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Antimicrobial Agents and Chemotherapy, May 2002, p. 1574-1576, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1574-1576.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Macrolide and Tetracycline Resistance and Molecular Relationships of Clinical Strains of Streptococcus agalactiae

Esther Culebras,* Iciar Rodriguez-Avial, Carmen Betriu, Montserrat Redondo, and Juan J. Picazo

Department of Clinical Microbiology, Hospital Universitario San Carlos, 28040 Madrid, Spain

Received 8 October 2001/ Returned for modification 14 December 2001/ Accepted 24 January 2002


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ABSTRACT
 
Mechanisms for tetracycline and macrolide resistance in 54 isolates of erythromycin-resistant Streptococcus agalactiae were analyzed by PCR. The erm(B), erm(A), and mef(A) genes, either alone or in combination, were detected in all the erythromycin-resistant isolates. The tet(M) and tet(O) genes were responsible for tetracycline resistance. Random amplification of polymorphic DNA indicated different clonal origins of the isolates.


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TEXT
 
Group B streptococcal infections are a leading cause of neonatal mortality and also affect pregnant women and the elderly. ß-Lactam agents are the treatment of choice for these infections, but macrolides and related drugs provide useful alternative therapy for allergic patients. Streptococcus agalactiae is considered to be susceptible to ß-lactam antimicrobial agents, but the emergence of strains resistant to macrolides and tetracycline has been increasingly reported (15, 17).

The mechanisms of erythromycin resistance in S. agalactiae include target site modification and active drug efflux. Target modification is conveyed by the action of a family of methyltransferase enzymes encoded by the erm genes. The erm genes found in S. agalactiae are erm(B) and erm(A). Both genes may be inducibly or constitutively expressed (14). Active drug efflux is mediated by the mef(A) gene and causes resistance to 14- and 15-membered macrolide compounds (4).

Tetracycline resistance genes are often found on the same mobile unit as erythromycin resistance genes (22). In a variety of gram-positive and gram-negative species, erm(B) is frequently found linked with tet(M) (23). Seventeen different tetracycline resistance determinants have been characterized to date. Most of these determinants code either for a protein which pumps tetracycline out of the cell or for a ribosomal protection protein which protects the ribosomes from the action of tetracycline (2).

The purpose of this study was to investigate the phenotypic and genotypic distribution of erythromycin-resistant S. agalactiae isolates and to explore the clonality of these isolates by molecular methods. Rates of tetracycline resistance and the genes responsible were also investigated.

(Results of this study were presented at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 2001.)

From 1992 through 2000 a total of 109 S. agalactiae (55 erythromycin-susceptible and 54 erythromycin-resistant) isolates were collected in the Microbiology Department of the Hospital Clínico San Carlos. In order to avoid duplication, only one isolate per patient was studied. The clinical sources were as follows: skin and soft tissues (57 isolates), urine (29 isolates), vagina (12 isolates), blood (5 isolates), upper respiratory tract (4 isolates), and abdomen (2 isolates). Isolates were identified by a commercial latex agglutination technique (Slidex Strepto B; bioMérieux, Marcy L'Etoile, France).

All S. agalactiae isolates were tested for antibiotic resistance. MICs of erythromycin (Abbott, Madrid, Spain), clindamycin (Pharmacia & Upjohn, Barcelona, Spain), and tetracycline (Sigma Chemical Co., St. Louis, Mo.) for all the erythromycin-resistant strains were determined by the agar dilution method in accordance with NCCLS guidelines (18, 19). Plates were incubated overnight at 37°C under 5% CO2. The resistance phenotypes of all erythromycin-resistant isolates were determined by the double-disk diffusion method using erythromycin (15 µg) and clindamycin (2 µg) disks as described elsewhere (24).

For the detection of different resistance genes, DNA of S. agalactiae was extracted as previously described (3). DNAs of resistant isolates were amplified by using primers specific for the erm(A), erm(B), erm(C), and mef(A) genes. The sequences of the primer sets and PCR conditions were as previously described (14, 26). All isolates were also tested for the presence of the tet(K), tet(L), tet(M), and tet(O) tetracycline resistance determinants (27).

Epidemiologic typing of S. agalactiae isolates was performed with two different primers (M13 and H2) and Ready-to-Go Analysis Beads (Pharmacia Biotech) as described by Seppälä et al. (25), and the same criteria were used to interpret and compare the patterns.

