Spain14-5 international multiresistant Streptococcus pneumoniae clone resistant to fluoroquinolones and other families of antibiotics

Emilio Pérez-Trallero1,2,*, José M. Marimón1, Alberto Gonzalez1 and Luis Iglesias1

1 Servicio de Microbiología, Hospital Donostia, Paseo Dr. Beguiristain s/n, 20014 San Sebastián (Gipuzkoa); 2 Departamento de Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad del País Vasco, San Sebastián, Spain

Received 17 October 2002; returned 20 November 2002; revised 25 November 2002; accepted 26 November 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Spain14-5 international multiresistant clone was initially described as resistant to penicillin, tetracycline, chloramphenicol and trimethoprim/sulfamethoxazole. In Gipuzkoa, Spain, Streptococcus pneumoniae isolated from 16 patients, and determined by PFGE and multilocus sequence typing to belong to the Spain14-5 clone, showed further resistance to fluoroquinolones (all strains had point mutations in the parC and gyrA genes). In addition, most strains showed resistance to amoxicillin (MIC >= 8 mg/L), cefotaxime (MIC >= 2 mg/L), macrolides and lincosamides. Two strains were resistant to rifampicin (MIC 8 mg/L). The multiresistance observed in these isolates converts the Spain14-5 clone into one of the most, if not the most, multiresistant of the international clones described.

Keywords: rifampicin resistance, erythromycin resistance, mechanism of resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Until the beginning of the 1980s, most Streptococcus pneumoniae were susceptible to penicillin and other antibiotics. Shortly after penicillin resistance was detected among S. pneumoniae, it was clear that this resistance was associated with resistance to two or more classes of antibiotics. These multidrug-resistant isolates belonged to a restricted number of serotypes that finally led to the description and characterization of the major multidrug-resistant international clones.1 The number of reports on multidrug-resistant S. pneumoniae clones has been increasing in the last few years. Coffey et al.2 initially described the Spain14-5 clone in 1996 and its multiresistance included penicillin (but not amoxicillin or cefotaxime), tetracycline, chloramphenicol and trimethoprim/sulfamethoxazole, with a few strains showing resistance to erythromycin.

In this article, the isolation and characterization of the S. pneumoniae Spain14-5 clone strains whose resistance was expanded to new families of antibiotics are described.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
S. pneumoniae were identified to species level by their colony morphology, optochin sensitivity and bile solubility. Serotyping was carried out by the Quellung reaction (Quellung antisera, Staten Seruminstitut, Copenhagen). MICs of antibiotics and criteria for susceptibility and resistance were as described by the NCCLS.3 The agar dilution method4 for macrolides and fluoroquinolones was carried out in order to expand the range of dilutions available in the broth microdilution trays. Molecular typing methods [PFGE, BOX-PCR and multilocus sequence typing (MLST)] of the isolates were carried out according to previously described protocols.1 The PFGE patterns were analysed using the Diversity Database fingerprinting software version 2 (Bio-Rad) and a dendrogram was constructed by the unweighted pair group method with arithmetic averages, the Dice coefficient and a position tolerance of 1%. Isolates with patterns varying by three or less bands were considered to represent the same PFGE type, which nearly corresponds to a genetic relatedness of >85%.5

Both current1 and previously considered reference strains of the Spain14-5 international clone (strain MS22, ATCC 700902, and strain VH14, ATCC 700672) were used as controls. The presence of the mef(A), erm(B) and erm(A) [erm(TR)] genes was studied by PCR as described previously.6 Point mutations at Ser-79 in the parC and at Ser-81 in the gyrA genes, known to confer fluoroquinolones resistance, were studied by a PCR-RFLP assay7 and were confirmed by gene sequencing. Mutations in the rpoB gene responsible for rifampicin resistance were studied by sequencing in the two rifampicin-resistant isolates, as described previously.8


