1 Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006, Logroño; 2 Laboratorio de Microbiología, Hospital San Millán, Logroño, Spain
Received 10 March 2003; returned 27 April 2003; revised 21 May 2003; accepted 29 May 2003
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Abstract |
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Methods: Clonal relationships were studied for streptococci of groups A (GAS, n = 30), B (GBS, n = 34), C (GCS, n = 4) and G (GGS, n = 4) by means of PFGE. Streptococcal isolates were obtained from a 450-bed hospital in Spain during the following periods: GAS (19962001), GBS (19992001), GCS and GGS (19972000).
Results: Twenty-two of the 30 GAS isolates showed the M resistance phenotype and harboured the mef(A) gene. Five unrelated PFGE patterns were identified among these 22 GAS isolates. Sixteen of them, obtained during four different years of the study, showed one of the two predominant PFGE patterns. The remaining eight GAS isolates showed the MLSB resistance phenotype, and four unrelated PFGE patterns were detected. All but one erythromycin-resistant GBS showed the MLSB resistance phenotype, and an erm gene was identified in all cases [erm(B) or erm(A)]. Twenty-two unrelated PFGE patterns were demonstrated among 25 GBS with the MLSB resistance phenotype; the remaining eight MLSB GBS isolates could not be typed by PFGE. The eight erythromycin-resistant GGS and GCS isolates of this study presented seven unrelated PFGE patterns. GGS and GCS strains showed an MLSB resistance phenotype and had the erm(A) gene.
Conclusions: High clonal diversity was detected in this series of erythromycin-resistant GBS, whereas lower diversity was seen in the GAS isolates.
Keywords: streptococci, erythromycin resistance, PFGE
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Introduction |
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The phenotypes and the mechanisms of macrolidelincosamidestreptogramin B (MLSB) resistance in a series of group A, B, C and G streptococci recovered in La Rioja (Spain) had been analysed by our group in previous studies.5,6 The purpose of the present work is the genotypic analysis of Spanish erythromycin-resistant isolates of ß-haemolytic streptococci collected during 19962001, as it builds on previous reports5,6 by adding data from PFGE.
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Material and methods |
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A total of 72 erythromycin-resistant ß-haemolytic streptococci were included in this study [30 group A streptococci (GAS), 34 group B streptococci (GBS), four group C streptococci (GCS) and four group G streptococci (GGS)]. These isolates were recovered from clinical samples in the Hospital San Millán of La Rioja (Spain). The GAS were recovered during two periods: 20002001 (n = 17; all erythromycin-resistant GAS recovered in this period were included) and 19961998 (n = 13; randomly selected from the total erythromycin-resistant GAS of this period). The GBS were obtained during 19992001 (n = 34; all erythromycin-resistant GBS of this period) and the GCS and GGS during 19972000 (n = 8; all erythromycin-resistant GCS and GGS of this period). The MLSB resistance phenotypes and genotypes of some of these isolates have been reported previously (11 GAS, 20 GBS, one GCS and three GGS).5,6 In the present study, MLSB resistance phenotypes and resistance mechanisms have been analysed for the remaining 37 streptococci.
MLSB resistance phenotypes and mechanisms
Susceptibility testing was performed for erythromycin, clindamycin, spiramycin and virginiamycin (Sigma, St Louis , MO, USA) using a disc diffusion method on MuellerHinton agar (Difco, Detroit, MI, USA) supplemented with 5% sheep blood. Erythromycin-resistant isolates were classified into two phenotypes: M (erythromycin-resistant and clindamycin-susceptible) and MLSB (erythromycin- and clindamycin-resistant). The constitutive or inducible character of the MLSB resistance phenotype (cMLSB or iMLSB) was deduced as previously indicated.5
PCR detection of erm(A), erm(B), erm(C), mef(A), erm(TR) and msr(A) genes was carried out as previously reported.6 In accordance with the nomenclature for macrolide and MLSB resistance determinants recommended by Roberts et al.7 in 1999, we considered that positive PCR amplicons obtained using either erm(A) and/or erm(TR) primers corresponded with the presence of the erm(A) gene.
PFGE clonal analysis
Genomic DNA was digested either with SfiI (GAS) or with SmaI (GBS, GCS and GGS). PFGE was performed using a CHEF-DR II system (Bio-Rad Laboratories, Richmond, CA, USA). The running parameters were as follows: voltage, 5 V/cm; run time, 22 h; initial switch time, 5 s; final switch time, 45 s. A bacteriophage ladder (Bio-Rad) was used as a size marker. PFGE banding patterns were analysed and compared visually. Isolates were classified as indistinguishable, closely related, possibly related or unrelated.
