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Journal of Clinical Microbiology, December 1998, p. 3514-3519, Vol. 36, No. 12
School of Biological Sciences,
Received 15 July 1998/Returned for modification 3 September
1998/Accepted 24 September 1998
In this paper we demonstrate the advantages of a new molecular
typing procedure, multilocus sequence typing, for the unambiguous characterization of penicillin-resistant pneumococci. The sequences of
~450-bp fragments of seven housekeeping genes were determined for 74 penicillin-resistant Taiwanese isolates of Streptococcus pneumoniae (MIC of penicillin > 0.5 µg/ml). The
combination of alleles at the seven loci defined an allelic profile for
each strain, and a dendrogram, based on the pairwise mismatches in allelic profiles, grouped 86% of the isolates into one of three penicillin-resistant clones for which the MICs of penicillin were 1 to
2 µg/ml. Isolates within each clone had identical alleles at all
seven loci or differed at only a single locus, and the fingerprints of
their pbp1A, pbp2B, and pbp2X genes
were uniform. Isolates of the Taiwan-19F clone and the Taiwan-23F clone
were resistant to penicillin, tetracycline, and erythromycin but were susceptible to chloramphenicol. A second serotype 23F clone and serotype 19F variants of this clone were resistant to penicillin, tetracycline, chloramphenicol, and, in some cases, erythromycin. Comparisons of the allelic profiles of the three major clones with
those of reference isolates of the known penicillin-resistant clones
showed that the Taiwan-19F and Taiwan-23F clones were previously undescribed, whereas the second serotype 23F clone was
indistinguishable from the Spanish multidrug-resistant serotype 23F
clone. Single isolates of the Spanish penicillin-resistant serotype 9V
clone and the Spanish multidrug-resistant serotype 6B clone were also identified in the collection.
Isolates of Streptococcus
pneumoniae with intermediate-level penicillin resistance (MIC Molecular characterization of penicillin-resistant pneumococci requires
an assessment of the overall genetic relatedness of the isolates and
the relatedness of their penicillin-binding protein (pbp)
genes (6). Several different techniques, including
multilocus enzyme electrophoresis (20, 21, 24, 26),
pulsed-field gel electrophoresis (18, 23, 26), PCR with
repetitive element primers (9, 19, 31), and restriction
fragment end labeling (11), have been used to assess the
genetic relatedness of isolates; analysis of pbp genes is
normally carried out by high-resolution fingerprinting of the
PCR-amplified pbp1A, pbp2B, and pbp2X
genes (5). By these approaches, penicillin-resistant
isolates that are identical (or very similar) in overall genotype and
which have the same allelic forms of the three pbp genes can
be assigned as members of the same clonal group.
These molecular typing studies have identified a number of clones of
highly penicillin-resistant pneumococci, some of which have spread
globally (6, 30). The most extensively studied clones are
those that appear to have emerged within Spain (5, 7, 9, 11, 20,
24, 26). Predominant among these are the major Spanish
multidrug-resistant serotype 6B (26), 14 (7), and
23F (20) clones (MMSp6B, MMSp14, and MMSp23F, respectively) and the major penicillin-resistant serotype 9V clone
(MPSp9V) (5, 24). Variants of these clones that differ in
serotype have also emerged (5, 9, 11, 22) by recombinational exchanges at the capsular biosynthetic locus (8), and some of these have become prevalent (e.g., the serotype 19F variant of the
MMSp23F clone [7]). Other clones have been found to predominate in countries that have a high incidence of
antibiotic-resistant pneumococci, e.g., Hungary (21),
Slovakia (23), South Africa (25), and parts of
the United States (18).
The currently used methods for assessing the overall relatedness of
resistant isolates each have advantages and disadvantages but are
generally adequate for a local epidemiology study, in which a
laboratory typically wishes to identify clusters of identical isolates
(clones) within a local population of penicillin-resistant pneumococci.
However, these methods are less satisfactory when a laboratory wishes
to establish whether an identified penicillin-resistant clone is unique
to their locality or has been identified previously in other countries
(global epidemiology). For global epidemiology, the currently used
molecular typing methods produce data that are poorly portable between
laboratories as the methods are predominantly based on comparisons of
DNA fragment patterns on agarose gels.
