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. 1999 Oct;37(10):3210–3216. doi: 10.1128/jcm.37.10.3210-3216.1999

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, Brian G Spratt 1,*
PMCID: PMC85530  PMID: 10488179

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.

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.

TABLE 1.

Serotype distribution, penicillin resistance, and diversity of isolates of S. pneumoniae from patients with meningitis

Serotype n Pena No. of STs No. of clonesb
6B 12 12 6 2
3 9 0 2 2
18C 9 0 6 2
14 9 9 7 1
19F 7 4 6 2
23F 7 7 4 1
15B 4 1 3 1
9V 4 4 1 1
12F 4 0 1 1
19A 4 1 2 1
11A 4 0 3 1
4 4 0 4 1
8 4 0 2 1
33F 3 0 1 1
20 3 0 3 0
15A 2 2 1 1
16F 2 0 1 1
23A 2 0 2 1
Otherc 13 0 13
Total 106 40 68 21
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a

Pen, penicillin resistant. 

b

Clones are defined as isolates with the same ST plus those that differ at a single locus. 

c

The 13 serotypes that were represented by only a single isolate are grouped together. 

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.

TABLE 2.

Properties of S. pneumoniae isolates from patients with meningitisa

ST Strain no. Allelic profile
Serotype Yr of isolation MIC (μg/ml) of:
Comments
aroE gdh gki recP spi xpt ddl Pen Ery Tet Cm Ctx
156 MA9754 7 11 10 1 6 8 1 9V 1997 2 0.25 0.5 2 1 France9V-3 clone
156 MA9883 7 11 10 1 6 8 1 9V 1998 1 0.06 0.25 2 0.5 France9V-3 clone
156 MA9892 7 11 10 1 6 8 1 9V 1998 1 0.06 0.25 2 0.5 France9V-3 clone
156 MA98111 7 11 10 1 6 8 1 9V 1998 1 0.12 0.25 2 0.5 France9V-3 clone
156 MA9717 7 11 10 1 6 8 1 14 1997 1 0.06 0.25 2 0.25 France9V-3 clone
156 MA9735 7 11 10 1 6 8 1 14 1997 0.5 0.12 0.5 2 0.5 France9V-3 clone
156 MA9756 7 11 10 1 6 8 1 14 1997 1 0.12 0.5 2 0.5 France9V-3 clone
44 MA9747 1 11 10 1 6 8 1 14 1997 2 0.12 0.5 2 1 Single-locus variant of the France9V-3 clone
144 MA98100 7 5 10 1 6 8 1 14 1998 2 0.06 0.25 2 4 Single-locus variant of the France9V-3 clone
167 MA9720 7 11 10 1 4 8 1 14 1997 0.5 0.06 0.25 2 0.25 Single-locus variant of the France9V-3 clone
265 MA98114 7 34 10 1 6 8 1 14 1998 1 0.12 0.25 2 0.5 Single-locus variant of the France9V-3 clone
90 MA9714 5 6 1 2 6 3 4 6B 1997 0.5 128 16 16 0.25 Spain6B-2 clone
90 MA9716 5 6 1 2 6 3 4 6B 1997 0.5 0.5 16 2 0.25 Spain6B-2 clone
90 MA9733 5 6 1 2 6 3 4 6B 1997 1 8 64 2 0.5 Spain6B-2 clone
90 MA9888 5 6 1 2 6 3 4 6B 1998 1 128 32 8 0.5 Spain6B-2 clone
93 MA9877 5 6 1 15 6 3 4 6B 1998 1 128 64 16 0.5 Single-locus variant of the Spain6B-2 clone
93 MA9880 5 6 1 15 6 3 4 6B 1998 1 128 32 16 0.5 Single-locus variant of the Spain6B-2 clone
94 MA9896 5 6 1 2 6 3 54 6B 1998 2 128 64 16 1 Single-locus variant of the Spain6B-2 clone
81 MA9725 4 4 2 4 4 1 1 19F 1997 1 128 32 16 0.5 Spain23F-1 clone
