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Antimicrobial Agents and Chemotherapy, October 2001, p. 2955-2957, Vol. 45, No. 10
Molecular Genetics Laboratory, Instituto de
Tecnologia Química e Biológica, Universidade Nova de
Lisboa, Oeiras, Portugal1; Microbiology
Department, School of Medicine, Universidad
Complutense,2 and Medical Department,
GlaxoSmithKline Pharmaceuticals,3 Madrid, Spain;
and Laboratory of Microbiology, The Rockefeller University,
New York, New York4
Received 12 April 2001/Returned for modification 7 June
2001/Accepted 9 July 2001
Analysis of the pulsed-field gel electrophoretic profiles of 82 pneumococcal isolates with reduced susceptibility to ciprofloxacin (RSC) and of 90 co-occurring susceptible isolates indicates a considerable genetic diversity among isolates with RCS and points to a
close relation between the two groups. This finding suggests that
pneumococci with RCS emerge through independent mutational events.
The recent emergence and increase in
incidence of Streptococcus pneumoniae clinical isolates with
reduced susceptibility to fluoroquinolone antibiotics (2, 7,
9) has raised questions concerning the mechanisms through which
this trait is acquired and spread within populations of S. pneumoniae. The issue is whether the resistant isolates represent
only a few clonal types or a genetically diverse set of strains that
emerged through the antibiotic's selective pressure upon existing
bacterial clones. These two alternatives have different implications as
to the choice of the most appropriate interventions to curtail further
increase in the number of resistant isolates.
Bacterial strains.
S. pneumoniae isolates were selected
from two previous studies performed in Spain (1, 6), where
2.22 defined daily doses of quinolones per 1,000 inhabitants were
prescribed in 1997 (19). Strains were collected during
1996 to 1999 in 20 different hospitals from patients suffering from
community-acquired respiratory infections. Of the 179 pneumococci
identified with reduced susceptibility to ciprofloxacin (MICs PFGE.
Total DNA was prepared, and chromosomal DNA fragments
generated by SmaI digestion were separated by pulsed-field
gel electrophoresis (PFGE) as previously described (16).
PFGE patterns were assigned by visual inspection of the
macro-restriction profiles, using accepted criteria (17).
Profiles displayed by at least two isolates were assigned letters
arbitrarily, except with previously recognized clones, which were named
according to the newly recommended nomenclature (10)
(Table 1).
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2955-2957.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Streptococcus pneumoniae Isolates
with Reduced Susceptibility to Ciprofloxacin in Spain: Clonal Diversity
and Appearance of Ciprofloxacin-Resistant Epidemic Clones
ABSTRACT
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4 µg/ml) (RSC), 82 were selected for molecular typing. All but five of
these 82 isolates were from adults; 63 were from the respiratory tract,
14 were from blood, and 5 were from the middle ear. An additional 90 strains were also selected from among isolates susceptible to
ciprofloxacin (MIC = 1 µg/ml) collected at the same surveillance
sites and during the same surveillance period. Most of these 90 isolates were from the respiratory tract, 22 were from blood, and 12 were from the middle ear. The ciprofloxacin-susceptible isolates were
selected to match as closely as possible the characteristics of the
isolates with RSC in terms of antimicrobial resistance profile and
serogroup. For 63.4% of the 82 isolates with RSC the ciprofloxacin MIC
was 4 µg/ml, for 18.2% the MIC was 8 µg/ml, for 15.8% the MIC was 16 µg/ml, and for 3.6% the MIC was >16 µg/ml. Regarding their distribution by serogroup, the most frequent serogroups were 19 (19.2%); 3 (13.2%); 9, 14, and 23 (each 12.0%); 6 (8.4%);
and 18 (4.8%). Nontypeable strains accounted for 3.6% of the
isolates, and the remaining 14.8% belonged to other serogroups.
TABLE 1.
Diversity of microbiological profiles among the 172 isolates analyzeda
B, E, and Keach of which was restricted to a single hospital.
Serogroup and PFGE type. In Table 1 are identified isolates that, in spite of sharing the same clonal type, as defined by PFGE, expressed different serotypes. Particularly interesting is the acquisition of capsular type 3 by a representative of the France9V-3 clone, since change to this serotype by a representative of the Spain23F-1 clone was shown to have a profound impact on virulence (12). These isolates are believed to result from a capsular switch in vivo through transformation or transduction with foreign DNA (3, 12, 14).
