The Drug Resistance Profile of Mycobacterium abscessus Group Strains from Korea

BACKGROUND: Bacteria of the Mycobacterium abscessus group are the second most common pathogens responsible for lung disease caused by nontuberculous mycobacteria in Korea. There is still a lack of studies investigating the genetic mechanisms involved in M. abscessus resistance to antibiotics other than clarithromycin. This study investigated the characteristics of drug resistance exhibited by M. abscessus clinical isolates from Korea. METHODS: We performed drug susceptibility testing for a total of 404 M. abscessus clinical strains. Subspecies were differentiated by molecular biological methods and examined for mutations in drug resistance-related genes. RESULTS: Of the 404 strains examined, 202 (50.00%), 199 (49.26%), and 3 (0.74%) strains were identified as M. abscessus, M. massiliense, and M. bolletii, respectively. Of the 152 clarithromycin-resistant strains, 6 possessed rrl mutations, while 4 of the 30 amikacin-resistant strains contained rrs mutations, and 5 of the 114 quinolone-resistant strains had gyr mutations. All mutant strains had high minimal inhibitory concentration values for the antibiotics. CONCLUSIONS: Our results showed the distribution of the strains with mutations in drug resistance-related genes was low in the M. abscessus group. Furthermore, we performed drug susceptibility testing and sequence analyses to determine the characteristics of these genes in the M. abscessus group.

Optimal Combination of VNTR Typing for Discrimination of Isolated Mycobacterium tuberculosis in Korea / 결핵및호흡기질환

BACKGROUND: Variable-number tandem repeat (VNTR) typing is a promising method to discriminate the Mycobacterium tuberculosis isolates in molecular epidemiology. The purpose of this study is to determine the optimal VNTR combinations for discriminating isolated M. tuberculosis strains in Korea. METHODS: A total of 317 clinical isolates collected throughout Korea were genotyped by using the IS6110 restriction fragment length polymorphism (RFLP), and then analysed for the number of VNTR copies from 32 VNTR loci. RESULTS: The results of discriminatory power according to diverse combinations were as follows: 25 clusters in 83 strains were yielded from the internationally standardized 15 VNTR loci (Hunter-Gaston discriminatory index [HGDI], 0.9958), 25 clusters in 65 strains by using IS6110 RFLP (HGDI, 0.9977), 14 clusters in 32 strains in 12 hyper-variable VNTR loci (HGDI, 0.9995), 6 clusters in 13 strains in 32 VNTR loci (HDGI, 0.9998), and 7 clusters in 14 strains of both the 12 hyper-variable VNTR and IS6110 RFLP (HDGI, 0.9999). CONCLUSION: The combination of 12 hyper-variable VNTR typing can be an effective tool for genotyping Korean M. tuberculosis isolates where the Beijing strains are predominant.

Increased Tuberculosis Burden Due to Demographic Transition in Korea from 2001 to 2010 / 결핵및호흡기질환

BACKGROUND: Notified tuberculosis (TB) cases in Korea have not decreased over the last decade (2001-2010). METHODS: To clarify the reasons, we analyzed an annual report on notified tuberculosis patients and age-specific population drift in Korea. RESULTS: Compared to the age-specific notified TB cases between 2001 and 2010, distinctive features in notified TB cases and new cases increased markedly in people aged 45-54 years and in patients over 65 years old, whereas those between 15-34 years in 2010 decreased drastically. In particular, notified TB individuals over 65 years old occupied 29.6% of the cases in 2010, which was 1.5 times higher than that in 2001. The main reason not to decrease in notified TB patients for the last decade (2001-2010) was due to the increasing elderly population as well as the aging of baby boomers, which have a higher risk of TB development. CONCLUSION: Korea needs to pay attention to the older population in order to successfully decrease the burden of TB in the future.

