key: cord-0985653-ckryfoex authors: Pfaller, Michael A; Li, Li; Liu, Qingmei; Zhang, Jay; Huband, Michael D; Lindley, Jill M; Mendes, Rodrigo E title: In vitro activity of a novel aminomethylcycline antibacterial (KBP-7072), a third-generation tetracycline, against clinical isolates with molecularly characterized tetracycline resistance mechanisms date: 2021-12-14 journal: JAC Antimicrob Resist DOI: 10.1093/jacamr/dlab177 sha: b4a0eee646d3bcac679944c001a6f5351c2f94d3 doc_id: 985653 cord_uid: ckryfoex OBJECTIVES: This study evaluated the in vitro activity of KBP-7072 against 413 contemporary surveillance isolates, including subsets with known tetracycline resistance genes. MATERIALS: In total, 105 Klebsiella pneumoniae (51 tetracycline resistant), 103 Escherichia coli (52 tetracycline resistant), 103 Staphylococcus aureus (51 tetracycline resistant) and 102 Streptococcus pneumoniae (51 tetracycline resistant) isolates were included. These isolates were tested by broth microdilution using fresh media. CLSI/EUCAST breakpoints were applied, except for tigecycline and omadacycline, which used FDA criteria. RESULTS: KBP-7072 (MIC(50), 0.06 mg/L), tigecycline (MIC(50), 0.12 and 0.25 mg/L) and omadacycline (MIC(50), 0.12 and 0.5 mg/L) showed similar MIC(50)s for tetracycline-susceptible and -resistant S. aureus. Other tetracycline comparators had their MIC(50) increased 64- to 256-fold by tet. For S. pneumoniae, KBP-7072 (MIC(50/90), ≤0.015/0.03 mg/L) showed the lowest MICs, which remained unchanged for tetracycline-susceptible or -resistant isolates [mostly tet(M)]. Similar MICs were observed for omadacycline (MIC(50/90), 0.03–0.06/0.06 mg/L) and tigecycline (MIC(50/90), 0.03/0.03 mg/L) in the S. pneumoniae population. Tetracycline-susceptible and -resistant E. coli [94.2% tet(A)/tet(B)], KBP-7072 (MIC(90), 0.25 and 1 mg/L, respectively) and tigecycline (MIC(90), 0.25 and 0.5 mg/L) showed similar MIC(90)s. KBP-7072 (MIC(50/90), 0.25/0.5 mg/L) and tigecycline (MIC(50/90), 0.5/0.5 mg/L) had the lowest MIC for tetracycline-susceptible K. pneumoniae. The MIC for KBP-7072 (MIC(50/90), 1/4 mg/L) and tigecycline (MIC(50/90), 1/2 mg/L) increased 2- to 8-fold for tetracycline-resistant K. pneumoniae, which mostly produced Tet(A). CONCLUSIONS: KBP-7072 activity was minimally affected by the presence of acquired tetracycline genes. Antimicrobial resistance (AMR) among Gram-positive cocci (GPC) and Gram-negative bacilli (GNB) is now a well-recognized impediment to medical progress. 1, 2 Selection of bacterial pathogens with intrinsic or acquired mechanisms of resistance (MOR) to antimicrobial agents is facilitated by excessive and frequently inappropriate use of broad-spectrum antibacterial agents to the extent that entire classes of agents have been rendered useless clinically. [1] [2] [3] [4] [5] The threat of AMR to healthcare has been exacerbated recently by widespread use of antimicrobial therapies as part of the package of clinical care for coronavirus disease 2019 (COVID-19). 6 Increasing rates of resistance to b-lactams, fluoroquinolones, macrolides and older tetracyclines (doxycycline, minocycline and tetracycline) in recent years among Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli and Klebsiella pneumoniae pose serious challenges for providing effective treatment of community-and hospital-associated infections. [1] [2] [3] [7] [8] [9] S. aureus, E. coli and Klebsiella spp. represent the top three causes of healthcare-associated infections (HAI) 2 and S. pneumoniae is a leading cause of community-acquired bacterial pneumonia (CABP). 10 The increase in infections caused by MDR (resistant to one or more agents in at least three different classes of agents) bacteria poses significant problems for individuals with serious and potentially life-threatening infections given the few available and effective therapeutic options. 1, 2, 4 The tetracycline class of antimicrobial agents has been in use clinically for more than 60 years. 3, 5, 11 These agents continue to be V C The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons. org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com used clinically for treatment of a variety of GPC and GNB bacterial infections, including those intracellular pathogens as well as protozoans. 3, 5, 11 The broad use of the tetracycline class of agents has resulted in the development of resistance to the older members, especially tetracycline and doxycycline, by genes that are often associated with mobile genetic elements. 3, 5, 11 Tetracycline resistance in both GPC and GNB is mostly due to the acquisition of genes encoding efflux, ribosomal protection proteins and tetracyclineinactivating enzymes, but target site alterations and the overexpression of intrinsic efflux pumps do also occur. 3, 12, 13 Semisynthetic derivatives of tetracycline, such as omadacycline, tigecycline and eravacycline with improved potency against MDR GPC and GNB, including against bacteria with acquired tetracycline-resistance genes, have been approved for clinical use. 3 Most recently, an additional aminomethylcycline, KBP-7072, has been developed and is undergoing early-stage clinical development for the treatment of acute bacterial skin and skin structure infection (ABSSSI), CABP and complicated intraabdominal infections (cIAI). 