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Journal of Clinical Microbiology, October 1999, p. 3210-3216, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Three Major Spanish Clones of
Penicillin-Resistant Streptococcus pneumoniae Are the Most
Common Clones Recovered in Recent Cases of Meningitis in
Spain
Mark C.
Enright,1
Asunción
Fenoll,2
David
Griffiths,3 and
Brian
G.
Spratt1,*
Wellcome Trust Centre for the Epidemiology of
Infectious Disease, Department of Zoology, University of Oxford, Oxford
OX1 3FY,1 and Department of
Microbiology, John Radcliffe Hospital, University of Oxford, Oxford
OX3 9DU,3 United Kingdom, and
Laboratorio de Referencia de Neumococos, Servicio de
Bacteriología, Centro Nacional de Microbiología,
Instituto de Salud Carlos III, 28220 Majadahonda, Madrid,
Spain2
Received 28 April 1999/Returned for modification 22 June
1999/Accepted 6 July 1999
 |
ABSTRACT |
One hundred six isolates of Streptococcus pneumoniae
recovered in Spain from patients with meningitis in 1997 and 1998 were characterized by multilocus sequence typing. A heterogeneous collection of genotypes was associated with meningitis in Spain: 65 different sequence types were resolved and, even at a genetic distance of 0.43, there were 37 distinct lineages. Thirty-eight percent of the isolates,
including all isolates of serotypes 6B, 9V, 14, and 23F, were resistant
to penicillin, and 24% of the isolates were members of the three major
Spanish penicillin-resistant or multidrug-resistant clones of serotypes
6B, 9V, and 23F or serotype variants of these clones. These three
clones (MICs, 1 to 2 µg of penicillin/ml) were the most common clones
associated with pneumococcal meningitis in Spain during 1997 and 1998. Only two of the other clones associated with meningitis were penicillin resistant (MICs, 0.12 to 0.5 µg/ml). One of the two most prevalent penicillin-susceptible clones causing meningitis (serotype 3) has not
been detected outside of Spain, whereas the other (serotype 18C) has
been recovered from patients with meningitis in the United Kingdom, The
Netherlands, and Denmark. The prevalence of meningitis caused by
isolates of the three major Spanish penicillin-resistant or multiply
antibiotic-resistant clones, which are now globally distributed, is
disturbing and clearly establishes their ability to cause
life-threatening disease.
| |
INTRODUCTION |
Spain was one of the first countries
to report a high prevalence of penicillin-resistant and multiply
antibiotic-resistant Streptococcus pneumoniae (3,
27). Penicillin resistance among invasive pneumococci from Spain
was first recognized in the 1970s, and the incidence of resistance
among these isolates has increased steadily, from 6% in 1979 to 44%
in 1989 (11). In the 1990s, 55% of invasive isolates from
children and 37% from adults are resistant to penicillin (MIC 
0.12 µg/ml), and 8.4% of all invasive isolates have high-level
penicillin resistance (MIC 2 µg/ml) (12). As in
other countries, penicillin resistance in Spain is predominantly found
among pneumococci of serogroups 6, 9, 14, 19, and 23 (12),
which are typically associated with carriage and disease in children
(21); almost all isolates of these serotypes from Spain are
currently resistant to penicillin (12).
Molecular studies have shown that three major clones of
penicillin-resistant pneumococci have been prevalent in Spain since the
1980s, and all of these have now spread globally (8, 14, 15, 18,
19, 22-24, 26). The major Spanish serotype 23F and 6B clones
(Spain23F-1 and Spain6B-2 in the newly
recommended nomenclature) are resistant to penicillin (MICs, 0.75 to 2 µg/ml), tetracycline, and chloramphenicol (19, 24). The
major Spanish-French penicillin-resistant 9V clone (now called
France9V-3, as this clone may have originated in France) is
resistant to penicillin (MICs, 0.75 to 2 µg/ml) and
trimethoprim-sulfamethoxazole but susceptible to tetracycline and
chloramphenicol (4, 13). In recent years, resistance to
penicillin has increased among isolates of serogroups 19 and 14 from
Spain (12), and this is due to the increase in frequency of
serotype 14 variants of the France9V-3 clone (2,
7) and serotype 19F variants of the Spain23F-1 clone
(5), as well as the emergence of a multiresistant serotype
14 clone (Spain14-5) (5).
