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Antimicrobial Agents and Chemotherapy, June 1999, p. 1480-1483, Vol. 43, No. 6
Laboratoire de Microbiologie,
Received 30 November 1998/Returned for modification 2 February
1999/Accepted 17 March 1999
The genetic relatedness of French isolates of Streptococcus
pneumoniae highly resistant to amoxicillin (MIC, In France, penicillin resistance
rates in Streptococcus pneumoniae of up to 42.8% were
reported in 1996 (7). Penicillin-resistant S. pneumoniae isolates show increased resistance to other For this report, we analyzed 29 clinical isolates of S. pneumoniae for which the amoxicillin MICs were DNA ribotyping was performed as previously described (1)
after digestion with HindIII and EcoRI
(Boehringer Mannheim, Mannheim, Germany). pbp1a,
pbp2b, and pbp2x were PCR amplified from
chromosomal DNA by using the primers previously described (2, 6,
14). HinfI and DdeI-StyI were
used for fingerprinting pbp1a, DdeI and AluI were used for pbp2b, and DdeI and
HinfI were used for pbp2x. After digestion of the
amplified fragments (pbp1a, nucleotides 786 to 3194;
pbp2b, nucleotides 812 to 2317; pbp2x,
nucleotides 478 to 2533), electrophoresis was performed on 6%
polyacrylamide gels which were stained with ethidium bromide, and the
DNA bands were visualized with a UV transilluminator. All patterns were analyzed with Bio-Gene V.96 computer software (Vilber Lourmat, Marne-la-Vallée, France). For each DNA fingerprint, patterns obtained with the two enzymes were combined. These combined patterns were used for the similarity estimation and cluster analysis, in which
the similarity among strains was estimated by the Dice comparison, and
the clustering of strains was determined by the unweighted pair group method.
The serotypes of and MICs for the clinical isolates studied are
presented in Table 1. The 29 SPA4 isolates belong to only four
serotypes: 23F (n = 11), 6B (n = 7), 14 (n = 9), and 19F (n = 2). The MICs of
amoxicillin for all of these isolates were equal to or 1 to 2 dilutions
higher than the MICs of penicillin. As demonstrated by the cefotaxime
MICs, eight of these isolates were resistant to the extended-spectrum
cephalosporins according to the NCCLS criteria, and the cefotaxime MICs
for six of them were Digestion with HindIII generated 18 different patterns
among the 43 isolates and 10 patterns among the 29 SPA4 isolates.
Digestion with EcoRI generated 15 different patterns among
the 43 isolates and 10 patterns among the 29 SPA4 isolates. Combination
of the results obtained with the two enzymes generated 24 ribotypes
among the 43 isolates and 14 ribotypes among the 29 SPA4 isolates. A dendrogram constructed from the cluster analysis shows estimates of the
genetic relationships among the 24 ribotypes (Fig.
1). Among the 11 serotype 23F SPA4
isolates, 9 were genotypically indistinguishable or closely related
(>70% homology) to the serotype 23F French and Spanish clones. The
remaining two serotype 23F isolates exhibited slightly different
patterns which placed them near the 23F intermediate strains. The
serotype 14 SPA4 isolates were indistinguishable or closely related
(>90% homology) to the serotype 9V/14 resistant French clone
previously described (4). The seven serotype 6B SPA4
isolates were indistinguishable from the serotype 6B Spanish clone and
closely related to the current French penicillin-resistant 6B strain.
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Emergence in France of Multiple Clones of Clinical
Streptococcus pneumoniae Isolates with High-Level Resistance
to Amoxicillin
ABSTRACT
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References
4 µg/ml,
equal to or exceeding those of penicillin) was investigated by
molecular fingerprinting. The results suggest that high-level
resistance to amoxicillin has emerged within preexisting
penicillin-resistant clones.
TEXT
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-lactam antibiotics. However, MICs of amoxicillin and extended-spectrum cephalosporins (cefotaxime and ceftriaxone) are generally about equal
to or two to four times less than the MIC of benzylpenicillin (17). Thus, these drugs are currently recommended for
the treatment of pneumococcal infections. Recently,
pneumococci with high-level resistance to amoxicillin
(amoxicillin MICs, 4 µg/ml) and/or extended-spectrum cephalosporins
(cefotaxime MICs, 4 µg/ml) have been identified in France. The
emergence of such strains is of considerable concern, because it
further limits the available options for the therapy of severe
pneumococcal infections.
4 µg/ml (SPA4)
recovered from 29 patients in different cities widely separated
geographically across France by rRNA gene restriction pattern analysis
to determine their overall genetic relatedness and examined DNA
fingerprinting of the pbp1a, pbp2b, and
pbp2x genes to determine the relatedness of the
pbp genes. For comparison, 11 isolates with various levels of susceptibility to amoxicillin (MICs, <4 µg/ml) representative of
the strains currently recovered in France were also studied: 3 French
derivatives of the serotype 23F penicillin-resistant pandemic clone
(4), 1 member of the serotype 9V/14 penicillin-resistant clone (4), 1 member of the serotype 6B penicillin-resistant clone, and 6 penicillin-intermediate strains (serotypes 23F, 14, and
19F). In addition, two Spanish penicillin-resistant clones (serotype
23F and 6B) and the penicillin-susceptible reference strain R6 were
included in the study. These isolates and their relevant properties are
listed in Table 1. Penicillin,
amoxicillin, cefotaxime, and ceftriaxone MICs were determined by the
dilution method on Mueller-Hinton agar supplemented with 5% sheep
blood according to the National Committee for Clinical Laboratory
Standards (NCCLS) guidelines (16). Serotyping was performed
by coagglutination with antiserum-coated latex particles.