14 Modifications at the C-9 position provide activity even in the presence of ribosomal protection proteins and efflux pump mechanisms. 14 Whereas eravacycline, omadacycline and tigecycline have been extensively investigated, 3 little has been published regarding the in vitro spectrum and potency of KBP-7072, including strains with defined tetracycline-resistance mechanisms. The present study investigated the in vitro activity of KBP-7072 against a subset of key target pathogens (S. aureus, S. pneumoniae, E. coli and K. pneumoniae) that were molecularly characterized for acquired tetracycline-resistance mechanisms. A comparative analysis of newer-generation tetracycline derivatives (omadacycline and tigecycline) with older-generation tetracyclines (doxycycline, minocycline and tetracycline) is also provided. A global collection of 413 tetracycline-resistant and -susceptible isolates recovered from various infections in 119 medical centres located in 34 countries during the SENTRY Surveillance Program for 2015-19 were included (Table S1 , available as Supplementary data at JAC-AMR Online). [7] [8] [9] Tetracycline-resistant isolates (205) were previously selected for genome sequencing and screened for various resistance mechanisms, and were known to carry acquired tetracycline-resistance genes. A similar number of tetracycline-susceptible isolates (208) were also selected. The collection of isolates included: 103 S. aureus Antimicrobial susceptibility testing Isolates were tested for susceptibility to KBP-7072, doxycycline, minocycline, omadacycline, tetracycline and tigecycline by broth microdilution following CLSI M07 15 guidelines. Results were interpreted using CLSI, 16 EUCAST 17 and FDA 18 breakpoint criteria. Frozen-form broth microdilution panels were manufactured by JMI Laboratories (North Liberty, IA, USA) and contained fresh CAMHB (2.5%-5% lysed horse blood added for testing streptococci). KBP-7072 and omadacycline were provided by KBP Biosciences Co. Ltd (Jinan, China). Quality assurance was performed by concurrently testing CLSI-recommended quality control reference strains (S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and S. pneumoniae ATCC 49619). Tetracycline-resistant isolates had their total genomic DNA extracted by the fully automated Thermo Scientific TM KingFisher TM Flex Magnetic Particle Processor (Cleveland, OH, USA), which was used as input material for library construction. DNA libraries were prepared using the Nextera TM library construction protocol (Illumina, San Diego, CA, USA) following the manufacturer's instructions and were sequenced on MiSeq Sequencer platforms at JMI Laboratories. FASTQ format sequencing files for each sample set were assembled independently using de novo assembler SPAdes 3.11.1. Inhouse software was applied to align the assembled sequences against a curated database containing known tetracycline-resistance genes with the purpose of screening. 19, 20 Results and discussion The geographic distribution of tetracycline-resistant isolates and tetracycline-resistance genes associated with each of the species included in this study is detailed in Table S1 . Table 2) . KBP-7072 had MIC 50 values of 0.12 mg/L and 0.25 mg/L when tested against tetracycline-susceptible and -resistant E. coli, respectively (Table 1) Table 2 ). In contrast, the MIC for both KBP-7072 (MIC 50/90 , 1/4 mg/L) and tigecycline (MIC 50/90 , 1/2 mg/L) increased 2-to 8-fold for tetracycline-resistant K. pneumoniae, which mostly produced Tet(A) ( Table 2) . The tetracycline-resistance mechanisms observed here represent those encoded by common genes carried by GPC and GNB, Pfaller et al. Table 1 . KBP-7072 activity against characterized isolates JAR although the presence of the overexpression of intrinsic efflux pumps and target site alterations were not investigated, which could be considered as a study limitation. In addition, the isolates included in this study were initially selected for molecular characterization for reasons other than the screening of tetracycline-resistance genes. Thus, isolates and their respective resistance genes are exclusively presented here for the purpose of in vitro activity analysis and do not represent their actual occurrences in these populations. Moreover, isolates from the Latin American and Asia-Pacific regions were underrepresented. Further analyses are required to complete this in vitro assessment of activity for KBP-7072. Based on MIC 90 values, KBP-7072 was active in vitro against tetracycline-susceptible and -resistant isolates with potencies remaining somehow stable between each respective group. This stable MIC profile observed for KBP-7072 between tetracyclinesusceptible and -resistant isolates resembled that of tigecycline. Thus, this stable in vitro activity of KBP-7072 in the presence of various tet genes warrants further development of this investigational agent as an additional option for the treatment of infections caused by MDR GPC and GNB. This study was performed by JMI Laboratories and supported by KBP Biosciences, which included funding for services related to preparing this manuscript. Table S1 ). c All isolates carried tet(M), except for 1 strain with a tet(32). Pfaller et al. 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