Pathogenicity of enterotoxigenic Escherichia coli in Caenorhabditis elegans as an alternative model host.

Enterotoxigenic Escherichia coli (ETEC), one of the diarrheagenic E. coli, is the most common cause of diarrhea in developing country and in travelers to those areas. In this study, Caenorhabditis elegans was used as an alternative model host to evaluate ETEC infections. The ETEC strain ETEC1, which was isolated from a patient with diarrhea, possessed enterotoxins STh, LT1, and EAST1 and colonization factors CS2 and CS3. Live ETEC1 shortened the life span and body size of C. elegans in association with increased expression of enterotoxin genes and intestinal colonization. In contrast, heat-killed ETEC1 did not affect the life span of C. elegans. Caenorhabditis elegans infected with ETEC1 showed upregulated expression of genes related to insulin-like peptides and host defense responses. These results suggest that ETEC1 exhibits pathogenicity through intestinal colonization and enterotoxin production in C. elegans. This system is useful as an ETEC infection model.

Impacts of Bacillus subtilis var. natto on the lifespan and stress resistance of Caenorhabditis elegans.

AIM: Bacillus subtilis var. natto is used in the production of natto, a typical Japanese fermented soybean food. Although the probiotic attributes and health-related effects of B. subtilis var. natto have been reported, the effect on longevity remains unknown. In the present study, the effects of B. subtilis var. natto strains on lifespan extension and the molecular mechanisms governing the prolongevity were examined using Caenorhabditis elegans as a model animal. METHODS AND RESULTS: Synchronized 3-day-old (young adult) worms were fed Escherichia coli OP50 (control) or a subcloned isolate of B. subtilis var. natto Miyagino strain (MI-OMU01) and subjected to lifespan, survival against pathogens and abiotic stress resistance assays. Notably, the lifespan of worms fed MI-OMU01 was significantly longer than that of the animals fed OP50. Moreover, MI-OMU01 increased the resistance of C. elegans to several stressors, including UV irradiation, H2O2, and Cu2+. CONCLUSIONS: Genetic and gene expression analyses using mutant animals suggested that MI-OMU01 extended the lifespan of worms in TIR-1/SARM, p38 MAPK, and insulin/IGF-1 signaling pathway-dependent manners.

Attempt of thyX gene silencing and construction of a thyX deleted clone in a Mycobacterium bovis BCG.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. This study assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. A thyX deletion mutant strain was successfully constructed from that strain, which reinforces the non-essentiality of thyX under a certain genetic background.

Analytical estimation of maximum fraction of infected individuals with one-shot non-pharmaceutical intervention in a hybrid epidemic model.

BACKGROUND: Facing a global epidemic of new infectious diseases such as COVID-19, non-pharmaceutical interventions (NPIs), which reduce transmission rates without medical actions, are being implemented around the world to mitigate spreads. One of the problems in assessing the effects of NPIs is that different NPIs have been implemented at different times based on the situation of each country; therefore, few assumptions can be shared about how the introduction of policies affects the patient population. Mathematical models can contribute to further understanding these phenomena by obtaining analytical solutions as well as numerical simulations. METHODS AND RESULTS: In this study, an NPI was introduced into the SIR model for a conceptual study of infectious diseases under the condition that the transmission rate was reduced to a fixed value only once within a finite time duration, and its effect was analyzed numerically and theoretically. It was analytically shown that the maximum fraction of infected individuals and the final size could be larger if the intervention starts too early. The analytical results also suggested that more individuals may be infected at the peak of the second wave with a stronger intervention. CONCLUSIONS: This study provides quantitative relationship between the strength of a one-shot intervention and the reduction in the number of patients with no approximation. This suggests the importance of the strength and time of NPIs, although detailed studies are necessary for the implementation of NPIs in complicated real-world environments as the model used in this study is based on various simplifications.

Oral cleaning habits and the copy number of periodontal bacteria in pregnant women and its correlation with birth outcomes: an epidemiological study in Mibilizi, Rwanda.

