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Antimicrob Agents Chemother. Jun 2008; 52(6): 2260–2262.
Published online Mar 31, 2008. doi:  10.1128/AAC.01270-07
PMCID: PMC2415785

Mechanisms of Macrolide Resistance among Streptococcus pneumoniae Isolates from Russia[down-pointing small open triangle]

Abstract

Among 76 macrolide-nonsusceptible Streptococcus pneumoniae isolates collected between 2003 and 2005 from Central Russia, the resistance mechanisms detected in the isolates included erm(B) alone (50%), mef alone [mef(E), mef(I), or a different mef subclass; 19.7%], or both erm(B) and mef(E) (30.3%). Isolates with dual resistance genes [erm(B) and mef(E)] belonged to clonal complex CC81 or CC271.

Antimicrobial resistance in Streptococcus pneumoniae, especially to macrolides, has been a matter of growing concern in the last two decades due to increasing numbers of reports on the failures of treatment of infections caused by macrolide-resistant pneumococcal isolates (4, 10). The most prevalent mechanisms of macrolide resistance in S. pneumoniae are target modification due to ribosomal methylation [associated with the erm(B) or the erm(A) gene] and a macrolide-specific efflux mechanism encoded by closely related mef genes (15). A new subclass of mef genes, mef(I), has recently been recognized in S. pneumoniae (2). The emergence and dissemination of macrolide-resistant S. pneumoniae strains with both erm(B) and mef genes are also new challenges (6). The goals of this study were to analyze the phenotypic characteristics, mechanisms of resistance, and clonal relationships among macrolide-resistant S. pneumoniae isolates from Russia.

(Parts of this work were presented at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 14 to 17 September 2006, abstr. C2-432.)

A total of 823 isolates of S. pneumoniae from the central and northwestern regions of Russia (Moscow, n = 618; St. Petersburg, n = 174; Yaroslavl, n = 31) were obtained from 2003 to 2005. Only one isolate per patient was included in the study. Isolates were from sputum and other lower respiratory tract specimens (56.6%), upper respiratory tract specimens (37.9%), sterile body sites (2.2%), and other or unspecified sources (3.3%). The patient age groups were ≤3 years (15.6%), 4 to 16 years (32.5%), 17 to 65 years (23.4%), >65 years (12.9%), and no age data available (15.7%). Among all patients, 56% were males, 39.5% were females, and no data were available for 4.5%.

MIC testing was performed by the broth microdilution method, as recommended by the Clinical and Laboratory Standards Institute (1). S. pneumoniae ATCC 49619 was used as a control strain. Ninety-one (11.1%) isolates were nonsusceptible to erythromycin (MICs ≥ 1 μg/ml). Only three isolates were resistant to macrolides alone; all other isolates were multidrug resistant. Associated resistance to penicillin G, tetracycline, chloramphenicol, co-trimoxazole, and levofloxacin was observed in 64.5%, 84.2%, 50.0%, 80.2%, and 3.3% of the macrolide-nonsusceptible isolates, respectively. Resistance to three or more classes of antibacterials was detected in 81.5% of macrolide-nonsusceptible isolates. Seventy-six erythromycin nonsusceptible isolates were sent to the National Reference Center for Streptococci (Aachen, Germany), where the results of susceptibility testing were confirmed, detection of macrolide resistance genes was carried out, and serotyping and multilocus sequence typing (MLST) were performed. Macrolide resistance phenotypes were characterized by the triple-disk diffusion test with erythromycin, clindamycin, and rokitamycin disks (14).

Genomic DNA from clinical isolates was used as the template for PCR assays. The oligonucleotide primers used are described in Table Table1.1. DNA sequence analysis of the mef genes was performed with the same primers used for PCR and an ABI Prism 3100 genetic analyzer (Applied Biosystems). The original database entries for mef(A), mef(E), and mef(I) (GenBank accession numbers U70055, U83667, and AJ971089, respectively) were used for the alignment of the nucleotide sequences and the discrimination of mef genes.

