Several reports show that GAS resistance to macrolides and tetracyclines are high some countries such Spain and continue to increase; indeed, they have become clinically problematic.
In Europe, the most northerly countries (with the exception of Finland) have reported low levels of resistance (<4%)  while strong resistance has been reported from Mediterranean countries such as Italy (22,6%), France (22.4%), Greece (24.0%), Spain (21.3%) and Portugal (26.6%) [6–10]. This values contrast with those of Israel (1.8%) and Iran (0.2%) [11, 12].
In our study, 32.8% of isolates showed resistance to macrolides. Efflux pumps (M phenotype) are one of the major mechanisms conferring resistance to macrolide antibiotics, and streptococci making use of this system have been commonly reported from European countries, Argentina, the USA and Canada [5, 13–15]. The M phenotype has been identified as predominant in several Spanish studies, reaching a rate of 95.6% in a multicentre study undertaken in 1998 or 64.5% in an extensive national multicenter surveillance study in 2006–2007 [16, 17]. In the present population, the efflux system was also the main macrolide resistance mechanism seen, being manifested by 76.9% of isolates.
cMLSB phenotype, another common phenotype reported in Europe , was displaced by the M phenotype in several European countries from 1990 [10, 19]. In our study, cMLSB phenotype was the second most commonly encountered (20.3%) like SAUCE project carried out in 2006–2007 . In this last report, flutuations in the rates of resistance to macrolides are observed (1996–1997: 26.7%; 1998–1999: 20.4%; 2001–2002: 24.3; 2006–2007: 19%) meanwhile there is an increasing trend in the prevalence of MLSB phenotype from 14% in 2001–2002 to 35.5% in 2006–2007 .
Among Spanish isolates of this work, iMLSB phenotype was minority (2.7%) in contrast to Norway (75%) (1993–2002) or Bulgaria (57.7%) (1993 – 2002) where it was reported the most prevalent phenotype .
A gene-phenotype correlation previously described was also noticed [3, 9]. mef(A) and erm(B) were predominant in isolates with the M and cMLSB phenotype respectively, whereas all isolates with the iMLSB phenotype harboured the erm(A) gene.
The mef(A) gene responsible for the M phenotype was detected in all but three of the present Spanish isolates with that phenotype. One of these three isolates showed none of the genes studied. In the remaining two, msr(D) was observed alone or in combination with erm(A). In these last two cases, the msr(D) gene might be only one of the determinants responsible for the M phenotype. msr(D) and mef(A) have been placed in the same genetic element [8, 20], suggesting that the proteins they encode may act as a dual efflux system. However, it has also been suggested that the msr(D)-encoded pump can function independently of the mef-encoded protein .
The erm(B) gene responsible for the cMLSB phenotype was identified in all but three of the present isolates with this phenotype. None of genes tested could be amplified in two isolates, indicating that other resistance genes must be involved. The remaining isolate harboured erm(A) and mef(A). In this case, erm(A) may be responsible for the cMLSB phenotype since alterations in the regulatory region of the gene have been identified that induce constitutive expression .
An ample macrolide resistance genes combination was identified, specifically fourteen genotypes. Interestingly, single genotypes could show one or several phenotypes, a phenomenon reported by other authors [5, 10]. One of these, erm(B)/msr(D)/mef(A) genotype showed M and MLSB phenotypes in 25 and 8 isolates respectively, while the erm(B)/erm(TR)/msr(D)/mef(A) genotype showed all three macrolide resistance phenotypes. Nowadays, this correlation between genotype and phenotype is not well understood.
In our erythromycin-resistant population (295), the 6 most common emm/types: emm4T4 (39.3%), emm75T25 (14.6%), emm28T28 (13.2%), emm6T6 (9.8%), emm12T12 (6.8%) and emm11T11 (4.1%) have been previously associated with macrolide resistance in numerous reports [6, 10, 12, 14]. emm28 and emm4 have been reported the most common in Europe (2003–2004) , and to be responsible for an increase in erythromycin resistance among GAS in Spain, Finland and Quebec . emm12 is the main resistant emm type in Germany, Greece, Italy, Portugal, Israel [10, 12, 13] and the second one in the United States, being surpassed only by emm75 .
