Background
The emergence and rapid transmission of antibiotic-resistance genes among
Staphylococcus aureus (
S. aureus) strains pose serious public health challenges worldwide [
1]. The erythromycin ribosome methylase (
erm) genes encode proteins that methylate adenine residues A2058/2059 in the peptidyl transferase region of 23S rRNA domain V, and are responsible for macrolide, lincosamide, and streptogramin B (MLSB) antibiotic resistance [
2]. In some regional reports, the frequency of methicillin-resistant
S. aureus (MRSA) with macrolide resistance was over 90% while methicillin-sensitive
S. aureus (MSSA) rose gradually to over 40% [
3]. The rapid transmission and broad antibiotic resistance spectrum of
erm have greatly limited the clinical utility of traditional macrolides such as erythromycin and azithromycin.
The
Erm gene-mediated resistant strains exhibit two antimicrobial resistance phenotypes, constitutive MLSB (cMLSB) and inducible MLSB (iMLSB). These phenotypes can be distinguished by D-zone test and are due to different molecular regulatory mechanisms. The translation initiation of
erm in the iMLSB strain is inhibited due to the sequestration of its mRNA ribosome-binding site by the leader peptide as well as dependent on inducers like erythromycin binding to the leader peptide, by which constrains its role and releases the mRNA ribosome-binding site. In contrast, cMLSB strains allow for direct and timely inducer independent translation of transcripts, in that variations in the leader peptide gene sequence abolish its translation [
4,
5]. Through this mechanism, the cMLSB phenotype confers resistance to macrolides, lincosamides, and streptogramin B, while the iMLSB phenotype is resistant to macrolides and streptogramin B but sensitive to lincosamides.
The novel agent, solithromycin, has been reported to be effective against erythromycin-resistant strains and have a formidable antibacterial effect with an extensive antibacterial spectrum. The minimum inhibitory concentrations (MICs) of solithromycin in drug-resistant or multi-drug-resistant strains of clinical isolates (methicillin-resistant
S. aureus and macrolide-lincosamide-streptogramin B-resistant
Streptococcus pneumoniae) are comparable or generally lower than those of other regularly used antibiotics, such as linezolid or vancomycin. The antibacterial spectrum of solithromycin includes
Haemophilus influenzae,
Moraxella catarrhalis, beta-hemolytic streptococci,
Legionella,
Bordetella pertussis,
Chlamydophila pneumoniae, and
S. aureus. However,
S. aureus strains with a solithromycin MIC over 32 mg/L have already emerged, the mechanism of which is unknown [
6‐
8].
In order to address this unknown mechanism, we conducted antimicrobial susceptibility testing of solithromycin against erm-positive and -negative strains, analysed the differences in MIC distribution among strains, and explored the potential basis of solithromycin resistance.
Discussion
Limited data shows that solithromycin has a more potent antimicrobial activity against a variety of bacteria than traditional and novel marolides such as erythromycin and telithromycin. According to previous reports, solithromycin MIC
50/90 values were 0.008/0.12 mg/L for
S. pneumoniae, 0.06/0.12 mg/L for
Moraxella catarrhalis, 0.015/0.03 mg/L for beta-hemolytic streptococci
, 1/2 mg/L for
Haemophilus influenzae, 0.06/0.06 mg/L for MSSA, and 0.06/> 32 mg/L for MRSA [
7]. Solithromycin exhibits different in vitro antimicrobial activity against
S. aureus. Assessing this difference is critical because the effect of solithromycin on
S. aureus in China is unclear. In this study, we found several specific features of solithromycin susceptibility in
S. aureus strains from China. First, the solithromycin MIC
50/90 values for the
erm-positive and -negative
S. aureus strains were 2/> 16 mg/L and 0.125/0.25 mg/L, respectively, indicating that the solithromycin resistant strains were mainly
erm-positive. Second, MSSA and MRSA have similar solithromycin MIC
50/90 values with no significant difference (2/> 16 mg/L), which is different from a previous investigation, in which solithromycin resistance was predominate in MRSA strains [
7]. Third, the MIC
50 values for the iMLSB strains were dramatically lower than those of the cMLSB strains, suggesting that the
erm-mediated cMLSB phenotype increases solithromycin MICs and is a signature pattern for solithromycin resistance. To the best of our knowledge, no other reports have demonstrated that the cMLSB phenotype in
S. aureus predicts solithromycin resistance. Although strains with the cMLSB phenotype primarily exhibited solithromycin resistance, a few cMLSB strains did show low solithromycin MIC values. The majority of
S. aureus strains with the iMLSB phenotype were sensitive to solithromycin, but whether possession of the
erm gene increases the risk for solithromycin resistance under antibiotics pressure needs to be determined.
