Staphylococcus aureus is an important nosocomial pathogen that is associated with various infections, such as skin and skin structure infections, endocarditis, and bloodstream infections (Pérez-Montarelo et al., 2018). S. aureus is one of the major and most fatal causes of bacteremia, and has a mortality rate of almost 20%. Almost half of the patients with S. aureus bacteremia will develop complicated bacteremia (Guimaraes et al., 2019). Recently, the emergence of multidrug-resistant (MDR) superbugs, such as methicillin-resistant and vancomycin-resistant S. aureus, has become a significant threat to public health (Gasch et al., 2014; McGuinness et al., 2017). The methicillin-resistant S. aureus (MRSA) is considered a prioritized nosocomial pathogen by the World Health Organization (WHO; Serral et al., 2021). Overall, S. aureus infections are considered a significant clinical challenge.

The tetracycline class of antimicrobial agents has been clinically used for more than 60 years (Roberts, 2003). And they are continued to treat various serious infections caused by Gram-positive and-negative pathogens, including MRSA (Grossman, 2016). With their extensive use in clinical settings, resistance to old tetracyclines, especially doxycycline and tetracycline, is increasing worldwide (Roberts, 2003, 2005; Grossman, 2016). New generation tetracycline agents, such as eravacycline, omadacycline, and tigecycline, can be used as a treatment option for bacterial infections owing to their broad-spectrum antimicrobial activity.

Tigecycline is a semisynthetic glycylcycline with broad-spectrum antimicrobial agents (Doan et al., 2006). In 2005, it was approved by the US Food and Drug Administration (FDA) approval for the treatment of various serious infections, such as adults with complicated intra-abdominal infections and community-acquired bacterial pneumonia (Noskin, 2005). Eravacycline and omadacycline with modifications at the C-9 position are newer generation tetracycline agents similar to tigecycline. They were approved by the FDA in 2018 for the treatment of polymicrobial MDR infections (Solomkin et al., 2019; Morrissey et al., 2020; Zhanel et al., 2020). Similar to other tetracyclines, these new generation tetracycline agents inhibit bacterial protein synthesis by binding to the 30S ribosomal subunit. Eravacycline, omadacycline and tigecycline have broad-spectrum antibacterial activity against Gram-positive and Gram-negative microorganisms. However, the antimicrobial activity of these tetracyclines against S. aureus has not been comprehensively studied.

Many mechanisms of underlying tetracyclines resistance have been proposed. The resistance is mostly due to the acquisition of tetracycline resistance genes and mutation of ribosomal protection proteins (Nguyen et al., 2014; Beabout et al., 2015; Grossman, 2016; Linkevicius et al., 2016). Overexpression of efflux pumps has been reported both in Gram-positive and Gram-negative pathogens (Truong-Bolduc et al., 2015; Fiedler et al., 2016; Zhanel et al., 2016). Mutations or overexpression of the MepRAB efflux pump contributes to the decreased susceptibility to tigecycline (McAleese et al., 2005; Fang et al., 2020). Recent studies have revealed that a branched-chain amino acid transport system II carrier protein affects eravacycline and omadacycline susceptibility in S. aureus (Bai et al., 2019; Wang et al., 2020). Moreover, a recent study revealed a novel tet(L) efflux pump that confers resistance to eravacycline and tigecycline resistance in Staphylococcus (Wang et al., 2021). However, the potential contribution of these resistance factors of tetracyclines to the development of resistance to eravacycline and omadacycline in S. aureus is not completely known. In addition, multi-locus sequence typing (MLST), which was first established by Maiden et al. (1998), is now the most frequently used method to monitor epidemiology and investigate evolution of pathogens due to its high discriminatory power and comparability (Li T. et al., 2022). The distribution of sequence types (STs) profiles was unclear in tetracyclines-resistant (eravacycline/omadacycline/tigecycline-resistant) S. aureus Taken together, the results of this study will give insight into the prevalence and molecular epidemiology characteristics of tetracyclines-resistant S. aureus using MLST method.