Intracellular Signaling Pathways that Regulate Macrophage Chemokine Expression in Response to Mycobacterium abscessus

Mycobacterium abscessus (Mabc) is an emerging human pathogen. Less is known about the host immune response to Mabc than to M. tuberculosis. Here, we examined the intracellular signaling pathways that govern the expression of chemokines including (C-C motif) ligand 2 (CCL2) and (C-X-C motif) ligand 2 (CXCL2) in macrophages after infection with Mabc. Specifically, Mabc triggered the generation of reactive oxygen species (ROS) and the production of CCL2 and CXCL2 in murine bone marrow-derived macrophages (BMDMs). Mabc-induced CCL2, but not CXCL2, was dependent on the generation of ROS. Toll-like receptor (TLR) 2, MyD88, but not TRIF, was required for Mabc-induced CCL2 and CXCL2 expression. Additionally, Mabc infection significantly induced nuclear factor (NF)-kappaB nuclear translocation and luciferase activity. The activation of NF-kappaB was required for Mabc-induced CCL2, but not CXCL2 expression. Moreover, Mabc-induced ROS generation was required for NF-kappaB activation. Treatment of BMDMs with Mabc rapidly induced the activation of mitogen-activated protein kinase (MAPKs) pathways. Interestingly, CCL2 expression was dependent on the activation of JNK and ERK1/2 pathways, whereas it was negatively regulated by the p38 MAPK pathway. In contrast, Mabc-dependent CXCL2 expression was not regulated by MAPK pathways. These data suggest that intracellular ROS generation is required for innate and inflammatory responses during Mabc infection of macrophages.

Differentiation between Mycobacterium bovis and Mycobacterium tuberculosis Infection / 대한내과학회지

No abstract available.

The Present and Future of Molecular Epidemiology in Tuberculosis

Molecular epidemiology has been initiated for the confirmation of transmission link among tuberculosis patients. IS6110 restriction fragment length polymorphisms (RFLP) technique has been used as an excellent tool to discriminate Mycobacterium tuberculosis isolates, especially in tuberculosis (TB) outbreak in the population. IS6110 RFLP has the most discriminatory power for the M. tuberculosis isolates with high copy number of IS6110 like Korean isolates. Spoligotyping using spacers of direct repeat is useful to distinguish Beijing strains which are found widely in Eastern Asia, from non-Beijing strains. It is known that Beijing strains are more virulent, apt to be drug resistant than non-Beijing strains. Strain typing techniques of Mycobacterium tuberculosis has lead to the development of phylogenetic classification. Variable number tandem repeat (VNTR) of M. tuberculosis is another good target for strain typing. The technique using VNTR is rising as an alternative tool to overcome disadvantages of IS6110 RFLP which is time consuming in the sense that it takes longer time to process from the culture positive bacilli, and has the intrinsic difficulties in objectification of the results. The combination of many VNTR loci enhances discriminatory power to become equal to that of IS6110 RFLP. On the other hand, the optimal VNTR combination differs from one country to another due to different dominant clade. Large sequence polymorphisms (LSP) and single nucleotide polymorphisms (SNP) are important tools for the classification of the phylogeny of M. tuberculosis complex. Many previous reports indicate that the depending upon the type of strains, the ways of transmission of disease, the way to get infected with disease and the development of drug resistance conditions are variable. Therefore, the molecular epidemiology of M. tuberculosis has become more important for tuberculosis control in the world. It will be possible to set up tuberculosis-tailored policy after the characterization of M. tuberculosis by molecular epidemiologically.

Five Rare Non-Tuberculous Mycobacteria Species Isolated from Clinical Specimens / 결핵및호흡기질환

BACKGROUND: Recently, the rate of infections with non-tuberculous mycobacteria (NTM) has been increasing in Korea. Precise identification of NTM is critical to determination of the pathogen and to target treatment of NTM patients. METHODS: Sixty-eight unclassified mycobacteria isolates by rpoB PCR-RFLP assay (PRA) collected in 2008 were analyzed by National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) search after sequencing of 16S rRNA, hsp65, rpoB genes. RESULTS: Nineteen strains of 68 isolates were specified as species after sequencing analysis of 3 gene types. We found 3 M. lentifulavum, 5 M. arupense, 4 M. triviale, 4 M. parascrofulaceum, and one M. obuense. One M. tuberculosis and another M. peregrinum were mutated at the Msp I recognition site needed for rpoB PRA. The remaining 49 isolates did not coincide with identical species at the 3 kinds genes. CONCLUSION: Sequencing analysis of 16S rRNA, hsp65, rpoB was useful for identification of NTM unclassified by rpoB PRA.

