Soon Nang Park

Application of rpoB and Zinc Protease Gene for Use in Molecular Discrimination of Fusobacterium nucleatum Subspecies

ABSTRACT Fusobacterium nucleatum is classified into five subspecies that inhabit the human oral cavity (F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, and F. nucleatum subsp. animalis) based on several phenotypic characteristics and DNA-DNA hybridization patterns. However, the methods for detecting or discriminating the clinical isolates of F. nucleatum at the subspecies levels are laborious, expensive, and time-consuming. Therefore, in this study, the nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and zinc protease gene were analyzed to discriminate the subspecies of F. nucleatum. The partial sequences of rpoB (approximately 2,419 bp), the zinc protease gene (878 bp), and 16S rRNA genes (approximately 1,500 bp) of the type strains of five subspecies, 28 clinical isolates of F. nucleatum, and 10 strains of F. periodonticum (as a control group) were determined and analyzed. The phylogenetic data showed that the rpoB and zinc protease gene sequences clearly delineated the subspecies of F. nucleatum and provided higher resolution than the 16S rRNA gene sequences in this respect. According to the phylogenetic analysis of rpoB and the zinc protease gene, F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme might be classified into a single subspecies. Five clinical isolates could be delineated as a new subspecies of F. nucleatum. The results suggest that rpoB and the zinc protease gene are efficient targets for the discrimination and taxonomic analysis of the subspecies of F. nucleatum.

Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12)

To develop novel antibiotic peptides useful as therapeutic drugs, a number of analogues were designed to increase the hydrophobic helix region either by Trp-substitution or net positive charge increase by Lys-substitution, from HP(2-9)-ME(1-12). The antibiotic activities of these peptides were evaluated using bacterial (Salmonella typhimurium, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus), fungi (Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans), tumor and human erythrocyte cells. The substitution of Lys for Thr at position 18 and 19 of HP(2-9)-ME(1-12) (HM5) increased activity against Proteus vulgaris and fungal strains without hemolysis. In contrast, substitution of Trp for Lys and Thr at positions 2, 15 and 19 of HP(2-9)-ME(1-12), respectively (HM3 and HM4), decreased activity but increased hemolysis against human erythrocytes. This suggests that an increase in positive charge increases antimicrobial activity whereas an increase in hydrophobicity by introducing Trp residues at C-terminus of HP(2-9)-ME(1-12) causes a hemolytic effect. Circular dichroism spectra suggested that the α-helical structure of these peptides plays an important role in their antibiotic effect but that the α-helical property is not connected with the enhanced antibiotic activity.

Detection of methicillin or vancomycin-resistant Staphylococcus aureus from dental hospital

본 연구는 조선대학교 치과병원의 진료환경 및 진료요원으로부터 기회감염성 병원체로 알려진 methicillin 또는 vancomycin 저항성 황색포도상 구균 (methicillin-or vancomycin-resistant Staphylococcus aureus. MRSA or VRSA)의 존재 여부를 조사하여, 이를 광주지역 개원치과와 비교분석을 통해 현재 조선대학교 치과병원의 MRSA와 VRSA의 오염정도를 파악하고자 하였다. 이를 위해 진료실 환경 및 진료요원으로부터 분리한 S. aureus 균주들의 8종 항생제에 대한 감수성 조사를 시행하고, 기존에 알려진 항생제 내성 유전자 존재 여부를 PCR법을 이용하여 확인하였다. 그 결과, 조선대학교 치과병원의 진료요원에서 채취한 샘플 중 1개 (2.3%), 개원 치과에서는 2명 (10%)의 진료요원의 샘플에서 S. aureus가 분리되었으며, 진료환경에서는 두 곳 모두에서 S. aureus가 검출되지 않았다. 조선대학교 치과병원과 개원치과에서 분리된 S. aureus는 amoxicillin, penicillin G, ciprofloxacin clindamycin, vancomycin에 내성을 보이며 oxacillin, cefuroxime에는 균주에 따라 감수성 또는 내성을 보였다. 조선대학교 치과병원에서 분리된 S. aureus는 erythromycin과 clindamycin에 내성 유전자인 ermA가 존재하였으며, 개원치과에서 분리된 3개의 S. aureus 중 2개에서 penicillin과 oxacillin에 내성 유전자 mecA가 존재하는 것으로 나타났다. Vancomycin 내성 유전자인 vanA, vanB는 어떠한 샘플에서도 검출되지 않았다. 이상의 결과를 종합할 때, 본 연구는 조선대학교 치과병원과 개원치과의 S. aurues분포 및 MRSA 또는 VRSA의 존재여부를 조사하여 MRSA와 VRSA의 확산예방을 위한 치과진료 환경의 개선과 적절한 항생제 사용에 대한 기초 자료를 제공할 것으로 사료된다. 【The purpose of this study was to obtain the basic information for the improvement of dental environment by investigating the presence of methicillin- or vancomycin-resistant Staphylococcus aureus (MRSA or VRSA) isolated from dental health care workers (DHCWs) and environment of the Chosun University Dental Hospital (CUDH) and a private dental clinic (control group). Staphylococcus aureus (S. aureus) was isolated from anterior nares of 42 DHCWS and 38 sites, unit chairss, x-ray devices, computers, etc., at 10 departments of the CUDH and 20 DHCWs and 11 sites at the private dental clinic. S. aureus was isolated on mannitol salt agar plate and confirmed by PCR with S. aureus species-specific primer. Antimicrobial susceptibility test of clinical isolates of S. aureus against several antibiotics including methicillin (oxacillin) was performed by investigating minimum inhibitory concentration (MIC) using broth microdilution assay. In addition, PCR was performed to detect the methicillin- or vancomycin-resistant gene. The data showed that one strain of S. aureus was isolated from DHCWs of the CUDH and three strains of S. aureus was isolated from 3 samples of the private dental clinic, respectively. All of the isolates from the CUDH and the private dental clinic had resistance to penicillin G, amoxicillin and vancomycin and susceptibility to oxacillin and ciprofloxacin. The S. aureus strains were already obtained the resistance to penicillin G and amoxicillin. These results suggest that two dental clinics were under relatively safe environment.】