NGUYEN THI LE THUY

Tsukuba Repository

黄色ブドウ球菌における薬剤耐性の水平伝達の研究

Study on Horizontal Transfer of Drug
Resistance in Staphylococcus aureus
著者 NGUYEN THI LE THUY
発行年 2017
その他のタイトル 黄色ブドウ球菌における薬剤耐性の水平伝達の研究
学位授与大学 筑波大学 (University of Tsukuba)
学位授与年度 2017
報告番号 12102甲第8346号
URL http://hdl.handle.net/2241/00149993
    
 
 
 
論文の要旨 
Abstract of thesis 
 
Purpose 
Staphylococcus aureus is the leading cause of nosocomial infections in hospitals and healthcare 
settings. Its ability to rapidly acquire the resistance to antimicrobial compounds by horizontal gene 
transfer (HGT) has been the global concern. There are three major mechanisms responsible for HGT 
in bacteria: conjugation, transduction and transformation. Conjugation is responsible for the transfer of 
DNA from one living cell to another and is mediated by special structures including conjugative pili. 
Transduction is mediated by bacterial virus (bacteriophage), and is considered as the major route to 
acquire new DNA in S. aureus. Transformation is through the direct uptake of free DNA by the cells that 
developed the ‘competence’ state. Although transduction and conjugation have been studies for 
decades, natural transformation was only recently described in S. aureus, and little is known about its 
regulatory mechanisms. The finding of the natural transformation also raised the necessary to re-
evaluate the evolutionary ability of S. aureus. In this context, this study aimed to clarify some aspects 
of the HGT in S. aureus. First, the applicant aimed to specify the major transmission modes of the 
氏     名 NGUYEN THI LE THUY  
学 位 の 種 類       E 博士（ 人間生物学  ） 
A学 位 記 番 号       E 博甲第   8346   号 
A学位授与年月       E 平成 29年 7月 25日 
A学位授与の要件       E 学位規則 第４条第１項該当（昭和２８年４月１日文部省令第９号） 
審 査 組 織 グローバル教育院 
学位論文題目  Study on Horizontal Transfer of Drug Resistance in 
Staphylococcus aureus 
(黄色ブドウ球菌における薬剤耐性の水平伝達の研究 ) 
 
 （職名） （学位） （氏名） 
主   査 筑波大学教授 医学博士 柳沢 正史 
副   査 筑波大学教授(連携大学院) 医学博士 狩野 繁之 
副   査 筑波大学教授(連携大学院) 獣医学博士 宮田 桂司 
副   査 筑波大学教授 博士（人間・環境
学） 
森川 一也 
 linezolid resistance gene, cfr (chloramphenicol/florfenicol resistance), from Staphylococcus epidermidis 
strains isolated in Spanish hospitals to S. aureus strains isolated in Japan in which cfr has not been 
detected. Second, the applicant explored for the factor(s) that affect the natural transformation, focusing 
on a series of antibiotics.  
 
Results 
Dissemination of linezolid resistance through HGT 
    To investigate the conjugative transmission of cfr gene, 16 clinical MRSA (Methicillin-resistant S. 
aureus) isolated in Japan and two S. epidermidis strains (SE45 and SE50) from Spanish hospital were 
used as recipients and donors respectively. The pSCSF7-like plasmid carrying cfr gene responsible for 
the resistance was transferred from the clinical S. epidermidis isolates to a series of MRSA strains by 
filter mating method. The transmission efficiencies was different depend on the donor as well as on the 
recipient. 
   The applicant further tested whether the plasmid would be able to disseminate among S. aureus 
strains. Although the conjugation was previously believed as the only mechanism of the cfr 
dissemination, the results demonstrated that transduction could be considered as an alternative 
pathway for the cfr transmission among MRSA strains. Importantly, the whole plasmid was transferred 
in these transmissions. The natural transformation of cfr was undetectable, suggesting its low relevance 
in the cfr transmission, although there might be unknown conditions more suitable for detecting natural 
transformation. 
Antibiotics modulate transformation 
      In S. aureus, the natural competence is regulated by a sigma factor – SigH, which is tightly 
controlled to be expressed in a minor subpopulation. In the competence-inducing medium, 
transformation frequency in the wild type strain is hardly detectable (<10-11) and is low even if SigH was 
artificially overexpressed (~ 10-9). Taking into account that S. aureus has a prominent ability to swiftly 
acquire resistance, the applicant considered that there could be the favorable conditions to develop 
competence for transformation, and investigated the effect of antibiotics on the transformation efficiency 
in SigH-expressing strain (N315ex w/oφ h). In this strain, the prophage was eliminated to exclude the 
possibility of phage-dependent “pseudo-competence” DNA transfer, which is distinct from the natural 
competence. SigH is expressed by a plasmid, pRIT-sigH. Strikingly, the applicant found that cell wall-
affecting antibiotics such as bacitracin, fosfomycin and vancomycin, but not β-lactams, increased the 
transformation frequency. Mitomycin C suppressed transformation in a concentration-dependent 
manner. Ciprofloxacin, norfloxacin and streptomycin had no significant effect. Further investigations on 
the external physical damages to the cell wall (by silica beads or lysostaphin, an enzyme that cleaves 
S. aureus cell walls) did not facilitate the transformation. The results suggested that effects of cell wall-
affecting antibiotics on transformation might involve certain complex cellular activity. 
 
