In vitro Activity of the Novel Tetracyclines Derivative, Zifanocycline Against Mycobacterium abscessus

Introduction

M. abscessus is a non-tuberculous mycobacterium that can be classified into three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense.¹ There is a growing report of M. abscessus infections occurring in populations with cystic fibrosis, as well as those without it, around the world.^2–4 M. abscessus exhibits a high level of intrinsic resistance to most classes of antibiotics, rendering the infections often difficult to treat, and displays poor tolerance to the limited available antibiotic options.⁵ A comprehensive analysis of results from treatments for pulmonary disease caused by M. abscessus showed that the cure rate was merely 45%, with M. abscessus subsp. massiliense (57%), demonstrating a higher cure rate than M. abscessus subsp. abscessus (33%).⁶ Consequently, the threat posed by M. abscessus to human health is escalating, highlighting the urgent need for the discovery or development of new anti-infective agents.

Zifanocycline represents a significant advancement as a broad-spectrum third-generation tetracycline that is specifically classified as aminomethylcycline. This antibacterial agent is currently in the developmental phase to address several critical medical conditions, including community-acquired bacterial pneumonia (CAP), acute bacterial skin and skin structure infections (ABSSSI), and complicated intra-abdominal infections (cIAI).^7,8 Zifanocycline effectively overcomes many common tetracycline resistance mechanisms by inhibiting the normal functioning of bacterial ribosomes. The 3-methyl-3-azabicyclo[3.1.0]hexane side chain that is unique to KBP-7072 could play a role in overcoming some forms of tetracycline resistance, comparable to that of Tigecycline´s C9 extension, which restricts the access of ribosomal protection proteins (RPP), like Tet(M), to the primary tetracycline binding site.⁹ The unique C9 extension of this compound results in a specific interaction pattern with the 30S ribosomal subunit and the ability to form direct, stable interactions with the phosphate groups of 16S rRNA, setting it apart from the third-generation tetracycline such as tigecycline.^9,10 This differentiation not only highlights the unique structural features of the compound but also serves to expand the range of potential interactions within the primary tetracycline-binding pocket.¹⁰ Such distinct interactions could lead to variations in the efficacy and development of novel mechanisms of action against bacterial targets, which could enhance the therapeutic application of this compound in the treatment of antibiotic-resistant infections.

Zifanocycline has completed Phase 1 clinical development as of December 2015, which evaluated its safety, tolerability, pharmacokinetics, and multiple ascending doses in healthy subjects.^11,12 The pharmacokinetic/pharmacodynamic (PK/PD) index, particularly the area under the curve of concentration over time (AUC) the minimum inhibitory concentration (MIC), has a strong correlation with effectiveness.¹³ The pharmacokinetic results obtained from animal studies align with the findings from both single- and multiple-ascending-dose trials conducted with healthy subjects, thereby confirming the effectiveness of zifanocycline for once-daily administration via oral and intravenous routes in clinical investigations.^14,15 However, the antibacterial activity of zifanocycline against M. abscessus has not been systematically evaluated. In this study, we evaluated the in vitro efficacy of zifanocycline and 13 other antimicrobial compounds against 67 unique clinical isolates of M. abscessus that were sourced from the Affiliated Hospital of Jiaxing University between 2019 and 2024.

Materials and Methods

Collection and Culture Conditions of M. abscess Isolates

The Affiliated Hospital of Jiaxing University serves as the designated tuberculosis hospital in the region. This study involved sputum and bronchoalveolar lavage fluid samples from patients exhibiting clinical symptoms of lower respiratory tract infections who visited the hospital between 2019 and 2024. Each sample was incubated at 37°C in Middlebrook 7H10 broth enriched with 10% oleic acid-albumin-dextrose-catalase (Becton Dickinson and Company, USA). Non-tuberculous mycobacteria (NTM)-positive cultures were identified using the MODI-TOF MS mass spectrometer (Mérieux, France) and were preliminarily classified as strains of M. abscessus. A total of 67 strains of M. abscessus were collected for this study, with duplicate strains (ie, multiple strains from the same patient) excluded. These isolates were obtained through standard diagnostic tests and patient care processes and were not specifically isolated for this study, thus exempting the need for additional patient-informed consent. This study has received approval from the Institutional Ethics Committee of the Jiaxing University Affiliated Hospital (Ethics Approval Number: 2024-LY-400).

