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Antimicrobial Agents and Chemotherapy, June 2008, p. 1964-1969, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.01487-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Polyclonal Population Structure of Streptococcus pneumoniae Isolates in Spain Carrying mef and mef plus erm(B)

Elia Gómez G. de la Pedrosa,¹ María-Isabel Morosini,¹ Mark van der Linden,² Patricia Ruiz-Garbajosa,¹ Juan Carlos Galán,¹ Fernando Baquero,¹ Ralf René Reinert,² and Rafael Cantón^1*

Servicio de Microbiología, Hospital Universitario Ramón y Cajal, and CIBER-ESP, Madrid, Spain,¹ Institute for Medical Microbiology, National Reference Center for Streptococci, University Hospital (RWTH), Aachen, Germany²

Received 15 November 2007/ Returned for modification 4 February 2008/ Accepted 15 March 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The population structure (serotypes, pulsed-field gel electrophoresis [PFGE] types, and multilocus sequencing types) of 45 mef-positive Streptococcus pneumoniae isolates [carrying mef alone (n = 17) or with the erm(B) gene n = 28)] were studied. They were selected from among all erythromycin-resistant isolates (n = 244) obtained from a collection of 712 isolates recovered from different Spanish geographic locations in the prevaccination period from 1999 to 2003. The overall rates of resistance (according to the criteria of the CLSI) among the 45 mef-positive isolates were as follows: penicillin G, 82.2%; cefotaxime, 22.2%; clindamycin, 62.2%; and tetracycline, 68.8% [mainly in isolates carrying erm(B) plus mef(E); P < 0.001]. No levofloxacin or telithromycin resistance was found. Macrolide resistance phenotypes (as determined by the disk diffusion approximation test) were 37.7% for macrolide resistance [with all but one due to mef(E)] and 62.2% for constitutive macrolide-lincosamide-streptogramin B resistance [cMLS_B; with all due to mef(E) plus erm(B)]. Serotypes 14 (22.2%), 6B (17.7%), 19A (13.3%), and 19F (11.1%) were predominant. Twenty-five different DNA patterns (PFGE types) were observed. Our mef-positive isolates were grouped (by eBURST analysis) into four clonal complexes (n = 18) and 19 singleton clones (n = 27). With the exception of clone Spain^9V-3, all clonal complexes (clonal complexes 6B, Spain^6B-2, and Sweden^15A-25) and 73.6% of singleton clones carried both the erm(B) and the mef(E) genes. The international multiresistant clones Spain^23F-1 and Poland^6B-20 were represented as singleton clones. A high proportion of mef-positive S. pneumoniae isolates presented the erm(B) gene, with all isolates expressing the cMLS_B phenotype. A polyclonal population structure was demonstrated within our Spanish mef-positive S. pneumoniae isolates, with few clonal complexes overrepresented within this collection.

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Macrolide resistance among Streptococcus pneumoniae clinical isolates has risen to prominence. The rate of resistance to macrolides is even higher than that to penicillin, particularly in Spain and some other European countries, such as France, Poland, Greece, and Portugal (17, 23). In most European countries, erythromycin resistance is mainly due to the presence of the erm(B) gene. This gene encodes an rRNA methylase responsible for the macrolide-lincosamide-streptogramin B resistance (MLS_B) phenotype and is associated with conjugative transposons. The clonal dispersion of erm(B)-positive S. pneumoniae isolates and the horizontal transfer of transposable elements carrying this determinant have been associated with the increase in the rate of erythromycin resistance in this organism (4, 12). Erythromycin resistance may also be associated with the expression of efflux pumps encoded by mef genes that endow the macrolide resistance (M) phenotype (7). This phenotype has traditionally been more prevalent in North America than in Europe (7, 12).

