). Of the 39 ICE positive isolates that were ApR and ß-lactamase positive, 31 yielded PCR amplicons for TEM-1 and none yielded amplicons for ROB-1. Eight failed to amplify with either of the sets of primers TEM-1 or ROB-1. Determination of the likely genetic basis for ß-lactamase activity in these eight strains is beyond the scope of this study and deserves further investigation. Of the 22 TcR resistant ICE containing isolates 17 yielded PCR amplicons to tet(B). All, but two yielded PCR products containing tetC or tetD, therefore suggesting the TcR resistance phenotype is associated with Tn10. The mechanism of resistance for the five TcR isolates, for which no PCR products were produced, is unknown and is also beyond the scope of this work. These observations suggest that ICEs associate largely with Tn10 encoded TcR and TEM-1 ß-lactamase-encoded ampicillin resistance. Whether other ß-lactamase genes or tetracycline resistance genes are found on ICEs needs to be investigated.
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Table 2. Distribution of ICE sequences among resistance phenotypes
ApR, ampicillin resistant; ß-lac+, ß-lactam positive; TetR, tetracycline resistant; ß-lac–, ß-lactam negative.
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Among the 339 distinguishable isolates cultured in this study, 8 species were identified (7 of these being Haemophilus spp. and 1 Actinobacillus actinomycetemcomitans), as listed in Table 3. ICEs were only found in a similar proportion among the two species H. influenzae and H. parainfluenzae as follows: complete ICEs in 8.2 and 9.95 %, and incomplete ICEs in 1.8 and 4.3 %, respectively. The possibility that ICEs may occur among species other than H. influenzae and H. parainfluenzae cannot be ruled out as this study was underpowered for detection among the other species.
The occurrence of ICE containing haemophili among study subjects was as follows: 20 children (33 %) contained at least one complete ICE and 11 children (18 %) at least one incomplete ICE; therefore, 31 children (51 %) carried ICE sequences among culturable haemophili. In three of the children, both complete and incomplete ICEs were observed. Three different study subjects yielded strains with indistinguishable ICEs present in two genotypically different host haemophili, consistent with recent horizontal transfer of the ICE between haemophili in the nasopharynx. Two other genotypically indistinguishable ICEs were found in distinguishable host haemophili, though in different children, also suggesting horizontal transfer of these ICEs, though, more distantly in the past. These data suggest there is frequent transfer of ICEs and this is consistent with the rapid flux of ICE genes, and indicates that these elements may rapidly spread through populations. This may indeed be what happened in the 1970s when resistance rapidly rose from low to high frequency among H. influenzae (Syriopoulou et al., 1978) over a relatively short time.
There were two distinguishable lineages of int, one lineage, int1, similar to ICEHin1056, and the other, int2, which diverged by 26 % at the nucleotide level (as determined by NCBI BL2SEQ), see Fig. 2. These two lineages of int distributed unequally between H. influenzae and H. parainfluenzae; int1 of the ICEHin1056 lineage being significantly associated with H. influenzae (2=19.95, P<0.001). This finding is discussed further below.
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Fig. 2. Neighbour-joining tree of int nucleotide sequences indicating the two lineages differing by 26 %. Underlined strains indicate int from ICEs found in H. influenzae, including H. haemolyticus. int1 refers to the ICEHin1056 lineage and int2 refers to the ICEHin299 lineage. Bootstrap values are indicated on the branch nodes.
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Each of the partial gene sequences orf1, orf11, orf43 and orf51 present in the complete ICEs were aligned and the resultant trees compared as represented in Fig. 3. The topology of each of these trees differed markedly. There was no evidence that specific sequence types associated with either of the species H. influenzae or H. parainfluenzae. These features would be consistent with a single gene pool circulating between the two Haemophilus spp. and undergoing frequent recombination. The tree generated by alignment of the concatenated sequences, including those of ICEHin299 (a previously described H. influenzae type b isolate from Athens, Greece) (Dimopoulou et al., 1997) and ICEHin1056, both found in H. influenzae type b hosts isolated from cases of meningitis, clustered with those found among H. parainfluenzae (see Fig. 4). This suggests that the antibiotic resistance ICEs found in H. influenzae type b share the same gene pool as those occurring in the commensal H. parainfluenzae flora.
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Fig. 3. Neighbour-joining trees produced from nucleotide sequence alignments of orf1 (a), orf11 (b), orf43 (c) or orf51 (d). Strain HT16B2, which lacked detectable int, is indicated with an asterisk. Underlined strains indicate ICEs found in H. influenzae including H. haemolyticus. Bootstrap values are indicated on the branch nodes. , Strains associated with the ICEHin1056 int lineage, int1.