Relation between clonal types and ciprofloxacin MIC. The 82 S. pneumoniae clinical isolates with RSC presented 44 different PFGE patterns. Of these, 29 patterns were unique to isolates with RSC and were each represented by a single isolate. Three internationally disseminated clones were identified among both isolates with RSC and susceptible isolates (Table 1). Additionally, other isolates identified in this study sharing the same PFGE pattern and serotype were susceptible and showed RSC (clones A, F, I, and J). Moreover, three clones, with the exception of the internationally disseminated clones, presented different resistance levels (clones A, C, and K) (Table 1).
Surprisingly, an increase in ciprofloxacin MIC did not cause a reduction in the diversity of PFGE profiles observed. We identified 32 different PFGE patterns among the 52 isolates for which the ciprofloxacin MIC was 4 µg/ml, 12 patterns among the 15 isolates for which the MIC was 8 µg/ml, and 7 patterns among the 13 isolates for which the MIC was 16 µg/ml. None of the three isolates for which the MIC was >16 µg/ml had identical PFGE profiles (Table 1). This situation is in contrast to what is observed with penicillin resistance, with which we find a smaller number of clonal types among highly resistant isolates (18). Ciprofloxacin was introduced into therapeutic practice in Spain in 1988. Notwithstanding, genetic diversity among ciprofloxacin-resistant S. pneumoniae is still widespread among clinical isolates collected from 1996 through 1999, and the same PFGE profiles can be found among isolates with RSC and susceptible isolates. The findings suggest that ciprofloxacin-resistant pneumococci are the products of independent mutational events selected by the drugs from among a diverse population of pneumococci, presumably during treatment with these antibiotics. Most likely the mechanism of resistance involves alteration of the quinolone targets through either mutation (8, 11, 13) or transformation with genes derived from other organisms (5, 20), explaining the observed clonal diversity. The fact that approximately 93% of the strains for which MICs are4 µg/ml from the original collections were isolated from adults correlates well with the exclusive use of fluoroquinolones among
adult and not pediatric patients (2, 6, 9). The five
pediatric isolates with RSC studied in detail do not show an
overrepresentation of the international clones frequently isolated from
children; one isolate belonged to the France9V-3
clone and two belonged to the Spain23F-1 clone,
but two others presented unique PFGE types.
In addition to the genetic diversity of the pneumococcal isolates with
RSC, equally impressive was the fact that a large proportion25 of the
82 isolates or 30%of the pneumococci with RSC belonged to two
internationally spread multidrug-resistant epidemic clones: France9V-3 and Spain23F-1.
In spite of their high numbers, there is no overrepresentation of these
clones among isolates with RSC in relation to the number of sensitive
isolates selected to be as similar as possible in terms of antibiotype
and serotype in our sample (Table 1). This finding suggests that the
high prevalence of these clones among isolates with RSC reflects their
prevalence in the population, i.e., these clones are not more
represented than would be expected if we assume that isolates with RSC
are being selected from existing clones. The epidemicity of these
clones (4, 15) suggests that dissemination of
ciprofloxacin resistance through these isolates is a plausible scenario.
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ACKNOWLEDGMENTS |
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We thank Alexander Tomasz for critical reading of and suggestions concerning the manuscript and Lorenzo Aguilar (Medical Department, GlaxoSmithKline) for technical support.
Partial support for this work was provided by contract PRAXIS/P/SAU14051/1998 from Junta Nacional de Investigação Científica e Tecnológica. L.A. was supported by a grant from the Universidad Complutense de Madrid, Madrid, Spain. M.R. was the recipient of a fellowship from Fundação para a Ciência e Tecnologia (BPD/20185/99).
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FOOTNOTES |
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* Corresponding author. Mailing address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, R. da Qta. Grande 6, Oeiras, Portugal. Phone: 351-21-446 9800. Fax: 351-21-442 8766. E-mail: ramirma{at}itqb.unl.pt.
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REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Baquero, F.,
J. A. Garcia-Rodriguez,
J. Garcia de Lomas,
L. Aguilar, and the Spanish Surveillance Group for Respiratory Pathogens.
1999.
Antimicrobial resistance of 1,113 Streptococcus pneumoniae isolates from patients with respiratory tract infections in Spain: results of a 1-year (1996-1997) multicenter surveillance study.
Antimicrob. Agents Chemother.
43:357-359 |
| 2. |
Chen, D. K.,
A. McGeer,
J. C. de Azavedo, and D. E. Low.
1999.
Decreased susceptibility of Streptococcus pneumoniae to fluoroquinolones in Canada.
N. Engl. J. Med.
341:233-239 |
| 3. | Coffey, T. J., M. C. Enright, M. Daniels, J. K. Morona, R. Morona, W. Hryniewicz, J. C. Paton, and B. G. Spratt. 1998. Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae. Mol. Microbiol. 27:73-83[CrossRef][Medline]. |
| 4. |
Enright, M. C.,
A. Fenoll,
D. Griffiths, and B. G. Spratt.
1999.