Identification of M. abscessus Subspecies and Determination of the erm41 Gene Polymorphism

Genomic DNA was extracted from positive cultures of M. abscessus strains using a magnetic bead-based bacterial genomic DNA extraction kit (Sangon Biotech, Shanghai, China). Whole-genome sequencing was conducted using the same 150-bp paired-end Illumina NovaSeq X Plus platform. The sequence data were subjected to de novo assembly using SPAdes software,¹⁶ which is specifically designed for assembling short sequencing reads into longer contigs. ANI Calculator (https://www.ezbiocloud.net/tools/ani) was used to determine Average Nucleotide Identity (ANI) values. For the ANI comparison, the reference genomes included M. abscessus subsp. massiliense strain GO 06 (GenBank: GCA_000277775.2), M. abscessus subsp. bolletii BD (GenBank: GCA_003609715.1), and M. abscessus subsp. abscessus ATCC 19977 (GenBank: GCA_000069185.1). Typically, the ANI value for the same species is >99%. The ResFinder database was used to analyze M. abscessus, leading to the identification of erm41.¹⁷ Using the erm41 gene sequence from M. abscessus subsp. abscessus ATCC 19977 as a reference, all strains were aligned with SnapGene software to determine the C/T polymorphism at the 28th nucleotide of the erm41 gene.

Antimicrobial Susceptibility Tests

Susceptibility testing for the drug was performed according to the guidelines outlined in the 2018 CLSI M24 document.¹⁸ Single colonies of M. abscessus were selected from M7H10 agar plates, diluted to a McFarland standard of 0.5 using sterile saline, and further adjusted to a bacterial burden of 1×10⁵ to 5×10⁵ CFU/mL with cation-adjusted Mueller-Hinton broth (CAMHB). Amikacin, moxifloxacin, linezolid, bedaquiline, and clofazimine were purchased from Adooq Biosciences (Adooq Bioscience, Nanjing, China). Ciprofloxacin, trimethoprim–sulfamethoxazole, cefoxitin, tigecycline, omadacycline, zifanocycline, doxycycline, imipenem, and clarithromycin were purchased from MedChemExpress (MCE, Shanghai, China). Zifanocycline was dissolved and diluted with deionized water. Bedaquiline and clofazimine were first solubilized with dimethyl sulfoxide (DMSO) and then diluted with deionized water. For other drugs, dissolution and dilution were performed in accordance with the operating procedures specified in CLSI M100.¹⁹ All antibacterial agents were serially diluted in a 1:2 ratio in a 96-well microtiter plate using CAMHB, and 100 μL of the bacterial suspension in CAMHB was added to each well. The final concentrations ranged from 0.015 to 32 mg/L for zifanocycline, tigecycline, omadacycline, bedaquiline, clofazimine, and trimethoprim-sulfamethoxazole; 0.03 to 64 mg/L for doxycycline, moxifloxacin, ciprofloxacin, and clarithromycin; 0.06 to 128 mg/L for amikacin, linezolid, and imipenem; and 0.12 to 256 mg/L for cefoxitin. The drug-susceptible plates were incubated at 37°C for 3–4 days until noticeable growth was observed in the control wells. In this study, Mycobacterium smegmatis strain ATCC 700686 and Staphylococcus aureus strain ATCC 29213 were used as reference strains to ensure the quality of the tested pharmaceutical products. The MICs of clarithromycin were evaluated after incubation periods of 3 and 14 days to determine the presence of inducible resistance to clarithromycin. M. abscessus ATCC 19977 was used as the reference strain for MIC comparison. Quality control and result interpretation for all drugs were conducted according to breakpoints established in the 2018 CLSI M62 guidelines.²⁰

Synergistic Antimicrobial Susceptibility Tests

The in vitro combined effects of zifanocycline with seven commonly used antimycobacterial drugs—namely, clarithromycin, cefoxitin, linezolid, bedaquiline, amikacin, imipenem, and moxifloxacin—were evaluated using the broth microdilution checkerboard assay. This study utilized five clinical isolates of M. abscessus that were chosen randomly. A 96-well sterile microplate was used, with each antimicrobial agent starting at twice the MIC and serially diluted in sterile CAMHB, typically across eight dilutions. Fifty microliters of each dilution was arranged in rows and columns of the plate. Subsequently, 100 μL of the bacterial suspension was carefully added to a sterile microplate to ensure a final concentration of 5 × 10⁵ CFU/mL. The microplate was then incubated for 72 h. At the end of the incubation period, the MIC was determined as the concentration that successfully inhibited bacterial growth. The fractional inhibitory concentration index (FICI) was calculated based on the difference in MIC values before and after drug combination. The formula for FICI was expressed as follows: FICI = [(MIC of zifanocycline used in combination/MIC of zifanocycline alone) + (MIC of the second antibiotic used in combination/MIC of the second antibiotic when used alone)]. If the FICI is ≤0.5, synergy is indicated; if it falls between 0.5 and 4, it is considered indifference; and if it is greater than 4, it suggests antagonism.