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The isolates were from sputum or bronchial aspirate, of 16 patients sampled between 1993 and the end of 2001. The majority of the diagnoses were exacerbations of chronic bronchitis; there were eight cases of pneumonia. Except for two HIV-infected patients aged 31and 36 years that were infected in 1993, the patients were all elderly (ages ranged from 66 to 90 years, mean 79 years) and were infected between 1999 and the end of 2001. Overall, these 16 patients yielded 24 isolates within an interval equal to or greater than 3 weeks. The PFGE patterns of all the isolates of these 16 patients showed >95% similarity compared with the pattern of the reference strains of the Spain14-5 international clone studied. By MLST, all the strains belonged to two sequence types: ST17 (allelic profile 1-5-4-11-9-3-47) and ST18 (allelic profile 1-5-4-11-9-3-16) (Table 1). By BOX-PCR, these isolates showed two different patterns, arbitrarily named variant I and variant II, which differed by only two bands (Figure 1). Isolates belonging to variant I showed the same BOX-PCR pattern as the ATCC reference strains and were found only in three patients. Two of these patients were HIV-infected young men with pneumonia and the other was an 80-year-old patient with bronchitis. The strains of the two HIV patients showed resistance to penicillin, tetracycline, chloramphenicol, trimethoprim/sulfamethoxazole, macrolides, lincosamides, fluoroquinolones and rifampicin (Table 1).


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Table 1.  MICs (mg/L) for 16 representative clinical isolates (one per patient) and the two reference strains of S. pneumoniae Spain14-5
 


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Figure 1. BOX-PCR (a) and PFGE (b) patterns of six S. pneumoniae serotype 14 clinical isolates and three reference strains of two multidrug-resistant international clones. Lanes 1 and 2: clinical isolates ST17, BOX-PCR pattern II. Lanes 3 and 4: clinical isolates ST17, BOX-PCR pattern I. Lane 5: clinical isolate ST18, BOX-PCR pattern I. Lanes 6 and 7: BOX-PCR pattern I of Spain14-5 reference strains VH14 (ATCC 700672) and MS22 (ATCC 700902), respectively. Lane 8: clinical isolate serotype 14 variant of the international clone Spain9v-3. Lane 9: reference strain of the Spain9v-3 clone, TL7-93 (ATCC 700671). Lane 10: molecular weight control (100 bp ladder for BOX-PCR and 50 kb ladder for PFGE).

 
The remaining 21 S. pneumoniae isolates from 13 patients, four of which had multiple (two to four) isolates of the same strain separated by 3 weeks to five and a half months, showed the BOX-PCR variant II pattern. All the isolates with the variant II BOX-PCR pattern showed resistance to amoxicillin, cefotaxime, macrolides, lincosamides and fluoroquinolones.

Point mutations at Ser-79 in the parC and at Ser-81 in the gyrA genes were found in all isolates. In all erythromycin-resistant isolates, the presence of the erm(B) gene was detected and two rpoB mutations (Thr-318->Ile and His-425->Asn) were found in the two rifampicin-resistant isolates. All isolates from the 16 patients were susceptible to vancomycin (MIC <= 0.5 mg/L), quinupristin/dalfopristin (MIC <= 1 mg/L) and linezolid (MIC <= 2 mg/L). Telithromycin MICs for all isolates were <=0.5 mg/L.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the introduction of penicillin in S. pneumoniae therapy, pneumococci have progressively adapted to and acquired resistance to the newer antimicrobials introduced in the treatment of their infections. Four different serotype 14 strains are included nowadays among the major international antimicrobial-resistant clones of S. pneumoniae defined by the Pneumococcal Molecular Epidemiology Network (PMEN):1 clone 5 (Spain14-5), clone 3 (Spain9v-3-14 or variant 14 of the clone Spain9v-3), clone 9 (England14-9) and clone 10 (CSR14-10). In our region (northern Spain) the clone Spain14-5 was isolated almost with the same frequency as the serotype 14 variant of the clone Spain9v-3 (E. Pérez-Trallero, J. M. Garcia-Arenzana, J. M. Marimón & A. Gonzalez, unpublished results) although it is known that the Spain9v-3-14 clone has a globally wider spread.1

Isolates belonging to the most common clones usually share identical susceptibility patterns, but regional selective pressure in different parts of the world can add resistance to other antibiotics. The susceptibility referred to for the Spain14-5 clone by the PMEN1 showed resistance to a high number of antimicrobials. Now resistance to new families of antibiotics, and resistance to cefotaxime and amoxicillin shown by the majority of the isolates turns it into the international clone with one of the widest, if not the widest, spectrum of antibiotic resistance among all the clones described.