In recent years, the presence of both the erm(B) and the mef(E) genes in S. pneumoniae clinical isolates has been increasingly recognized, but they are more prevalent in Asian countries than in Europe and the United States (10, 11). Despite the interest in this association on the evolution of macrolide and ketolide resistance, very few studies have investigated the population biology of a collection of isolates recovered during different time periods and/or from different geographic origins (13, 26). In Spain, population structure studies have shown that resistance in S. pneumoniae international clones, such as clones Spain^9V-3 and England¹⁴-9, are mainly associated with those endowed with the M phenotype (1, 2), whereas clones Spain^23F-1 and Spain^6B-2 are associated with isolates endowed with the MLS_B phenotype (14). This type of analysis with S. pneumoniae isolates with both the erm(B) and the mef genes has not been reported. In the present study, the population structure and clonal relatedness of 45 mef-positive isolates [62.2% of which had both the erm(B) and the mef genes] identified within a collection of 712 S. pneumoniae isolates recovered from different Spanish hospitals from 1999 to 2003 were fully investigated. Moreover, the corresponding resistance phenotypes and antibiotic susceptibility patterns were also studied.

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Bacterial isolates. Seven hundred twelve S. pneumoniae clinical isolates (244 isolates nonsusceptible to erythromycin) recovered during the prevaccination period (in Spain, the conjugate heptavalent vaccine was accepted for use in 2002, but it was rarely used until 2004) were studied. Isolates were prospectively collected from 14 Spanish hospitals representing 14 different geographic areas during the fall and winter seasons of 1999 through 2003. Among these strains, a total of 45 mef-positive isolates were detected, and all of them were selected for further studies. The origins of these 45 isolates are included in Table 1. A total of 26.6% (12 of 45) of these isolates were of pediatric origin.

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TABLE 1. Typing characteristics of 45 mef-positive Streptococcus pneumoniae isolates recovered in Spain

Susceptibility testing and erythromycin resistance phenotypes. The MICs of penicillin G, cefotaxime, erythromycin, clindamycin, telithromycin, tetracycline, and levofloxacin were determined by the broth microdilution method, according to Clinical Laboratory Standards Institute (CLSI) guidelines (3). Incubation was performed at 35°C in ambient air. S. pneumoniae ATCC 49619 was used as the reference strain in each run. The breakpoints were those established by the CLSI guidelines (3). The phenotypic detection of erm induction was performed by using the macrolide (erythromycin)-clindamycin disk diffusion approximation test, as described previously (17).

Detection of erythromycin resistance genes. A real-time PCR approach was carried out for detection of the erm and mef genes among erythromycin-resistant isolates by using the conditions described previously (20). A subsequent scheme of multiplex PCR was followed to differentiate between the mef(A) and the mef(E) genes. Specific primers designed for this purpose were forward primer mefAF (5'-AATACAACAATTGGAAACTT-3'), forward primer mefEF (5'-AAGGAGTTGTGGTTCTGA-3'), and a reverse primer for both the mef(A) and the mef(E) genes, primer mefR (5'-AATCGTGTAAATCATTGG-3'). The expected sizes of the PCR products were 1,080 kb for mef(A) and 480 kb for mef(E). The PCR amplification mixture of 25 µl contained 15 mM Tris-HCl, 50 mM KCl (pH 8.0), 25 mM MgCl₂, 100 µM of each nucleotide, 0.15 pmol of the two forward primers mefAF and mefEF, 0.3 pmol of reverse primer mefR, 1.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), and 1 µl of genomic DNA. The PCR conditions (PTC-100 thermocycler; MJ Research Inc., Watertown, MA) comprised an initial denaturation step at 94°C for 12 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and elongation at 72°C for 1 min. After the amplification cycles, a final elongation step of 10 min at 72°C was included.

Population structure. Serotyping was performed with the Neufeld Quellung reaction by using antisera provided by the Statens Seruminstitut (Copenhagen, Denmark). Pulsed-field gel electrophoresis (PFGE) was performed as previously described by del Campo et al. (6). Briefly, chromosomal DNA was prepared by following the standard protocol for gram-positive bacteria, with some modifications (6). The DNA was restricted with the SmaI endonuclease (Amersham Biosciences Europe GmbH, Freiburg, Germany). Electrophoresis was performed with CHEF DR-III equipment (Bio-Rad, Birmingham, United Kingdom) for 23 h at 14°C, and the following settings were applied: 6 V/cm and 1 to 30 s. The PFGE patterns obtained were compared with those for clones established by the Pneumococcal Molecular Epidemiology Network (16).