81 MA98113 4 4 2 4 4 1 1 23F 1998 2 0.12 16 16 2 Spain23F-1 clone
81 MA9868 4 4 2 4 4 1 1 23F 1998 1 0.12 32 16 0.5 Spain23F-1 clone
81 MA9879 4 4 2 4 4 1 1 23F 1998 2 8 32 16 1 Spain23F-1 clone
85 MA98102 4 4 2 4 4 1 47 23F 1998 2 0.06 1 16 1 Single-locus variant of the Spain23F clone
85 MA98118 4 4 2 4 4 1 47 23F 1998 1 0.06 32 16 1 Single-locus variant of the Spain23F clone
189 MA9887 8 4 2 4 4 1 1 23F 1998 1 0.06 16 8 1 Single-locus variant of the Spain23F clone
260 MA9737 26 9 15 14 9 16 19 3 1997 0.015 4 0.5 2 0.015
260 MA9753 26 9 15 14 9 16 19 3 1997 0.015 0.12 0.25 2 0.015
260 MA9866 26 9 15 14 9 16 19 3 1998 0.015 0.12 0.25 2 0.015
260 MA9870 26 9 15 14 9 16 19 3 1998 0.015 0.06 0.25 2 0.015
260 MA9881 26 9 15 14 9 16 19 3 1998 0.015 0.12 0.25 2 0.015
260 MA9890 26 9 15 14 9 16 19 3 1998 0.015 0.06 0.25 2 0.015
113 MA979 7 2 1 1 10 1 21 18C 1997 0.015 0.06 0.25 2 0.015
113 MA9863 7 2 1 1 10 1 21 18C 1998 0.015 0.12 0.5 2 0.015
113 MA9889 7 2 1 1 10 1 21 18C 1998 0.015 0.06 0.25 2 0.015
110 MA9727 7 2 1 5 10 1 21 18C 1997 0.015 0.06 0.25 2 0.015
122 MA9721 7 2 29 1 10 1 21 18C 1997 0.015 0.06 0.25 2 0.015
262 MA9893 28 2 1 1 10 1 21 18C 1998 0.015 0.06 0.25 2 0.015
218 MA9719 10 20 14 1 6 1 29 12F 1997 0.015 0.06 0.25 2 0.015
218 MA9724 10 20 14 1 6 1 29 12F 1997 0.015 0.12 0.5 2 0.015
218 MA98105 10 20 14 1 6 1 29 12F 1998 0.015 0.06 0.5 4 0.015
218 MA9876 10 20 14 1 6 1 29 12F 1998 0.015 0.12 0.25 2 0.015
135 MA9732 7 5 4 12 6 20 46 6B 1997 0.12 0.06 0.5 2 0.12
135 MA9743 7 5 4 12 6 20 46 6B 1997 0.12 128 4 4 0.06
135 MA9744 7 5 4 12 6 20 46 6B 1997 0.12 128 8 4 0.12
136 MA9882 7 5 4 1 6 20 46 6B 1998 0.25 64 64 16 0.25
53 MA98103 2 5 1 11 16 3 14 8 1998 0.015 128 0.5 2 0.015
53 MA9862 2 5 1 11 16 3 14 8 1998 0.015 0.25 0.25 1 0.015
53 MA9864 2 5 1 11 16 3 14 8 1998 0.015 0.12 16 4 0.015
56 MA9875 2 5 1 11 16 44 14 8 1998 0.015 0.12 0.25 2 0.015
202 MA9729 8 16 19 15 6 40 26 19A 1997 0.015 128 32 16 0.03
202 MA9742 8 16 19 15 6 40 26 19A 1997 0.015 128 64 4 0.015
202 MA9861 8 16 19 15 6 40 26 19A 1998 0.015 0.25 64 1 0.015
196 MA9895 8 11 19 15 6 40 26 19A 1998 0.5 128 32 4 0.5
100 MA971 5 12 29 12 9 39 18 33F 1997 0.015 0.06 0.25 2 0.015
100 MA9869 5 12 29 12 9 39 18 33F 1998 0.03 0.12 0.25 4 0.015
100 MA9873 5 12 29 12 9 39 18 33F 1998 0.015 0.12 0.25 2 0.015
101 MA9734 5 13 1 1 6 41 18 15B 1997 0.015 0.12 0.5 2 0.015
101 MA9867 5 13 1 1 6 41 18 15B 1998 0.03 0.12 0.5 4 0.015
101 MA98112 5 13 1 1 6 41 18 15C 1998 0.015 0.12 0.25 2 0.015
180 MA977 7 15 2 10 6 1 22 3 1997 0.015 0.06 0.25 2 0.015
180 MA9878 7 15 2 10 6 1 22 3 1998 0.015 0.06 0.25 2 0.015
180 MA9885 7 15 2 10 6 1 22 3 1998 0.015 0.06 0.25 2 0.015
177 MA9711 7 14 4 12 1 1 14 19F 1997 0.03 0.06 64 2 0.03
