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Antimicrobial Agents and Chemotherapy, August 2001, p. 2375-2377, Vol. 45, No. 8
Department of Medical Microbiology,
University Hospital Maastricht, Maastricht,1 and
Research Laboratory for Infectious Diseases, National
Institute of Public Health and the Environment, 3720 BA
Bilthoven,2 The Netherlands
Received 19 October 2000/Returned for modification 28 November
2000/Accepted 17 May 2001
Twenty-two unrelated erythromycin-resistant anginosus group strains
(3.2% resistance rate) were assessed for mechanisms of resistance.
Streptococcus anginosus accounted for 16 of the 22 isolates. Fifteen isolates harbored the erm(B) gene. The
erm(TR) and the mef(E) genes were carried by
two isolates each. In three isolates, none of these resistance genes
was detected by PCR.
The antimicrobial susceptibility
patterns of the Streptococcus anginosus group (AG) of
streptococci, previously designated the "Streptococcus
milleri" group, vary according to geography (7, 11,
16), and periodic review of local susceptibility data has been
recommended (7, 11). Some authors have reported increasing
resistance rates of AG isolates against macrolides and clindamycin
(7, 16, 25).
Macrolides, lincosamides, and streptogramins are grouped together as
MLS antibiotics. Two MLS antibiotic resistance phenotypes are
recognized in streptococci (20, 23, 24). The first confers resistance to macrolides, lincosamides, and streptogramin B and is
caused by methylation of the target site in the 23S rRNA subunit by an
adenine-N6-methyltransferase encoded by an
erm (erythromycin ribosome methylase) gene
(15). The phenotypic expression of MLSB
resistance may be inducible (MLSBi phenotype) or
constitutive (MLSBc phenotype). The other phenotype,
designated the M phenotype, causes resistance to 14- and 15-membered
macrolides only and is caused by an active drug efflux system
(23) encoded by the genes mef(A) (macrolide efflux) and mef(E) in Streptococcus pyogenes and
Streptococcus pneumoniae respectively (3, 24).
In the present study, we wanted to determine the prevalence of MLS
resistance among our collection of AG isolates and to assess the
mechanisms of this resistance.
The streptococci studied were nonrepetitive AG isolates consecutively
collected from clinical specimens submitted for culture at the
microbiology department of the University Hospital of Maastricht from
September 1995 to June 1999. They were recovered and identified as
previously described (8), and species were identified by hybridization with 16S rRNA oligonucleotide probes in a reverse line
blot assay (9, 10).
Screening for erythromycin and clindamycin resistance was carried out
by the agar dilution method as recommended by the NCCLS (11, 17,
18). Isolates that were intermediate or resistant to one or both
antibiotics were selected for determination of resistance type,
together with the erythromycin-resistant AG isolates described
previously (11).
For determination of the resistance phenotype, MICs of erythromycin,
roxithromycin, azithromycin, and clindamycin were determined with
E-test strips (Oxoid, Basingstoke, United Kingdom) and susceptibility to lincomycin and tylosin was assessed by a disk method (Rosco, Taastrup, Denmark). Inducible resistance was assessed with the double-disc test (20).
For resistance genotype determination, DNAs of the isolates were
amplified with primers specific to the erm(A),
erm(B), and erm(C) genes (12), the
mef(A) and mef(E) genes (23), and
the erm(TR) gene (14). For all PCR
amplifications, PCR buffer containing 1.5 mM MgCl2 was
used. Nucleotide triphosphates were used at a concentration of 0.04 mM,
and 0.01 U of Taq polymerase was added to each reaction
mixture. Amplifications were performed in a Perkin-Elmer Cetus GeneAmp
PCR System 9600 (Perkin-Elmer, Norwalk, Conn.), and the PCR protocols
used were those described previously (12, 14, 23). The PCR
products generated by the mef(A)-mef(E) and erm(TR) primers were directly sequenced by a
fluorescent-sequence method (Amersham Pharmacia Biotechnology,
Piscataway, N.J.). To assess the clonal identities of the isolates,
typing was performed by amplified fragment lenght polymorphism (AFLP)
analysis as previously described (22).