BACKGROUND: Since 1996, many studies have reported that periodontal disease during pregnancy may be a risk factor for preterm birth and low birth weight; however, in Africa, periodontal disease is considered a non-high-priority disease. In addition, there are few dental facilities in rural Rwanda; thus, the oral condition of pregnant women has not been investigated. The objective of this study was to assess the tooth brushing habits of pregnant women in rural Rwanda and evaluate whether periodontal bacteria in the oral cavity of pregnant women are related to birth outcomes or oral cleaning habits. METHODS: A questionnaire survey and saliva collection were conducted for pregnant women in the catchment area population of Mibilizi Hospital located in the western part of Rwanda. Real-time PCR was performed to quantitatively detect total bacteria and 4 species of periodontal bacteria. The relationship of the copy number of each bacterium and birth outcomes or oral cleaning habits was statistically analyzed. RESULTS: Among the participants, high copy numbers of total bacteria, Tannerella forsythia, and Treponema denticola were correlated with lower birth weight (p = 0.0032, 0.0212, 0.0288, respectively). The sex ratio at birth was higher in women who had high copy numbers of Porphyromonas gingivalis and T. denticola during pregnancy (p = 0.0268, 0.0043). Furthermore, regarding the correlation between oral cleaning habits and the amount of bacteria, the more frequently teeth were brushed, the lower the level of P. gingivalis (p = 0.0061); the more frequently the brush was replaced, the lower the levels of P. gingivalis and T. forsythia (p = 0.0153, 0.0029). CONCLUSIONS: This study suggested that improving tooth brushing habits may reduce the risk of periodontal disease among pregnant women in rural Rwanda. It also indicated that the amount of bacteria is associated with various birth outcomes according to the bacterial species. Both access to dental clinics and the oral cleaning habits of pregnant women should be important considerations in efforts to alleviate reproductive-related outcomes in rural Africa.

Periodontal disease is known to cause many complications. For instance, pregnant women with periodontal disease are at increased risk of preterm birth and delivering low-birth-weight infants. However, the importance of oral care during pregnancy is not an important consideration in rural Africa, where preterm birth rates and low-birth-weight rates are particularly high. Moreover, even the oral hygiene status of pregnant women has not been assessed in such areas. In this study, we focused on the amount of periodontal bacteria that cause periodontal disease and investigated the relationship between the amount of bacteria and birth outcomes. Our findings indicate that tooth brushing guidance for pregnant women and improved access to dental clinics in rural Africa may contribute to reduced rates of preterm birth and low birth weight.

INTENSIVE TREATMENT OF A CAPTIVE BORNEAN ELEPHANT (ELEPHAS MAXIMUS BORNEENSIS) INFECTED WITH MYCOBACTERIUM CAPRAE IN JAPAN.

In 2015, an estimated 17-year-old female Bornean elephant (Elephas maximus borneensis) at Fukuyama Zoo in Japan exhibited anorexia and significant weight loss. Pan-susceptible Mycobacterium tuberculosis complex (MTBC) was isolated from vaginal discharge, oral mucus, urine, and fecal samples by culture. The isolate was identified as Mycobacterium caprae by genetic analysis. Isoniazid, pyrazinamide, and levofloxacin were administered rectally. Body weight increased to normal, but subsequently decreased again. Elevation of liver enzymes occurred, likely related to the increase in isoniazid dosage. After recovery from side effects, the elephant's weight increased further. However, isoniazid-resistant M. caprae was isolated from oral mucus after anti-tuberculosis drug treatment for 9 mo. The regimen was changed to rifampicin, pyrazinamide, ethambutol, and levofloxacin, administered orally or rectally. The 18-mo treatment was completed in October 2018. This elephant has shown no clinical sign since. No MTBC-positive sample had been obtained as of March 2020.

Emergence of vancomycin- and teicoplanin-resistant Enterococcus faecium via vanD5-harbouring large genomic island.

BACKGROUND: Treatment of VRE is of clinical concern. While certain numbers of vanD-type VRE have been isolated, only two vanD5-harbouring Enterococcus faecium isolates have been reported in Canada and Japan. METHODS: We report the isolation of vanD5-type E. faecium and the first ever determination of the whole-genome sequence to investigate the possible mechanisms of the acquisition of the vanD5 gene cluster in E. faecium. RESULTS: Two vanD5-harbouring vancomycin-resistant E. faecium were isolated from the skin (SMVRE19) and faeces (SMVRE20) of a patient with a skin ulcer in Japan. The isolates exhibited vancomycin and teicoplanin MIC values of 128 mg/L, whilst the previous isolates of vanD5-harbouring E. faecium were only resistant to vancomycin. SMVRE19 and SMVRE20 were clones related to ST18, which is also seen in vanA- and vanB-type VRE. These isolates harboured an insertion element, ISEfm1, in the ddl gene, similar to a previously described teicoplanin-resistant vanD3-type E. faecium. The vanD5 gene cluster was integrated into the SMVRE20 chromosome as a part of a large genomic island (approximately 127 kb), similar to other recently spreading vanD variants in the Netherlands. The genomic island shared the greatest similarity with a part of the Blautia coccoides genome sequence, except for the region surrounding the vanD gene cluster. CONCLUSIONS: This study reports that emergence of vancomycin- and teicoplanin-resistant vanD5-type E. faecium occurred via acquisition of the vanD5 cluster and ISEfm1 insertion into ddl. Considering the genetic similarity between the various VRE strains, the current study should serve as a warning against the spread of vanD5-type VRE.

