Distribution of mefA-containing genetic elements in Italian isolates of Streptococcus pyogenes

Objectives: Erythromycin resistance M phenotype in Group A Streptococci (GAS, Streptococcus pyogenes) is due to an efflux system coded by the mefA gene, which is carried by a mobile genetic element, named either Phi10394.4 or Tn1207.3. This element is essentially composed of a transposon inserted into a prophage and its chromosomal insertion site has been mapped into the comEC gene. The aim of the present study was to analyze the genetic localization of the mefA gene by determining the general structure variability of the genetic element and its insertion site into the bacterial chromosome in 124 erythromycin resistant S. pyogenes clinical Italian isolates. Methods: comEC gene was amplified in all isolates to test for its integrity. Negative reaction was taken as an indication of the insertion of the mobile genetic element. Subsequently, a set of oligonucleotide primers was constructed and used in PCR experiments to screen for the presence of distinctive parts along the entire sequence of the Phi10394.4/Tn1207.3 chimeric element (i.e. flanking regions and R-6, mefA, ABC-transporter, umuCmucB, type II methylase genes). Results: 20 out of 124 isolates (16.1%) were negative to the comEC gene amplification. The analysis of the insertion point flanking regions indicated that the Phi10394.4/Tn1207.3 element is present and inserted into the comEC gene. 9.7% of the population showed a mixed genotypic pattern, where comEC gene was amplified but the prophagic element was still present. This result was additionally confirmed by sequencing of the PCR products obtained. The remaining comEC positive population (74.2%) was Phi10394.4/Tn1207.3 negative, as none of the mobile element genes tested was amplified. Conclusions: A very low portion of the population (16.1%) has the mefA gene carried by the Phi10394.4/ Tn1207.3 prophage element inserted into the comEC gene. About a tenth of the population contains an intact form of the comEC gene while presenting a Phi10394.4/ Tn1207.3 positive pattern. Unexpectedly, the vast majority of isolates (74.2%) has the mefA gene vectored by neither forms of the prophagic chimeric element previously described. However, since it has been largely demonstrated that streptococci efflux mediated resistance can be transferred among bacteria by means of a transformation process, other mobile genetic elements should be present in Streptococcus pyogenes that act as molecular hosting vector for the mefA gene