In vitro activities of Eravacycline against 336 isolates collected from 2012 to 2016 from 11 teaching hospitals in China

Background In China multidrug-resistant bacteria pose a considerable threat to public health. Antimicrobial resistance has weakened the effectiveness of many medicines widely used today. Thus, discovering new antibacterial drugs is paramount in the effort to treat emerging drug-resistant bacteria. Methods Eravacycline, tigecycline and other clinical routine antibiotics were tested by reference broth micro-dilution method against 336 different strains collected from 11 teaching hospitals in China between 2012 and 2016. These isolates included Enterobacteriaceae, non-fermentative, Staphylococcus spp., Enterococcus, and a number of fastidious organisms. The strains involved in this study possess the most important drug resistance characteristics currently known in China. Drug resistant bacteria such as those producing extended spectrum β-lactamases (ESBL) and carbapenemases (KPC-2 and NDM-1), and those exhibiting colistin resistance (mcr-1) and tigecycline were included in this study. Additionally, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), β-lactamase positive Haemophilus influenzae, and penicillin resistant Streptococcus pneumoniae (PRSP) were also included. Results Eravacycline exhibited good efficacy against all the strains tested, especially for organisms with ESBLs, carbapenemases, and mcr-1 gene compared with tigecycline and other antibiotics tested. The MIC values of eravacycline against carbapenemase producing Enterobacteriaceae and OXA-23-producing A. baumannii were much lower than the MIC values of other antibiotics. MRSA, VRE, β-lactamase positive Haemophilus influenza, and PRSP were sensitive to eravacycline in every strain tested. Furthermore, in most strains tested, the MICs of eravacycline were two to four-fold lower than the MICs of tigecycline. Conclusions Eravacycline has shown potent antibacterial activity against common and clinically important antibiotic-resistant pathogens. The MIC distribution of eravacycline was generally lower than that of tigecycline which demonstrates that this new drug is potentially more effective than the existing medications. Electronic supplementary material The online version of this article (10.1186/s12879-019-4093-1) contains supplementary material, which is available to authorized users.


Background
In China, microbial resistance to presently administered antimicrobial agents is increasing steadily owing to the emergence of novel resistance mechanisms in the microbes [1,2]. Multidrug-resistant bacterium causes a considerable threat to public health. Antimicrobial resistance weakened the effectiveness of many medicines widely used today [3]. Thus discovering new antibacterial drugs are required to combat the threat of these emerging resistant bacteria. Eravacycline (TP-434 or 7fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline) is a novel broad-spectrum synthetic tetracycline antibiotic being developed for the treatment of severe life-threatening infections, including those that are resistant to current broad-spectrum antibiotics [4]. Eravacycline has already been proven effective against some clinically important antibiotic-resistant pathogens, including gram-positive and gram-negative aerobic and anaerobic pathogens [5,6]. Moreover, eravacycline was found to be safer and more effective than carbapenems in patients with complicated intra-abdominal infection (cIAI) during global phase 3 clinical trials (NCT01844856 and NCT02784704) [5,7]. Additionally, there is a clinical development plan in place to introduce it into China to address bacterial drug resistance. The targets of eravacycline include complicated intra-abdominal infection (cIAI), complicated urinary tract infection (cUTI), and pulmonary infections caused by other susceptible pathogens. Tigecycline is a relatively new competing drug for eravacycline, imipenem, meropenem, and colistin in the treatment of carbapenemresistant Enterobacteriaceae. The present study was designed to evaluate the in vitro activities of eravacycline against panels of clinical bacterial pathogens, with or without remarkable resistance factors, which were collected in recent years and were similar to pathogenic bacteria that this drug was designed to treat. This study was designed to prove the in-vitro efficacy of eravacycline (presented by minimum inhibitory concentration, MIC) against major target pathogens in China, which will be used to support further clinical development of eravacycline within China.