Interferon-gamma Release Assay among Tuberculin Skin Test Positive Students in Korean High Schools / 결핵및호흡기질환

BACKGROUND: There are several active tuberculosis (TB) cases in Korean high schools each school year. The risk of transmission in schools is extremely high due to the considerable time spent in closed classrooms. We evaluated the control of latent tuberculosis infection in Korean high schools. METHODS: When a student was identified with active TB, tuberculin skin testing was performed on their classmates and on students in their same school grade. When a student had a positive tuberculin skin tests (TST), they underwent follow-up testing with QuantiFERON-TB Gold In-Tube (QFT). The manufacturer recommended a cut-off of 0.35 IU/mL to determine QFT positivity was applied. RESULTS: A total of 131 pulmonary tuberculosis (TB) patients were included based on the criteria for screening TB contacts in the National Tuberculosis Control Program. Seventy-five (57.2%) students tested smear positive. TST were performed on 7,109 students who were classmates of, or in the same grade as, a TB patient. Of the contacts, 1,231 students (17.3%) were TST positive and they were screened with QFT. Six hundred-sixty-six (55.0%) of the tested students returned a positive QFT result and the rate of positivity was significantly associated with the increasing size of TST indurations (p<0.0001). CONCLUSION: The use of QFT resulted in approximately 45% of TST positive students not being given chemoprophylaxis.

Clarithromycin Susceptibility Testing of Mycobacterium avium Complex Using 2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride Microplate Assay with Middlebrook 7H9 Broth

A series of 119 Mycobacterium avium complex isolates were subjected to clarithromycin susceptibility testing using microplates containing 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC). Among 119 isolates, 114 (95.8%) were susceptible to clarithromycin and 5 were resistant according to the new and the standard method. STC counts the low cost and reduces the number of procedures needed for susceptibility testing.

Clarithromycin Susceptibility Testing of Mycobacterium avium Complex Using 2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride Microplate Assay with Middlebrook 7H9 Broth

A series of 119 Mycobacterium avium complex isolates were subjected to clarithromycin susceptibility testing using microplates containing 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC). Among 119 isolates, 114 (95.8%) were susceptible to clarithromycin and 5 were resistant according to the new and the standard method. STC counts the low cost and reduces the number of procedures needed for susceptibility testing.

Detection of Clarithromycin-resistant Strains from Clinical Isolates of Mycobacterium abscessus / 결핵및호흡기질환

BACKGROUND: Mycobacterium abscessus is the most pathogenic and drug-resistant rapid-growing mycobacterium. Clarithromycin or azithromycin are the only regular oral antimycobacterial agents that have an effect on M. abscessus. We tried to detect the clarithromycin-resistant strains from the clinical isolates of M. abscessus. METHODS: We tried to isolate the clarithromycin-resistant strains from 220 clinical isolates of M. abscessus by performing using reverse hybridization assay (RHA) and the broth microdilution test (BMT). RESULTS: Seven resistant strains (3.2%) from all the tested clinical isolates were detected by BMT. Three of these resistant strains were also detected by RHA and it was confirmed that they had point mutants. CONCLUSION: These results showed that clarithromycin resistance in M. abscessus clinical isolates is related to a point mutation and other unknown mechanisms.

Use of Molecular Identification Analysis in a Case of Intra-familial Transmission of Tuberculosis / 결핵및호흡기질환

No abstract available.

Drug Resistance Rate of New Pulmonary Tuberculosis Patients Treated from the Private Sector in 2003~2005 / 결핵및호흡기질환

BACKGROUND: Surveillance of TB drug resistance (DR) is essential for providing information on the magnitude and trends in resistance, for developing treatment guidelines and for monitoring the effect of interventions. Up to now national surveys of drug resistance of M. tuberculosis have been conducted four times since 1994 among patients registered at health centers. The purpose of this study is to estimate the prevalence of primary drug resistance among new cases identified in private sector, and to compare it with the previous national drug resistance surveys. METHODS: The study collected results of drug susceptibility testing (DST) performed at the Korean Institute of Tuberculosis by the request of private sector from January 2003 to December 2005, and then finally selected new cases for the analysis from the database of Korean TB Surveillance (KTBS) by matching patients' name and social identification numbers. RESULTS: Of the 5,132 new patients included in the study, 689 (13.4%) patients were found to have drug resistance at least one drug, 530 patients (10.3%) were isoniazid resistant, 195 patients (3.8%) were multi-drug resistant (MDR), and 21 patients (0.4%) were extensively drug resistant (XDR). The rate of drug resistance tended to decrease annually but it was not statistically significant. When compared with previous national DR surveys in 2003 and in 2004 respectively, they were not significantly different. CONCLUSION: The prevalence of DR among new cases managed in the private sector did not show significant difference from that of new patients registered in the public sector in the same year.