We recently developed a procedure that provides a highly portable
approach to global epidemiology, multilocus sequence typing (MLST)
(17). In this procedure the sequences of ~450-bp internal fragments of seven housekeeping genes are obtained for each bacterial isolate and the alleles at each of the seven loci define an allelic profile or sequence type. MLST therefore uses the well-established principles of multilocus enzyme electrophoresis but assigns alleles directly via DNA sequencing, rather than indirectly from the
electrophoretic mobilities of their gene products. A great advantage of
MLST is that sequence data are unambiguous and electronically portable, so that any laboratory can compare the allelic profiles of their isolates with those held in a central MLST database on the World Wide
Web via the Internet. MLST was originally described and validated for
meningococci (17) but has subsequently been developed and validated for pneumococci (10). In this paper we show how
MLST can be used to identify clusters of related isolates (clones) among a population of penicillin-resistant pneumococci and to compare
the resistant isolates with those found in other countries.
Pneumococcal isolates.
The 74 penicillin-resistant isolates
were recovered from patients in six hospitals in Taiwan between 1993 and 1997 (Table 1). Of the isolates, 33 were from Taichung Veterans
General Hospital (TCVGH), 26 were from Mackay Memorial Hospital (MMH),
9 were from Taipei Veterans General Hospital (TPVGH), and 2 each were
from Hsinchu Hospital (HCH), Cathay General Hospital (CGH), and
Kaohsiung Veterans General Hospital (KSVGH). A total of 19 isolates
(26%) were from blood, cerebrospinal fluid (CSF), or pleural effusion; 26 isolates (35%) were from sputum; and the remainder were mostly from
pus, the nasopharynx, or the middle ear. The isolates from TCVGH and
MMH represented all of the penicillin-resistant isolates for which MICs
were >0.5 µg/ml obtained, predominantly from patients with disease,
during 1993 to 1997 and the first half of 1997, respectively. Those
from the other hospitals were a random selection of similar isolates
provided to us by C.-P. Fung. Serotyping was carried out by the
quellung reaction with antisera from the Statens Serum Institut,
Copenhagen, Denmark. MICs were determined by the E test (AB Biodisk,
Solna, Sweden).
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 using the primers described
previously (10). The gene fragments were sequenced on both
strands, by using the same primers, on an ABI 377 Prism automated
sequencer with dRhodamine-labeled terminators (PE Applied Biosystems).
For each gene, the sequences were compared with each other and with
those in our pneumococcal MLST database (http://mlst.zoo.ox.ac.uk) by
using the Macintosh computer program Sequence Output
(http://epunix.biols.susx.ac.uk/biols/Biochem/Molbiol/). Sequences
were assigned as known alleles if they were identical to alleles in our
database or as new alleles if they differed in sequence from any of the
known alleles. No weighting was given to the degree of sequence
divergence between alleles, since, in the absence of knowledge of the
proportion of allelic changes that are due to recombination rather than
mutation, we cannot say that alleles differing at many sites are any
more distantly related than those that differ at a single site. The
alleles at each of the seven loci defined the allelic profile, or
sequence type, and the relatedness of isolates was determined by
constructing a tree by the unweighted pair group method with arithmetic
means (UPGMA) from the matrix of pairwise differences between the
allelic profiles by using Statistica software (StatSoft, Tulsa, Okla.), as described elsewhere (10, 17).
Analysis of pbp genes.
Fingerprints of the
pbp1A, pbp2B, and pbp2X genes were
obtained as described previously (5), except that DNA
fragments were detected on polyacrylamide gels by staining with
ethidium bromide rather than by end labeling and autoradiography. For
each pbp gene, isolates that produced identical patterns of
DNA fragments were assigned the same allele number (7).
Nucleotide sequence accession numbers.
The nucleotide
sequences described in the present work have been assigned GenBank
accession no. AJ233886 and AJ233896.
Properties of penicillin-resistant isolates.
All 74 strains
were resistant to >0.5 µg of penicillin/ml, and all but one of them
(TW47) were multidrug resistant (Table 1). Resistance to
erythromycin (MIC
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Three Major Clones of Multiply
Antibiotic-Resistant Streptococcus pneumoniae in
Taiwanese Hospitals by Multilocus Sequence Typing
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
0.12 µg/ml) or high-level resistance (MIC 2 µg/ml) have
been reported from many countries in recent years (1, 6,
30). In an increasing number of countries a high proportion of
pneumococcal isolates from carriers and patients with disease are
penicillin resistant or multiply antibiotic resistant (1,
30).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 µg/ml), tetracycline (MIC 8 µg/ml), and chloramphenicol was found in 97, 97, and 31% of the
isolates, respectively.
