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Journal of Clinical Microbiology, March 2000, p. 977-986, Vol. 38, No. 3
Wellcome Trust Centre for the Epidemiology of
Infectious Disease, Department of Zoology, University of Oxford, Oxford
OX1 3FY, United Kingdom
Received 21 July 1999/Returned for modification 13 November
1999/Accepted 27 November 1999
Multilocus sequence typing was used to characterize isolates of the
major Spanish clones of penicillin-resistant and
multiple-antibiotic-resistant Streptococcus pneumoniae.
Isolates of the multidrug-resistant Spanish serotype 23F clone and
serotype variants of this clone either had identical allelic profiles
or their allelic profiles differed from this typical allelic profile at
only one of the seven housekeeping loci. Similarly, isolates of the
Spanish serotype 6B and 14 clones and the penicillin-resistant serotype
9V clone (and serotype variants of this clone) each had the same
allelic profiles or profiles that differed at a single locus.
Multilocus sequence typing therefore allows resistant pneumococci to be
assigned to the Spanish clones if they have the typical allelic profile of the clone or if their profiles differ from that profile at a single
locus. A few resistant isolates that had allelic profiles typical of
that of a Spanish clone or whose profiles differed from that of the
typical profile at only a single locus possessed penicillin-binding
protein pbp1a, pbp2b, or pbp2x
genes that differed from those that are characteristic of the clone. In
most cases these isolates could be assigned as variant members of the
clone. Since almost all serotype 9V isolates have very similar
genotypes, independently emerging penicillin-resistant clones of this
serotype will inevitably appear to be similar by molecular typing
procedures. Analysis of the pbp genes, in addition to
multilocus sequence typing (or any other molecular typing procedure),
is therefore required to assign isolates unambiguously to the
penicillin-resistant Spanish serotype 9V clone.
Spain was among the first countries
to report a high incidence of antibiotic-resistant Streptococcus
pneumoniae (3). Penicillin-resistant and
multidrug-resistant isolates were first detected in the late 1970s, and
their prevalence increased rapidly during the 1980s to reach a peak in
1989 of about 44% of invasive isolates with penicillin resistance
(15). Since 1989, the incidence of penicillin-resistant pneumococci (MICs, Multidrug-resistant clones of serotypes 23F and 6B and a
penicillin-resistant clone of serotype 9V have been common in Spain since the 1980s (10, 12, 35), and more recently, a
multidrug-resistant serotype 14 clone has been identified
(5). According to the newly recommended nomenclature, the
latter clones are now designated Spain23F-1,
Spain6B-2, France9V-3 (as this clone may have
emerged within France), and Spain14-5. The first three
clones have been detected in many countries and on different continents
and can be considered globally distributed (2, 19, 20, 25, 26, 28,
29, 31, 34).
Studies from other countries that have a high incidence of
penicillin-resistant pneumococci have identified additional distinctive clones which have presumably emerged within these countries (17, 28, 30). In addition to the increasing number of
penicillin-resistant clones that are being recognized, variants of the
major clones that differ in serotype are commonly observed. For
example, serotype 19F variants of the Spain23F-1 clone
(4) and serotype 14 variants of the France9V-3
clone (2) are now widely disseminated (7-9, 19).
Penicillin-resistant isolates are assigned to the same clone if they
have identical or very similar overall genotypes and identical altered
forms of the penicillin-binding protein pbp1a, pbp2b, and pbp2x genes (10). Allelic
variants of these pbp genes are usually assigned by gene
fingerprinting (4), but a number of different methods have
been used to compare the overall relatedness of resistant pneumococci
(19, 26, 29, 31, 36). As increasing numbers of
penicillin-resistant clones are defined and as the genotypes of clones
diversify and their serotypes change, the assignment of isolates to
these clones becomes more difficult and a method is required that can
unambiguously assign resistant isolates to known clones or new clones
without the need for laboratories to exchange reference strains.
Multilocus sequence typing (MLST) is a procedure that fulfils these
criteria as it produces unambiguous data that can be transmitted electronically via the Internet (24, 32). By this procedure the nucleotide sequences of ~450-bp internal fragments of seven housekeeping genes are determined for each isolate. The different sequences at each locus are assigned as distinct alleles, and for each
isolate, the alleles at the seven loci define the allelic profile or
sequence type. MLST can resolve billions of pneumococcal genotypes, and
the probability that two isolates will have the same allelic profile by
chance is extremely low (11). The method is highly portable,
as any laboratory can compare the sequences at the seven loci in their
isolates with those in a central database on the World Wide Web
(http://mlst.zoo.ox.ac.uk) and can obtain the allelic profile of each
isolate. The relationship of these isolates to the known
penicillin-resistant clones can then be established by comparing these
allelic profiles with those in the central database, which contains the
allelic profiles of the known penicillin-resistant clones (11, 28,
32).
