Production of the Monoclonal Antibodies against Bartonella henselae Isolated from a Korean Patient

길세희,강재승 대한미생물학회 2012 Journal of Bacteriology and Virology Vol.42 No.1

Bartonellosis is spotlighted recently as an emerging zoonosis and Bartonella henselae is reported to be the main infectious agent. In Korea, however, few studies have been made on the epidemiology and microbiology on bartonellosis. Thus, this study was conducted to produce a new monoclonal antibody that can be used for identifying B. henselae. In order to prepare monoclonal antibodies against B. henselae, we inoculated mice with the isolated strain from Korean patient and performed cell fusion experiment. The selected hybridoma clones produced monoclonal antibodies which showed positive immunofluorescence staining of bacteria and specific protein bands in western blot analysis. In order to examine whether these antibodies could be used for the identifying and quantifying Bartonella, we performed confocal microscopy and flow cytometry using the new antibodies. These monoclonal antibodies can be used as a useful tool in further researches on the biology of Bartonella.

국내 환자에서 분리된 Orientia tsutsugamushi에서 doxycycline 내성

김은실,김미경,이혜명,길세희,정문현,이진수,강재승 대한감염학회 2008 Infection and Chemotherapy Vol.40 No.5

Background : Doxycycline has been the drug of choice for the treatment of scrub typhus. However, scrub typhus with poor response to doxycycline was first reported in Thailand in 1996. To date, it is not known whether doxycycline resistant scrub typhus is present in Korea. To investigate the doxycycline resistance of scrub typhus in Korea, we performed an experiment to evaluate the degree of growth inhibition of Orientia tsutsugamushi at the minimal inhibitory concentration (MIC) of doxycycline. Materials and Methods : 37 isolates of Orientia tsutsugamushi were collected from Korean patients with scrub typhus during 10 year period (from 1997 to 2007). 34 isolates consisted of Boryong serotype or related serotypes. These strains were stained with FS15, a monoclonal antibody against the linear epitope on the protein located at the outer membrane of O. tsutsugamushi. Three isolates were confirmed as serotype Gilliam, which was stained by anti-Gilliam antibody. The antibody- stainable 35 isolates were incubated in media containing 0.1 and 0.2 µg/mL of doxycycline for 3 days. Then the growth indices of Orientia were measured by flow cytometry. If the growth index was greater than 10%, the isolate was tentatively classified as an isolate resistant to doxycycline and its MIC was measured. Results : Demographic and treatment data were similar to the results of previous reports. Infection was mainly observed in Inchon and the Chungcheongnam province. No patient showed delayed response to antibiotics. Of 35 hospitalized patients, 4 (11.4%) were managed at the intensive care unit, and 1 (2.9%) died of nosocomial pneumonia. All the 2 patients whose isolates were serotype Gilliam Yonchon/Ikeda-like strain were managed at the intensive care unit. Six of the 34 isolates from Korean patients exhibited growth indices greater than 10% at the MIC of doxycycline; however, the MICs of these isolates did not exceed 0.2 µg/mL. One isolate of serotype Gilliam showed an MIC of 0.0125 ug/mL, which seemed to be underestimated due to weak fluorescence of the anti-Gilliam antibody. Conclusions : Although the number of tested isolates is small, insensitivity of scrub typhus to doxycycline is not a major reason for treatment failure in Korea. Serotype Gilliam, especially Yochon/ Ikeda-like strain, may be associated with severe form of scrub typhus. More studies concerning the antibiotic susceptibility for serotype Gilliam are warranted. Background : Doxycycline has been the drug of choice for the treatment of scrub typhus. However, scrub typhus with poor response to doxycycline was first reported in Thailand in 1996. To date, it is not known whether doxycycline resistant scrub typhus is present in Korea. To investigate the doxycycline resistance of scrub typhus in Korea, we performed an experiment to evaluate the degree of growth inhibition of Orientia tsutsugamushi at the minimal inhibitory concentration (MIC) of doxycycline. Materials and Methods : 37 isolates of Orientia tsutsugamushi were collected from Korean patients with scrub typhus during 10 year period (from 1997 to 2007). 34 isolates consisted of Boryong serotype or related serotypes. These strains were stained with FS15, a monoclonal antibody against the linear epitope on the protein located at the outer membrane of O. tsutsugamushi. Three isolates were confirmed as serotype Gilliam, which was stained by anti-Gilliam antibody. The antibody- stainable 35 isolates were incubated in media containing 0.1 and 0.2 µg/mL of doxycycline for 3 days. Then the growth indices of Orientia were measured by flow cytometry. If the growth index was greater than 10%, the isolate was tentatively classified as an isolate resistant to doxycycline and its MIC was measured. Results : Demographic and treatment data were similar to the results of previous reports. Infection was mainly observed in Inchon and the Chungcheongnam province. No patient showed delayed response to antibiotics. Of 35 hospitalized patients, 4 (11.4%) were managed at the intensive care unit, and 1 (2.9%) died of nosocomial pneumonia. All the 2 patients whose isolates were serotype Gilliam Yonchon/Ikeda-like strain were managed at the intensive care unit. Six of the 34 isolates from Korean patients exhibited growth indices greater than 10% at the MIC of doxycycline; however, the MICs of these isolates did not exceed 0.2 µg/mL. One isolate of serotype Gilliam showed an MIC of 0.0125 ug/mL, which seemed to be underestimated due to weak fluorescence of the anti-Gilliam antibody. Conclusions : Although the number of tested isolates is small, insensitivity of scrub typhus to doxycycline is not a major reason for treatment failure in Korea. Serotype Gilliam, especially Yochon/ Ikeda-like strain, may be associated with severe form of scrub typhus. More studies concerning the antibiotic susceptibility for serotype Gilliam are warranted.