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Results and discussion |
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Twenty-two out of the 30 GAS (73%) showed the M resistance phenotype and had the mef(A) gene. The remaining eight isolates showed the MLSB resistance phenotype (six cMLSB and two iMLSB). Five unrelated PFGE patterns (A, B, C, D and E) and one closely related to clone A (pattern A1) were identified in the series of 22 isolates with the M resistance phenotype after SfiI digestion (Table 1). One of these two patterns, pattern A or B, was recognized in 16 out of 22 isolates with the M resistance phenotype recovered in different years (from 1996 to 2001). It is interesting that pattern B was detected in one isolate recovered in 1996, in eight isolates in 1997 and also in four GAS obtained in 2000. Two patterns (F and G) were detected in six GAS with the cMLSB phenotype recovered in different years (pattern F in 2000 and 2001; pattern G in 1997 and 1998). The erm(B) and/or the erm(A) genes were detected in the four isolates with pattern F.
The two GAS isolates with pattern G showed a negative PCR result with the primers for the different MLSB resistance genes [erm(A), erm(B), erm(C), mef(A), erm(TR) and msr(A)] and both of them expressed the cMLSB resistance phenotype. Recently, new macrolide resistance mechanisms, resulting from ribosomal mutations, were detected in Streptococcus pyogenes isolates carrying the iMLSB resistance phenotype.8
Two unrelated patterns (H or I) were identified for the two GAS with the iMLSB resistance phenotype, which carried the erm(A) gene [detected with the erm(TR) primers], alone or in association with erm(B) gene.
Only a few clones accounted for most erythromycin-resistant GAS in La Rioja, and nearly 50% of GAS isolates (13 out of 30) fell into clone B. Similar results were obtained by other authors,13 and a comparable experience was noted in Quebec (Canada) where a single clone represented 65% of the erythromycin-resistant strains.9 However, in that case, all the isolates showed an iMLSB resistance phenotype and harboured the erm(A) gene [detected with the erm(TR) primers], whereas in European countries the M phenotype is more frequent and is associated with the mef(A) gene.1,2,5 Remarkably, there is a clear predominance of some clonal types isolated from community-acquired infections that have resulted from erythromycin-resistant GAS isolates. They may represent widespread clones whose population dynamics should be monitored carefully.
Group B streptococci
With respect to GBS, 33 out of the 34 isolates harboured the MLSB phenotype (97%) (13 iMLSB and 20 cMLSB). The resistance genes detected in these isolates were: erm(B) (20 isolates), erm(A) (12 isolates) and erm(A)+erm(B) (one isolate). Seventeen of these isolates gave 17 unrelated banding profiles (one pattern per isolate) designated AQ (Table 1 and Figure 1). Eight isolates with the MLSB phenotype collected in different years of sampling displayed four unrelated PFGE patterns (R, S, T and U) and one closely related to clone U (pattern U1) (Table 1). The remaining eight isolates with the MLSB resistance phenotype were non-typeable using the SmaI enzyme. The presence of unrestricted SmaI clones has been reported for Streptococcus pyogenes by different authors and an association with tetracycline susceptibility in this species has been suggested.3 In the period studied, only one isolate showed the M phenotype, resulting in an unrelated banding profile (Table 1). Considerable genetic diversity was found among erythromycin-resistant GBS isolates (23 patterns, two of them closely related among 25 erythromycin-resistant GBS), in agreement with data reported by other authors.10
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Clonal diversity was also found after analysing all the erythromycin-resistant GCS and GGS isolates recovered in this study during November 1996March 2001. Three unrelated PFGE patterns (A, B and C) and one closely related to clone C (pattern C1) were observed in the four GCS isolates studied (Figure 1). Each of the four GGS isolates belonged to an unrelated PFGE pattern, showing no clonal relationship. The erm(A) gene [detected with the erm(TR) primers] was found to be the genetic determinant of erythromycin resistance in all the GCS and GGS isolates. The polymorphism found among GCS and GGS in our study had also been reported in previous studies.4
In conclusion, erythromycin resistance in our area is a result of multiclonal dissemination within the GAS population, and two clones were predominant among GAS isolates recovered in different years. Regarding GBS, GCS and GGS, an extensive diversity of PFGE patterns was found among the erythromycin-resistant isolates of this study, suggesting a high degree of genetic variability. More erythromycin-resistant GGS and GCS isolates should be analysed in the future to have a clear idea of the clonal diversity in this specific group of microorganisms. Periodic surveillance of the clonal heterogeneity of erythromycin-resistant Streptococcus strains is necessary to avoid the spread of dominant PFGE patterns among human populations.
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Acknowledgements |
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Footnotes |
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References |
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2 . Pérez-Trallero, E., Urbieta, M., Montes, M. et al. (1999). Clonal differences among erythromycin-resistant Streptococcus pyogenes in Spain. Emerging Infectious Diseases 5, 23540.[ISI][Medline]
3 . Zampaloni, C., Vitali, L. A., Prenna, M. et al. (2002). Erythromycin resistance in Italian isolates of Streptococcus pyogenes and correlations with pulsed-field gel electrophoresis analysis. Microbial Drug Resistance 8, 3944.[CrossRef][ISI][Medline]
4 . Bert, F., Branger, C. & Lambert-Zechovsky, N. (1997). Pulsed-field gel electrophoresis is more discriminating than multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis for typing pyogenic streptococci. Current Microbiology 34, 2269.[CrossRef][ISI][Medline]
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