TABLE 1.
Properties of 74 Taiwanese
penicillin-resistant pneumococcia
Analysis of relatedness of isolates. The sequences of the seven housekeeping gene fragments were obtained from all 74 resistant isolates, and a dendrogram was constructed from the matrix of pairwise differences in their allelic profiles (Fig. 1). Sixteen different allelic profiles were distinguished among the 74 isolates, and, at a genetic distance of 0.2, there were three major clusters of isolates, which together included 65 of the isolates. The largest cluster (cluster 1) included 29 serotype 19F isolates, of which 28 had indistinguishable pbp1A, pbp2B, and pbp2X gene fingerprints (pattern A-A-A). All of these 29 isolates had the same MLST allelic profile (15-16-19-15-6-20-26) or differed from this profile at a single locus, and they were defined as the Taiwan-19F clone (Table 1). TW19 had the normal allelic profile but differed in having a variant pbp2X gene. Five of the six single-locus variants were identical and differed from the typical allelic profile at the ddl locus. The other single locus variant (TW14) differed at the recP locus. One other serotype 19F isolate (TW5) clustered with the Taiwan-19F clone, but it appeared to be less closely related as it had a variant pbp2B gene and also differed from the normal allelic profile at two of the seven housekeeping loci. All 29 isolates of the Taiwan-19F clone (and TW5) were resistant to penicillin (MICs, 0.75 to 2 µg/ml), tetracycline (MICs, 12 to 32 µg/ml), and erythromycin (MICs, 1.5 to 8 µg/ml). They were all susceptible to chloramphenicol (MICs < 4 µg/ml).
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1 µg/ml), and 4 isolates with
the typical allelic profile and all 5 isolates of the single-locus
variant were highly resistant (MIC > 256 µg/ml).
Cluster 3 contained 15 closely related isolates, which were all
serotype 23F, except strain TW80, which was serotype 19F. Fourteen
isolates gave indistinguishable pbp1A, pbp2B, and
pbp2X gene fingerprints (pattern A-C-B); had the same
allelic profile (15-29-4-21-30-1-14), except TW4, which differed at the
spi locus; and were defined as the Taiwan-23F clone (Table
1). All of these isolates were resistant to penicillin (MICs, 0.75 to 2 µg/ml), erythromycin (MICs > 256 µg/ml), and tetracycline
(MICs, 24 to 64 µg/ml) but were susceptible to chloramphenicol
(MICs 
4 µg/ml). The other strain in this cluster (TW47) was
considered a variant of the Taiwan-23F clone as it differed from the
typical allelic profile at only a single locus (ddl), but it
had a variant pbp2B gene fingerprint and was susceptible to
erythromycin and tetracycline (Table 1).
Two highly penicillin-resistant serotype 23F isolates (MICs, 1 to 2 µg/ml) from different hospitals had identical allelic profiles
(10-13-6-1-38-1) and identical pbp2B and pbp2X
gene fingerprints (their pbp1A genes could not be amplified
with our normal primers). Both isolates were resistant to erythromycin
and tetracycline but were susceptible to chloramphenicol. The other six
penicillin-resistant isolates each had unique allelic profiles.
Relationship of Taiwanese penicillin-resistant isolates to those from other countries. The allelic profiles of the 74 Taiwanese isolates were compared with those in our MLST database, which included isolates of the major Spanish penicillin-resistant and multidrug-resistant clones (MPSp9V, MMSp6B, MMSp14, MMSp15F, and MMSp23F), isolates of the highly cephalosporin-resistant serotype 23F U.S. clone (18), and the highly penicillin-resistant serotype 14 clone from Slovakia (23), as well as yet-undescribed resistant clones from South America and Eastern Europe and individual resistant isolates from a number of other countries. The Taiwan-19F and Taiwan-23F clones were not closely related to any of the previously characterized penicillin-resistant clones. However, two high-level penicillin-resistant serotype 19F isolates in our MLST database, recovered from blood cultures from patients with pneumonia in the same London hospital in 1995 (4 months apart), had the same allelic profile as the majority of the isolates of the Taiwan-19F clone (15-16-19-15-6-20-26).