The significance of penicillin resistance for most nonmeningeal
infections is doubtful, but pneumococcal meningitis caused by
penicillin-resistant isolates cannot reliably be treated with penicillin (9, 16). Currently, in Spain, approximately 43% of pneumococci from cerebrospinal fluid (CSF) are resistant to penicillin (12), causing serious problems in the treatment
of pneumococcal meningitis. In this paper we use multilocus sequence typing (MLST [10, 17, 25]) to characterize a
collection of 106 pneumococci obtained in recent cases of meningitis in
Spain and show that the Spain23F-1, Spain6B-2,
and France9V-3 clones are the three most common clones
causing meningitis. Approximately one in four cases of meningitis in
Spain is now caused by one of these penicillin-resistant clones.
| |
MATERIALS AND METHODS |
Bacterial strains.
All S. pneumoniae isolates
were from children or adults with pneumococcal meningitis and were
consecutive CSF isolates received during 1997 and 1998 from Spanish
hospitals by the Pneumococcal Reference Laboratory at the Instituto de
Salud Carlos III, Madrid, Spain. The isolates were serogrouped at the
Pneumococcal Reference Laboratory and were serosubtyped in Oxford,
England, using the Quellung reaction with sera obtained from the
Statens Seruminstitut, Copenhagen, Denmark. The serotypes of isolates
that were identical or closely related in genotype but which differed
in serotype were rechecked. The MICs of antibiotics were determined by
agar dilution according to the guidelines of the National Committee for
Clinical Laboratory Standards (20).
The 106 isolates obtained in recent cases of meningitis in Spain were
compared with 274 isolates from patients with invasive disease in eight
other countries (10), with reference isolates of each of the
known penicillin-resistant clones (28), and with all
isolates within the pneumococcal MLST database (3a).
MLST.
MLST was carried out as described previously
(10). Briefly, internal fragments of the aroE,
gdh, gki, recP, spi,
xpt, and ddl genes were amplified by PCR from
chromosomal DNA with the primer pairs described by Enright and Spratt
(10). The amplified fragments were directly sequenced in
each direction with the primers that were used for the initial
amplification and, after editing, the sequences at each locus were
compared with all of the known alleles at that locus. Sequences that
were identical to a known allele were assigned the same allele number,
and those that differed from any known allele, even at a single
nucleotide site, were assigned new allele numbers. The assignment of
alleles at each locus was carried out with the software at the
pneumococcal MLST web site (3a).
The alleles at each of the seven loci provide the allelic profile of
each isolate and also define their sequence type (ST). Allelic profiles
are shown as the alleles at each of the seven loci, in the order
aroE, gdh, gki, recP,
spi, xpt, and ddl (e.g., 4-4-2-4-4-1-1 is the typical allelic profile of the Spain23F-1 clone).
The allelic profiles of the Spanish isolates were compared with each
other, with other isolates from invasive disease (10), and
with all isolates in the pneumococcal MLST database, using the software
at the MLST website (see above). The relatedness among the isolates was
represented as a dendrogram, constructed by the unweighted pair group
method with arithmetic averages (UPGMA), from the matrix of pairwise
differences in the allelic profiles (10, 17).
Nucleotide sequence accession numbers.
The nucleotide
sequences reported here have been submitted to the EMBL and GenBank
databases under accession no. AJ 252518 to AJ 252549.
| |
RESULTS AND DISCUSSION |
Properties of the 106 pneumococci from patients with
meningitis.