By the agar dilution method, 11 (3.2%) of 342 isolates were resistant
to erythromycin. This resistance rate was slightly higher than the
2.6% resistance rate previously found in our laboratory (11). It was comparable to data reported from Argentina
(2) but lower than those reported from Spain (14.3 and
17.7%) and the United States (13.6%) (7, 16, 25).
The 11 previously identified erythromycin-resistant isolates
(11) were added to the present ones, resulting in a total
of 22 isolates included for further characterization. Table
1 lists the resistance phenotypes and
genotypes matched by species. S. anginosus accounted for the
largest proportion of erythromycin-resistant isolates (P = 0.015). Other studies of smaller numbers of isolates demonstrated
only slight differences among the three AG species in terms of MLS
resistance rates (2, 7, 16, 25).
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2375-2377.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Prevalence of Macrolide Resistance Genes in
Clinical Isolates of the Streptococcus anginosus
("S. milleri") Group
ABSTRACT
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TABLE 1.
Identification to the species level and characterization
of 22 erythromycin-resistant AG strains
For 15 isolates assigned to the MLSBc phenotype, the MICs of erythromycin were >256 mg/liter and for 1 isolate the MIC was 8 mg/liter. For four MLSBi phenotype isolates, the MICs of erythromycin were 1, 3, 12, and >256 mg/liter, respectively, and these isolates showed inducible resistance to both clindamycin and tylosin. For the two M phenotype isolates, the MIC of erythromycin was 4 mg/liter. For all MLSBc phenotype strains, the MICs of clindamycin were >256 mg/liter.
Among the erythromycin-resistant isolates analyzed in this study, the erm(B) gene was predominant, which is in agreement with a previous study (4). The results of the sequence analysis showed that the PCR products generated by the mef(A)-mef(E) and erm(TR) primers were identical to the mef(E) and erm(TR) genes, respectively. mef genes have been demonstrated in a limited number of isolates of the viridans group, including one AG isolate (1, 19). mef genes have accounted for the rapid emergence of erythromycin resistance in S. pyogenes and S. pneumoniae in several countries (6, 21), and therefore their presence in AG isolates is of concern.
The erm(TR) gene has been found in S. pyogenes (5) and in large-colony group G streptococci (13), but, to our knowledge, it has not been demonstrated in AG isolates before. Three isolates assigned to the MLSB phenotype did not react with any of the primers chosen in the PCR amplification. We are conducting further studies to reveal the genetic mechanism of this resistance.
The numbers of isolates recovered from various body sites were as follows: abdomen, eight; genital tract, five; head and neck region, five; thoracic cavity, two; blood, one. For one isolate, complete clinical data were not available. AFLP analysis showed that all of the isolates had unique genotypes. There was no relationship between the Lancefield group, pattern of hemolysis, or site of isolation of the isolates on the one hand and the resistance genotype on the other hand.
In conclusion, this study demonstrated the presence of different erythromycin resistance genes, including the erm(B), erm(TR), and mef(E) genes, in a series of clinical AG isolates. The present data highlight the need for periodic surveillance of the rates of the antimicrobial resistance and its mechanism in these streptococci.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the following colleagues for providing control strains: H. Seppälä, National Public Health Institute, Turku, Finland, for S. pyogenes strains KOT R44 and KOT R59 (MLSBi phenotype), KUO R21 and ROV R367 (MLSBc phenotype), and KOT R45 and KOT R46 (M phenotype); S. Schwartz, Institut für Kleintierforschung der Bundesforschungsanstalt für Landwirtschaft, Celle, Germany, for S. aureus control strains [erm(A) and erm(C) genes]; L. Jenssen, Danish Veterinary Laboratory, Copenhagen, Denmark, for E. faecalis JH2-2::Tn1545 [control strain for erm(B) detection], J. Sutcliffe, Department of Infectious Diseases, Pfizer Central Research, Groton, Conn., for S. pyogenes 02C1064 and S. pneumoniae 02J1175 [for mef(A)-mef(E) gene detection]. We thank C. Schot, E. Cornelissen, and K. Spee for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. Phone: 31-43-387 46 44. Fax: 31-43-387 66 43. E-mail: JJA{at}LMIB.azm.nl.
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Antimicrobial Agents and Chemotherapy, August 2001, p. 2375-2377, Vol. 45, No. 8
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2375-2377.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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