The typical allelic profile of the serotype 19F and 23F isolates within cluster 2 was identical to that of the MMSp23F clone (4-4-2-4-4-1-1). As expected, the antibiotic resistance profile of these Taiwanese isolates and their pbp1A, pbp2B, and pbp2X gene fingerprints corresponded to those of a reference isolate of the MMSp23F clone (7). One serotype 6B isolate (TW25) was assigned by MLST to the MMSp6B clonal complex, and one serotype 9V isolate (TW50) was assigned to the MPSp9V clonal complex. In both cases the isolates had the characteristic pbp1A, pbp2B, and pbp2X gene fingerprints of the corresponding Spanish clones (7) and differed from their typical allelic profiles at only a single locus (Table 1). The antibiotic resistance profile of the Taiwanese isolate of the MMSp6B clone was typical of this clone (26). This single locus variant of the MMSp6B clone has also been found in Thailand (32). The Taiwanese isolate of the MPSp9V clone was resistant to penicillin and tetracycline, whereas members of this clonal complex are normally tetracycline susceptible. The four other penicillin-resistant isolates, which each had unique allelic profiles, were not closely related to strains in our MLST database.| |
DISCUSSION |
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Materials & Methods
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Discussion
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Penicillin-resistant pneumococci have increased dramatically in
frequency in the Far East, and several countries, including South
Korea, Japan, and Hong Kong, have recently reported resistance rates of
>40% (1, 4, 14-16, 27). The incidence of
antibiotic-resistant pneumococci in Taiwanese hospitals has also
increased greatly during the 1990s. In Taipei (northern Taiwan), <10%
of isolates from children with pneumococcal disease were penicillin
resistant in 1991 and 1992 (13), and in southern Taiwan,
12% of clinical isolates from all age groups were penicillin resistant
from 1990 to 1993 (12). However, in 1994 and 1995, 45% of
isolates from children in Taipei were penicillin-resistant
(13), and 71% of isolates from children in Kaohsiung were
penicillin resistant from 1995 to 1997, with 58% of these isolates
exhibiting high-level resistance (3). Resistance to
erythromycin and tetracycline is also particularly prevalent in Taiwan
(>60% of isolates [2, 3, 12]), and almost all
penicillin-resistant isolates are resistant to at least two other
classes of antibiotic (3, 12). In the present study, almost
all of the penicillin-resistant isolates were resistant to tetracycline
(MIC 8 µg/ml), and erythromycin (MIC 1 µg/ml), with
40% of all isolates being highly erythromycin resistant (MICs > 256 µg/ml). Resistance to chloramphenicol was 31% but, with one
exception, was restricted to isolates of the MMSp23F and MMSp6B clones
that have been imported into Taiwan.
We chose to use MLST to identify clusters of penicillin-resistant
pneumococci from Taiwan that are closely related in their overall
genotype. MLST provides a powerful method for the characterization of
penicillin-resistant pneumococci as it has a very high discriminatory power, which results from the presence of a large number of alleles at
each of the seven housekeeping loci (10). The average number of alleles per locus for the ~500 pneumococcal isolates currently in
the MLST database is >30, providing the ability to distinguish >307 (22 billion) genotypes. The expected frequency at
which any allelic profile will occur by chance can be estimated from
the products of the frequencies of each allele in the pneumococcal
population (10). For example, on the basis of allele
frequency data from our pneumococcal MLST database, the allelic
profiles of the three major penicillin-resistant clones in Taiwan would
each be expected to occur by chance at a frequency of
<10
10. The high discriminatory power therefore makes it
very unlikely that unrelated isolates will by chance have identical, or
even similar, allelic profiles (10).
In this study isolates that had the same allelic profile or that differed at one locus were considered to be members of the same clonal complex. According to this criterion there were three major multidrug-resistant clones among the 74 penicillin-resistant isolates from Taiwanese hospitals. This clustering of isolates by MLST was validated by the demonstration that (with minor exceptions) isolates within each cluster had the same pbp1A, pbp2B, and pbp2X gene fingerprints and serotypes. One isolate of the Taiwan-23F clone and five isolates of the MMSp23F clone were serotype 19F, and these presumably represent serotype variants of these clones. Serotype 19F variants of the MMSp23F clone are commonly encountered in Spain (7) and have also been reported in other countries, including South Korea (19) and Thailand (11).
