Eravacycline activity against clinical S. aureus isolates from China: in vitro activity, MLST profiles and heteroresistance

Mortality rates for patients with Staphylococcus aureus (S. aureus) infections have improved only modestly in recent decades and S. aureus infections remain a major clinical challenge [1]. Indeed, multi-drug resistant S. aureus has been reported worldwide and its rates are increasing, particularly MRSA, where only last resort antibiotics can be used, such as tigecycline, daptomycin, and linezolid [2]. The recently developed antibiotic eravacycline (erava; a.k.a. TP-434), a synthetic fluorocycline, was shown to be active against numerous Gram-positive, Gram- negative, and anaerobic bacteria, including those with acquired tetracycline (Tet)-specific efflux pumps, ribosomal protection factors, and multi-drug resistance mechanisms [3–5]. However, data regarding the efficacy of erava against clinical S. aureus isolates, especially from China, are quite limited [3–7]. Moreover, studies showing excellent in vitro antimicrobial activity of erava against S. aureus, enterococci, and streptococci, among others, revealed the rare occurrence of antimicrobial resistance [3, 8].

The development of heteroresistance in bacteria under antibiotic pressure often leads to antimicrobial treatment failure, and vancomycin heteroresistance in S. aureus in particular has become a serious threat for the induction of resistance that can make treatment difficult [9]. Furthermore, in recent years, strains of several microbial species have exhibited troubling heteroresistance to tigecycline [10, 11], which is structurally similar to erava.

Emergent mutations in genes encoding several 30S ribosome components, including 16SrRNA and ribosomal S10, have been found to correlate with reduced susceptibility to tigecycline [8, 12–20]. Tigecycline resistance in S. aureus has been reported to correlate closely with overexpression of a MATE (multidrug and toxic compound extrusion) family efflux pump encoded by mepA in passaged mutants, and some Escherichia coli or Enterococcus faecium isolates expressing high levels of the Tet-specific resistance factors Tet(M), Tet(K), and Tet(B) have been found to have tigecycline minimum inhibitory concentration (MIC) values two to four-fold to that in the controls [8, 12]. Tigecycline nonsusceptibility and heteroresistance risk in several Gram-negative bacteria species have been reported to correlate with expression levels of several efflux pumps and cell envelope proteins [21–23]. To date, erava efficacy appears to be mostly unaffected by common Tet resistance factors [3, 4]. However, the potential effects of Tet resistance factors on the development of erava resistance or heteroresistance in S. aureus, including cross-resistance to tigeycline, needs to be further studied.

Carbonyl cyanide m-chlorophenylhydrazine (CCCP) is a protonophore that exhibits reversible binding and transmembrane transport of protons across the cell membrane, leading to membrane depolarization that can diminish the electrochemical gradient and thereby reduce ATP production by ATP synthase [10, 21–23]. It has been used to investigate how protonophore activity can affect the potency of critical reserve antibiotics, including carbapenem, colistin, and tigecycline [21–23]. Phe-Arg-β- naphthylamide (PAβN) is a highly active broad-spectrum inhibitor of bacterial resistance-nodulation-division efflux pumps [21]. It remains to be determined how CCCP and PAβN may affect erava MICs.

The overall aim of this study was to examine the characteristics of erava antimicrobial activity and potential risks of erava resistance mechanisms. Toward this aim, we assessed the in vitro activity of erava against clinical S. aureus isolates from China and estimated the frequency of erava heteroresistance with population analysis profiling (PAP). Multilocus sequence typing (MLST) was performed to identify the molecular genotype of the isolates in our sample. We then used polymerase chain reaction (PCR) amplification to detect mutations in genes encoding Tet target sites in heteroresistance-derived S. aureus clones. Finally, we employed CCCP and PAβN experimental assays to examine the role of cell envelope and efflux pump proteins in erava heteroresistance.

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 [3–5]. 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 [29–31]. 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 [3–7, 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 [24–26, 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, 12–20]. 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 [21–23]. 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 [16–18]. 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.

In conclusion, erava exhibited excellent in vitro activity against clinical S. aureus isolates from China, overcoming the presence of Tet factors, including the Tet(K) and Tet(L) efflux pumps and the ribosome protection protein Tet(M). However, the risk of erava heteroresistance in S. aureus, especially in MSSA with MICs ≥0.5 mg/L, is worthy of concern. MSSA had higher MICs and a higher frequency of erava heteroresistance than MRSA in this study. S. aureus with erava MICs of 1.0 mg/L did not have 30S ribosome subunit mutations and the MICs of heteroresistance-derived MSSA and MSSA with erava MICs ≥1.0 mg/L could be reduced by CCCP or PAβN. Further studies are needed to elucidate the mechanisms by which efflux pump and cell envelope proteins may enable erava heteroresistance development and MIC creep in S. aureus.