Molecular Detection of the Macrolide Efflux Gene: To Discriminate or Not To Discriminate between mef(A) and mef(E)

The presence of a novel macrolide efflux system in streptococci was first described and firmly established in 1996 by Sutcliffe et al. (81). This system was phenotypically recognized and characterized to confer low-level resistance (MICs, 1 to 32 μg/ml) to 14- and 15-membered macrolides but not to 16-membered macrolides, lincosamides, or streptogramin B (or their analogues). This phenotypic pattern of antibiotic resistance was referred to as M-type resistance and is in contrast to the MLSB phenotype, which confers constitutive high-level resistance (MICs, ≥256 μg/ml) to macrolides, lincosamides, as well as streptogramin B. In the same year Clancy et al. (16) identified the gene responsible for this novel efflux system in Streptococcus pyogenes, and it was designated mef(A). This gene was deposited in the public DNA databases and could be considered the reference sequence for the mef(A) gene (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"U70055","term_id":"1669856","term_text":"U70055"}}U70055). Tait-Kamradt et al. (84) later identified a similar gene in Streptococcus pneumoniae that at the time was designated mef(E). Likewise, this gene could be considered the reference sequence for mef(E) (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"U83667","term_id":"1800300","term_text":"U83667"}}U83667). The coding sequences of these two genes appeared to share 90% identity at the DNA level (Fig. ​(Fig.1). 1). Remarkably, in contrast to what might be expected due to the degeneracy of the genetic code, they share only 88% identity at the protein level (48 mismatches in a protein of 405 amino acids). The encoded proteins are strongly hydrophobic, apparent integral membrane proteins with 12 transmembrane segments (16). Because of the high degree of similarity between the mef(A) and the mef(E) genes, Roberts et al. (74) suggested in a minireview that both genes be referred to as just a single class, mef(A). The result of this recommendation would be that if there would be a need to discriminate between the two genes, the recommended nomenclature would be something like mef(A) subclass mef(A) to indicate mef(A) and mef(A) subclass mef(E) to indicate mef(E). In order to increase the readability, we prefer to use the original names mef(A) and mef(E) throughout this minireview. FIG. 1. Alignment of the mef(A) and mef(E) nucleotide sequences and the corresponding amino acid sequences. All sequences were taken from the original database entries [GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"U70055","term_id":"1669856","term_text":"U70055"}} . In a number of reports it has since been shown that a number of marked differences between mef(A) and mef(E) exist. For instance, the genetic elements carrying mef(A) or mef(E) and their contexts have been studied by Santagati et al. (75), Gay and Stephens (29), and Del Grosso et al. (23) and were shown not only to be quite different but also to behave quite differently. The two genes have disseminated markedly differently and are being recognized in an ever growing number of microbial species. At present, both the mef(A) and the mef(E) genes have unambiguously been identified in five streptococcal species, whereas mef(E) has been identified in five more streptococcal species and in nine additional nonstreptococcal species (Table ​(Table1). 1). Furthermore, Amezaga et al. (5) reported that the MICs for mef(A)-containing S. pneumoniae isolates were significantly higher than those for mef(E)-containing isolates. This indicates that, despite the high degree of homology between the two genes, in the context of the genome in which they are embedded, the differences between them are sufficient to impose different susceptibility characteristics on the strains carrying the genes. The existence of these differences between the two genes has prompted others to suggest that the difference between the two genes be maintained (23, 55) and may have been one reason(s) why others also continued using the names mef(A) and mef(E) after publication of the nomenclature minireview (5, 12, 13, 15, 19, 23, 35, 54, 55, 66). TABLE 1. Dissemination of mef genes among microbial species As a result of this, there is no widespread consensus about the nomenclature for the mef genes in the present literature. For readers unaware of this, this may give rise to conflicting interpretations of the available literature and resources on the subject. In this minireview, we outline how the use of different mef gene nomenclatures has created considerable confusion in the field. We also review the current resources on the subject: we performed a search for mef gene sequences in public DNA databases and looked at methods for the detection of mef genes in clinical isolates and tools that can be used to discriminate between mef(A) and mef(E). This information was then used to review the data in the literature with respect to reported genes versus the actual genes that were studied, given the information in the methods sections.