In this study, we investigated and compared the in vitro antimicrobial efficacy of eravacycline, omadacycline, and tigecycline against 1,017 clinical S. aureus isolates. Resistance determinants and STs profiles of the tetracyclines-resistant isolates were further investigated using polymerase chain reaction (PCR). Furthermore, genetic mutations in the MepRAB efflux pump and 30S ribosome units and expression of mepA, tet38, tet(K) and tet(L) were also determined by sequencing, cloning experiment, efflux pump inhibition and quantitative real-time PCR (RT-qPCR).

Studies have shown that eravacycline, omadacycline, and tigecycline are broad-spectrum antibiotics, which act on several Gram-positive and-negative organisms (Zhanel et al., 2018; Dowzicky and Chmelařová, 2019; Xiao et al., 2020). Consistent with previously published studies (Li et al., 2020; Xiao et al., 2020), an excellent antimicrobial activity of these tetracycline agents was observed against the 1,017 S. aureus isolates. In the present study, we compared the antimicrobial effects of them, the results showed that eravacycline and tigecycline, especially eravacycline, had more power antimicrobial effect than omadacycline against tested S. aureus. Furthermore, out of the 1,017 S. aureus, we successfully screened 41 tetracyclines-resistant isolates. Three tetracyclines co-resistant strains were observed among them, revealing a cross-resistance toward eravacycline, omadacycline, and tigecycline in the clinical S. aureus isolates, which was also observed in Streptococcus agalactiae (Li et al., 2020; Li G. et al., 2022). Hence, it is important to monitor and detect the resistance of tetracyclines, in order to prevent their cross-resistance leading to treatment failure. We will focus on the cross-resistance mechanism of these new generation tetracycline agents in future studies.

New generation tetracycline agents can overcome the resistance mechanisms of tetracycline and also exhibit antibacterial activity against strains containing tetracycline resistance genes, including tet(K), tet(L), tet(M), tet(O), and tet(S) (Nguyen et al., 2014; Linkevicius et al., 2016; Zhanel et al., 2018). In this study, eravacycline and tigecycline exhibited great antibacterial activity against clinical S. aureus isolates harboring the tetracycline resistance genes. However, eravacycline, omadacycline, or tigecycline were not able to against the isolates (JP3349, JP4612 and JP5113) harboring tet(K) + tet(L) + tet(M). The finding implies that the coexistence of multiple tetracycline resistance genes may be related to the resistance to new generation tetracycline agents in S. aureus isolates, which is similar to the findings of Boukthir et al. in enterococci (Boukthir et al., 2020). Furthermore, among the 41 tetracyclines-resistant isolates, strains harboring tet(K) alone or in combination with other tetracycline resistance genes were predominant. Therefore, the effect of tet(K) on tetracyclines susceptibility needs further studied. Consistent with previous studies (Zhang et al., 2018; Bai et al., 2019), tet(O) and tet(S) were not detected in the tested strains and need further investigation in other S. aureus isolates.

The analysis of 41 tetracyclines-resistant isolates showed that the accumulation of several resistance mechanisms resulted in tetracyclines resistance. Accumulating evidence showed that amino acid substitutions at S10 might be associated with reduced tetracyclines susceptibility (Beabout et al., 2015; Bai et al., 2019; Wang et al., 2020). We also detected different mutation sites (such as M1L and Y58D) in S10 in the tetracyclines-resistant isolates. K57M was found in both tetracyclines-resistant and-susceptible strains, suggesting that it was not associated with tetracyclines resistance (Fang et al., 2020). Consistent with the findings of Wang et al. (Wang et al., 2020), no mutations were detected in the 30S ribosome protein S3, illustrating that S3 was not the predominant factor contributing to tetracyclines resistance in S. aureus.