In Vitro Antimicrobial Susceptibility of Mycobacterium abscessus in Korea

Mycobacterium abscessus is the second most common etiology of pulmonary disease caused by nontuberculous mycobacteria in Korea. Although antimicrobial susceptibility tests are important for appropriate patient management in M. abscessus lung disease, the tests have never been investigated in Korea. Seventy-four isolates of M. abscessus recovered from patient respiratory samples were tested against eight antimicrobial agents following the guidelines set forth by the National Committee for Clinical Laboratory Standards. Of the parenteral antibiotics, amikacin (99%, 73/74) and cefoxitin (99%, 73/74) were active against most isolates. Imipenem (55%, 36/66) and tobramycin (36%, 27/74) had activity against moderate number of isolates. Of the oral antibiotics, clarithromycin (91%, 67/74) was active against the majority of isolates. Moxifloxacin (73%, 54/74) and ciprofloxacin (57%, 42/74) had activity against a moderate number of isolates. Doxycycline was the least active, inhibiting only 7% (5/74) of isolates. In conclusion, the variations in susceptibility within M. abscessus isolates to currently available antimicrobials suggest that the antimicrobial susceptibilities of any clinically significant M. abscessus isolate be needed individually.

The Interval Between Initiation of Anti-tuberculosis Treatment in Patients with Culture-positive Pulmonary Tuberculosis and Receipt of Drug-susceptibility Test Results

Although mycobacterial culture and the subsequent drug-susceptibility test (DST) for anti-tuberculosis (TB) drugs take several months to complete using solid media, there are no reports on the turnaround times of these tests under clinical conditions. The aim of this study was to determine the interval between initiation of anti-TB treatment and receipt of DST requested at an outpatient clinic. We prospectively enrolled patients with culture-positive pulmonary TB at Seoul National University Hospital from September 2002 to December 2004. Patients were followed up monthly. Mycobacterial cultures were done using Ogawa media at Seoul National University Hospital. DST were performed at the Korean Institute of Tuberculosis. Of the 104 patients enrolled, 54 were male. The median age was 41 yr. The median interval from initiation of anti-TB treatment to receipt of mycobacterial culture results by clinicians was 37 days (range, 0-89 days). The median interval from initiation of treatment to confirmation of DST by requesting clinicians was 80.5 days (range, 28-145 days). Clinicians only received the results of DST more than two months after initiation of treatment when they followed up patients monthly and mycobacterial culture was performed using solid media.

Evaluation of the Broth Microdilution Method Using 2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride for Rapidly Growing Mycobacteria Susceptibility Testing

As the incidence of nontuberculous mycobacterial infection has been increasing recently in Korea, the importance of drug susceptibility test for clinical isolates of mycobacteria has become larger. In this study we determined the antimicrobial susceptibility patterns of clinical isolates of M. fortuitum and M. abscessus in Korea, and evaluated the efficacy of a modified broth microdilution method using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC), in terms of its ability to provide accurate and easy-to-read minimal inhibitory concentration (MIC) endpoints for the susceptibility testing of rapidly growing mycobacteria. Most isolates of M. fortuitum and M. abscessus in Korea are susceptible or intermediately susceptible to amikacin, cefoxitin, ciprofloxacin, and clarithromycin. Many isolates of M. fortuitum are susceptible to doxycycline, sulfamethoxazole, and imipenem, while many M. abscessus isolates are resistant to these drugs. In the present study, the modified broth microdilution method using STC was found to be reliable, easy to read, and inexpensive for M. fortuitum and M. abscessus susceptibility testing. The modified colorimetric MIC testing method using STC was proven to be a useful surrogate for RGM antibiotic susceptibility testing.

Pulmonary Disease Caused by Mycobacterium xenopi: The First Case in Korea

Mycobacterium xenopi is a nontuberculous mycobacterium (NTM) that rarely causes pulmonary disease in Asia. Here we describe the first case of M. xenopi pulmonary disease in Korea. A 66-year-old man was admitted to our hospital with a 2-month history of productive cough and hemoptysis. His past medical history included pulmonary tuberculosis 44 years earlier, leading to a right upper lobectomy. Chest X-ray upon admission revealed cavitary consolidation involving the entire right lung. Numerous acid-fast bacilli were seen in his initial sputum, and M. xenopi was subsequently identified in more than five sputum cultures, using molecular methods. Despite treatment with clarithromycin, rifampicin, ethambutol, and streptomycin, the infiltrative shadow revealed on chest X-ray increased in size. The patient's condition worsened, and a right completion pneumonectomy was performed. The patient consequently died of respiratory failure on postoperative day 47, secondary to the development of a late bronchopleural fistula. This case serves as a reminder to clinicians that the incidence of NTM infection is increasing in Korea and that unusual NTM are capable of causing disease in non-immunocompromised patients.

