We observed excellent in vitro erava efficacy against all 328 clinical S. aureus isolates from China examined in this study, with erava MIC50/MIC90 values of 0.25/0.25 mg/L for MRSA and 0.25/0.5 mg/L for MSSA. These findings are consistent with previous reports indicating that staphylococci, streptococci, and enterococci, regardless of concurrent resistance phenotypes, have typically been found to have erava MIC50 and MIC90 values ≤0.25 mg/L, with no more than a two-fold MIC50 to MIC90 difference [3,4,5]. The highest erava MICs reported previously for clinical MRSA and MSSA isolates were 4.0 mg/L and 0.5 mg/L, respectively, with higher-end values being found for hospital-acquired MRSA, as opposed to community-acquired MRSA and MSSA [3]. Relative to our MRSA isolates, our MSSA isolates had a lower frequency of Tet resistance, but a higher frequency of isolates with erava MICs ≥0.5 mg/L. Moreover, our findings of both MRSA and MSSA isolates with erava MICs ≥0.5 mg/L, and of a few MSSA isolates with erava MICs of 1 mg/L indicate higher erava MICs among MSSA than MRSA from this region and are especially worthy of concern.
The aforementioned erava MIC data differ from the reported data previously showing higher erava MICs in MRSA than MSSA [3, 4]. These differences could be due, at least in part, to sample and regional variation given that the molecular and antimicrobial susceptibility characteristics of S. aureus are known to vary across regionsand the very limited volume of data that have been reported regarding erava effects on S. aureus [28, 29]. Previously, there has been a focus on the antimicrobial susceptibility of erava mainly in the multi-drug resistant S. aureus, including MRSA as well as vancomycin-resistant and linezolid-resistant isolates. Our sample did not include vancomycin-resistant or linezolid-resistant S. aureus due to their low frequency in China; however, we did encounter a high frequency of erythromycin- resistant S. aureus. Further comparative studies of the antimicrobial activity of erava in MRSA versus MSSA are needed.
Recent reports have suggested that vancomycin, teicoplanin, daptomycin, and linezolid MIC creep in MRSA may be associated with clonality [29,30,31]. In the present data, erava MICs ≥0.5 mg/L were strongly represented among ST239 and ST59 MRSA isolates, but generally diversified among MSSA isolates though relatively well represented among ST398-MSSA isolates. The relationship of S. aureus clonality with erava susceptibility should be examined in large samples across different regions.
Recent evidence indicates that new-generation drugs of Tet class, including tigeycline and erava, can overcome the Tet-specific resistance mechanisms, including the efflux pumps such as Tet(K) and Tet(L) and the ribosome protection protein such as Tet(M). Tet(K) and Tet(L), constituted by 14 transmembrane segments as monovalent cation-H+ antiporters, are the most common Tet-specific efflux pumps in clinical Gram-positive isolates and play an important role in microbial coping with alkali stress, sodium stress, and potassium insufficiency [8]. Tet(M), a common and well-characterized ribosomal protection protein, catalyzes the GTP-dependent release of Tet from ribosomes [8]. Worthy of our concern, a recent study has linked tigecycline resistance in E. faecalis to Tn916-associated constitutive overexpression and increased copy numbers of Tet(M) and Tet(L) [14]. In this context, it is noteworthy that our data showed good in vitro activity of erava against clinical S. aureus isolates harboring Tet(M), Tet(L) and Tet(K) in both MRSA and MSSA, indicating that erava has the potential to overcome the common tetracycline specific resistance mechanisms. Moreover, our data showed a disassociation between Tet resistance trends and erava MIC creep.
Erava, being a new Tet class drug, has not yet completed its phase III clinical trial and does not yet have its own susceptibility breakpoints recommended by CLSI [3,4,5,6,7, 24]. Consequently, we referred conservatively to the tigecycline susceptibility MIC breakpoint for S. aureus of 0.5 mg/L, which was derived from the US Food and Drug Administration [24,25,26, 28]. The available data thus far suggest that erava is two- to four-fold more active than tigecycline against common clinically important Gram-positive aerobic species [3, 4]. Because erava is not yet in use clinically in China and erava MICs of 0.5 mg/L were observed often in both MRSA and MSSA, we hypothesized that all of these S. aureus isolates would be susceptible to erava, and thus we referred to the MIC susceptibility breakpoint to define heteroresistant mother strains with subpopulations able to grow in the presence of 1.0 mg/L erava. Evaluation of heteroresistance is useful for probing antimicrobial resistance risk. None of the MSSA isolates with erava MIC values of ≤0.125 or 0.25 met our criteria for heteroresistance, whereas we did observe heteroresistance risk among MSSA with erava MIC values of 0.5 mg/L, suggesting that we should be on alert for erava MIC creep and the potential emergence of erava resistance. Although no MRSA were found to be exhibiting erava heteroresistance, it should be noted that six MRSA isolates had erava MIC values of 0.5 mg/L, underscoring the need for further screening of wider samples of MRSA isolates for erava heteroresistance.
Like other Tet class, erava inhibits bacterial protein synthesis by binding to the 30S ribosomal subunit [3, 8], and genetic mutations affecting the 30S ribosomal subunit (i.e., 16SrRNA and ribosome proteins S3 and S10) have been shown to confer resistance to tigecycline [3, 12,13,14,15,16,17,18,19,20]. Although we found heteroresistance-derived clones with elevated erava MICs and clinical MSSA with MIC values of 1.0 mg/L, none of these clones had 30S ribosomal subunit mutations. The potential relationship between Tet target site mutations with erava heteroresistance and relatively high MICs in S. aureus warrants further exploration given the ample evidence linking tigecycline heteroresistance in Gram-negative bacteria to cell envelope and efflux pump proteins [10, 21, 23]. The presently observed suppressing effects of PAβN and CCCP on the erava and tigecycline MICs of heteroresistance-derived S. aureus clones and clinical isolates with MICs of 1.0 mg/L suggest that efflux pumps and cell envelopes also contribute to erava heteroresistance in S. aureus [21,22,23]. Importantly, our finding of higher MICs for tigecycline than for erava in erava heteroresistance-derived clones is suggestive of possible tigecycline cross-resistance. Furthermore, the suppressing effects of CCCP and PAβN on tigecycline MICs further implicates efflux pump/cell envelope components in tigecycline MIC creep.
Upregulation of MepA, a multidrug-resistant efflux pump, has been shown to confer tigecycline resistance in S. aureus [16,17,18]. Furthermore, in strain SA984 S. aureus, the erava MIC increased from 0.004 mg/L in a MepA-negative parent isolate to 0.016 mg/L in S. aureus expressing MepA, whereas MepA addition increased tigecycline MICs from 0.016 mg/L to 1.0 mg/L, pointing to a negligible effect of MepA on erava resistance relative to its effect on tigecycline resistance [3, 8, 16]. The molecular mechanisms by which efflux pumps and cell envelope components participate in the MIC creep of these new Tet class drugs need to be further studied.