177 MA9723 7 14 4 12 1 1 14 19F 1997 0.03 0.06 8 2 0.03
179 MA9874 7 14 40 12 1 1 14 19F 1998 0.03 128 64 4 0.03
62 MA9728 2 5 29 12 16 3 14 11A 1997 0.03 0.06 0.25 2 0.015
62 MA9751 2 5 29 12 16 3 14 11A 1997 0.015 0.12 0.25 2 0.015
55 MA9750 2 5 1 12 16 3 14 11A 1997 0.015 0.12 0.25 2 0.015
88 MA974 5 5 7 7 8 5 7 19F 1997 0.12 0.06 32 8 0.12 Minor penicillin-resistant serotype 19F clone
89 MA9748 5 5 7 7 8 5 1 19F 1997 0.5 0.06 64 2 0.25 Minor penicillin-resistant serotype 19F clone
106 MA9897 5 16 7 7 8 5 7 19F 1998 0.25 128 32 16 0.25 Minor penicillin-resistant serotype 19F clone
30 MA9710 1 5 27 20 1 1 1 16F 1997 0.015 0.06 0.25 2 0.015
30 MA9749 1 5 27 20 1 1 1 16F 1997 0.015 0.12 0.25 2 0.015
63 MA9718 2 5 36 12 17 21 14 15A 1997 0.12 128 64 2 0.06
63 MA9757 2 5 36 12 17 21 14 15A 1997 0.12 128 64 4 0.06
133 MA9726 7 5 1 1 10 1 45 18C 1997 0.015 0.12 0.5 2 0.015
133 MA9730 7 5 1 1 10 1 45 18C 1997 0.015 0.06 0.25 2 0.015
247 MA9738 16 13 4 5 6 10 14 4 1997 0.015 0.06 0.5 4 0.015
248 MA9736 16 13 4 5 6 39 14 4 1997 0.015 0.06 0.5 2 0.015
42 MA9758 1 8 9 9 6 4 6 23A 1997 0.015 0.06 64 16 0.015
264 MA98110 1 33 9 9 6 4 6 23A 1998 0.015 0.12 32 8 0.015
17 MA9745 1 5 4 11 9 3 47 14 1997 1 128 32 16 0.5 Single-locus variant of the Spain14-5 clone
23 MA9860 1 5 4 5 35 3 8 14 1998 1 128 0.25 2 0.5
32 MA9891 1 6 4 5 15 1 20 10F 1998 0.015 0.06 0.25 2 0.015
45 MA9741 1 12 1 23 15 26 14 18C 1997 0.015 0.06 0.25 2 0.015
64 MA9898 2 6 6 12 16 3 14 11A 1998 0.015 0.12 0.5 4 0.015
69 MA9739 2 8 37 4 6 1 1 9N 1997 0.015 0.06 0.5 2 0.015
70 MA9872 2 13 1 4 6 12 1 13 1998 0.015 0.06 0.25 2 0.015
72 MA9752 2 13 2 4 9 4 1 24F 1997 0.015 0.06 0.25 2 0.015
97 MA9740 5 7 4 2 10 1 27 10A 1997 0.015 0.06 0.25 2 0.015
103 MA9759 5 13 38 1 6 1 18 15B 1997 0.06 0.12 0.25 2 0.06
104 MA9894 5 13 41 2 6 3 4 6B 1998 0.5 128 64 16 0.25
105 MA98101 5 15 4 1 6 1 6 25 1998 0.015 0.06 0.25 2 0.015
123 MA9884 7 2 40 1 10 1 45 17F 1998 0.015 0.06 0.25 2 0.015
142 MA9899 7 5 8 6 6 12 8 23F 1998 0.5 0.12 0.5 2 0.12
216 MA9871 10 16 32 1 15 1 6 20 1998 0.015 0.06 0.25 2 0.015
224 MA975 10 25 8 6 25 6 8 6A 1997 0.06 0.06 0.25 2 0.06
225 MA9755 10 32 6 24 33 28 18 28A 1997 0.015 0.12 0.5 4 0.015
228 MA9886 12 8 1 5 17 4 20 1 1998 0.015 0.06 0.25 2 0.015
229 MA9865 12 13 1 5 6 1 53 15B 1998 2 128 64 2 1
235 MA9731 15 8 8 18 15 1 31 20 1997 0.015 0.06 32 16 0.015
241 MA9712 25 31 4 16 32 28 44 18A 1997 0.015 0.06 0.25 2 0.015
244 MA98104 16 2 4 1 6 10 18 4 1998 0.015 0.06 0.25 2 0.015
259 MA976 24 5 35 1 15 12 43 4 1997 0.015 0.06 0.5 4 0.015
263 MA98109 2 11 2 1 17 1 1 20 1998 0.015 0.25 0.25 2 0.015
266 MA98116 16 35 1 16 9 45 17 28 1998 0.015 0.06 0.5 4 0.015
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a