The activities of the three antimicrobial agents tested against erythromycin-resistant and erythromycin-susceptible S. agalactiae strains are summarized in Table 1. As expected, MICs of clindamycin were lower for erythromycin-susceptible (range, 0.03 to 0.06 µg/ml) than for erythromycin-resistant (range, 0.03 to >64 µg/ml) S. agalactiae isolates. It should be stressed that for 13 erythromycin-resistant S. agalactiae isolates, clindamycin MICs were higher than erythromycin MICs. Rates of tetracycline resistance in erythromycin-resistant and -susceptible isolates (87 and 72%, respectively) were very similar, as were the MICs at which 50 and 90% of isolates were inhibited (MIC50 and MIC90, respectively) (Table 1).


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  		TABLE 1. Activities of erythromycin, clindamycin, and tetracycline against 54 erythromycin-resistant and 55 erythromycin- susceptible S. agalactiae isolates

The numbers of strains with various macrolide-resistant phenotypes, and the genes associated, are given in Table 2. Most (53.7%) of the erythromycin-resistant S. agalactiae isolates showed macrolide-lincosamide-streptogramin B cross-resistance, whereas 37.0 and 9.3% of the strains had the inducible and M phenotypes, respectively.


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  		TABLE 2. Distribution of erythromycin resistance genes among 54 erythromycin-resistant S. agalactiae isolates categorized according to erythromycin resistance phenotype and erythromycin and clindamycin MIC ranges

The mef(A) gene was detected by PCR in all isolates with the M resistance phenotype, either alone (two strains) or associated with the erm(A) gene (three strains). The erm(B) gene was detected either alone (16 isolates) or associated with either erm(A) (18 isolates) or mef(A) (2 strains).

Genes responsible for tetracycline resistance were determined in erythromycin-resistant isolates. Forty-seven erythromycin-resistant S. agalactiae isolates were also resistant to tetracycline; 31 of these had tet(M), and 8 had tet(O). The tet(L) and tet(K) genes did not appear in any of the strains. In 8 isolates, we were not able to find any of the tetracycline-resistant genes tested for. It is possible that these isolates carried another tet determinant.

The most widely distributed tetracycline resistance determinant in gram-positive bacteria is tet(M) (21). The tet(O), tet(K), and tet(L) genes also appear in gram-positive microorganisms but are much less common. As expected, the majority of our isolates harbored the resistance determinant tet(M). We found tet(M) associated with all macrolide-resistant genes.

Resistance to macrolides and tetracyclines in Streptococcus pneumoniae has been assumed to be mainly due to the presence of a conjugative transposon that encodes erm(B) in addition to tet(M) (5). However, in group C and group G streptococci this association between erythromycin and tetracycline resistance determinants was not found (14). From this study, it seems that tetracycline resistance in S. agalactiae is not linked to erythromycin resistance.

Mechanisms of erythromycin and tetracycline resistance in S. pneumoniae and Streptococcus pyogenes have been widely investigated (7, 9, 10, 12, 13, 16), but there are few studies of S. agalactiae, and those are focused only on erythromycin resistance (1, 6, 8, 11, 20). In agreement with the findings of these previous studies, we observed that erythromycin resistance in S. agalactiae was mainly associated with the presence of the erm(B) gene. erm(A) was the second most common gene found. A high percentage of strains in our collection (44.5%) carried more than one macrolide resistance gene. These data agree with some of the previous studies (6, 20). Nevertheless, the association of mef(A) with erm(A) in S. agalactiae was first detected in our study. Novel subphenotypes of macrolide-lincosamide-streptogramin B resistance due to the concomitant presence of erm and mef genes have recently been described for S. pyogenes (12, 13) and S. agalactiae (6). However, the correspondence with our findings appears to be only partial. The erythromycin MIC range for our strains was 1 to 4 µg/ml, and three of these strains were fully susceptible and did not show inducible resistance to clindamycin.

Our collection of S. agalactiae strains was very heterogeneous with regard to macrolide MICs, the presence of erythromycin resistance determinants, and association between different tetracycline- and macrolide-resistant genes. A similar heterogeneity was found when we analyzed the clonal relationships of isolates.