Is splenectomy for dissecting splenic hilar lymph nodes justified for scirrhous gastric cancer?

BACKGROUND: Splenectomy for dissecting splenic hilar lymph nodes (#10) should be avoided for most gastric cancer, considering the high morbidity and lack of any survival benefit, but it is often selected for scirrhous gastric cancer because this type frequently invades the whole stomach and lymph nodes. Splenectomy is necessary for dissecting #10; however, the survival benefit of dissecting #10 is unclear. METHODS: Patients who had scirrhous gastric cancer and underwent D2 total gastrectomy with splenectomy at National Cancer Center Hospital, Japan, between 2000 and 2011 were retrospectively analyzed. The therapeutic value index was calculated by multiplying the metastatic rate of each nodal station and the 5-year survival of patients who had metastasis to each node. RESULTS: In total, 137 patients were eligible for the present study. The most frequent metastatic node was #3(58%), followed by #4d(46%), #1(35%), #4sb(23%), #6(22%), #7(21%), #4sa(18%), #10(15%), #2(14%), #11p(14%), #11d(13%), #9(13%), and #8a(11%). These lymph nodes had a metastatic rate of more than 10%. The node station with the highest index was #3(18.9), followed by #4d(14.1), #1(10.8), #4sa(6.11), #4sb(6.06), #10(5.09), #7(4.39), #11d(4.36), #11p(4.06), #2(2.93), #8a(2.18), and #9(1.45). The index of #10 exceeded that of #2, #7, #8a, and #9, which are the key nodes dissected in D2. CONCLUSION: The metastatic rate of the splenic hilar lymph nodes was relatively high, and the therapeutic index was the sixth highest among the 15 regional lymph nodes included in D2 dissection. Splenectomy for dissecting splenic hilar lymph nodes would be justified for scirrhous gastric cancer.

Antenatal Care Visits and Adverse Pregnancy Outcomes at a Hospital in Rural Western Province, Rwanda.

In many economically developing countries, and especially in the rural regions of sub-Saharan African coun-tries, there have been only limited investigations into the association between antenatal care (ANC) and adverse pregnancy outcomes. We obtained information on ANC and pregnancy outcomes between 2011 and 2016 from hospital files of pregnant women (n = 4,960) served at a rural hospital in Rwanda, and we examined the associa-tions between their ANC visits and the adverse pregnancy and neonatal outcomes by using univariate and mul-tivariate logistic regression models to estimate the odds ratios (ORs) and 95% confidence intervals (CIs). Most of the pregnant women had ≥ 4 ANC visits, but 39% (n = 1,911) did not have ≥ 3 visits before delivery. The prev-alence of low birth weight (LBW) and that of preterm birth (PTB) were 12% and 9.9%, respectively. Compared to the women who attended only one ANC visit, those who attended ≥ 4 ANC visits had lower risks of LBW (OR 0.20; 95%CI: 0.11-0.36) and PTB (OR 0.28; 95%CI: 0.11-0.76). Frequent ANC visits were also associ-ated with better postnatal outcomes of the newborns. Encouraging women to attend ANC visits before delivery can markedly reduce PTB-related and LBW-related complications, especially in resource-limited settings.

Host Range-Associated Clustering Based on Multilocus Variable-Number Tandem-Repeat Analysis, Phylotypes, and Virulence Genes of Atypical Enteropathogenic Escherichia coli Strains.