Multilocus sequence typing (MLST) scheme for S. pneumoniae was carried out as described previously (9). Clusters of related sequence types (STs) were grouped into clonal complexes (CCs) by use of the eBURST program (http://www.mlst.net). STs were clustered with BioNumerics software (version 4.0; Applied Maths, Sint-Martens-Latem, Belgium) by using a categorical coefficient and a graphing method called the minimum-spanning tree, as described previously (24).

Statistical analysis. Statistical associations were analyzed by the chi-square test. Differences were considered statistically significant when the two-tailed P value was less than 0.05.

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Macrolide resistance genes, phenotypes, and susceptibility patterns. Within the entire S. pneumoniae population (n = 712), 244 isolates were nonsusceptible to erythromycin. Among those isolates, 80.7% carried the erm(B) gene as the sole genetic determinant and showed the MLS_B phenotype. In addition, 45 (18.4%) of the erythromycin-resistant isolates carried a mef-type gene, and of these, 44 isolates had the mef(E) gene and the remaining one had the mef(A) gene. It is of note that 28 of 45 mef-positive isolates also carried the erm(B) gene (62.2%). In two isolates displaying erythromycin and clindamycin MICs of 0.5 mg/liter, neither the erm(B) gene nor the mef gene was detected. The constitutive MLS_B phenotype was observed in all 28 isolates carrying both the mef and the erm(B) genes, whereas the M phenotype was detected in the remaining 17 isolates that carried only one of the mef genes alone. No temporal or local geographic association among the mef-positive or the mef- plus erm(B)-positive isolates was observed (data not shown).

The overall rates of resistance to penicillin G, cefotaxime, tetracycline, and clindamycin among all 45 mef-positive isolates were 82.2% [35.5% for mef-positive isolates plus 46.7% for the mef- plus erm(B)-positive isolates], 22.2% [17.7% for the mef-positive isolates plus 4.4% for the mef- plus erm(B)-positive isolates], 68.8% [13.3% for the mef-positive isolates plus 55.5% the mef- plus erm(B)-positive isolates], and 62.2% [0% for the mef-positive isolates plus 62.2% for the mef- plus erm(B)-positive isolates], respectively. Neither telithromycin resistance nor levofloxacin resistance was found (MIC ranges, 0.03 to 1 mg/liter and 0.25 to 2 mg/liter, respectively).

Population structure in mef-positive S. pneumoniae isolates. The serotype distribution among the 45 mef-positive isolates was as follows: serotype 14, 22.2%; serotype 6B, 17.7%; serotype 19A, 13.3%; serotype 19F, 11.1%; serotype 11A, 6.6%; serotype 9V, 6.6%; serotype 15A, 6.6%; serotype 23F, 4.4%; and other serotypes, 11.1%. With a single exception, all isolates belonging to serotype 14 (n = 10) harbored the mef gene alone. Six different PFGE patterns were found among the mef(E)-positive isolates, with 58.8% of them belonging to the Spain^9V-3 clone. Among the erm(B)- plus mef(E)-positive isolates, 19 different PFGE patterns were found, with the Sweden^15A-25, Spain^6B-2, Spain^23F-1, Poland^6B-20, and Norway^NT-42 clones represented. Analysis of the MLST results by use of the eBURST program showed that our mef-positive isolates were grouped into 4 CCs and 19 singleton clones (Fig. 1). The serotypes, the PFGE type distribution according to analysis with the eBURST program, and the corresponding STs are shown in Table 1.

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FIG. 1. Clustering of 30 STs identified among 45 mef-positive Streptococcus pneumoniae isolates by use of the minimum-spanning tree. Each circle represents an ST, and the type number is indicated in the circle. The area of each circle corresponds to the number of isolates. Thick, short, solid lines connect single-locus variants; thin, longer, solid lines connect double-locus variants; black dotted lines connect STs which differ at three loci; and gray dotted lines connect STs that differ in more than three loci. CCs and international resistant clones (Pneumococcal Molecular Epidemiology Network) are indicated.

The CCs grouped 18 of the mef-positive isolates; 10 of them carried the mef(E) gene as the sole resistance determinant and belonged to the Spain^9V-3 international clone, and 8 isolates carried both the erm(B) and the mef(E) genes. The latter isolates were grouped into the Spain^6B-2 international clone (n = 3); the Sweden^15A-25 clone (n = 2); and CC-6B, which includes two different STs (ST135 and ST1638, which is a double-locus variant of ST135) (n = 3).