A total of 35 different randomly amplified polymorphic DNA (RAPD) patterns were found among the 54 erythromycin-resistant S. agalactiae strains studied. RAPD-PCR typing with both the M13 and H2 primers resulted in 27 unique fingerprint patterns. Of the remaining 27 isolates, 2 different profiles appeared in 4 isolates each, 2 appeared in 3 isolates each, and 3 different profiles appeared in 2 isolates each. One DNA profile was demonstrated in 7 isolates.

We conclude that, among S. agalactiae strains, resistance to erythromycin and tetracycline in our area is due to multiclonal dissemination of resistance within the streptococcal population rather than to the epidemic spread of single clones.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from Comunidad Autónoma de Madrid (CAM 08.2/0005/1999.1) and by a grant from the Fondo de Investigación Sanitaria (FISS 99/0434), Madrid, Spain.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Clinical Microbiology, Hospital Universitario San Carlos, Martín Lagos s/n, 28040 Madrid, Spain. Phone: 34 913303269. Fax: 34 913303478. E-mail: eculebras{at}cbm.uam.es. Back


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REFERENCES
 
    1
  1. Arpin, C., H. Daube, F. Tessier, and C. Quentin. 1999. Presence of mefA and mefE genes in Streptococcus agalactiae. Antimicrob. Agents Chemother. 43:944-946.[Abstract/Free Full Text]
    2. 2
  2. Chopra, I., P. M. Hawkey, and M. Hinton. 1992. Tetracyclines, molecular and clinical aspects. J. Antimicrob. Chemother. 29:245-277.[Free Full Text]
    3. 3
  3. Chung, W. O., C. Werckenthin, S. Schwarz, and M. C. Roberts. 1999. Host range of the ermF rRNA methylase gene in bacteria of human and animal origin. J. Antimicrob. Chemother. 43:5-14.[Abstract/Free Full Text]
    4. 4
  4. Clancy, J., J. Petitpas, F. Dib-Haij, W. Yuan, M. Cronan, A. V. Kamath, J. Bergeron, and J. A. Retsema. 1996. Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes. Mol. Microbiol. 22:867-879.[CrossRef][Medline]
    5. 5
  5. Courvalin, P., and C. Carlier. 1986. Transposable multiple antibiotic resistance in Streptococcus pneumoniae. Mol. Gen. Genet. 205:291-297.[CrossRef][Medline]
    6. 6
  6. De Mouy, D., J. D. Cavallo, R. Leclercq, and R. Fabre. 2001. Antibiotic susceptibility and mechanisms of erythromycin resistance in clinical isolates of Streptococcus agalactiae: French multicenter study. Antimicrob. Agents Chemother. 45:2400-2402.[Abstract/Free Full Text]
    7. 7
  7. Doherty, N., K. Trzcinski, P. Pickerill, P. Zawadzki, and C. G. Dowson. 2000. Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 44:2979-2984.[Abstract/Free Full Text]
    8. 8
  8. Fitoussi, F., C. Loukil, I. Gros, O. Clermont, P. Mariani, S. Bonacorsi, I. Le Thomas, D. Deforche, and E. Bingen. 2001. Mechanisms of macrolide resistance in clinical group B streptococci isolated in France. Antimicrob. Agents Chemother. 45:1889-1891.[Abstract/Free Full Text]
    9. 9
  9. Giovanetti, E., M. P. Montanari, F. Marchetti, and P. E. Varaldo. 2000. In vitro activity of ketolides telithromycin and HMR 3004 against Italian isolates of Streptococcus pyogenes and Streptococcus pneumoniae with different erythromycin susceptibility. J. Antimicrob. Chemother. 46:905-908.[Abstract/Free Full Text]
    10. 10
  10. Giovanetti, E., M. P. Montanari, M. Mingoia, and P. E. Varaldo. 1999. Phenotypes and genotypes of erythromycin-resistant Streptococcus pyogenes strains in Italy and heterogeneity of inducibly resistant strains. Antimicrob. Agents Chemother. 43:1935-1940.[Abstract/Free Full Text]
    11. 11
  11. Hsueh, P. R., L. J. Teng, L. N. Lee, S. W. Ho, P. C. Yang, and K. T. Luh. 2001. High incidence of erythromycin resistance among clinical isolates of Streptococcus agalactiae in Taiwan. Antimicrob. Agents Chemother. 45:3205-3208.[Abstract/Free Full Text]
    12. 12