We demonstrate that most isolates of each of the major Spanish clones
have the same allelic profile or have a profile that differs from this
typical allelic profile at a single locus, allowing resistant
pneumococci to be assigned unambiguously to these clones via the Internet.
Pneumococcal isolates.
The pneumococci included in this
study were multiple-antibiotic-resistant pneumococci from Spain
(5), penicillin-resistant pneumococci from Taiwan
(28), and multiple-antibiotic-resistant serotype 6B isolates
assigned to the Spain6B-2 clone by Hermans et al.
(19). In addition, we studied a collection of 429 isolates
from patients who recently had cases of invasive disease, including 274 isolates from eight countries described by Enright and Spratt
(11) and 106 isolates from Spain (12). These
isolates have the prefix M. We also searched the MLST database, which
includes the allelic profiles of over 700 pneumococci.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of the Major Spanish Clones of
Penicillin-Resistant Pneumococci via the Internet Using Multilocus
Sequence Typing
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
0.1 µg of penicillin/ml) has remained fairly constant, with penicillin resistance occurring in about 40% of all
isolates (16).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Genetic relatedness of isolates. The nucleotide sequences of ~450-bp internal regions from the aroE, ddl, gdh, gki, recP, spi, and xpt genes were amplified by PCR with the primers described previously (11). Both strands of the gene fragments were directly sequenced by using the primers that were used for the PCR and an ABI 377 Prism automated sequencer with BigDye terminators (Perkin-Elmer Applied Biosystems, Foster City, Calif.). For each isolate, the allelic profiles were obtained from the sequences at the seven loci, and the allelic profiles were compared with those in our pneumococcal MLST database by using the software at the pneumococcal MLST website (http://mlst.zoo.ox.ac.uk). The relatedness among isolates was visualized as a dendrogram, constructed from the matrix of pairwise differences between the allelic profiles by using the unweighted pair group method with arithmetic averages (UPGMA).
Analysis of pbp genes. The pbp1a, pbp2b, and pbp2x genes were amplified from chromosomal DNA by PCR with the primers and conditions described previously (5). The pbp gene fragments were gel purified with a Qiagen QIAEX II Agarose gel extraction kit (Qiagen Ltd., Crawley, United Kingdom), and 50 ng of the purified fragments was used in a second 50-µl PCR mixture (25 cycles) that contained 0.5 µg of each primer (as described above), deoxynucleoside triphosphates (100 µM), rhodamine (R110)-labeled dUTP (1 µM), PCR buffer (Qiagen), and 2.5 U of Qiagen Taq DNA polymerase. The fluorescently labeled pbp gene fragments were ethanol precipitated, resuspended in 40 µl of distilled water, digested for 1 h with AluI (5 U; total reaction volume, 50 µl), ethanol precipitated, and resuspended in 30 µl of distilled water. One microliter of the digested fragments was mixed with 2.3 µl of loading buffer containing TAMRA-labeled GeneScan-500 internal molecular size markers (Perkin-Elmer Applied Biosystems), and the fragments were fractionated on a 4% denaturing polyacrylamide gel with an ABI Prism 377 DNA sequencer with virtual filter set C. The DNA fragment patterns were compared by using Genescan Analysis software (PE Applied Biosystems).
The fingerprints obtained by AluI digestion of the pbp genes of from isolates assigned by MLST to the major resistant Spanish clones were compared to the fingerprints obtained by AluI digestion of pbp genes from previously characterized reference isolates of each clone (5). Those with identical AluI digestion fingerprints were assigned the same allele numbers as those reported previously (5). Isolates that had pbp fingerprints which differed from those typical of the clone were assigned new allele numbers. As these variant pbp fingerprints were not compared with the AluI digestion fingerprints of each of the many alleles identified previously (5), the variant alleles were assigned roman (rather than arabic) numerals.| |
RESULTS |
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Materials and Methods
Results
Discussion
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Analysis of isolates of the Spain23F-1 clone.