Isolates are grouped into STs, which have identical allelic profiles, and clones, which also include isolates that differ at a single locus. Pen, penicillin; Ery, erythromycin; Tet, tetracycline; Cm, chloramphenicol; Ctx, cefotaxime.  

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).

FIG. 1.

FIG. 1

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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 France9V-3 (11 isolates; typical allelic profile, 7-11-10-1-6-8-1; ST 156), Spain6B-2 (7 isolates; typical allelic profile, 5-6-1-2-6-3-4; ST 90), and Spain23F-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 Spain23F-1 clone was serotype 19F, and four isolates with the typical allelic profile of the France9V-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 France9V-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 France9V-3 clone (MA98100) had an atypically high level of resistance to cefotaxime (MIC, 4 μg/ml). Isolates assigned to the Spain6B-2 clone were resistant to penicillin (MICs, 0.5 to 2 μg/ml), tetracycline, and, with two exceptions, chloramphenicol. All but one of the Spain6B-2 isolates were resistant to erythromycin. One isolate assigned to the Spain23F-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 (Spain14-5), 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 Spain6B-2 clone (differing at two of seven loci).

The penicillin-resistant France9V-3, Spain6B-2, and Spain23F-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.

TABLE 3.

Rank order of prevalence of STs of 380 isolates of S. pneumoniae obtained in recent cases of serious invasive disease

STa n Serotypeb Countriesc Resistance to:d
Allele no.
Pen Ery aroE gdh gki recP spi xpt ddl
9 21 14/19F (1) A/GB S R/S (4) 1 5 4 5 5 1 8
191 20 7F GB/D/F/N/U S S 8 9 2 1 6 1 17
156 13 9V/14 (4)/NT (1) GB/C/D/U/SP R S/R (2) 7 11 10 1 6 8 1
180 11 3 GB/C/D/N/SP S S 7 15 2 10 6 1 22
138 11 6B GB/D/S S S 7 5 8 5 10 6 14
124 11 14 A/GB/C/F/N/S S S 7 5 1 8 14 11 14
218 10 12F GB/C/D/U/SP S S 10 20 14 1 6 1 29
113 9 18C/18B (1) GB/N/SP S S 7 2 1 1 10 1 21
227 8 1 GB/D S S 12 5 13 5 17 4 20
53 6 8 GB/N/SP S S 2 5 1 11 16 3 14
205 6 4/NT (1) A/C/D/S S S 10 5 4 5 13 10 18
247 6 4 D/N/SP S S 16 13 4 5 6 10 14
260 6 3 SP S S 26 9 15 14 9 16 19
81 5 23F/19F (1) GB/SP R S/R (2) 4 4 2 4 4 1 1
90 5 6B A/SP R R/S (1) 5 6 1 2 6 3 4
199 5 19A/15B (1) GB/N S S 8 13 14 4 17 4 14
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a

STs containing fewer than five isolates are not shown. 

b

Some STs contained isolates of more than one serotype. The predominant serotype is shown first, followed by the minority serotype(s), with the numbers of isolates of these serotypes in parentheses. 

c

A, Australia; C, Canada; D, Denmark; F, Finland; N, The Netherlands; GB, Great Britain; S, Sweden; U, Uruguay; SP, Spain. 

d

In some cases, isolates of the same ST were either resistant (R) or susceptible (S) to erythromycin. The predominant susceptibility category is given first, and the numbers of isolates in the minority category are shown in parentheses. Pen, penicillin; Ery, erythromycin; NT, nontypeable. 

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 (Spain23F-1, Spain6B-2, and France9V-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.