Table 1 shows the
serotype distribution of 106 isolates obtained in recent cases of
pneumococcal meningitis. The distribution was similar to that found
among CSF isolates from Spain from 1990 to 1996 (12) except
that serotypes 11A and 33F, which were previously rare, appear to be
slightly overrepresented in our sample.
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TABLE 1.
Serotype distribution, penicillin resistance, and
diversity of isolates of S. pneumoniae from patients
with meningitis
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|
Forty of the isolates (38%) were resistant to penicillin, and eight of
these (7.5%) were highly resistant to penicillin (Table
2). All of the highly
penicillin-resistant isolates, and two
of the isolates for
which the MICs were 1 µg of penicillin/ml,
were
resistant to cefotaxime (MIC > 0.5 µg/ml). All isolates of
serotypes 6B, 14, 23F, 9V, and 15A and four of the seven serotype
19F
isolates were penicillin resistant. Resistance to tetracycline
(MIC > 2 µg/ml), chloramphenicol (MIC > 4 µg/ml), and
erythromycin
(MIC > 0.25 µg/ml) was present in 33, 20, and 24%
of the isolates,
respectively.
Genetic relatedness among the isolates from patients with
meningitis.
The sequences of the internal fragments of the seven
housekeeping genes were determined for the 106 isolates, and their
allelic profiles were assigned. The genetic relationships among the
isolates are shown in Fig. 1. As was
found previously (10), serious pneumococcal infections are
caused by a relatively heterogeneous collection of isolates: 65 different STs were distinguished among the 106 isolates and, at a
genetic distance of 0.43 (corresponding to different sequences at three
of the seven loci), there were 37 distinct lineages. Nineteen STs
contained at least two isolates, and five STs included at least four
isolates (Table 2).
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FIG. 1.
Dendrogram showing the relatedness of pneumococci from
patients with meningitis. The dendrogram was constructed by the UPGMA
method from the matrix of differences in the allelic profiles of the
106 isolates. STs that contain at least three isolates are labeled.
Isolates that are resistant to penicillin are marked as follows: *,
MIC = 0.12 or 0.25 µg/ml; **, MIC = 0.5 or 1 µg/ml;
***, MIC > 1 µg/ml. The rectangles show clusters of
closely related STs that, with three exceptions, expressed the same
serotype. The serotypes of the isolates of these STs are shown, and the
numbers of isolates with the serotypes are shown in parentheses. The
cross-hatched rectangles denote the three major penicillin-resistant or
multiply antibiotic-resistant clones (Spain23F-1,
Spain6B-2, and France9V-3). The serotypes of
the penicillin-resistant isolates that have unique STs, including the
isolates assigned to the Spain14-5 clone, are indicated.
|
|
Analysis of the variability in the allelic profiles of isolates of the
major Spanish penicillin-resistant and multiply antibiotic-resistant
clones has shown that well-characterized members of these clones
are
usually identical in allelic profile but may occasionally
differ at one
of the seven loci (
28). We therefore designate
pneumococci
which have identical nucleotide sequences at all seven
loci as STs and
those which include isolates that have the same
allelic profile (ST),
plus isolates that differ at a single locus,
as clones. Using these
criteria, there were 15 clones that included
at least three isolates
among the 106 pneumococci (Table
2).
There was good congruence between genotypes and serotypes (Fig.
1).
Sixteen of the 19 STs that contained more than one isolate
were uniform
in serotype and, except for the three STs that contained
isolates of
differing serotype (see below), all isolates within
clones also had the
same serotype (Table
2). The good congruence
between serotypes and
genotypes among invasive pneumococci was
found previously
(
10). Isolates of the same serotype are, however,
not
necessarily closely related in genotype (Table
2). This has
been
interpreted to imply that, in the long term, the genes that
specify
serotype are distributed horizontally among the pneumococcal
population, although in the short term pneumococcal clones emerge
and
start to diversify with only occasional changes of serotype
(
10).