Statistical Analysis

All data were presented using Microsoft Excel 2021. Descriptive statistical methods were used to calculate the resistance rates and related variables of Mycobacterium abscessus (M. abscessus) isolates. The results were expressed as counts and percentages. All statistical analyses were performed using SPSS Version 20 (IBM Corp., Armonk, NY, USA).

Result

In vitro Activity of Zifanocycline and Comparators Against 67 M. abscessus Isolates

Zifanocycline exhibited potent antibacterial activity against 67 isolates of M. abscessus, with MIC₅₀ as 0.06 mg/L and MIC₉₀ as 0.25 mg/L, respectively. At a concentration of 0.5 mg/L, zifanocycline inhibited the growth of all tested isolates (Figure 1).

Figure 1 MIC distribution of zifanocycline, tigecycline, and omadacycline for 67 M. abscessus isolates. The column indicated by the MIC50 arrow shows the MIC50 result for zifanocycline, and the column indicated by the MIC90 arrow shows the MIC90 result for zifanocycline.

In comparison to other broad-spectrum tetracycline antibiotics, zifanocycline (MIC₉₀, 0.25 mg/L) demonstrated a 2-fold lower MIC₉₀ than both tigecycline (MIC₉₀, 0.5 mg/L) and omadacycline (MIC₉₀, 0.5 mg/L). Additionally, the MIC₉₀ values for both bedaquiline and clofazimine against M. abscessus isolates were found to be 0.5 mg/L, indicating antibacterial activity comparable to that of tigecycline and omadacycline. Amikacin (100% susceptibility) and linezolid (100% susceptibility) exhibited excellent antibacterial activity against M. abscessus isolates in vitro with MIC₅₀ and MIC₉₀ values of 2 mg/L, 4 mg/L, and 2 mg/L, 8 mg/L, respectively. The tested isolates demonstrated moderate susceptibility to cefoxitin (52.24%susceptibility), according to CLSI breakpoints. However, ciprofloxacin (4.48% susceptibility), moxifloxacin (25.37% susceptibility), trimethoprim-sulfamethoxazole (35.82% susceptibility), doxycycline (2.99% susceptibility), and imipenem (0% susceptibility) displayed comparatively low antibacterial activities against the tested isolates based on CLSI breakpoints (Table 1).

Table 1 In vitro Activities of Zifanocycline and Comparators Against 67 M. abscessus Isolates

The MIC₅₀ and MIC₉₀ of zifanocycline for 57 isolates from the M. abscessus subsp. abscessus erm41 T28 genotype, which confers induced resistance to clarithromycin, were consistently 0.06 mg/L and 0.25 mg/L (Table 2).

Table 2 In vitro Antimicrobial Activity of Zifanocycline, Tigecycline, and Omadacycline Against M. abscessus with Different erm41 Genotypes

Zifanocycline is Compatible with the Most Commonly Utilized Drugs for Treating M. abscessus Infections

The compatibility of zifanocycline with seven commonly used antimycobacterial drugs—namely imipenem, clarithromycin, moxifloxacin, linezolid, cefoxitin, bedaquiline, and amikacin—was evaluated in vitro through the broth microdilution checkerboard titration method and five randomly selected clinical isolates of M. abscessus. Indifference effects were observed between zifanocycline and the aforementioned antimycobacterial drugs (Table 3).

Table 3 The Index of Fractional Inhibitory Concentration for Zifanocycline Used Alongside Drugs That are Frequently Employed in the Clinical Treatment of M. abscessus Infections

Discussion

M. abscessus is the second most common non-tuberculous mycobacterial (NTM) pathogen responsible for pulmonary diseases and is widely recognized as one of the most antibiotic-resistant mycobacteria.^5,21 Research reports indicate an overall increase of 64.7% in infections caused by M. abscessus, along with a 25% increase in disease incidence.²¹ The cumulative mortality rates for patients with M. abscessus pulmonary disease at 5, 10, and 15 years are 11.4%, 29.8%, and 50.0%, respectively, representing an exceptionally high mortality rate for a disease that is not typically classified as an infectious disease.²²

The existing multidrug chemotherapy regimens containing macrolides have demonstrated limited efficacy in the treatment of M. abscessus, underscoring an urgent need for the development of novel therapeutic strategies.²³ To date, several innovative agents that may exhibit antibacterial activity against M. abscessus have been evaluated, including the quinolone sitafloxacin and the oxazolidinones linezolid, tedizolid, and contezolid. Sitafloxacin has shown superior antibacterial activity against M. abscessus (MIC₅₀/MIC₉₀, 1/2 mg/L) compared to other quinolones.²⁴ Although linezolid has been proven to possess good in vitro and in vivo activity against M. abscessus, its use as a long-term oral treatment option is limited due to side effects such as myelosuppression and peripheral neuropathy.^25,26 Although tedizolid appears to be safer regarding myelosuppression and neurotoxicity, and contezolid in terms of myelosuppression,^27–29 further clinical research is necessary to confirm their safety for long-term use.