REFERENCES

  • 1.Barnes D M, Whittier S, Gilligan P H, Soares S, Tomasz A, Henderson F W. Transmission of multidrug-resistant serotype 23F Streptococcus pneumoniae in group day care: evidence suggesting capsular transformation of the resistant strain in vivo. J Infect Dis. 1995;171:890–896. doi: 10.1093/infdis/171.4.890. [DOI] [PubMed] [Google Scholar]
  • 2.Camou T, Hortal M, Tomasz A. The apparent importation of penicillin-resistant capsular type 14 Spanish/French clone of Streptococcus pneumoniae into Uruguay in the early 1990’s. Microb Drug Res. 1998;4:219–224. doi: 10.1089/mdr.1998.4.219. [DOI] [PubMed] [Google Scholar]
  • 3.Casal J. Antimicrobial susceptibility of Streptococcus pneumoniae: serotype distribution of penicillin-resistant strains in Spain. Antimicrob Agents Chemother. 1982;22:222–225. doi: 10.1128/aac.22.2.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3a.Chan, M.-S., M. C. I. Maiden, and B. G. Spratt. 26 July 1999, revision date. Multilocus sequence typing database. [Online.] http://mlst.200.ox.ac.uk. [6 August 1999, last date accessed.]
  • 4.Coffey T J, Dowson C G, Daniels M, Zhou J, Martin C, Spratt B G, Musser J M. Horizontal transfer of multiple penicillin-binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae. Mol Microbiol. 1991;5:2255–2260. doi: 10.1111/j.1365-2958.1991.tb02155.x. [DOI] [PubMed] [Google Scholar]
  • 5.Coffey T J, Berrón S, Daniels M, Garcia-Leoni M E, Cercenado E, Bouza E, Fenoll A, Spratt B G. Multiply-antibiotic-resistant Streptococcus pneumoniae recovered from Spanish hospitals (1988–1994): novel major clones of serotypes 14, 19F and 15F. Microbiology. 1996;142:2747–2757. doi: 10.1099/13500872-142-10-2747. [DOI] [PubMed] [Google Scholar]
  • 6.Coffey T J, Enright M C, Daniels M, Morona J K, Morona R, Hryniewicz W, Paton J C, Spratt B G. Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae. Mol Microbiol. 1998;27:73–83. doi: 10.1046/j.1365-2958.1998.00658.x. [DOI] [PubMed] [Google Scholar]
  • 7.Coffey T J, Daniels M, Enright M C, Spratt B G. Serotype 14 variants of the Spanish penicillin-resistant serotype 9V clone of Streptococcus pneumoniae arose by large recombinational replacements of the cpsA-pbp1a region. Microbiology. 1999;145:2023–2031. doi: 10.1099/13500872-145-8-2023. [DOI] [PubMed] [Google Scholar]
  • 8.Corso A, Severina E P, Petruk V F, Mauriz Y R, Tomasz A. Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates causing respiratory disease in the United States. Microb Drug Res. 1998;4:325–337. doi: 10.1089/mdr.1998.4.325. [DOI] [PubMed] [Google Scholar]
  • 9.Crook D W M, Spratt B G. Multidrug resistance in Streptococcus pneumoniae. Br Med Bull. 1998;54:593–608. doi: 10.1093/oxfordjournals.bmb.a011713. [DOI] [PubMed] [Google Scholar]
  • 10.Enright M C, Spratt B G. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology. 1998;144:3049–3060. doi: 10.1099/00221287-144-11-3049. [DOI] [PubMed] [Google Scholar]
  • 11.Fenoll A, Martín Bourgon C, Muñoz R, Vicioso D, Casal J. Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates causing systemic infections in Spain, 1979–1989. Rev Infect Dis. 1991;13:56–60. doi: 10.1093/clinids/13.1.56. [DOI] [PubMed] [Google Scholar]
  • 12.Fenoll A, Jado I, Vicioso D, Pérez A, Casal J. Evolution of Streptococcus pneumoniae serotypes and antibiotic resistance in Spain: update (1990–1996) J Clin Microbiol. 1998;36:3447–3454. doi: 10.1128/jcm.36.12.3447-3454.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Gasc A M, Geslin P, Sicard A M. Relatedness of penicillin-resistant Streptococcus pneumoniae serogroup 9 strains from France and Spain. Microbiology. 1995;141:623–627. doi: 10.1099/13500872-141-3-623. [DOI] [PubMed] [Google Scholar]
