Pulsed-field gel electrophoresis (PFGE) analysis

PFGE profiles from all erythromycin resistant strains were obtained as previously described.²³ The genomic DNA was digested with the SmaI restriction enzyme (New England Biolabs, Ipswich, MA, USA) and electrophoresis was performed in a CHEF DR III system (Bio-Rad Laboratories, USA) using the following program: switch time 1–30 s during 23 h with a 120° angle at a temperature of 11.3 °C and a voltage gradient of 6 V/cm. The Lambda Ladder PFG marker kit (New England Biolabs, USA) was used as the DNA size marker. The gels were stained with ethidium bromide and digitally photographed using a Scorpion SCOR-14SOM scanner (DNR Bioimaging System, Jerusalém, Israel) under ultraviolet light. The images were analyzed using Gel ComparII^® software (Applied Maths, Belgium). Electrophoretic profiles were compared according to the guidelines proposed by Tenover et al.²⁴ The Dice coefficient (95%) and a 1% position tolerance were used to analyze the similarities in the band patterns among the electrophoretic profiles. The unweighted pair group method using the arithmetic average was used to gather electrophoretic profiles into polymorphism patterns, also referred to as clusters. The polymorphism patterns were defined grouping profiles that showed >70% dendrogram identity.