Methods
In the present study, a total of 336 different clinical isolates, were routinely collected from 11 teaching hospitals representing the south, north, northwest, east, and middle regions of mainland China between 2012 and 2016, and tested (list of the hospitals can be found in Additional file 1). After re-identification with the typical biochemical reaction of each organism, the strains were stored in a Microbank tube and placed in a refrigerator at − 80 degrees Celsius before test. All organisms and their associated drug resistance factors are detailed in Table 1. MIC measurements were performed via the reference broth microdilution method as described by the Clinical and Laboratory Standards Institute (CLSI) M7-A9 (2012) [8]. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were utilized as quality controls in MIC testing of gram-negative bacteria. Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were utilized as quality controls in MIC testing of gram-positive bacteria. Streptococcus pneumoniae ATCC 49619, Haemophilus influenzae ATCC 49247 and Haemophilus influenzae ATCC 49766 were used as quality controls during MIC testing of the fastidious organisms. Tigecycline, the major comparator for eravacycline, imipenem, meropenem and colistin to treat carbapenemresistant Enterobacteriaceae and Acinetobacter baumannii, were selected in the panel of antibiotics to be tested. We evaluated eravacycline with a gradient concentration of 0.002-16 mg/L against common clinical gram-negative bacilli, gram-positive cocci, and fastidious organisms collected from our previous studies [9][10][11][12][13], including Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae), Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae and Haemophilus influenzae. Antibiotic solutions for susceptibility testing were freshly prepared according to the manual of CLSI [8]. A scatter plot of eravacycline versus tigecycline was drawn for each species of bacteria, to reveal the relationship between the two antibiotics in different organisms. All the results related to resistant genes were readily available, directly from our previous researches [12][13][14]. Statistical analyses and data visualization were done with R (version 3.4.4) and ggplot2 package (version 2.2.1).

Results
In vitro activity of eravacycline was evaluated against 336 strains of clinically significant species, with many exhibiting resistance factors (Table 1). In most of the strains tested, the MIC 50 and MIC 90 values for eravacycline were lower than that of tigecycline and other comparable antibiotics tested for each organism/phenotypic group. Furthermore, eravacycline was highly effective against all of the organisms tested, regardless of resistance factors.
For Enterobacteriaceae bacteria, the MIC values of eravacycline varied with the resistance characteristics, especially for K. pneumoniae. The MIC 50 values of eravacycline against E. cloacae and E. coli were much lower than the values of other comparable drugs, especially in strains with resistance phenotypes ( Table 2). For K. pneumoniae, the MIC distribution of eravacycline differed depending on the drug resistance features. K. pneumoniae strains which were ESBL-positive (n = 10), kpc-2-positive (n = 9) and NDM-1-positive (n = 3), had similar MIC distributions. The MIC 50 value of eravacycline against strains with the above three resistance mechanisms is 0.5 mg/L, and the MIC90 values were 1 mg/L, 2 mg/L and 1 mg/L respectively.
K. pneumoniae strains resistant to tigecycline were susceptible to eravacycline at higher MIC 50 values of 8 mg/L, while the MIC 90 was equivalent to that of tigecycline at 16 mg/L. For mcr-1 positive strains, the MIC 50 of eravacycline was 1 mg/L compared with 16 mg/L for tigecycline, while the MIC 90 of eravacycline and tigecycline was equivalent at 16 mg/L. The MIC 50 (0.5 mg/L) and MIC 90 (2 mg/L) values of eravacycline against carbapenem-resistant K. pneumoniae, were much lower than those of other antibiotics such as imipenem, meropenem, cephalosporins, and fluoroquinolones. The MIC distributions for K. pneumoniae of different resistant phenotypes to eravacycline, tigecycline, and other clinically common antibiotics are presented in Table 3.
MIC distributions for A. baumannii also varied by resistance characteristics. A. baumannii isolates were tigecycline resistant and showed slightly elevated MIC 50 and MIC 90 for eravacycline at 2 mg/L. OXA-23-producing A. baumannii isolates have a MIC 50 of 1 mg/L and MIC 90   Table 4.     Table 5.
In the results obtained for Enterococcus spp. it was found that MIC 50 and MIC 90 of eravacycline for E. faecalis were both 0.032 mg/L. The MIC 50 and MIC 90 of eravacycline for E. faecium were 0.016 mg/L and 0.032 mg/L. For Vancomycin-Resistant Enterococci (VRE) strains, the MIC 50 and MIC 90 were identical with that of vancomycinsusceptible E. faecium strains. For other antibiotics, the values are presented in Table 6. In general, for grampositive bacteria with varying resistance factors, eravacycline demonstrated substantial antibacterial activity.  For fastidious strains, including 20 S. pneumoniae isolates and 20 H. influenzae isolates, eravacycline showed high antimicrobial activities against S. pneumoniae with MIC 50 (0.008 mg/L) and MIC 90 (0.008 mg/L), there was no difference with eravacycline distribution between PRSP (Penicillin-resistant S. pneumoniae) and PSSP (Penicillin-sensitive S. pneumoniae) strains (Table 7). For H. influenzae the MIC 50 and MIC 90 were 0.064 mg/L and 0.125 mg/L, and they were the same in both β-lactamase-positive and βlactamase-negative strains (Table 8).
A jittered scatter plot was drawn using the MIC values of eravacycline and tigecycline involving all the strains tested. A clear pattern was found showing that most of the MIC values of tigecycline are higher than the corresponding MIC values of eravacycline (in many cases by 2 to 4 fold). For all of the clinical isolates tested, except for Staphylococcus spp. and S. maltophilia, more points are located above the diagonal y = x line, suggesting that eravacycline has lower MIC distribution than tigecycline (Fig. 1). For Staphylococcus spp. and S. maltophilia the points were distributed on both sides of the diagonal