Characterization of penicillin-resistant isolates causing
meningitis.
The allelic profiles of the 106 isolates were compared
with those of reference isolates of the known penicillin-resistant and
multiply antibiotic-resistant clones included in the pneumococcal MLST
database (3a). Isolates that exactly matched the typical allelic profile of the known antibiotic-resistant clones, or which differed at a single locus, were assigned as members of these clones.
The three major clones (Fig.
1 and Table
2) were identified as the
France
9V-3 (11 isolates; typical allelic profile,
7-11-10-1-6-8-1; ST
156), Spain
6B-2 (7 isolates; typical
allelic profile, 5-6-1-2-6-3-4; ST 90),
and Spain
23F-1 (7 isolates; typical allelic profile, 4-4-2-4-4-1-1; ST 81)
clones. One
isolate with the typical allelic profile of the Spain
23F-1
clone was serotype 19F, and four isolates with the typical
allelic
profile of the France
9V-3 clone and three single-locus
variants of this clone were serotype
14. Serotype variants of these two
clones have been reported previously
(
1,
4,
8,
14) and have
been shown to be due to large
recombinational exchanges at the capsular
biosynthetic locus which
determines pneumococcal serotype (
6,
7).
The MICs for the isolates assigned to the France
9V-3 clone
were 0.5 to 2 µg of penicillin/ml; all of the isolates were
susceptible
to erythromycin, tetracycline, and chloramphenicol. One
serotype
14 single-locus variant of the France
9V-3 clone
(MA98100) had an atypically high level of resistance
to cefotaxime
(MIC, 4 µg/ml). Isolates assigned to the Spain
6B-2 clone
were resistant to penicillin (MICs, 0.5 to 2 µg/ml),
tetracycline,
and, with two exceptions, chloramphenicol. All but
one of the
Spain
6B-2 isolates were resistant to erythromycin. One
isolate assigned
to the Spain
23F-1 clone was susceptible to
tetracycline, but otherwise they were
all resistant to penicillin
(MICs, 1 to 2 µg/ml), tetracycline,
and chloramphenicol; two isolates
were also resistant to
erythromycin.
One multiply antibiotic-resistant serotype 14 isolate (MA9745)
was assigned to the Spanish multiply antibiotic-resistant
serotype
14 clone (Spain
14-5), as its allelic profile
differed from the typical allelic
profile of this clone
(1-5-4-11-9-3-16) at only a single locus,
and three isolates (STs 88, 89, and 106) were assigned to the
Spanish intermediate-level
penicillin-resistant serotype 19F clone
(
5).
Four intermediate-level penicillin-resistant serotype 6B isolates (STs
135 and 136; MICs, 0.12 to 0.25 µg/ml) and two serotype
15A isolates
(ST 63; MICs, 0.12 µg/ml) were not closely related
to any of the
known clones of penicillin-resistant pneumococci.
Similarly, the single
highly penicillin-resistant serotype 15B
isolate (ST 229) was not
closely related to any isolate in the
MLST database. The single
isolates for which the MICs were 0.5
to 1 µg of penicillin/ml (STs
23, 104, and 142) were also not
members of known penicillin-resistant
clones, although the serotype
6B isolate of ST 104 was related to the
Spain
6B-2 clone (differing at two of seven
loci).
The penicillin-resistant France
9V-3,
Spain
6B-2, and Spain
23F-1 clones were the
most commonly recovered clones causing meningitis
in Spain during
1997 and 1998 (Table
2). These clones are considered
to show high-level
resistance to penicillin (MIC > 1 µg/ml), although
the MICs for
members of these clones are typically between 1 and
2 µg of
penicillin/ml. Together, 23.6% of the isolates from patients
with
meningitis were members of these three
clones.
Comparison with invasive isolates from other countries.