Twenty-seven isolates were grouped into singleton clones which included some international resistant clones, such as Spain^23F-1 [two isolates harbored both the erm(B) and the mef(E) genes], Poland^6B-20 and Norway^NT-42 [one isolate each carried both the erm(B) and the mef(E) genes], and England¹⁴-9 [represented by the only isolate that carried the mef(A) gene detected in this study]. With the exception of tetracycline (MIC, 4 mg/liter), this isolate was susceptible to all antibiotics tested. ST276 (a single-locus variant of the Denmark¹⁴-32 clone), ST549, and ST62 grouped more than one isolate each (four, three, and three isolates, respectively), while the other STs were each represented by a single isolate. The relationship between the STs and the macrolide resistance genes is shown in Table 1.

Serotype distribution according to analysis with the eBURST program showed that even though strains of some serotypes belonged to specific international clones, such as serotypes 6B, 14, 19A, and 15A, these serotypes were also found among the isolates grouped as singleton clones. The serotype distribution according to analysis with the eBURST program is shown in Table 1.

Coresistance to penicillin (MIC range, 0.12 to 4 mg/liter) was found among the isolates grouped into CCs. The only isolate intermediate for cefotaxime (MIC, 4 mg/liter) belonged to CC-Spain^9V-3. This isolate also showed increased MICs for levofloxacin (2 mg/liter). This was also found among the CC-Spain^6B-2 and CC-6B isolates. Only eight isolates (all of them belonging to CC-Spain^9V-3) were susceptible to tetracycline. The susceptibility patterns among the CCs is shown in Table 2. Some differences in susceptibilities among the isolates grouped as singleton clones were observed between the mef-positive isolates and the isolates containing both erm(B) and mef(E).

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TABLE 2. Susceptibility patterns of isolates belonging to different clonal complexes

Coresistance to penicillin G was more prevalent among the isolates carrying erm(B) plus mef(E) (89.2%) than among the mef(E)/mef(A)-positive (70.5%) isolates. The same trend, but with a high degree of statistical significance (P < 0.001), was observed for the rate of tetracycline resistance among the isolates positive for both erm(B) plus mef(E) (92.8%) compared with that among the mef(E)/mef(A)-positive (29.4%) isolates. The rates of cefotaxime resistance were similar between the two groups. The susceptibility patterns of the singleton clones according to the macrolide resistance genotype are shown in Table 3.

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TABLE 3. Susceptibility patterns of singleton clones

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Typing studies are useful for providing an understanding of the epidemiology and spread of resistant bacteria as well as establishing control protocols against epidemics (9). For S. pneumoniae, capsular typing is commonly used in clinical laboratories and has demonstrated that particular serotypes, such as serotypes 23F and 6B, are related to the worldwide spread of penicillin G-resistant isolates (5, 21). PFGE is useful as a means of observing recent changes, and MLST is valuable for discriminating the variations that slowly accumulate among the bacterial population (9). These techniques have been used in our study with all 45 erythromycin-resistant S. pneumoniae isolates carrying mef genes recovered during a prospective study in different Spanish geographic areas. With the aid of the MLST technique, the mef-positive isolates among our Spanish S. pneumoniae isolates were demonstrated to be polyclonal, with few CCs overrepresented within the collection studied.

Resistance to macrolides in S. pneumoniae dramatically increased in Spain during the prevaccination period from 1999 to 2003 (17, 23). In our collection, 34.3% of the isolates were resistant to erythromycin, mainly due to the presence of the erm(B) gene. In recent years, an increase in the prevalence of efflux mechanisms (M phenotype) has been observed (13, 20). Among the macrolide-resistant isolates in our collection, the M phenotype was found in 6.9% of the isolates, and all of them carried a mef gene. However, the presence of this gene was even higher, since 11.5% (28 of 244 isolates) of the isolates with a constitutive MLS_B phenotype concomitantly presented the erm(B) gene. This value is higher than that previously reported from other studies in Spain, in which mef isolates did not have the erm(B) gene (2). Such an increasing association between the mef(E) and the erm(B) genes was unexpected in a country with a high incidence of S. pneumoniae isolates harboring the erm(B) gene. It is indeed difficult to understand the possible selective advantage of these isolates harboring both determinants, as erm(B) alone provides higher MICs than those apparently needed to resist the actions of macrolides. In other countries, the increased prevalence of isolates carrying both determinants has been related to the spread of specific clones, such the Taiwan^19F-14 clone, and members of a specific CC, CC-271, which includes ST271, ST236, and ST320 (8, 10). It is of note that ST271 was also found in our collection, but it was found as a singleton clone not related to the other STs (Fig. 1). In our study, the presence of isolates with both the erm(B) and the mef(E) genes was associated with three different CCs and 14 singleton clones (Table 1). These results demonstrated the nonclonal nature of the population studied and that horizontal gene transfer processes might have occurred, as may have the selection of resistant S. pneumoniae isolates harboring the mef(E) and the erm(B) genes.