  • 14.Hermans P W M, Sluijter M, Dejsirilert S, Lemmens N, Elzenaar K, vanVeen A, Goessens W H F, DeGroot R. Molecular epidemiology of drug-resistant pneumococci: toward an international approach. Microb Drug Res. 1997;3:243–251. doi: 10.1089/mdr.1997.3.243. [DOI] [PubMed] [Google Scholar]
  • 15.Klugman K P, Coffey T J, Smith A, Wasas A, Myers M, Spratt B G. Cluster of an erythromycin-resistant variant of the Spanish multiply resistant 23F clone of Streptococcus pneumoniae in South Africa. Eur J Clin Microbiol. 1994;13:171–174. doi: 10.1007/BF01982193. [DOI] [PubMed] [Google Scholar]
  • 16.Klugman K P. Epidemiology, control, and treatment of multiresistant pneumococci. Drugs. 1996;52(Suppl. 2):42–46. doi: 10.2165/00003495-199600522-00009. [DOI] [PubMed] [Google Scholar]
  • 17.Maiden M C J, Bygraves J A, Feil E, Morelli G, Russell J E, Urwin R, Zhang Q, Zhou J, Zurth K, Caugant D A, Feavers I M, Achtman M, Spratt B G. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA. 1998;95:3140–3145. doi: 10.1073/pnas.95.6.3140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.McGee L, Klugman K P, Friedland D, Lee H J. Spread of the Spanish multi-resistant serotype 23F clone of Streptococcus pneumoniae to Seoul, Korea. Microb Drug Res. 1997;3:253–257. doi: 10.1089/mdr.1997.3.253. [DOI] [PubMed] [Google Scholar]
  • 19.Muñoz R, Coffey T J, Daniels M, Dowson C G, Laible G, Casal J, Hakenbeck R, Jacobs M, Musser J M, Spratt B G, Tomasz A. Intercontinental spread of a multiresistant clone of serotype 23F Streptococcus pneumoniae. J Infect Dis. 1991;164:302–306. doi: 10.1093/infdis/164.2.302. [DOI] [PubMed] [Google Scholar]
  • 20.National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. Wayne, Pa: National Committee for Clinical Laboratory Standards; 1997. [Google Scholar]
  • 21.Scott J A G, Hall A J, Dagan R, Dixon J M S, Eykyn S J, Fenoll A, Hortal M, Jette L P, Jorgensen J H, Lamothe F, Latorre C, MacFarlane J T, Shlaes D M, Smart L E, Tauney A. Serogroup-specific epidemiology of Streptococcus pneumoniae—associations with age, sex, and geography in 7,000 episodes of invasive disease. Clin Infect Dis. 1996;22:973–981. doi: 10.1093/clinids/22.6.973. [DOI] [PubMed] [Google Scholar]
  • 22.Shi Z-Y, Enright M C, Wilkinson P, Griffiths D, Spratt B G. Identification of three major clones of multiply antibiotic-resistant Streptococcus pneumoniae in Taiwanese hospitals using multilocus sequence typing. J Clin Microbiol. 1998;36:3514–3519. doi: 10.1128/jcm.36.12.3514-3519.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sibold C, Wang J, Henrichsen J, Hakenbeck R. Genetic relationships of penicillin-susceptible and -resistant Streptococcus pneumoniae strains isolated on different continents. Infect Immun. 1992;60:4119–4126. doi: 10.1128/iai.60.10.4119-4126.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Soares S, Kristinsson K G, Musser J M, Tomasz A. Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980’s. J Infect Dis. 1993;168:158–163. doi: 10.1093/infdis/168.1.158. [DOI] [PubMed] [Google Scholar]
  • 25.Spratt B G. Multilocus sequence typing: molecular typing of bacterial pathogens in an era of rapid DNA sequencing and the Internet. Curr Opin Microbiol. 1999;2:312–316. doi: 10.1016/S1369-5274(99)80054-X. [DOI] [PubMed] [Google Scholar]
  • 26.Tarasi A, Chong Y, Lee K, Tomasz A. Spread of the serotype 23F multidrug-resistant Streptococcus pneumoniae clone to South Korea. Microb Drug Res. 1997;3:105–109. doi: 10.1089/mdr.1997.3.105. [DOI] [PubMed] [Google Scholar]
  • 27.Tomasz A. Antibiotic resistance in Streptococcus pneumoniae. Clin Infect Dis. 1997;24:S85–S88. doi: 10.1093/clinids/24.supplement_1.s85. [DOI] [PubMed] [Google Scholar]
  • 28.Zhou, J., and B. G. Spratt. Unpublished data.

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