The three major Spanish clones of penicillin-resistant Streptococcus pneumoniae are the most common clones recovered in recent cases of meningitis in Spain.
J. Clin. Microbiol.
37:3210-3216 |
| 5. |
Ferrandiz, M. J.,
A. Fenoll,
J. Linares, and A. G. De La Campa.
2000.
Horizontal transfer of parC and gyrA in fluoroquinolone-resistant clinical isolates of Streptococcus pneumoniae.
Antimicrob. Agents Chemother.
44:840-847 |
| 6. |
Garcia-Rey, C.,
L. Aguilar,
F. Baquero, and the Spanish Surveillance Group for Respiratory Pathogens.
2000.
Influences of different factors on prevalence of ciprofloxacin resistance in Streptococcus pneumoniae in Spain.
Antimicrob. Agents Chemother.
44:3481-3482 |
| 7. |
Ho, P. L.,
T. L. Que,
D. N. Tsang,
T. K. Ng,
K. H. Chow, and W. H. Seto.
1999.
Emergence of fluoroquinolone resistance among multiply resistant strains of Streptococcus pneumoniae in Hong Kong.
Antimicrob. Agents Chemother.
43:1310-1313 |
| 8. |
Jones, M. E.,
D. F. Sahm,
N. Martin,
S. Scheuring,
P. Heisig,
C. Thornsberry,
K. Kohrer, and F. J. Schmitz.
2000.
Prevalence of gyrA, gyrB, parC, and parE mutations in clinical isolates of Streptococcus pneumoniae with decreased susceptibilities to different fluoroquinolones and originating from worldwide surveillance studies during the 1997-1998 respiratory season.
Antimicrob. Agents Chemother.
44:462-466 |
| 9. |
Linares, J.,
A. G. de la Campa, and R. Pallares.
1999.
Fluoroquinolone resistance in Streptococcus pneumoniae.
N. Engl. J. Med.
341:1546-1548 |
| 10. |
McGee, L.,
L. McDougal,
J. Zhou,
B. G. Spratt,
F. C. Tenover,
R. George,
R. Hakenbeck,
W. Hryniewicz,
J. C. Lefevre,
A. Tomasz, and K. P. Klugman.
2001.
Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network.
J. Clin. Microbiol.
39:2565-2571 |
| 11. | Munoz, R., and A. G. De La Campa. 1996. ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype. Antimicrob. Agents Chemother. 40:2252-2257[Abstract]. |
| 12. | Nesin, M., M. Ramirez, and A. Tomasz. 1998. Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo. J. Infect. Dis. 177:707-713[Medline]. |
| 13. |
Pan, X. S., and L. M. Fisher.
1996.
Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.
J. Bacteriol.
178:4060-4069 |
| 14. | Ramirez, M., and A. Tomasz. 1999. Acquisition of new capsular genes among clinical isolates of antibiotic-resistant Streptococcus pneumoniae. Microb. Drug Resist. 5:241-246[Medline]. |
| 15. |
Sa-Leao, R.,
A. Tomasz,
I. S. Sanches,
S. Nunes,
C. R. Alves,
A. B. Avo,
J. Saldanha,
K. G. Kristinsson, and H. de Lencastre.
2000.
Genetic diversity and clonal patterns among antibiotic-susceptible and -resistant Streptococcus pneumoniae colonizing children: day care centers as autonomous epidemiological units.
J. Clin. Microbiol.
38:4137-4144 |
| 16. | Soares, S., K. G. Kristinsson, J. M. Musser, and A. Tomasz. 1993. Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s. J. Infect. Dis. 168:158-163[Medline]. |
| 17. | Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239[Medline]. |
| 18. | Tomasz, A., A. Corso, E. P. Severina, G. Echaniz-Aviles, M. C. Brandileone, T. Camou, E. Castaneda, O. Figueroa, A. Rossi, and J. L. Di Fabio. 1998. Molecular epidemiologic characterization of penicillin-resistant Streptococcus pneumoniae invasive pediatric isolates recovered in six Latin-American countries: an overview. Microb. Drug Resist. 4:195-207[Medline]. |
| 19. | Tovar, M. R., and A. R. Bremon. 1998. Consumo de quinolonas en el medio extrahospitalario en Espana. Bol. Epidemiol. Semanal Inst. Salud Carlos III 6:265-267. |
| 20. | Varon, E., and L. Gutmann. 2000. Mechanisms and spread of fluoroquinolone resistance in Streptococcus pneumoniae. Res. Microbiol. 151:471-473[Medline]. |
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