Atypical enteropathogenic Escherichia coli (aEPEC) strains (36 Japanese and 50 Bangladeshi) obtained from 649 poultry fecal samples were analyzed by molecular epidemiological methods. Clermont's phylogenetic typing showed that group A was more prevalent (58%, 50/86) than B1 (31%, 27/86). Intimin type ß1, which is prevalent among human diarrheal patients, was predominant in both phylogroups B1 (81%, 22/27) and A (70%, 35/50). However, about 95% of B1-ß1 strains belonged to virulence group I, and 77% of them were Japanese strains, while 17% (6/35) of A-ß1 strains did. Multilocus variable-number tandem-repeat analysis (MLVA) distributed the strains into 52 distinct profiles, with Simpson's index of diversity (D) at 73%. When the data were combined with those of 142 previous strains from different sources, the minimum spanning tree formed five zones for porcine strains, poultry strains (excluding B1-ß1), strains from healthy humans, bovine and human patient strains, and the B1-ß1 poultry strains. Antimicrobial resistance to nalidixic acid was most common (74%) among the isolates. Sixty-eight percent of them demonstrated resistance to ≥3 antimicrobial agents, and most of them (91%) were from Bangladesh. The strains were assigned into two groups by hierarchical clustering. Correlation matrix analysis revealed that the virulence genes were negatively associated with antimicrobial resistance. The present study suggested that poultry, particularly Japanese poultry, could be another reservoir of aEPEC (phylogroup B1, virulence group I, and intimin type ß1); however, poultry strains seem to be apart from patient strains that were closer to bovine strains. Bangladeshi aEPEC may be less virulent for humans but more resistant to antibiotics.IMPORTANCE Atypical enteropathogenic Escherichia coli (aEPEC) is a diarrheagenic type of E. coli, as it possesses the intimin gene (eae) for attachment and effacement on epithelium. Since aEPEC is ubiquitous even in developed countries, we previously used molecular epidemiological methods to discriminate aEPEC as a human pathogen. The present study assessed poultry as another source of human diarrheagenic aEPEC. Poultry could be the source of aEPEC (phylogroup B1, virulence group I, and intimin type ß1) found among patient strains in Japan. However, the minimum spanning tree (MST) suggested that the strains from Japanese poultry were far from Japanese patient strains compared with the distance between bovine and patient strains. Bangladeshi avian strains seemed to be less diarrheagenic but are hazardous as a source of drug resistance genes.

Mycobacterium caprae Infection in Captive Borneo Elephant, Japan.

In 2016, disseminated tuberculosis caused by Mycobacterium caprae was diagnosed in a captive Borneo elephant in Japan. The bacterium was initially identified from clinical isolates. An isolate collected during a relapse showed isoniazid monoresistance and a codon 315 katG mutation.

Whole genome analysis of a multidrug-resistant Streptococcus pneumoniae isolate from a patient with invasive pneumococcal infection developing disseminated intravascular coagulation.

Multidrug-resistant Streptococcus pneumoniae strains were isolated from blood and sputum of a patient with disseminated intravascular coagulation in Sapporo city, Japan. These antibiograms were only susceptible to vancomycin, linezolid, daptomycin, some carbapenems, and some fluoroquinolones. Identical antibiograms, serotypes (19F), and sequence types (ST10017) suggested a shared origin of these isolates. Only one ST10017 strain has been isolated in the same city in Japan previously (2014), and the 2014 isolate is still susceptible to macrolides. The whole genome of the blood-derived isolate was sequenced. The strain harbored resistance mutations in parC, gyrA, pbp1a, pbp2a, pbp2b, and pbp2x, and harbored the resistance genes, ermB and tetM. The nucleotide sequences of parC and pbp2x genes of strain MDRSPN001 were clearly different from those of other S. pneumoniae strains and were similar to those of oral streptococci strains. These findings suggest that strain MDRSPN001 has been rapidly and drastically evolving multidrug resistance by gene replacement and accumulation of genes originating from other strains, such as oral streptococci, Streptococcus mitis.

Mycobacterium tuberculosis Transmission among Elderly Persons, Yamagata Prefecture, Japan, 2009-2015.

In many countries with low to moderate tuberculosis (TB) incidence, cases have shifted to elderly persons. It is unclear, however, whether these cases are associated with recent Mycobacterium tuberculosis transmission or represent reactivation of past disease. During 2009-2015, we performed a population-based TB investigation in Yamagata Prefecture, Japan, using in-depth contact tracing and 24-loci variable-number tandem-repeat typing optimized for Beijing family M. tuberculosis strains. We analyzed 494 strains, of which 387 (78.3%) were derived from elderly patients. Recent transmission with an epidemiologic link was confirmed in 22 clusters (70 cases). In 17 (77.3%) clusters, the source patient was elderly; 11 (64.7%) of the 17 clusters occurred in a hospital or nursing home. In this setting, the increase in TB cases was associated with M. tuberculosis transmissions from elderly persons. Prevention of transmission in places where elderly persons gather will be an effective strategy for decreasing TB incidence among predominantly elderly populations.

Mycolactone-producing Mycobacterium marinum infection in captive Hong Kong warty newts and pathological evidence of impaired host immune function.