In addition, most of the erm(B) plus mef(E) isolates were also resistant to penicillin, a fact that has also been found among isolates in geographic areas where isolates with this dual genotype are prevalent (10). On the other hand, tetracycline resistance was significant among the isolates positive for both erm(B) and mef(E). This fact could be related to the presence of conjugative transposons, like Tn2010, recently described in these isolates (4, 8). The participation of this trait in the maintenance of these isolates should be investigated.

Within the population with both the erm(B) and the mef determinants that we studied, we were able to identify multiresistant international clones (www.sph.emory.edu/PMEN) circulating in Spain (2, 14, 18), including clones Spain^6B-2 and Sweden^15A-25 among the CCs and clones Spain^23F-1, Poland ^6B-20 and Norway^NT-42 as singletons. Both the Spain^6B-2 and Sweden^15A-25 clones have been shown to be overrepresented among S. pneumoniae isolates that are highly resistant to penicillin or that have an MLS_B phenotype (2, 14). Among the singleton clones, the Spain^23F-1 clone was scarcely represented in our collection (two isolates of serotypes 19A and 23F), as were the Poland^6B-20 and Norway^NT-42 clones, compared with their representations in other studies (2, 14). Moreover, we did not find the Spain¹⁴-5 clone, which also carries both genes and which has been found in some specific geographic areas of Spain over a 22-year period (15, 19). Among the isolates with the mef gene as the sole resistance determinant, only one isolate had the mef(A) subclass. This isolate belonged to the international resistant clone England¹⁴-9, also described in other European countries (1). The population of mef(E)-positive isolates was structured into four singleton clones and a CC belonging to the Spain^9V-3 international resistant clone with capsular switching into serotype 14, as previously described by Ardanuy et al. (1), and also into serotype 19A, as has been observed in other studies (25). A rate of penicillin resistance of only 14.2% was found among the mef(E)-positive singleton clones, and the penicillin MIC range for isolates of the CC-Spain^9V-3 clone was 0.12 to 4 mg/liter, in agreement with the findings of other Spanish studies (1).

In conclusion, the rate of macrolide resistance in our Spanish S. pneumoniae collection was high (34.3%). An increase in the presence of the mef gene compared with that in other studies was detected, but this was not associated with the M phenotype. Most of the mef-positive isolates also harbored the erm(B) gene (62.2% of the mef- positive isolates), with all of them showing the constitutive MLS_B phenotype. Among the isolates showing the M phenotype only, one carried the mef(A) subclass and belonged to the England¹⁴-9 clone, as previously described in Europe (1). Population structure analysis showed that the mef-positive isolates are grouped in different clones and CCs and not in only a few clones, as previously described in other geographic areas (1, 14).

 
E. Gómez G. de la Pedrosa is a recipient of a post-MIR contract from the Instituto de Salud Carlos III, Ministry of Health, Spain (ref. CM07/00147). Part of this study was funded by an unrestricted grant from Sanofi-Aventis and by CIBER-ESP (the Network Center for Biomedical Research in Epidemiology and Public Health) and the Microbial Sciences Foundation.

 
* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, and CIBER-ESP, Madrid 28034, Spain. Phone: 34-91-3368330. Fax: 34-91-3368809. E-mail: rcanton.hrc{at}salud.madrid.org

Published ahead of print on 24 March 2008.

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Antimicrobial Agents and Chemotherapy, June 2008, p. 1964-1969, Vol. 52, No. 6
0066-4804/08/$08.00+0     doi:10.1128/AAC.01487-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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