Another important resistance mechanism is the active efflux pump. The MepRAB efflux pump, a novel MATE family efflux pump, plays a key role in tigecycline resistance in S. aureus (McAleese et al., 2005; Fang et al., 2020), however, whether it affects eravacycline and omadacycline resistance is unclear. We first analyzed ta mutation in MepRAB efflux pump-encoding genes. The PCR results showed amino acid substitution mutations and premature termination in mepA were found, which might be correlated to tetracyclines resistance. In previous study, mepR mutations have already been related to tigecycline resistance in S. aureus isolates (Dabul et al., 2018). Conversely, we did not detect mutations in mepR, suggesting that it might not affect tetracyclines susceptibility in these tested isolates. Interestingly, the frameshift mutation in mepB was observed frequently in tetracyclines-resistant strains but not in tetracyclines-susceptible strains. Cloning experiment further confirmed that a frameshift mutation in mepB might contribute to omadacycline resistance in S. aureus. MepB has been considered a hypothetical protein with unknown functions until a study by Agah et al. revealed that mepB played a role in responding to antimicrobials by interacting with nucleic acids (McAleese et al., 2005; Agah et al., 2014). To the best of our knowledge, this is the first study to demonstrate that a frameshift mutation in mepB might mediate omadacycline resistance in S. aureus. Second, we also comprehensively analyzed the levels of the MepRAB efflux pump in the tetracyclines-resistant strains. We observed that the overexpression of MepRAB efflux pump-encoding genes affected tigecycline, eravacycline and omadacycline resistance.

The isolates were exposure in 1/2 × MIC tetracycline agents to understand the role of different efflux pumps. We found that, different efflux pump-encoding genes, especially tet(K), upregulated to varying degrees after the resistant strains were stimulated by 1/2 × MIC tetracyclines, suggesting that the active tet(K) efflux pump was primarily responsible for tetracyclines resistance (Bai et al., 2019; Wang et al., 2020). Although previous study showed that tnovel tet(L) efflux pump variants affected the susceptibility of eravacycline and tigecycline (Wang et al., 2021), Wang et al. and the present study showed that the tet(L) overexpression might not confer resistance to eravacycline (Wang et al., 2020), however, it might confer resistance to omadacycline and tigecycline in S. aureus. Besides conferring resistance to tetracycline (Chen and Hooper, 2018), our study revealed that the active tet38 efflux pump might be associated with resistance to eravacycline, tigecycline and omadacycline.

The MLST analyses showed that the most tetracyclines-resistant S. aureus belonged to ST5, a globally disseminated and highly pathogenic lineage (Hau et al., 2018), and ST764, a variant of the ST5 lineage.

Combined with drug sensitivity, we found that the ST5 and ST764 clonotype isolates were MDR strains. Previous studies have reported that ST5 and ST764 MRSA strains were hypervirulent and MDR, and dominated S. aureus infections in China (Takano et al., 2013; Jian et al., 2021). For the first time, our findings revealed that ST5 and its variant ST764 dominated tetracyclines-resistant S. aureus strains, suggesting that tetracyclines-resistant S. aureus strains might pose a serious clinical threat hence more attention should be paid to their prevention and control.

However, our work also has some limitations. Tetracyclines-resistant S. aureus strains were only collected from the same hospital, which might lead to deviation in our results. It is necessary to use other pathogen to verify our findings, as the efficacy and resistance mechanisms of tetracyclines and distribution of ST type might be different in different hospital. Besides, the cross-resistance mechanism of these new generation tetracyclines was not illustrated in this work. Thus, the resistance and cross-resistance mechanisms of these new generation tetracyclines should be further investigated.

To conclude, eravacycline and tigecycline showed more excellent antibacterial activity against S. aureus, including tetracycline-resistant isolates, than omadacycline did. Tetracyclines resistance resulted from an accumulation of several resistance mechanisms. The coexistence of multiple tetracycline resistance genes may contribute to the emergence of tetracyclines resistance. Moreover, mutations in mepA and S10 might play crucial role in tetracyclines resistance. Importantly, we reported that frameshift mutations in mepB contributed to reduced omadacycline susceptibility. Furthermore, mepA, tet38, tet(K) and tet(L) overexpression reduced tetracyclines susceptibility. Moreover, a major clonal dissemination type, ST5, and its variant ST764 were determined in most tetracycline-resistant strains, suggesting that these strains might possess the risk of clonal transmission and require further investigation.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

WZ participated in the design of the study, carried out the experiments, analyzed the data, and drafted the manuscript. XZ, YL, and YZ participated in experiments and data analyses. MX and SW provided the analysis of the results. YS participated in analysis and discussions. LC and TZ designed the study, participated in data analyses, and provided critical revisions of the manuscript. All authors contributed to the revision of the manuscript and approved the final version for submission.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.1043736/full#supplementary-material