The
allelic profiles of the 106 isolates from patients with meningitis were
compared with those of 274 isolates from patients with invasive disease
in the United Kingdom, Denmark, Holland, Canada, Uruguay, Australia,
Finland, and Sweden (10). Neither of the two most prevalent
STs associated with invasive disease in these countries (ST 9, serotype
14, and ST 191, serotype 7F) was detected in Spain.
The two most prevalent penicillin-susceptible clones among the Spanish
isolates, serotypes 3 (ST 260) and 18C (STs 113, 110,
122, and 262),
were each represented by six isolates (Table
2).
The serotype 3 clone
was not closely related to any of the 274
isolates from patients with
invasive disease in other countries
or to any other isolates in the
pneumococcal MLST database. The
serotype 18C clone has been identified
as a cause of invasive
disease in the United Kingdom, The Netherlands,
and Denmark. Interestingly,
all eight isolates from these countries
with the typical ST of
the serotype 18C clone (ST 113) were from
patients with meningitis.
Further studies will establish whether this
clone is predominantly
associated with
meningitis.
Of the penicillin-susceptible clones represented by four isolates, the
serotype 12F clone (ST 218) has been identified in
the United Kingdom,
Denmark, Uruguay, and Canada, and the serotype
8 clone (STs 53 and 56)
has been identified in the United Kingdom
and The Netherlands (Table
2), whereas the serotype 19A clone
(STs 202 and 196) was unique to
Spain. Of the penicillin-susceptible
clones represented by three
isolates, the serotype 3 clone (ST
180) has been identified in several
other countries (Table
2),
but none of the others corresponded to the
major invasive clones
identified by Enright and Spratt (
10).
The isolates of the serotypes
3, 8, and 12F clones, from countries
other than Spain, were recovered
from both patients with meningitis and
those with
septicemia.
Prevalence of the clones causing pneumococcal invasive disease in
nine countries.
The MLST data from the 106 isolates from patients
with meningitis in Spain were combined with those from the 274 isolates from patients with invasive disease in eight other countries
(10). Table 3 shows the most
common STs obtained from the analysis of the allelic profiles of these
380 invasive isolates.
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TABLE 3.
Rank order of prevalence of STs of 380 isolates of
S. pneumoniae obtained in recent cases of serious
invasive disease
|
|
Concluding remarks.
MLST provides molecular typing data that
allow different studies from the same laboratory, or from different
laboratories, to be easily combined and compared (10, 17,
25). Each isolate is unambiguously defined by its allelic profile
and, using the Internet, isolates with the same or closely related
allelic profile(s) can be searched for within a central database that
contains the allelic profiles and associated epidemiological data of
thousands of isolates. In this study, the major penicillin-resistant
and penicillin-susceptible clones associated with meningitis in Spain were identified by MLST and compared to those in the pneumococcal MLST
database and those in our earlier study of 274 invasive isolates from
eight developed countries (10).
In agreement with previous estimates (
12), nearly 40% of
recent isolates from patients with meningitis in Spain are resistant
to
penicillin. Several studies have estimated the prevalence of
the three
major Spanish penicillin- or multiply antibiotic-resistant
clones among
penicillin-resistant isolates (
5,
8), but we
are not aware
of any studies which estimate the prevalence of
these clones among all
isolates from patients with invasive disease.
The main conclusion of
this study is that approximately one in
four cases of meningitis in
Spain is now caused by the three major
penicillin-resistant, or
multiply antibiotic-resistant, Spanish
clones (Spain
23F-1,
Spain
6B-2, and France
9V-3). The fact that
isolates of these clones have become the most
prevalent causes of
pneumococcal meningitis in Spain underscores
the virulence of these
highly successful, globally distributed,
clones.
| |
ACKNOWLEDGMENTS |
This work was supported by the Wellcome Trust. B.G.S. is a
Wellcome Trust Principal Research Fellow.
We are most grateful to Derrick Crook and the Oxford Vaccine Group for
support and encouragement.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: WTCEID,
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