Discussion
As resistance to antibiotics grows worldwide, it becomes increasingly important to find new treatments for bacterial infections. In the present study, a new antibiotic eravacycline was compared to existing medications. Eravacycline demonstrated high in vitro activity against clinical isolates, including strains with specific resistant factors. Eravacycline was compared to a derivative of tigecycline, and in most cases presented with a lower MIC distribution for the majority of strains tested in this study. Since many years nosocomial pathogens, such as Enterobacteriaceae which are responsible for complicated intra-abdominal infection (cIAI) were increasing in frequency [15]. Moreover, cases of gram-positive cocci such as S. aureus, coagulase-negative staphylococci, and enterococci, the major causative organisms of complicated urinary tract infections (cUTI) were also increasing [16]. The emergence of multiple drug-resistant bacteria, such as Carbapenem resistant Enterobacteriaceae bacteria (CRE), Carbapenem-resistant Acinetobacter baumannii (CRAB) and Methicillin-resistant Staphylococcus aureus (MRSA), has compounded this problem significantly by increasing the difficulty of treatment, the proportion of failures, as well as the mortality rate of patients. Since Tigecycline and eravacycline belong to a different antibiotic class with a mechanism of action distinct from cephalosporins and carbapenem antibiotics, they can evade established resistance mechanisms of Enterobacteriaceae and exhibit higher efficacy against resistant bacteria. In this study, eravacycline showed high antibacterial activity against CRE strains, suggesting that eravacycline could be useful to treat complicated infections caused by CRE. Similarly, CRAB also shows resistance to antibiotics which were commonly used during the clinical practice. CRAB is the most notorious pathogen responsible for nosocomial infections in China at present [17][18][19]. This study found that the most effective drug for OXA-23 producing A. baumannii was colistin then eravacycline. Eravacycline also demonstrated high potency against OXA-23 producing A. baumannii, with a MIC 50 of 1 mg/L which was much lower than other antibiotics, except for colistin. Similar to eravacycline in structure and mechanism, tigecycline has been widely utilized in China for many years, and tigecycline-resistant strains have also emerged with the increase in use of this antibiotic [20,21]. In the present study, eravacycline also exhibited lower MIC distribution compared with tigecycline in tigecyclineresistant strains, suggesting that the mechanism which leads to tigecycline resistance does not inhibit the activity of eravacycline. Furthermore, high antibiotic potency against CRE and CRAB could make eravacycline a potential option to treat complex infections including respiratory and bloodstream infections. For Staphylococcus spp. the results were entirely different, with tigecycline values much lower than eravacycline. From the scatter plot we observed that the points are evenly distributed on both sides of the diagonal line (line: y = x). This may be either due to the combined effects of different resistance mechanisms, or potentially unknown resistance mechanisms. In addition, the total number of Staphylococcus spp. strains which were tested in this study was relatively small, which may cause random errors in the antibacterial activity of eravacycline. Thus, further validation utilizing different

Limitation and suggestion
The clinical isolates tested were limited by country as they were exclusively collected in China and within this country, these isolates were only obtained from 11 teaching hospitals. No strains from other hospitals were utilized. Therefore, many different clinical isolates remain untested. Thus, it is important that researchers reproduce our work in other countries with different isolates in order to understand the full spectrum of this new antibiotics' efficacy. The results of this study show that eravacycline has a positive application potential for the treatment of current drug-resistant bacterial infections. Considering the relatively small number of each organism and limited types of resistant phenotypes, the result of this study only partially represent the resistant phenotype encountered in real clinical practice, and additional studies are needed for a more comprehensive assessment of the antibacterial activity of eravacycline.

Conclusions
The results of this study proved that eravacycline possesses a broad spectrum of activity against a variety of gram-positive and gram-negative bacteria, including multi-drug resistant strains such as A. baumannii and carbapenem-resistant Enterobacteriaceae.

Additional file
Additional file 1: The list of committee and the institute to which it belongs for all hospitals that provided Administrative Consent to access or receive samples. This additional file list the committee (and the institute to which it belongs) for all hospitals that provided Administrative Consent to access or receive samples/data (DOCX 13 kb)