Journal of Antimicrobial Chemotherapy (2001) 47, 863-866
© 2001 The British Society for Antimicrobial Chemotherapy
Distribution of resistance genes tet(M), aph3'-III, catpC194 and the integrase gene of Tn1545 in clinical Streptococcus pneumoniae harbouring erm(B) and mef(A) genes in Spain
Department of Microbiology, University Hospital ‘Lozano Blesa’, San Juan Bosco 15, 50009 Zaragoza, Spain
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Abstract |
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The most prevalent macrolide resistance phenotype and genotype among pneumococcal isolates was the cMLSB phenotype [erm(B) or erm(B)/mef(A)] (91.3%). We studied the distribution of other resistance genes, tet(M), catpC194, aph3'-III, in these strains, seeing evolution at work in that some strains carried different combinations of resistance determinants. The most prevalent patterns associated with resistance to erythromycin [erm(B)] were resistance to tetracycline [tet(M)] and chloramphenicol (catpC194) (48.2%) or resistance to tetracycline [tet(M)] alone (42.2%). In our isolates of Streptococcus pneumoniae there was a strong association of the erm(B) and tet(M) genes with Tn1545-related elements.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Resistance to macrolides in pneumococci is generally mediated by methylation of 23S rRNA via erm(B) methylase, which can confer a macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype,1 or by drug efflux via mef(A), which confers resistance to 14- and 15- membered macrolides only.2 Resistance to MLSB, tetracycline, kanamycin and chloramphenicol has been linked to carriage of the conjugative transposon Tn1545.3 We have investigated the epidemiology of macrolide resistance genes in Streptococcus pneumoniae and the association of MLSB resistance with non-MLSB resistance genes: tet(M) (tetracycline and minocycline), catpC194 (chloramphenicol) and aph3'-III (kanamycin and related aminoglycosides). The presence of the conjugative transposon, Tn1545, and related elements was determined using a probe specific for intTn, the gene encoding the integrase required for the movement (transposition and conjugation) of Tn1545.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Bacterial strains
Seven-hundred and forty-four S. pneumoniae isolates, derived from 556 patients, were isolated from 1997 to 1999 at the University Hospital of Zaragoza, Spain. From these, we selected the most recent 100 erythromycin-resistant and 40 erythromycin-susceptible strains to study. The sources of the isolates were: sputum (59), blood (7), bronchial aspirate (41), cerebrospinal fluid (3) and other (30). Multiple isolates from the same patient were avoided. Serotyping of the isolates was by the Quellung reaction, with the use of 46 antisera provided by the Statens Seruminstitut (Copenhagen, Denmark).
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed by the agar dilution method, according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS).4 Erythromycin was incorporated into the medium in a log2 dilution series from 0.06 to 128 mg/L. The interpretative categories for each antibiotic were those recommended by the NCCLS. S. pneumoniae ATCC 49619 was used as quality control strain. Antibiotic susceptibility phenotypes were determined by disc diffusion tests; discs were purchased from Difco (Detroit, MI, USA) (erythromycin 15 µg, clindamycin 2 µg, azithromycin 15 µg, tetracycline 30 µg, minocycline 30 µg and chloramphenicol 30 µg) or made with compounds purchased from Sigma (St Louis, MO, USA) (kanamycin 500 µg, lividomycin 500 µg, butirosin 500 µg, neomycin 500 µg).
Detection of macrolide resistance genes by PCR and dot-blot hybridization
Total genomic DNA was extracted from S. pneumoniae strains, as described previously.5 Primers specific for the detection of erm(B), msr(A), mef(A), erm(A) [subclass erm(TR)] and the conditions for PCRs were as described.5,6 Dot-blot hybridization was performed by standard techniques.7 Probes specific for erm(B), erm(A), msr(A) or mef(A) were PCR amplicons obtained as described above.5,6 The probes were labelled with [
-32P]dCTP (Amersham SA, Les Ulis, France). Hybridization was carried out at 60°C overnight.7 Positive and negative controls from our strain collection were used to ensure probe specificity.