Macrolide and lincosamide resistance is a frequent event among other international multiresistant S. pneumoniae clones1,5 and five of the 18 strains included in the original description of the Spain14-5 clone were erythromycin resistant, although the mechanism of its resistance was not determined.2 In all the strains described here the erythromycin resistance was due solely to the presence of the erm(B) gene, with the mef(A) and erm(A) genes not being found. Fluoroquinolone resistance is still an infrequent event but may well increase in the near future. All the isolates described here were resistant to fluoroquinolones, and fluoroquinolone resistance has been described previously in the S. pneumoniae Spain23F-1 and Spain9v-3 clones.9 Eight years ago we made a short reference to the two isolates with resistance to rifampicin, but at that time they were not characterized as belonging to the Spain14-5 clone and nor were the mutations implicated in the mechanism of resistance studied.10 The His-425->Asn mutation found in our isolates has been described previously as conferring resistance to rifampicin in S. pneumoniae.8

Ketolides, of which telithromycin is the first to be registered for clinical use, and quinupristin/dalfopristin, which belongs to the macrolide–lincosamide–streptogramin B class, are new compounds that have retained activity against these strains even though they are resistant to macrolides and clindamycin. These drugs together with azalides and vancomycin represent the last therapeutic options against this clone with enhanced multidrug resistance.

MLST was a very useful method for the characterization of these multiresistant S. pneumoniae isolates. The clone was initially characterized by PFGE but the MLST proved to be a more discriminatory typing method as it allowed us to subdivide the clone into two ST types. Using the database available at the MLST web site (www.mlst.net), five different ST types belonging to the Spain14-5 clone can currently be found, ST18 and ST17 being the more common types in this database. Most of the strains presented in this work were ST17, which by BOX-PCR could be further subdivided into two varieties. The three different phenotypes of antibiotic resistance coincided with the three different molecular patterns (ST17 BOX-PCR I, ST17 BOX-PCR II and ST18 BOX-PCR I). Each type could represent a different lineage with a different spreading ability.

We hope that these multiresistant strains will not reach the pandemic diffusion that other S. pneumoniae belonging to other international multiresistant clones have reached. Nevertheless, it will be necessary to maintain a continuous monitoring of the resistant S. pneumoniae to warn against the spread of these organisms. In view of such a scene of antimicrobial resistance, the introduction of new vaccines or new immunization strategies is becoming more and more necessary for the future prevention of S. pneumoniae infections.


    Footnotes
 
* Corresponding author. Tel: +34-94-300-7046; Fax: +34-94-300-7063; E-mail: mikrobiol{at}terra.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . McGee, L., McDougal, L., Zhou, J., Spratt, B. G., Tenover, F. C., George, R. et al. (2001). Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network. Journal of Clinical Microbiology 39, 2565–71.[Abstract/Free Full Text]

2 . Coffey, T. J., Berron, S., Daniels, M., Garcia-Leoni, M. E., Cercenado, E., Bouza, E. et al. (1996). Multiply antibiotic-resistant Streptococcus pneumoniae recovered from Spanish hospitals (1988–1994): novel major clones of serotypes 14, 19F and 15F. Microbiology 142, 2747–57.[Abstract]

3 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA.

4 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

5 . Marimón, J. M., Iglesias, L., Vicente, D. & Perez-Trallero, E. (2003). Molecular characterization of erythromycin-resistant clinical isolates of the four major antimicrobial-resistant Spanish clones of Streptococcus pneumoniae (Spain23F-1, Spain6B-2, Spain9v-3, Spain14-5). Microbial Drug Resistance, in press.

6 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562–6.[Abstract]

7 . Pan, X. S., Ambler, S., Mehtar, S. & Fisher, L. M. (1996). Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 2321–6.[Abstract]

8 . Padayachee, T. & Klugman, K. P. (1999). Molecular basis of rifampin resistance in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 43, 2361–5.[Abstract/Free Full Text]

9 . McGee, L., Goldsmith, C. E. & Klugman, K. P. (2002). Fluoroquinolone resistance among clinical isolates of Streptococcus pneumoniae belonging to international multiresistant clones. Journal of Antimicrobial Chemotherapy 49, 173–6.[Abstract/Free Full Text]

10 . Garcia-Arenzana, J. M., Montes, M. & Perez-Trallero, E. (1994). Are rifampin-resistant Streptococcus pneumoniae strains a consequence of the increase in cases of tuberculosis? Clinical Infectious Diseases 19, 360–1.[ISI][Medline]