Most of erythromycin-resistant isolates were Sma-non-restricted (73.2%) due to the presence prophage-like elements that confer the M phenotype and harbour the mef(A) and msr(D) genes. These genetic elements encode a DNA-modifying methyltransferase that acts on the SmaI recognition sequence and renders DNA refractory to cleavage by SmaI . All but four of the present SmaI non-restricted isolates were susceptible to tetracycline and had an M phenotype. This suggests that these isolates carry mef(A) and msr(D) contained within a Tn1207.1 transposon inserted into a larger genetic element such as the Tn1207.3 or 58.8 kb chimeric element, flanked by the comEC gene from the Tn1207.3/Φ10394.4 family . In our study, all emm4T4 and all emm75T25 erythromycin-resistant isolates but one were SmaI non-restricted and had the M phenotype; together these accounted for 53.9% of the Spanish macrolide-resistant isolates. Several resistant clones previously described in Spain were identified [9, 10]. The emm4T4 Sfi1 (79) clone resembles to clone B described in 1999 . It was the most common in the present study, indicating it to still be circulating in Spain. This clone has a wide distribution, and it has recently been identified in Finland, Greece, Italy, England and Sweden . Clone C, previously identified in Spain, the United Kingdom and the United States  was not detected among the present isolates, although it might be related to the present clones emm4T4 Sfi4 and emm4T4 Sfi5.
The major macrolide-resistant clone emm75T25 Sfi12(41) was similar (additional band between 48.5 and 97 kb) to clone D described by Perez-Trallero et al. . The emm6T6 Sfi17 and emm84T25 Sfi22 clones might be associated with resistance since they were only observed in isolates resistant to erythromycin.
Regarding tetracycline resistance, we detected values of 6.8% between 1994 and 2006, indicating there to be no trend towards increased tetracycline in Spain. However, higher rates have been found in other countries such as Israel (23.6%), Denmark (33.7%), Portugal (38.7%) or Iran (42%) [10–12].
In this study, a predominance of genotype with both genes tet(M) and tet(O) (42.6%) was observed. But no Spanish reports citing the predominance of both genes appears to exist, tet(M) alone is usually the most common resistance determinant followed by tet(O) .
In the present tetracycline-population, emm77T28 was the main emm/T type. emm77 has been previously associated with resistance to tetracycline in Israel and Europe . In Italy and Norway, an emm77 clone has been reported that is characterised by its carrying tet(O) linked to erm(A)and being associated with the iMLSB phenotype . In the present study, the two co-resistant emm77T28 isolates showed genotypes different to those described by Palmieri et al. .
With regard to co-resistance, we found that all isolates (19) except one had the cMLSB macrolide resistance phenotype such as Greece (Athens) and Norway [5, 15]. In contrast, in Finland, iMLSB isolates showing co-resistance have reached rates of 93% . A correlation between the M phenotype and co-resistance has been also reported , but this was not detected in the present study.
Of the 19 co-resistant isolates, five carried tet(M)/erm(B) as their only resistance genes, suggesting they may carry conjugative transposons of the Tn916 family in which erm(B) and tet(M) are linked ,whereas 13 harboured tet(M)/erm(B) associated with other resistance genes. In the remaining isolate, the erm(B), mef(A), tet(M) and tet(O) genes were all detected. mef(A) and tet(O) linkage has been previously reported in co-resistant isolates [22, 25]. In the present work, mef(A) appeared associated with other macrolide resistance genes and linked to tet(M) (1 isolate) or to tet(M)/tet(O) (5). The main emm/T type detected in coresistant isolates was emm11T11 (57.8%). This emm/T type has previously been associated with co-resistance [9, 11] with an erm(B)/tet(M) clone prevalent among Spanish MLSB isolates . Four isolates with this genotype were found in the present work, but we can not confirm whether they belong to the above clone.