Previously characterized isolates of the Spain23F-1 clone
(5), including serotype 19A and 19F variants of this clone
(6, 7), were analyzed by MLST. All 16 of these isolates had
identical allelic profiles (profile 4-4-2-4-4-1-1) except for one
isolate (isolate C85) whose profile differed from this profile (Table 1). Additional isolates with the same
allelic profiles and isolates whose profiles differed from this typical
profile at a single locus were identified among a collection of 74 penicillin-resistant isolates from hospitals in Taiwan (28).
Among these Taiwanese isolates, 16 had the typical allelic profile (11 were of serotype 23F and 5 were of serotype 19F), and a further 5 isolates (all of serotype 23F) had identical profiles (profile
4-4-2-4-6-1-1) which differed from the typical allelic profile only at
the spi locus. All 21 of the Taiwanese isolates had the same
pbp1a, pbp2b, and pbp2x gene
fingerprints as the Spain23F-1 clone and the characteristic
antibiotic resistance profile of the Spain23F-1 clone
(28).
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Analysis of isolates of the Spain6B-2 clone.
The
allelic profiles of three previously characterized isolates of the
Spain6B-2 clone were identical (profile 5-6-1-2-6-3-4)
(Table 2). These isolates have been shown
to be indistinguishable by multilocus enzyme electrophoresis (MLEE) (16 loci were examined) and by pbp1a, pbp2b, and
pbp2x gene fingerprinting (5). One further
isolate (isolate SP3026) that has been assigned to the
Spain6B-2 clone also had the allelic profile mentioned
above.
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Analysis of isolates of the France9V-3 clone.
A
reference isolate (isolate SP665) of the France9V-3 clone
(4) had allelic profile 7-11-10-1-6-8-1 (Table
3). A second reference strain from France
(strain TL7/1993 [22]) had the identical allelic
profile. Fourteen isolates with this allelic profile were found among
the collection of 429 invasive isolates (those with an M prefix). All
of these isolates were resistant to penicillin. Nine were serotype 9V
and had the same pbp1a, pbp2b, and
pbp2x gene fingerprints as the reference isolates of the
France9V-3 clone. Two further penicillin-resistant serotype
9V isolates from Poland in the MLST database (isolates 181 and 337)
also had the same allelic profile and pbp gene fingerprints
as the France9V-3 clone.
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Analysis of isolates of the Spain14-5 clone.
Eighteen previously characterized isolates of the Spain14-5
clone were analyzed. All of these isolates have been shown to be closely related in terms of their overall genotypes and to have identical pbp1a, pbp2b, and pbp2x gene
fingerprints (5). Fourteen had the same allelic profile
(profile 1-5-4-11-9-3-16), three were identical single-locus variants
(profile 1-5-4-18-9-3-16), and one was a different single-locus variant
(profile 1-5-4-4-9-3-16) (Table 4).
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1 µg/ml), and although
their pbp genes were not examined, they are very unlikely to
be members of the Spain14-5 clone.
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DISCUSSION |
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Materials and Methods
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The identification of the major clones, minor clones, and unique isolates among a collection of penicillin-resistant S. pneumoniae isolates that have been recovered within a community over a short time period is usually relatively straightforward. Many molecular typing procedures can successfully identify the clusters of closely related isolates that indicate the predominant penicillin-resistant clones in such populations (10, 21, 35). However, determination of whether these clones are novel or correspond to penicillin-resistant clones that have been described previously is more problematic. The difficulty arises from the fact that several of the major penicillin-resistant clones probably arose in the 1970s, and as they have spread their genotypes have diversified and their serotypes have changed so that they are no longer uniquely defined by any of the molecular typing procedures that are commonly used.
A second problem arises because penicillin resistance has emerged predominantly in a small number of serotypes, typically those that are commonly carried by children (serotypes 6B, 9V, 14, 19F, 23F [27]), and although isolates of the same serotype are often only distantly related in terms of overall genotype (11), most isolates of a few serotypes (e.g., serotypes 9V and 7F) appear to have very similar genotypes (11, 18, 23, 33). The genotypes of two penicillin-resistant clones of one of the latter serotypes that emerge independently will therefore almost inevitably appear to be closely related. The similarity of genotypes therefore does not always imply that two penicillin-resistant isolates are descended from the same ancestral resistant isolate.
MLST provides a new way of defining the genotype of a penicillin-resistant isolate as a string of integers, the alleles at each of the seven housekeeping loci. Comparison of allelic profiles is less ambiguous than comparison of patterns of DNA fragments on agarose gels, but as with all molecular typing procedures, there can be some ambiguity in assigning penicillin-resistant isolates as members of a clone, as variants of the clone, or as genetically similar isolates that have emerged independently. This problem can largely be circumvented by analyzing the pbp1a, pbp2b, and pbp2x genes, as well as the overall genotype, as it is extremely unlikely that independently arising penicillin-resistant clones will have acquired indistinguishable mosaic pbp genes.