The Taiwan-19F clone was the most prevalent (39% of isolates) and has been present in Taiwan since at least 1993. The Taiwan-23F clone (19% of isolates) has been present since 1994. Both of these clones were recovered from three hospitals in Taiwan, and they appear to differ from previously described penicillin-resistant clones. This suggests that they may have emerged within the Far East, where the incidence of antibiotic-resistant pneumococci is now very high. The Taiwan-19F clone appears to have already spread intercontinentally, as two penicillin-resistant serotype 19F isolates from the United Kingdom that were indistinguishable from this clone were identified in our MLST database (10). All of the isolates of the Taiwan-19F and Taiwan-23F clones were resistant to erythromycin, although the MICs for the isolates of the latter clone were >256 µg/ml, presumably due to the presence of the ermAM genes, which encode an rRNA methylase, whereas the MICs for the former clone were 1.5 to 8 µg/ml and may result from drug efflux due to mefE (28).
The 21 isolates within cluster 2 were unambiguously identified as members of the MMSp23F clonal complex by MLST combined with pbp gene fingerprinting. Most of these isolates had an allelic profile identical to those of isolates typical of the MMSp23F clonal complex, but there was also a distinctive highly erythromycin-resistant single-locus variant of this clone with a different allele of the spi gene within Taiwan. The latter isolates are clearly members of the MMSp23F clonal complex, as they have the characteristic pbp1A, pbp2B, and pbp2X gene fingerprints and serotype of this clone. Isolates of the MMSp23F clone in Taiwan were, with one exception, resistant to erythromycin, although some of them had high-level resistance characteristic of ermAM and others had the lower levels of resistance characteristic of drug efflux (28). The prevalence of the MMSp23F clone in Taiwan is not surprising since in recent years it has become established globally and has been previously reported to be present in the Far East (11, 19, 29). We also identified single isolates of both the MMSp6B clone and the MPSp9V clone within Taiwan. In each case, the isolates differed at a single locus from the typical allelic profile of these clones, but they were clearly members of the clones as they had the characteristic pbp gene fingerprint patterns and serotypes.
More than 90% of the 74 penicillin-resistant isolates from Taiwan were serotype 19F or 23F, and a similar predominance of these two serotypes is found among highly penicillin-resistant isolates from other countries in the Far East (4, 14, 16, 27). Comparisons of strains from these countries with those described here are needed to determine the contribution of the Taiwan-19F and Taiwan-23F clones and serotype 19F and 23F isolates of the MMSp23F clone to the high prevalence of multidrug-resistant pneumococci in the Far East.
This paper demonstrates that MLST provides an unambiguous method for identifying clones within populations of penicillin-resistant pneumococci and is particularly suitable for comparing isolates or clones with those described previously. Characterization of a clone of interest by MLST is straightforward and only requires that the seven housekeeping gene fragments be amplified by PCR and sequenced on both strands, by using a single primer for each direction. The sequences can then be submitted electronically to our MLST Web site, where they can be compared with those in the database and an allelic profile can be assigned. The allelic profile of a penicillin-resistant clone can then be compared with the allelic profiles in the database, which includes those of each of the known penicillin-resistant clones, making it simple to ascertain whether the clone of interest is a known or a previously undocumented penicillin-resistant clone, without the need to obtain reference strains. For definitive characterization, MLST or any other method for comparing the overall relatedness of penicillin-resistant isolates should be combined with an analysis of the pbp genes since penicillin resistance may emerge independently on more than one occasion in closely related lineages.
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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. Z.-Y.S. was supported by the TCHVGH.
We thank Chang-Phone Fung for providing strains and Derrick Crook and the Oxford Vaccine Group for assistance with serotyping.
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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, Oxford OX1 3PS, United Kingdom. Phone: 1865-281301. Fax: 1865-281891. E-mail: brian.spratt{at}zoology.oxford.ac.uk.
Present address: Section of Infectious Disease, Taichung Veterans
General Hospital, Taichung, Taiwan.
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Discussion
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Journal of Clinical Microbiology, December 1998, p. 3514-3519, Vol. 36, No. 12
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