Drug Resistance Rates of Mycobacterium tuberculosis at a Private Referral Center in Korea

The goals of this study were to identify first-line drug resistance in new and previously treated tuberculosis (TB) cases and to determine risk factors for multidrugresistant TB (MDR-TB) at a private referral center in Korea. All patients with cultureconfirmed pulmonary TB over a 2-yr period between July 2002 and June 2004 were prospectively included in this study. In total, 637 patients were included; 512 (80.4%) were new cases, and 125 (19.6%) were previously treated cases. Resistance to at least one first-line drug was identified in 11.7% of new cases and 41.6% of previously treated cases. MDR-TB was detected in 3.9% of new cases and 27.2% of previously treated cases. The proportion of extensively drug-resistant TB among MDR-TB patients was 16.7% (9/54). Factors associated with MDR-TB included age under 45 yr, previous TB treatment, and the presence of cavitation on chest radiography. Rates of first-line drug resistance are high, particularly in previously treated patients, in the private sector in Korea. This underscores the need for an improved control program, coupled with early diagnosis of MDR-TB, to reduce the spread and development of resistance.

Analysis of DNA fingerprints of Mycobacterium Tuberculosis Isolates from Patients Registered at Health Center in Gyeonggi Province in 2004 / 결핵및호흡기질환

BACKGROUND: IS6110 DNA fingerprint is a very useful tool for investigating the transmission of tuberculosis. The aim of this study was to identify the epidemiological situations within a given area (one province). METHODS: The 681 Mycbobacterium tuberculosis isolates from patients, who were registered at health centers in Gyeonggi Province from May to December in 2004, were subjected to IS6110 DNA fingerprinting. Patients belonging to clusters were interviewed by health-workers to determine their previous contacts or household TB history. RESULTS: The number of IS6110 copies of the 681 isolates showed diverse fingerprint patterns from 0 to 21 of which the most prevalent copy number was 10 from 120 isolates (17.6%). Thirty-three isolates (4.8%) belonged to the K strain, and 128 isolates (18.8%) belonged to the K family. There were 180 (26.4%) isolates belonged belonging to fifty clusters, of which two clusters were within household transmission. Forty-three (23.9%) out of 180 patients resided in an area under the same health center control. The rate of clusters in those aged 60-70 was higher than in any other age group ( 95% CI of RR : 1.072 ~ 1.988). CONCLUSION: This is the first report of an epidemiological survey based on a whole province using a DNA fingerprinting technique for M. tuberculosis. These results will be helpful in developing a program or policies to prevent the transmission of TB.

Investigation of the Growth Rate Change in Recombinant BCG which was cloned Mycobacterium tuberculosis Adenylate Kinase Mutation Gene or Human Muscle-type Adenylate Kinase Synthetic Gene / 결핵및호흡기질환

BACKGROUND: Normal cell proliferation and viability is strongly depends on the availability of metabolic energy and the maintenance of the appropriate adenylate-nucleotide pools. Hypothetically, changes in adenylate kinase (AK) expression could therefore be associated with adaptation to altered growth characteristics or inversely altered growth characteristics of proliferating cells could drive the changes in the metabolic profile. This study investigated whether the expression of either AK1 or a Mycobacterium tuberculosis adenylate kinase mutant which has the same catalytic activity of AK1 could affect the growth rate of slow-growing BCG. METHOD: Recombinant BCGs, which were cloned the human muscle-type adenylate kinase synthetic gene (AK1) and adenylate kinase mutation gene (AKmtDM) of Mycobacterium tuberculosis into the Mycobacterium/E.coli expression vectors, were constructed. Recombinant BCGs and wild-type BCG were cultured in 7H9 media and the optical density at 600nm was measured at intervals of 2-3 days. RESULT: There wasn't the growth rate change induced by AK1 or AKmtDM expression in recombinant BCGs. CONCLUSION: The expression of AK1 or Mycobacterium tuberculosis adenylate kinase mutant in BCG does not affect the growth rate of BCG.