Recent studies have demonstrated that the novel tetracycline derivatives tigecycline, omadacycline, and eravacycline demonstrate more significant antibacterial activity against M. abscessus isolates.^30,31 One of the newest tetracycline derivatives, zifanocycline, exhibits superior in vitro antimicrobial activity against Acinetobacter baumannii compared to tigecycline and omadacycline.^7,8 However, its antimicrobial activity against M. abscessus remains unexplored. This study evaluated the in vitro antibacterial activity of zifanocycline and its comparator agents against 67 strains of M. abscessus. The results demonstrated that zifanocycline exhibited significant advantages over other comparator drugs, including β-lactams, fluoroquinolones, and sulfonamides. Zifanocycline exhibited a MIC₅₀ of 0.06 mg/L, which is 4-fold lower than that of omadacycline and 2-fold lower than that of tigecycline; its MIC₉₀ is 0.25 mg/L, which is 2-fold lower than those of both omadacycline and tigecycline. Compared with omadacycline, zifanocycline exhibited superior in vitro antibacterial activity against M. abscessus. Additionally, zifanocycline and omadacycline are available in both oral and intravenous formulations, and the oral formulation provides an advantage in improving patient compliance for the treatment of M. abscessus-related diseases that require long-term administration to achieve therapeutic efficacy. In clinical investigations for the treatment of M. abscessus infections, over 75% of patients experienced clinical improvement when treated with chemotherapy regimens that included omadacycline, with a median duration of relatively safe continuous use extending up to 6–8 months.^32,33 The primary adverse reactions attributed directly to this treatment were nausea and vomiting; however, a subset of patients tolerated these side effects well.³³ In a brief pharmacokinetic investigation, it was determined that zifanocycline is safe and well-tolerated when administered at daily doses of up to 200 mg.³⁴ However, there is a notable lack of clinical research regarding its long-term safety.

A notable intrinsic resistance determinant of M. abscessus is the inducible macrolide erm41 gene, which encodes a methylase that modifies the ribosome, the target of clarithromycin and azithromycin, the cornerstone drugs for treating M. abscessus infections.³⁵ The erm41 gene is non-functional in M. abscessus subsp. massiliense; however, in M. abscessus subsp. bolletii and M. abscessus subsp. abscessus, the 28th nucleotide of this gene exhibits a (C/T) polymorphism, with only the erm41 T28 variant mediating macrolide resistance.^1,36 Furthermore, M. abscessus demonstrates both intrinsic and acquired resistance to numerous antimicrobial agents, which are challenging to overcome.⁵ Consequently, infections caused by M. abscessus typically necessitate a combination of multiple drugs.³⁷ Nevertheless, multiple studies have indicated that the recommended multidrug therapy regimen, primarily consisting of macrolides, amikacin, and β-lactams, results in sputum culture conversion to negative in only 25% to 26% of patients with non-cystic fibrosis M. abscessus subsp. abscessus infections, followed by a high recurrence rate of 17% to 55%.^38–40 The MIC₅₀ and MIC₉₀ values of zifanocycline against M. abscessus subsp. abscessus with the erm41 T28 genotype were determined to be 0.06 mg/L and 0.25 mg/L, respectively, which aligns with the results obtained from the MIC₅₀ and MIC₉₀ of all isolates. Thus, macrolide-induced resistance does not compromise the in vitro antibacterial activity of zifanocycline. Synergistic antimicrobial susceptibility tests observed indifference effects between zifanocycline and seven other commonly used anti-mycobacterial drugs, which facilitates flexible adjustment in anti-mycobacterial treatment regimens to achieve optimal therapeutic effects.

Conclusion

In conclusion, zifanocycline shows considerable antibacterial effectiveness against M. abscessus and observes indifference effects when combined with other anti-M. abscessus treatments in vitro. Thus, zifanocycline could be a promising option for incorporation into new therapeutic regimens for M. abscessus.