Detection of intTn, tet(M), catpC194 and aph3'-III by dot-blot hybridization
The probes consisted of the 830 bp TaqI fragment of Tn1545 for intTn, the 850 bp ClaI–HindIII fragment of Tn1545 for tet(M), the 530 bp HpaII fragment of the enterococcal plasmid pJHI for aph3'-III and the 1160 bp ClaI fragment of the staphylococcal plasmid pC194 for catpC194.3 Purified DNA fragments generated by restriction enzyme digestion were labelled as described above. DNA from strains S. pneumoniae BM4200, Enterococcus faecalis BM4110::Tn1545 and E. faecalis BM4110::Tn916 were included as positive controls.3
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Antimicrobial susceptibility testing
Of the 556 non-redundant pneumococcal isolates, 195 (35.1%) were resistant to erythromycin (MICs 
1 mg/L). Of these, 178 (91.3%) exhibited a cMLSB phenotype and 17 (8.7%) exhibited an M phenotype. The prevalence of erythromycin-resistant isolates increased from 30% in 1997 to 42% in 1999. MICs of erythromycin were higher for strains with the cMLSB phenotype (8–128 mg/L) than for those with the M phenotype (1–16 mg/L); 85% of S. pneumoniae isolates with the cMLSB phenotype had erythromycin MICs 64 mg/L.
Macrolide resistance genes
The hybridization of genomic DNA with the erm(B) probe was positive for all 83 isolates with cMLSB phenotype and negative for all susceptible isolates and isolates with the M phenotype. The mef(A) probe hybridized with genomic DNA from all 17 isolates with M phenotypes as well as with genomic DNA from two isolates with a cMLSB phenotype. None of the susceptible isolates carried sequences that hybridized to the erm(B) and mef(A) probes. The two strains with the erm(B) and mef(A) genes had MICs to erythromycin >64 mg/L.
Non-MLS B resistance genes: tet(M), catpC194 and aph3'-III
Of the 100 erythromycin-resistant strains, 82% carry the tet(M) determinant, 44% the catpC194 determinant and 4% the aph3'-III gene. The association of these genes with macrolide resistance genes erm(B) and mef(A) is shown in Table I
. The distribution of resistance determinants among serogroups or serotypes (SG/STs) is given in Table II. The percentage association of these resistant determinants with erm(B) and mef(A) in S. pneumoniae were: tet(M) [92.8 versus 29.4%], catpC194 [50.6% versus 11.8%] and aph3'-III [4.8% versus 0%], respectively.
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Tn1545 conjugative transposon
The intTn probe hybridized with genomic DNA from 86.7% and 29.4% of the isolates harbouring erm(B) and mef(A)/tet(M), respectively (Table I
).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This study revealed that the most prevalent macrolide resistance type among pneumococcal isolates from the University Hospital of Zaragosa between 1997 and 1999 was the cMLSB phenotype [erm(B) or erm(B)/mef(A)]. We found that two strains harbouring both erm(B) and mef(A) genes are no more resistant to erythromycin than strains with erm(B) alone, indicating that the cMLSB phenotype is dominant. Knowledge of the prevalence and type of macrolide resistance may have therapeutic implications in view of the different levels of resistance.