The spontaneous mutation frequency is a simple and practical method to evaluate the resistance risk during antibiotic pressure [
15] and is used to assess the effect of cMLSB on solithromycin resistance in
S. aureus and the resistance risk for the iMLSB phenotype. The data demonstrate that harboring
erm genes predicts the risk for solithromycin resistance to antibiotic stress, as solithromycin-sensitive
S. aureus with cMLSB phenotype is at greater risk than with iMLSB phenotype, which were at greater risk than erythromycin-sensitive
S. aureus. This conclusion is consistent with previous findings reported by Pamela McGhee et al., who demonstrated that the degree of solithromycin resistance in
erm(B)-positive
S. pneumoniae and
Streptococcus pyogenes strains was greater than that in erythromycin-sensitive counterparts. However, this report did not demonstrate that
erm gene mediated cMLSB was a signature of solithromycin resistance [
16]. In summary, the solithromycin-sensitive strains of
S. aureus with cMLSB have an increased risk of resistance, which is far higher than that of strains with iMLSB or with erythromycin-sensitivity. It is well known that pharmacological effects are influenced by a number of factors including antimicrobial susceptibility and desirable pharmacokinetic and pharmacodynamic parameters, such as high bioavailability. Therefore, further evaluation is necessary to determine the clinical significance of solithromycin-sensitive strains with the iMLSB phenotype (and potentially high resistance mutation frequency) during solithromycin antibiotic pressure.
Like the first approved ketolide antibiotic, ie, telithromycin, several possible mechanisms may explain resistance: (1)
erm aberrance such as deletions and mutations in its promoter region, leader sequences, and coding sequences [
17‐
19]; (2) mutations in the 23S rRNA domains II or V including A138G, C724T, U754A, A2058G, and C2611U [
20,
21]; (3) variations in riboproteins L4 or L22 containing insertions, deletions, or mutations of amino acids [
22‐
24]; (4) over-expression of active efflux pumps like
mef [
25]. In order to determine whether the cMLSB phenotype is a major determinant of solithromycin resistance, other mechanisms of macrolide resistance are ruled out. First, the genetic mutations at drug binding sites, including the 23S rRNA gene and the genes encoding the ribosomal proteins L3 (
rplC), L4 (
rplD), and L22 (
rplV), showed no mutation at the target sites, indicating that the target-site mutations were unlikely to be involved in
erm gene-mediated resistance. Second, the participation of the efflux pumps was also excluded from the solithromycin resistance of
S. aureus with the cMLSB phenotype. With these exclusions, over-expression and/or genetic polymorphisms of
erm genes were candidates for the underlying mechanism(s) for solithromycin resistance in
S. aureus with the cMLSB phenotype. Whereas, no genetic polymorphism in
erm genes was found to explain the various in vitro antimicrobial activities of solithromycin with cMLSB and iMLSB. Moreover, in this study, the transcriptional expression of the
erm genes in the cMLSB strains with solithromycin resistance appeared to be even slightly lower than that in iMLSB strains with solithromycin sensitivity. In view of previous reports, erythromycin-induced
erm(B) expression was regulated at the translational but not the transcriptional level by a translational attenuation/arrest mechanism [
5,
26,
27]. Thus, differential
erm gene expression can still explain the results because
erm gene expression in the cMLSB strains was independent of the inducers and had a relatively higher protein level than in the iMLSB strains. In addition, the increased expression of
erm proteins may increase A2058-methylation in rRNA molecules, which is positively correlated with the up-regulation of ketolide MICs [
28]. Consequently, cMLSB
S. aureus strains with larger percentages of A2058-methylation had lower susceptibility to solithromycin than their counterparts iMLSB strains, which became self-evident. For the strains with solithromycin MICs of more than 16 mg/L in iMLSB isolates and the strains with solithromycin MICs of less than 1 mg/L in cMLSB isolates, the mechanism may be the incomplete methylation of A2058, regardless of the phenotype, but different degree of methylation among bacterial strains [
28].
Acknowledgements
Not Applicable.