국내 환자에서 분리된 Orientia tsutsugamushi에서 doxycycline 내성

김은실,김미경,이혜명,길세희,정문현,이진수,강재승 대한감염학회 2008 감염과 화학요법 Vol.40 No.5

Background : Doxycycline has been the drug of choice for the treatment of scrub typhus. However, scrub typhus with poor response to doxycycline was first reported in Thailand in 1996. To date, it is not known whether doxycycline resistant scrub typhus is present in Korea. To investigate the doxycycline resistance of scrub typhus in Korea, we performed an experiment to evaluate the degree of growth inhibition of Orientia tsutsugamushi at the minimal inhibitory concentration (MIC) of doxycycline. Materials and Methods : 37 isolates of Orientia tsutsugamushi were collected from Korean patients with scrub typhus during 10 year period (from 1997 to 2007). 34 isolates consisted of Boryong serotype or related serotypes. These strains were stained with FS15, a monoclonal antibody against the linear epitope on the protein located at the outer membrane of O. tsutsugamushi. Three isolates were confirmed as serotype Gilliam, which was stained by anti-Gilliam antibody. The antibody-stainable 35 isolates were incubated in media containing 0.1 and 0.2 ug/mL of doxycycline for 3 days. Then the growth indices of Orient/a were measured by flow cytometry. If the growth index was greater than 10%, the isolate was tentatively classified as an isolate resistant to doxycycline and its MIC was measured. Results : Demographic and treatment data were similar to the results of previous reports. Infection was mainly observed in Inchon and the Chungcheongnam province. No patient showed delayed response to antibiotics. Of 35 hospitalized patients, 4 (11.4%) were managed at the intensive care unit and 1 (2.9%) died of nosocomial pneumonia. All the 2 patients whose isolates were serotype Gilliam Yonchon/Ikeda-like strain were managed at the intensive care unit. Six of the 34 isolates from Korean patients exhibited growth indices greater than 10% at the MIC of doxycycline; however, the MICs of these isolates did not exceed 0.2 ug/mL. One isolate of serotype Gilliam showed an MIC of 0.0125 ug/mL, which seemed to be underestimated due to weak fluorescence of the anti-GiIIiam antibody. Conclusions : Although the number of tested isolates is small, insensitivity of scrub typhus to doxy-cycline is not a major reason for treatment failure in Korea. Serotype Gilliam, especially Yochon/Ikeda-like strain may be associated with severe form of scrub typhus. More studies concerning the antibiotic susceptibility for serotype Gilliam are warranted.

Cholestatic Hepatitis Caused by Tongyeong Strain of Orientia tsutsugamushi

주고운,김미경,김준미,강재승,정문현,길세희 대한감염학회 2009 Infection and Chemotherapy Vol.41 No.2

In Korea, the clinical manifestation of scrub typhus in humans appears to be severe in the northern regions of the country and mild in the southern areas. Studies on the geographic distribution of the serotype of Orientia tsutsugamushi revealed that the Boryong serotype is predominant in the southern Korean peninsula, whereas the Gilliam, Karp, and Gilliam-Karp serotypes prevail in the northern or central peninsula. We report a case of severe scrub typhus caused by the Japanese Gilliam type of Orientia tsutsugamushi in a 66-year-old woman from Tongyeong, a city located in the southern part of Korean peninsula. The patient was admitted to the hospital with fever on August 29th, 2001. Scrub typhus complicated by shock, pneumonitis, and encephalitis was diagnosed. Additionally, markedly elevated alkaline phosphatase levels was observed, which necessitated an ultrasonographic examination, which showed an ill-defined hyperechogenic mass on the right hepatic lobe. Liver biopsy showed cholestatic hepatitis and blood culture showed growth of O. tsutsugamushi. Gene sequencing of the amplicons of a polymerase chain reaction using primers for the amplification of the gene encoding the 56-kDa protein of O. tsutsugamushi revealed a nucleotide sequence that matched Yonchon strain of O. tsutsugamushi.