A major advantage of MLST is that the allelic profiles of pneumococcal isolates, including reference isolates of all of the known penicillin-resistant and multidrug-resistant clones, together with epidemiological data, can be stored in a single central database which can be interrogated remotely via the Internet (32). MLST can be used to identify the major clones among large collections of resistant pneumococci from a geographic region (28), but for many laboratories this may not be practical, and in this case MLST can be applied to a single example of each clone identified by other methods (e.g., pulsed-field gel electrophoresis). By using software available on the pneumococcal MLST website (http://mlst.zoo.ox.ac.uk), the sequences at the seven loci can be used to identify whether each of the identified penicillin-resistant clones is already known or whether the sequences define new clones. The allelic profiles of the isolates and the associated epidemiological data can be deposited in the pneumococcal database on the MLST website to provide a tool for global epidemiology and surveillance.
This paper addresses the variation in allelic profiles that is found among the Spanish clones of penicillin-resistant pneumococci (which are probably among the oldest and which are certainly the most widely disseminated of such clones), as this determines the ease with which they can be identified unambiguously by MLST.
Previously characterized isolates of each penicillin-resistant clone had the same allelic profile (the typical profile) or their profiles differed from this profile at only a single locus. For example, all isolates with the typical allelic profile of the Spain23F-1 clone and all single-locus variants expressed high-level resistance to penicillin and had the antibiotic resistance profile and the typical pbp gene fingerprints of this clone. The allelic profiles of none of the isolates in the database differed from the typical allelic profile of the Spain23F-1 clone at two loci, and all the isolates whose profiles differed at three loci were penicillin susceptible. The absence from the MLST database of penicillin-susceptible isolates that are closely related in genotype to the Spain23F-1 clone suggests that the parental serotype 23F isolate from which the Spain23F-1 clone emerged may have had a rare genotype. The rarity of closely related genotypes allows an unambiguous identification of this clone by MLST, and a multidrug-resistant pneumococcus that has the typical allelic profile of the Spain23F-1 clone or whose profile differs from this profile at a single locus can confidently be assigned as a member of the Spain23F-1 clone.
Isolates of the Spain6B-2 clone that had the characteristic pbp gene fingerprints of this clone also had identical allelic profiles or profiles that differed from this profile at a single locus. However, in contrast to the Spain23F-1 clone, which had uniform pbp gene fingerprints, there was some variation among isolates of the Spain6B-2 clone in terms of their pbp2b fingerprints. Furthermore, one isolate (isolate GM41) whose allelic profile differed from the typical allelic profile of the Spain6B-2 clone at only a single locus had pbp1a, pbp2b, and pbp2x genes that were distinct from those found in this clone. This isolate was also very closely related to the Spain6B-2 clone by MLEE (5) and, on the basis of the criterion of genetic relatedness, would be assigned to the Spain6B-2 clone, although the differences in all three pbp genes suggest that it is not a member of the clone. This is probably an example of the independent emergence of penicillin resistance in a serotype 6B isolate that had a genotype similar to that of the Spain6B-2 clone.
Some of the penicillin-resistant serotype 6B isolates that we examined by MLST had been identified as members of the Spain6B-2 clone by using restriction fragment end labeling (19). Although most of these isolates were confirmed to be members of the Spain6B-2 clone by MLST, two isolates were clearly not members of this clone. These isolates differed from the Spain6B-2 clone at three of seven and six of seven loci, respectively, as determined by MLST and had completely different alleles at the pbp1a, pbp2b, and pbp2x genes (19). The six resistant serotype 6B isolates from Thailand were identical by MLST and were single-locus variants of the Spain6B-2 clone. These isolates were previously correctly identified by Hermans et al. (19) as members of the Spain6B-2 clone, but by restriction fragment end labeling, they were scattered across the part of the dendrogram which included the members of this clone. MLST was therefore able to identify in Thailand a distinctive variant of the Spain6B-2 clone which was not apparent by a method that depends on computer-assisted comparisons of DNA fragment sizes on gels. The identification of one multidrug-resistant serotype 6B isolate from Taiwan with the same allelic profile as the isolates from Thailand suggests that this distinctive variant of the Spain6B-2 clone has spread within Southeast Asia and also demonstrates the power of MLST to identify unambiguously a single isolate whose allelic profile matches a particular allelic profile within a database of any size.
