Objectives: In Streptococcus pyogenes (Group A Streptococcus, GAS), genes conferring efflux-mediated erythromycin (ERY) resistance (mefA/msr) are carried by functional prophages and may be associated with the tetracycline (TET) resistance determinant tetO. We sought to demonstrate the “ex vivo” lysogenic transfer of ERY and TET resistance genes among GAS. Methods: Forty-one ERY-susceptible GAS clinical isolates belonging to 12 different M-serotypes were used as recipients. The strain K56 (Mserotype 12) was used as a standard indicator to prepare lawns and as a model recipient, while the ERY- and TET-resistant strain m46 (Mserotype 4) was used as the donor of the tetO-mefA phage (phi-m46). The donor was induced with 0.2 mg/L mitomycin C. The culture supernatant was filtered, treated with DNAse/RNAse, concentrated by PEG/NaCl precipitation, and then added to cultures of the recipient strains. After incubation, these mixtures were either added to supplemented molten top agar and poured over BHI agar plates for the visualisation and counting of plaques or plated on BHI agar plus erythromycin for the selection of ERY-resistant lysogenic clones. PCR was used to check for the emm-type, mefA, and tetO genes before and after lysogenic transfer experiments, and to detect phi-m46 DNA in the supernatant of induced donor cultures. Results: The reference strain K56 and 85.4% (n = 35) of the clinical isolates acquired ERY/TET resistance when infected with purified phim46. All the M1 (n = 7), M12 (n = 6), M75 (n = 2), M18 (n = 1), M94 (n = 1) and a fraction of the M3 (5/7), M4 (1/3), M5 (1/2), M6 (4/7) strains were lysogenised and converted to the ERY/TET resistant phenotype. No lysogenic clones were isolated in the case of the M77, M78 and M89 recipients. Only M12 strains and the totality of them were phi-m46-sensitive. Conclusions: The erythromycin and tetracycline resistance determinants carried by phage-m46 can be efficiently transferred from a GAS strain to another by transduction. This is the first direct demonstration of phagemediated horizontal resistance gene transfer in S. pyogenes. In the set of strains analyzed, an M-serotype dependent barrier to lysogenic transfer was not observed. The exclusive lysis susceptibility of the M12 strains indicates that GAS with this serotype may represent the main responsible for the spread of this phage and the associated antibiotic resistance genes in S. pyogenes global population.

Bacteriophage-mediated transfer of erythromycin and tetracycline resistance genes among group A streptococci

VITALI, Luca Agostino;PETRELLI, Dezemona;PRENNA, Manuela;
2009

Abstract

Objectives: In Streptococcus pyogenes (Group A Streptococcus, GAS), genes conferring efflux-mediated erythromycin (ERY) resistance (mefA/msr) are carried by functional prophages and may be associated with the tetracycline (TET) resistance determinant tetO. We sought to demonstrate the “ex vivo” lysogenic transfer of ERY and TET resistance genes among GAS. Methods: Forty-one ERY-susceptible GAS clinical isolates belonging to 12 different M-serotypes were used as recipients. The strain K56 (Mserotype 12) was used as a standard indicator to prepare lawns and as a model recipient, while the ERY- and TET-resistant strain m46 (Mserotype 4) was used as the donor of the tetO-mefA phage (phi-m46). The donor was induced with 0.2 mg/L mitomycin C. The culture supernatant was filtered, treated with DNAse/RNAse, concentrated by PEG/NaCl precipitation, and then added to cultures of the recipient strains. After incubation, these mixtures were either added to supplemented molten top agar and poured over BHI agar plates for the visualisation and counting of plaques or plated on BHI agar plus erythromycin for the selection of ERY-resistant lysogenic clones. PCR was used to check for the emm-type, mefA, and tetO genes before and after lysogenic transfer experiments, and to detect phi-m46 DNA in the supernatant of induced donor cultures. Results: The reference strain K56 and 85.4% (n = 35) of the clinical isolates acquired ERY/TET resistance when infected with purified phim46. All the M1 (n = 7), M12 (n = 6), M75 (n = 2), M18 (n = 1), M94 (n = 1) and a fraction of the M3 (5/7), M4 (1/3), M5 (1/2), M6 (4/7) strains were lysogenised and converted to the ERY/TET resistant phenotype. No lysogenic clones were isolated in the case of the M77, M78 and M89 recipients. Only M12 strains and the totality of them were phi-m46-sensitive. Conclusions: The erythromycin and tetracycline resistance determinants carried by phage-m46 can be efficiently transferred from a GAS strain to another by transduction. This is the first direct demonstration of phagemediated horizontal resistance gene transfer in S. pyogenes. In the set of strains analyzed, an M-serotype dependent barrier to lysogenic transfer was not observed. The exclusive lysis susceptibility of the M12 strains indicates that GAS with this serotype may represent the main responsible for the spread of this phage and the associated antibiotic resistance genes in S. pyogenes global population.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11581/111511
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