TABLE 1.
Phenotypic and genotypic characteristics and origin of
the pneumococcal isolates in this study
4 µg/ml. Cefotaxime MICs were equal to or 1 dilution higher than penicillin MICs for seven isolates. Interestingly,
ceftriaxone MICs, which are generally equal to or 1 dilution less than
cefotaxime MICs (9), were 2 or 4 dilutions less than
cefotaxime MICs for four isolates (71969, 72830, 73234, and 73311).
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FIG. 1.
Dendrogram depicting genetic distance and overall
relatedness of pneumococcal isolates on the basis of rrn
RFLP data. a, isolates for which cefotaxime MICs were 4
µg/ml. See Table 1 for the other characteristics of the isolates.
(b, c, d, e, and
f are representative of the serotype 9V/14
penicillin-resistant French clone, the serotype 6B penicillin-resistant
Spanish clone, the serotype 6B penicillin-resistant French clone, the
French derivatives of the pandemic 23F multiresistant Spanish clone,
and the pandemic serotype 23F multiresistant Spanish clone,
respectively.)
By using the combination of the results of the restriction fragment
length polymorphism (RFLP) obtained with the two enzymes for each
pbp gene, 12 different RFLP patterns were found for
pbp1a, 19 were found for pbp2b, and 18 were found
for pbp2x. All clinical isolates gave pbp2b and
pbp2x gene patterns that differed from those of the
penicillin-susceptible strain R6. Only two intermediate-resistant isolates (7473 and 11758) gave the same pbp1a gene pattern
as R6. Eight different RFLP pbp1a patterns were found among
the SPA4 isolates. One of them was unique to 20 isolates and was
indistinguishable from that of 8 comparator isolates (Table 1). The
SPA4 isolates gave 11 different pbp2b patterns that were all
different from those of the isolates for which amoxicillin MICs were
<2 µg/ml. pbp fingerprinting revealed 10 different
patterns for pbp2x, all but 1 being distinct from those of
the comparator isolates (Table 1). The six isolates for which
cefotaxime MICs were 4 µg/ml exhibited six different
pbp1a gene patterns and four different pbp2b and
pbp2x gene patterns. With the combination of the ribotyping and pbp RFLP data, the following SPA4 isolates were
indistinguishable: 65127 and 72521 (serotype 23F); 72734, 72853, 73171, and 73202 (serotype 6B); and 73012, 73394, and 73529 (serotype 14).
In France, the penicillin-resistant S. pneumoniae isolates
(penicillin MICs, >1 µg/ml) belong to a limited number of serogroups (23, 9, 19, 6, and 14, representing 91.6% of all penicillin-resistant isolates) (7). The amoxicillin MICs for all of these
isolates were <4 µg/ml. Pneumococci for which amoxicillin MICs were
4 µg/ml have recently been detected in the United States
(3). During 1997, 29 SPA4 clinical isolates from 29 patients
and belonging to four different serotypes were isolated across France,
pointing to the dissemination of new clones. Among them, the cefotaxime MICs for six isolates belonging to two serotypes (23F and 14) were 4
µg/ml. Only a few isolates showing a high level of resistance to
extended-spectrum cephalosporins (MICs, 4 µg/ml) have previously been reported in Spain, the United States, and the United Kingdom (2, 11, 13, 15). The spread of such strains would compromise the use of these antibiotics in serious infections like meningitis (5, 12).
Based on the results of our overall genomic DNA analysis, SPA4 isolates are indistinguishable from or closely related to isolates of the predominant resistant clones of the same serotype currently found in France. This suggests that high-level resistance to amoxicillin has emerged de novo within preexisting clones. Four distinct lineages of SPA4 isolates were distinguished. The first group was highly related by serotyping and ribotyping to the pandemic multiresistant Spanish serotype 23F clone. A second lineage was related to the serotype 23F intermediate strains. Another was derived from the serotype 9V/14 resistant French clone, and the last was derived from the Spanish resistant clone 6B, which has spread to Iceland and the United Kingdom (18). Thus, for each SPA4 lineage, we identified a related amoxicillin-susceptible group. Inside each lineage, great heterogeneity was observed in the pbp patterns, pointing to multiplicity in the sequence modifications. Only pbp2b patterns were always different from those of the comparator isolates.
High-level resistance to penicillin was previously associated with
low-level resistance to amoxicillin and extended-spectrum cephalosporins. Indeed, treatment of infections due to
penicillin-resistant pneumococci has required a change from penicillin
to amoxicillin, cefotaxime, or ceftriaxone (10). Thus,
amoxicillin and extended-spectrum cephalosporins have been used
increasingly in France (8). The simultaneous occurrence of
high-level resistance to amoxicillin in several pneumococcal clones in
France suggests that amoxicillin-resistant pneumococci are likely to be
recovered with increasing frequency, given the strong selective
pressure resulting from extensive use of -lactams for the treatment
of pneumococcal infections in France. DNA sequencing of pbp
genes (especially pbp2b) is warranted to determine the
genetic modifications responsible for the high level of resistance to amoxicillin.
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FOOTNOTES |
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* Corresponding author. Mailing address: Service de Microbiologie, Hôpital R. Debré, 48 Bd. Sérurier, 75019 Paris, France. Phone: 33 (1) 40 03 23 40. Fax: 33 (1) 40 03 24 50. E-mail: edouard.bingen{at}rdb.ap-hop-paris.fr.
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