Acquired multiple antibiotic resistance in S. pneumoniae can result from the presence of a conjugative transposon, Tn1545, which carries multiple resistance determinants, erm(B), tet(M), aph3'-III and catpC194.3 In this study, we see evolution at work in that some strains carry different combinations of resistance determinants. Ninety-three per cent of the S. pneumoniae harbouring erm(B) also had the tet(M) determinant, while 51% also had the catpC194 determinant. Ten combinations of resistance patterns were distinguished. The two most prevalent resistance patterns were resistance to erythromycin [erm(B)], tetracycline [tet(M)] and chloramphenicol (catpC194) (48.2%) and resistance to erythromycin [erm(B)] and tetracycline [tet(M)] (42.2%). In our isolates there is a strong association of erm(B) and tet(M) with Tn1545-related elements. One strain carried erm(B) only, suggesting that Tn917 or a related element is present in this strain. Some strains resistant to erythromycin [erm(B)] and tetracycline [tet(M)] may carry the composite element Tn3872 or one similar to it.8
None of the S. pneumoniae isolates with M phenotype that harboured only the resistance gene mef(A) hybridized with the intTn probe. mef(A) has recently been shown to be part of a transposon. It is the promoter-proximal gene in a bicistronic operon, the second gene of which encodes a putative efflux pump with homology to the ABC transporter superfamily.9–10
Linkage of multiple antibiotic resistance genes on the same mobile element is of public health importance, because use of any one of the antibiotics to which the element confers resistance selects for retention of the transposon and, accordingly, multiple antibiotic resistance genes. In the respiratory microbiota, interspecies exchanges of resistance genes between S. pneumoniae and other streptococcal species, especially with subsequent antibiotic selection pressure, could select multidrug-resistant pneumococci.
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Acknowledgments |
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We thank Patrice Courvalin for supplying the probes tet(M), catpC194, aph3'-III and intTn. We also thank Joyce Sutcliffe for comments on the manuscript. This work was supported in part by a grant from the Fondo de Investigaciones Sanitarias of Spain (FIS 98/0733), a grant from the Gobierno de Aragón (Ref: P49/97) and a grant from Bayer-SEIMC (personal to C. S.).
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Notes |
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* Corresponding author. Tel: +34-976-556400; E-mail: c.seral.000{at}recol.es
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References |
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1 . Leclercq, R. & Courvalin, P. (1991). Bacterial resistance to macrolide, lincosamide, and streptogramin antibiotics by target modification. Antimicrobial Agents and Chemotherapy 35, 1267–72.
2 . Tait-Kamradt, A., Clancy, J., Cronan, M., Dib-Hajj, F., Wondrack, L., Yuan, W. et al. (1997). mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 41, 2251–5.[Abstract]
3
.
Poyart-Salmeron, C., Trieu-Cuot, P., Carlier, C. & Courvalin, P. (1991). Nucleotide sequence specific for Tn1545-like conjugative transposons in pneumococci and staphylococci resistant to tetracycline. Antimicrobial Agents and Chemotherapy 35, 1657–60.
4 . National Committee for Clinical Laboratory Standards. (1999). Methods for Dilution Antimicrobial Susceptibility Testing for Bacteria That Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.
5 . 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]
6
.
Seppälä, H., Skurnik, M., Soini, H., Roberts, M. C. & Huovinen, P. (1998). A novel erythromycin methylase gene (ermTR) in Streptococcus pyogenes. Antimicrobial Agents and Chemotherapy 42, 257–62.
7
.
Thakker-Varia, S., Jenssen, W. D., Moon-McDermott, L., Weinstein, M. P. & Dubin. D. T. (1987). Molecular epidemiology of macrolide-lincosamides-streptogramin B resistance in Staphylococcus aureus and coagulase-negative staphylococci. Antimicrobial Agents and Chemotherapy 31, 735–43.
8
.
McDougal, L. K., Tenover, F. C., Lee, L. N., Rasheed, J. K., Patterson, J. E., Jorgensen, J. H. et al. (1998). Detection of Tn917-like sequences within a Tn916-like conjugative transposon (Tn3872) in erythromycin-resistant isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42, 2312–8.
9
.
Santagati, M., Iannelli, F., Oggioni, M. R., Stefani, S. & Pozzi, G. (2000). Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 44, 2585–7.
10 . Gay, K. & Stephens, D. S. (2000). Structure of the mefE- containing macrolide efflux genetic assembly (Mega) in Streptococcus pneumoniae: a novel chromosomal insertion element. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Ontario, Canada, 2000. Abstract 1929, p. 118. American Society for Microbiology, Washington, DC.
Received 29 September 2000; returned 29 November 2000; revised 17 January 2001; accepted 7 March 2001
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