  12. Jasir, A., and C. Schalen. 1998. Survey of macrolide resistance phenotypes in Swedish clinical isolates of Streptococcus pyogenes. J. Antimicrob. Chemother. 41:135-137.[Abstract/Free Full Text]
    13. 13
  13. Jasir, A., A. Tanna, A. Efstratiou, and C. Schalen. 2001. Unusual occurrence of M type 77, antibiotic-resistant group A streptococci in southern Sweden. J. Clin. Microbiol. 39:586-590.[Abstract/Free Full Text]
    14. 14
  14. Kataja, J., H. Seppälä, M. Skurnik, H. Sarkkinen, and P. Huovinen. 1998. Different erythromycin resistance mechanisms in group C and group G streptococcci. Antimicrob. Agents Chemother. 42:1493-1494.[Abstract/Free Full Text]
    15. 15
  15. Lin, F. Y., P. H. Azimi, L. E. Weisman, J. B. Philips, J. Regan, P. Clark, G. G. Rhoads, J. Clemens, J. Troendle, E. Pratt, R. A. Brenner, and V. Gill. 2000. Antibiotic susceptibility profiles for group B streptococci isolated from neonates, 1995-1998. Clin. Infect. Dis. 31:76-79.[CrossRef][Medline]
    16. 16
  16. McDougal, L. K., F. C. Tenover, L. N. Lee, J. K. Rasheed, J. E. Patterson, J. H. Jorgensen, and D. J. LeBlanc. 1998. Detection of Tn917-like sequences within a Tn916-like conjugative transposon (Tn3872) in erythromycin-resistant isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42:2312-2318.[Abstract/Free Full Text]
    17. 17
  17. Morales, W. J., S. S. Dickey, P. Bornick, and D. V. Lim. 1999. Change in antibiotic resistance of group B streptococcus: impact on intrapartum management. Am. J. Obstet. Gynecol. 181:310-314.[CrossRef][Medline]
    18. 18
  18. National Committee for Clinical Laboratory Standards. 2000. Performance standards for antimicrobial disk susceptibility test; approved standard M2-A7, 7th ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.
    19. 19
  19. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically; approved standard M7-A5, 5th ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.
    20. 20
  20. Portillo, A., M. Lantero, I. Olarte, F. Ruiz-Larrea, and C. Torres. 2001. MLS resistance phenotypes and mechanisms in ß-haemolytic group B, C and G Streptococcus isolates in La Rioja, Spain. J. Antimicrob. Chemother. 47:115-116.[Free Full Text]
    21. 21
  21. Roberts, M. C. 1996. Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility and distribution. FEMS Microbiol. Rev. 19:1-24.[CrossRef][Medline]
    22. 22
  22. Roberts, M. C., W. O. Chung, and D. E. Roe. 1996. Characterization of tetracycline and erythromycin resistance determinants in Treponema denticola. Antimicrob. Agents Chemother. 40:1690-1694.[Abstract]
    23. 23
  23. Salyers, A. A., N. B. Shoemaker, A. M. Stevens, and L. Y. Li. 1995. Conjugative transposons: an unusual and diverse set of integrated gene transfer elements. Microbiol. Rev. 59:579-590.[Abstract/Free Full Text]
    24. 24
  24. Seppäla, H., A. Nissinen, Q. Yu, and P. Huovinen. 1993. Three different phenotypes of erythromycin-resistant Streptococcus pyogenes in Finland. J. Antimicrob. Chemother. 32:885-891.[Free Full Text]
    25. 25
  25. Seppälä, H., Q. He, M. Österblad, and P. Huovinen. 1994. Typing of group A streptococci by random amplified polymorphic DNA analysis. J. Clin. Microbiol. 32:1945-1948.[Abstract/Free Full Text]
    26. 26
  26. Sutcliffe, J., T. Grebe, A. Tait-Kamradt, and L. Wondrack. 1996. Detection of erythromycin-resistant determinants by PCR. Antimicrob. Agents Chemother. 40:2562-2566.[Abstract]
    27. 27
  27. Trzcinski, K., B. S. Cooper, W. Hryniewicz, and C. G. Dowson. 2000. Expression of resistance to tetracyclines in strains of methicillin-resistant Staphylococcus aureus. J. Antimicrob. Chemother. 45:763-770.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, May 2002, p. 1574-1576, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1574-1576.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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