The definition of the France9V-3 clone is likely to be slightly problematic by any typing procedure since the genotypes of all serotype 9V isolates (penicillin susceptible or penicillin resistant) appear to be closely related (11, 18), and this was the only Spanish resistant clone where there were penicillin-susceptible isolates which had allelic profiles that differed from those of the resistant clones at a single locus. Furthermore, three serotypes were represented and there was a poor congruence between serotype and genotype among the cluster of isolates that are most similar to the France9V-3 clone (Fig. 2), suggesting that changes of serotype may been more common among these isolates than among those of most other pneumococcal lineages (11). Examination of the pbp gene fingerprints is therefore essential for distinguishing isolates of the France9V-3 clone from genotypically similar penicillin-resistant isolates (e.g., isolate M295) that have emerged independently.
With one exception, all of the allelic variants of the France9V-3 clone that possessed allele 14 at the ddl locus were penicillin susceptible, whereas those with allele 1 were penicillin resistant. The ddl locus is known to be 783 bp downstream of pbp2b, and recombinational exchanges that replace the normal pbp2b gene of penicillin-susceptible isolates with that from related streptococcal species or from another penicillin-resistant pneumococcus often extend into or through ddl (13). A likely scenario is that a penicillin-susceptible serotype 9V clone (allelic profile 7-11-10-1-6-8-14) was the progenitor of the France9V-3 clone (allelic profile 7-11-10-1-6-8-1) and that, during the emergence of penicillin resistance, the recombinational exchange that introduced the mosaic pbp2b gene (probably from an isolate of the Spain23F-1 clone [4]) extended into ddl to result in a change from allele 14 to allele 1. The one exceptional isolate, which possessed ddl allele 14 but which was penicillin resistant, almost certainly emerged independently, as it possessed pbp1a, pbp2b, and pbp2x genes that differed from those of the France9V-3 clone.
Penicillin-resistant isolates that have the allelic profile 7-11-10-1-6-8-1 are clearly assigned as members of the France9V-3 clone. Penicillin-resistant isolates that differ from the typical allelic profile at a single locus are also likely to be members of the clone (although caution is required if they have a different ddl allele), especially if they are susceptible to chloramphenicol and tetracycline and resistant to trimethoprim-sulfamethoxazole. Comparison of the pbp gene fingerprints to those of a reference isolate of the France9V-3 clone is required for a definitive assignment to the clone.
All previously characterized isolates of the Spain14-5 clone that had the characteristic pbp gene fingerprints of this clone were identical by MLST or differed from the typical allelic profile at a single locus. Penicillin-resistant isolates can therefore be assigned as members of the Spain14-5 clone if they have the typical allelic profile of this clone or have a profile that differs from the typical profile at a single locus.
It is interesting that the allelic profiles of the penicillin-resistant and multidrug-resistant Spanish clones are not uniform and that the profiles of about 25% of the members of the Spanish clones differ from those of typical isolates of the Spanish clones at one of the seven housekeeping genes. The common ancestors of these clones must have emerged very recently on an evolutionary time scale and have certainly emerged since the introduction of penicillin in the 1940s, and it might be expected that the profiles of members of these very young clones would be identical at these neutral loci. The relatively rapid rate of clonal diversification in the resistant clones, which will make it increasingly difficult to define them unambiguously by MLST or any other molecular typing procedure, has been shown to be due to high rates of recombinational exchanges in the naturally transformable pneumococcus (14).
In conclusion, penicillin-resistant and multidrug-resistant isolates can be assigned to the major penicillin-resistant and multidrug-resistant Spanish clones by MLST if they have the typical allelic profile of the clone or if their profiles differ from the typical profile at a single locus. However, definitive assignment to a clone by MLST (or any other typing procedure) may require an analysis of their pbp1a, pbp2b, and pbp2x genes.
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ACKNOWLEDGMENTS |
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This work was supported by the Wellcome Trust. B.G.S. is a Wellcome Trust Principal Research Fellow.
We thank Tracey Coffey for allowing the use of unpublished data, David Griffiths and the Oxford Vaccine Group for serotyping, and Marcel Sluijter for providing pneumococcal isolates.
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FOOTNOTES |
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* Corresponding author. Mailing address: Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, South Parks Rd., Oxford OX1 3FY, United Kingdom. Phone: 44 1865 281301. Fax: 44 1865 281891. E-mail: brian.spratt{at}ceid.ox.ac.uk.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, March 2000, p. 977-986, Vol. 38, No. 3
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