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Fig. 4. Neighbour-joining tree of the concatenated nucleotide sequence for all four loci orf1, orf11, orf43 and orf51. No species-specific clustering of ICE sequences from H. influenzae or H. parainfluenzae is observed. The sequences of both int lineages are distributed across the diversity of the concatenated sequence. Underlined strains indicate H. influenzae including H. haemolyticus. The ICE in strain HT16B2 without int is indicated with an asterisk. The ICEHin1056 and ICEHin299, both found in H. influenzae type b strains isolated from cases of meningitis, are labelled as 1056 and 299, respectively. Bootstrap values are indicated on the branch nodes. , Strains associated with the ICEHin1056 int lineage, int1.
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This family of antibiotic resistance ICEs have been shown to be prevalent among the set of Greek children studied, are genetically diverse, form a large common gene pool, lack population structure and possess features suggestive of panmixia. As such, these data are inconsistent with the recent emergence (in the past 30 years) of a single lineage of ICE transferring to H. influenzae, for example, from a H. parainfluenzae, as a rare founding event in Greece and then spreading horizontally among these Greek H. influenzae. Rather the sequence diversity observed among the ICEs suggests a long-standing and shared evolutionary relationship between these ICEs and the host bacteria H. influenzae and H. parainfluenzae. Alternatively, the data are consistent with multiple acquisitions from a diverse source population other than the two species H. influenzae and H. parainfluenzae. The present study did not find evidence of such a third population, though it was insufficiently powered to exclude one of the other oropharyngeal Haemophilus spp. being the source.
Though these ICEs lack population structure, curiously, these elements consist of a region at the 3' end represented by int, which can be either of two different lineages that diverge by 26 % at the nucleotide level. The remaining ICE sequences, represented by orf1, orf11, orf43 and orf51, by contrast, differ by as little as 3 % at the nucleotide level, similar to the extent of nucleotide divergence observed in housekeeping genes of H. influenzae (Meats et al., 2003). In this sample from Greece, the int lineage, int1, similar to that of ICEHin1056 strongly associates with H. influenzae and is not found in H. parainfluenzae. However, the associated four non-int genes (orf1, orf11, orf43 and orf51) do not consist of two divergent lineages and appear to be part of the same gene pool undergoing frequent recombination, including with the two distinct int lineages. This is inferred from the lack of congruence in the topology of the neighbour-joining trees for each of the individual gene alignments, see Fig. 3. The relevance of the int1 lineage associating with H. influenzae is unclear and a better understanding may arise from population studies of larger collections of isolates from divergent geographical regions to see if this finding is largely unique to these Greek isolates. Another explanatory possibility that could be investigated in laboratory studies is a possible fitness advantage int1 possessing ICEs have for H. influenzae over H. parainfluenzae.
Given the sequence diversity found among these Greek isolates, how did resistance emerge in the 1970s? The findings reported here are consistent with the hypothesis suggested by Laufs that ICEs existed as cryptic elements in the pre-antibiotic era and then became ‘visible’ in the clinical laboratory when they acquired transposons (e.g. Tn3 and Tn10) that encoded antibiotic resistance (Jahn et al., 1979; Laufs et al., 1981). Whether this occurred independently in different parts of the world or whether there has been pandemic spread of ICEs worldwide from a few common sources can now be addressed. The former possibility would be apparent were ICE DNA sequence diversity geographically structured in the same way as found for Helicobacter pylori (Falush et al., 2003); while the latter would be associated with an even distribution of ICE sequence types worldwide, namely, a lack of geographical structure. This question can now be answered on collections of isolates from around the world using the techniques described in this report combined with more advanced phylogenetic analysis.
As these Haemophilus resistance ICEs have recently been shown to be related to a large family of syntenic genomic islands with deep evolutionary origins found among Proteobacteria (Mohd-Zain et al., 2004), these findings emphasize the association between antibiotic resistance genes or structures that evolved well before the antibiotic era. Though this has been recognized for resistance genes themselves (D'Costa et al., 2006; Levy, 2006), it has not been so apparent for the vectors. The family of Haemophilus ICEs clearly have distant evolutionary origins and this reinforces the deep origins of bacterial genes encoding and spreading antibiotic resistance.
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ACKNOWLEDGEMENTS
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We thank Dr Basiliki Arvanitidou, Dr Diogenis Spathopoulos and Dr Anastasia Grapsa for helping collect the strains. I. D. D. was funded by the Democritus University of Thrace and D. W. C. was on a Wellcome Trust leave fellowship for part of the work.
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INTRODUCTION
METHODS