TABLE 1.
Oligonucleotide primers used for conventional PCR in the study

The resistance genes detected and the associated phenotypes and serotypes are summarized in Table Table2.2. Only mef genes were detected in 15 isolates (19.7%). Twelve of these were positive by PCR with the MCP-L and MCP-S sets of primers. The 1,218-bp amplicons obtained with the MCP-L primers were used as templates for discrimination of the mef genes by DNA sequence analysis. Eight of the amplicons were mef(E); two were mef(I); and two amplicons had >99% similarity with a mef gene from Bacteroides ovatus (GenBank accession number AJ557257) but less than 95% similarity with mef(A), mef(E), and mef(I) subclass sequences. The sequences of the mef(E), mef(I), and the similar Bacteroides ovatus mef genes were similar to each other. The remaining three isolates had positive PCR results only with the MCP-S set of primers. Sequence analysis of the 263-bp amplicons from these isolates revealed the highest level of similarity (>99%) with the corresponding fragment of the mef(I) gene. These data support the hypotheses that the diversity of mef genes in S. pneumoniae may be much greater than is currently known and that the recently described mef(I) subclass may have spread in different geographical regions. We did not detect any relevant difference in susceptibility to macrolides between the mef(E) and the mef(I) isolates. The discrimination of mef genes may be more important when their association with different genetic elements (Tn1207.1, mega element [8] and the 5216IQ complex [13]) and the potential for horizontal transfer are considered. All mef-only isolates were of the macrolide resistance (M) phenotype. These mef-positive isolates belonged to different serotypes, with no serotype obviously predominating (Table (Table22).

TABLE 2.
Macrolide resistance genes, phenotypes, and serotypes of erythromycin-nonsusceptible isolates

As a single resistance determinant, the erm(B) gene was detected by PCR in 38 (50.0%) of the erythromycin-nonsusceptible isolates. A majority of the erm(B) isolates (n = 27) were indistinguishable on the basis of their inducible macrolide-lincosamide-streptogramin B resistance (iMcLS) phenotype by the triple-disk diffusion test (no significant zone of inhibition appeared around either the erythromycin or the clindamycin disk, and a zone of inhibition appeared around the rokitamycin disk). Serotype 6B predominated among isolates with the iMcLS phenotype. Two erm(B)-positive isolates had phenotypes similar to iMcLS, but without blunting of the zone of inhibition around the rokitamycin disk. The remaining nine isolates had the constitutive macrolide-lincosamide-streptogramin B resistance (cMLS) phenotype, and all of these were highly resistant to macrolides and clindamycin.

The dual combination of the ermB and the mef genes was detected in 23 isolates (30.3%), and among these, all mef genes were determined to be mef(E), as was reported for dual-mechanism isolates from the PROTEKT study (7). Twenty-one of the isolates from this group had the cMLS phenotype and two had the iMcLS-like phenotype, without blunting of the inhibition zone around rokitamycin. Serotype 23F predominated among isolates with the cMLS phenotype.

Eight epidemiologically unrelated isolates carrying both erm(B) and mef(E), obtained from different hospitals from 2003 to 2005, were randomly selected and analyzed by MLST, as described by Enright and Spratt (5). S. pneumoniae isolates carrying both the erm(B) and the mef(E) genes belonged to two clonal complexes (CCs), CC81 and CC271. CC81 was represented by four sequence type (ST) variants: ST81 (one isolate) and three new single-locus variants of ST81, ST2032 (two isolates), ST2033 (two isolates), and ST2576 (one isolate). Two CC271 isolates belonged to ST320. To our knowledge, this is the first description of a high prevalence of CC81 isolates carrying both erm(B) and mef(E) outside Asia or Africa. Such isolates of S. pneumoniae are usually reported from regions with a high prevalence of resistance to macrolides (30% or more) worldwide (3, 6, 11, 12), but in Russia the rate of resistance to macrolides is relatively low. From 1998 to 2003 the rates of resistance to macrolides in Moscow varied from 7.9% to 19% (9). The independent emergence in Russia of dual isolates seems to be unlikely among isolates with the erm(B) genotype, which (at least at present) is significantly more prevalent than the mef genotype among macrolide-resistant isolates of S. pneumoniae (50% and 19.7%, respectively). It is possible that isolates with both erm(B) and mef(E) reached Central Russia from Southeast Asia through the Russian Far East.

Nucleotide sequence accession numbers.

The sequences of the mef(E), mef(I), and the similar Bacteroides ovatus mef genes have been deposited in the GenBank database under accession numbers EU486997, EU486999, and EU486995, respectively. A representative of the 263-bp amplicon sequence was deposited in the GenBank database under accession number EU487001.

Acknowledgments

We thank the clinical microbiology laboratories in Central Russia for their cooperation and for providing the isolates and N. Neuberger for excellent technical assistance.

The study was supported in part by grant no. 2460/61 from the Biotechnology Engagement Program, U.S. Department of Health and Human Services, and the International Science and Technology Center.

Footnotes

[down-pointing small open triangle]Published ahead of print on 31 March 2008.

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