論文の内容の要旨

Understanding Processes that Contribute to Antimicrobial Resistance (AMR) in

Infectious DiseaseAgents (感染症治療薬に対する細菌の抵抗性に寄与するプロセスの解明)

氏名 ンウィボ ダニエル ドン

NWIBO, DANIEL DON

PART 1: Diversity of Antimicrobial Resistant Genes inMulti-Drug Resistant (MDR) Coagulase-Negative Staphylococciin Environment

The environment is a reservoir of antimicrobial resistant genes and mobile genetic elements that are actively involved in resistant gene mobilization and deployment. Accurate identification and proper antimicrobial susceptibility profiling of pathogens are necessary for tracking the epidemiology and treatment of infectious diseases. We are proposing the use of silkworm model for evaluation of novel drug candidates and investigating host-pathogen interaction processes (Nwibo et al, 20151). Previously, we established Enterococcus mundtii as a natural pathogen of silkworm larva. Antibiotic susceptibility profiling of E. mundtiis howed that the bacteria had MDR phenotype, though it was isolated in antibiotic free silkworm larva host (Nwibo et al, 20152); suggesting the role of the environmental genetic exchanges in mediation of antibiotic resistance. This study reports the isolation of MDR Gram-positive cocci harboring multiple antimicrobial resistant genes from the environment. During the course of experiment with silkworm larva infected with S. aureus; tetracycline, Tc resistant (TcR) colonies were isolated on agar plates supplemented with Tc. Tc resistance levels achieved by TcR strains were much greater than those achieved by in vitro S. aureus cultured isolates (at least 16-fold increase) as determined by micro-broth dilution method. TcR

isolates also displayed high resistance to other antimicrobial agents: kanamycin, erythromycin, norfloxacin, teicoplanin and ampicillin (Table 1-A), as well as intermediate resistance to oxacillin, cefcapene and cefditoren. In addition to clinically relevant antimicrobial agents, these isolates showed reduced susceptibility to benzalkonium chloride; a non-target specific biocidal disinfectant, which may allow for persistence in the environment and subsequent transmission on to a suitable host. To identify the MDR bacteria, genomic DNA of three (3) isolates were extracted and analyzed by Next-generation sequencing (NGS) using personal Genome Machine. Sequence result identified the isolated strains as Staphylococcus capitis and Staphylococcus haemolyticus. De novo assembly analysis of the NGS data was performed. Genomic annotation demonstrated the presence of multiple antibiotic resistant genes in the bacteria, suggesting the origin of the multiple antimicrobial resistant phenotypes (Table 1-B). This is the first study to describe the appearance of multiple antimicrobials resistant S. capitis (AYP1020) strain in a non-hospital setting. The presence of plethora of genes responsible for AMR indicates that S. haemolyticus (JCSC1435) and S. capitis (AYP1020) could present

potential threat to human health. Furthermore, such environmentally mobilized MDR bacteria can serve as a foothold towards transference of antimicrobial resistance conferring foreign genetic elements to other clinically relevant pathogens. PART 2: Colony-Spreading (CS) Contributes to Acquisition of Tetracycline Resistance (TcR) in Staphylococcus (S.) aureus Antimicrobial resistance is a critical challenge in clinical medicine underpinned by various molecular mechanisms. Previous reports indicate that colonies of S. aureus rapidly enlarge when cultured on soft agar (0.24%) medium. However, the role of CS in acquisition of antimicrobial resistance is unknown. In this study, whether CS facilitated acquisition of drug resistance in S. aureus was examined. When S. aureus cells were cultured on soft-agar supplemented with sub-inhibitory concentrations of tetracycline (Tc), inhibition of spreading was observed. Isolates from the spreading colonies showed decreased sensitivity against Tc as demonstrated by elevated minimum inhibitory concentration (MIC), determined by micro-broth dilution method. Tc resistance levels achieved by the CS cells were much greater than those achieved in absence of spreading (non-colony spreading conditions). Tc MICs of the isolates increased with spreading distance. Psmα genes deletion mutant (ΔPSMα) of S. aureus, which is known to possess attenuated CS phenotype, displayed reduced capacity to acquire Tc resistance on the soft agar containing sub-inhibitory concentration of Tc compared to wild type strain (Fig. 1-A & Fig. 1-B). MICs of Tc against clinically isolated tetracycline-sensitive spreading methicillin-sensitive Staphylococcus aureus andmethicillin-resistant Staphylococcus aureus also increased with spreading capacity. Four (4) CS isolates and wild type were analyzed by Next-generation sequencing using personal Genome Machine. Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type strains was performed by CLC genomics work bench, and the difference within coding regions between each set of strains was then extracted. Multiple chromosomal mutations including single nucleotide variations in the rpsJ gene encoding ribosomal protein S10, genes encoding acetyltransferase and hypothetical proteins commonly occurred in the four isolates examined. Moreover, total number of mutated genes positively correlated (R2=0.8494) with Tc MICs of the mutants (Fig. 1-C). In bacterial pathogens including S. aureus, ribosomal protein S10 is a common target of tetracycline and its chemical analogues and a marker for detecting tetracycline resistance. Taken together, these findings indicate for the first time that CS phenotype of S. aureus facilitates the acquisition of antibiotic resistance via accumulated chromosomal gene mutations, and this could be due to increased radical-induced intracellular mutagenesis created by stresses from both antibiotic pressure and closely-packed layers of spreading cells. Antagonizing colony spreading processes might provide novel therapeutic options against antimicrobial resistant S. aureus infection; which could be key in target-oriented drug development. Therefore, further investigation of the underlying mechanism of colony spreading diversity and its role in the emergence of antimicrobial resistance in S. aureus is needed for better understanding of S. aureusmulti-drug resistanceprocesses.

Reference (as cited and published by this author)

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Figure 1: Tetracycline, Tc Resistance Acquisition correlates with Accumulated Chromosomal Gene Mutation in Colony-Spreading S. aureus

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