 
 Discussion 
    The results obtained in this study showed that the cfr-carrying pSCFS7-like vectors could be 
efficiently transferred to clinical MRSA in Japan, the country in which this gene has not been detected. 
Thus, the result emphasizes the importance of the surveillance programs aiming at the early detection 
of the cfr gene in both S. aureus and the non-pathogenic bacteria such as coagulase-negative 
staphylococci including S. epidermidis, since they are considered to be the genetic reservoir available 
for S. aureus.  
     Regarding the natural transformation, complex mechanisms participate in the control of the 
competence development in species-specific manners, which have been well studied in model bacteria 
such as Streptococcus and Bacillus. In S. aureus, it was recently found that SigH plays a key role in 
competence development, but many aspects of the regulation in competence development and the 
following transformation are not known. As part of the efforts to gain insight into the regulation after SigH 
expression, the present study firstly showed the effects of cell wall-affecting reagents on transformation. 
The positive effects of cell wall-targeting antibiotics were not simply caused by damaging the cell wall 
integrity. Therefore, the involvement or certain cellular response(s) was predicted in the regulation of 
competence and transformation.  
The long-term goal in the studies of antibiotics resistance and their dissemination is to conquer 
the problem of antimicrobial resistance. To prevent and stop the dissemination of resistance, it is 
necessary to take it into account not only the dissemination among human society, but also the livestock 
and the environment, under the concept of “One health”. The present study provided the experimental 
evidence on the HGT mechanism regarding the cfr transmission, strongly supporting the idea that CNS 
species serves as the genetic pool of the antibiotics resistance. The finding of the effects of antibiotics 
on the natural transformation has a potential to be applied for the control of S. aureus evolution, but of 
course it requires further extensive studies. 
 
審査の要旨 
 Abstract of assessment result 
 
【批評 Review】 
The applicant provided experimental evidence that the conjugation-like system and the phage-mediated 
transduction are the potential transfer mechanisms of the linezolid resistance gene among 
staphylococcal species. Considering the high evolutionary potential of Staphylococcus aureus, the 
finding is relevant to predict the further dissemination of the linezolid resistance gene, which has not 
been detected in our country. The dissertation also describes the effects of antibiotics on another 
transfer mechanism, natural genetic transformation, which was recently demonstrated by her group. 
Although the transformation assay was carried out using the competent cells that artificially overexpress 
the transformation regulator, the finding that some antibiotics could suppress or facilitate the natural 
transformation implicated the potential effects of prescription on the S. aureus evolution. Further studies 
on horizontal gene transfer, including the mechanisms how antibiotics affect the natural transformation, 
would reveal the uncanny evolutionary ability of this important human pathogen. 
  
【最終試験の結果 Result】 
The final examination committee conducted a meeting as a final examination on 8 May, 2017. The 
applicant provided an overview of dissertation, addressed questions and comments raised during Q&A 
session. All of the committee members reached a final decision that the applicant has passed the final 
examination. 
 
【結論 Conclusion】 
The final examination committee approved that the applicant is qualified to be awarded Doctor of 
Philosophy in Human Biology. 
 
 
 


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黄色ブドウ球菌における薬剤耐性の水平伝達の研究

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