A mass mortality event of captive Hong Kong warty newts Paramesotriton hongkongensis with non-granulomatous necrotic lesions occurred in Taipei Zoo, Taiwan, in 2014. Clinically, the sick newts were lethargic and often covered with water mold Saprolegnia sp. on the skin of the body trunk or extremities. Predominant pathological findings were multifocal non-granulomatous necrotic lesions in the liver, spleen, and kidneys and severe skin infection with Saprolegnia sp., with deep invasion and involvement of underlying muscles. The possibility of ranavirus infection was ruled out by negative PCR results. Unexpectedly, abundant intralesional acid-fast positive bacilli were found in the necrotic lesions of the liver, spleen, and kidney in all 14 sick newts. PCR targeting the hsp65, ITS region, and partial 16S rRNA genes was performed, and the sequence identity from amplified amplicons of hsp65 and partial 16S rRNA genes was 100% identical to that of the corresponding gene fragment of Mycobacterium marinum. Further molecular investigations demonstrated that the current M. marinum was a mycolactone-producing mycobacterium with the presence of esxA/esxB genes. Mycolactone is a plasmid-encoded, immunosuppressive, and cytotoxic toxin. The possible immunosuppression phenomenon characterized by systemic non-granulomatous necrotic lesions caused by M. marinum and the unusual deep invasive infection caused by water mold might be associated with the immunosuppressive effect of mycolactone. Therefore, it should be noted that non-granulomatous necrotic lesions in amphibians can be caused not only by ranavirus infection but also by mycobacteriosis.

Application of Genomic Epidemiology of Pathogens to Farmed Yellowtail Fish Mycobacteriosis in Kyushu, Japan.

To investigate mycobacterial cases of farmed yellowtail fish in coastal areas of western Japan (Kagoshima, Kyushu), where aquaculture fisheries are active, Mycobacterium pseudoshottsii, the causative agent, was isolated from six neighboring fishing ports in 2012 and 2013. A phylogenetic ana-lysis revealed that the strains isolated from one fishing port were closely related to those isolated from other regions of Japan, suggesting the nationwide spread of a single strain. However, strains from Japan were phylogenetically distinct from those from the Mediterranean and the United States; therefore, worldwide transmission was not observed based on the limited data obtained on the strains exami-ned in this study. The present results demonstrate that a bacterial genomic ana-lysis of infected cases, a mole-cular epidemiology strategy for public health, provides useful data for estimating the prevalence and transmission pathways of M. pseudoshottsii in farmed fish. A bacterial genome ana-lysis of strains, such as that performed herein, may play an important role in monitoring the prevalence of this pathogen in fish farms and possible epidemics in the future as a result of international traffic, logistics, and trade in fisheries.

Multiplex agarose gel electrophoresis system for variable number of tandem repeats genotyping: analysis example using Mycobacterium tuberculosis.

As one genotyping method for Mycobacterium tuberculosis, variable number of tandem repeats (VNTR) is a promising tool to trace the undefined transmission of tuberculosis, but it often requires large equipment such as a genetic analyzer for DNA fragment analysis or CE system to conduct systematic analyses. For convenient genotyping at low cost in laboratories, we designed a multiplex PCR system that is applicable to agarose gel electrophoresis using fluorescent PCR primers. For tuberculosis genotyping by VNTR, the copy quantities of minisatellite DNA must be determined in more than 12 loci. The system can halve laborious electrophoresis processes by presenting an image of two VNTR amplicons on a single lane. No expensive equipment is necessary for this method. Therefore, it is useful even in developing countries.

[Evaluation of JATA(12)-variable number of tandem repeats as a marker of the source of tuberculosis outbreaks in Osaka].

OBJECTIVES: To evaluate the usefulness of the JATA (12)-variable number of tandem repeats (VNTR) system for identifying the source of Mycobacterium tuberculosis outbreaks. DESIGN; JATA(12)-VNTR genotyping was performed on M. tuberculosis isolates from a total of 206 patients in whom group infection was confirmed by epidemiological studies ("group infection"), as well as from 64 patient clusters in whom group infection was suspected but not confirmed ("non-group infection"). The patients were diagnosed in Osaka Prefecture from April 1999 to December 2011. RESULTS: All isolates from the "non-group infection" patients showed a unique VNTR pattern, whereas isolates from 185 (89.9%) "group infection" patients showed a common and group-specific JATA (12)-VNTR pattern. However, single-locus variants were observed in 1 (1.6%) "non-group infection" case and in 21 (10.2%) "group infection" cases. CONCLUSION: Tuberculosis in 248 (91.9%) of the 270 study patients could be correctly identified based on the genotyping of the isolates by using the JATA (12)-VNTR. If proper attention is paid to the single-locus variant, the JATA (12)-VNTR system would be a useful tool for identification of sources of tuberculosis outbreaks.

[Effectiveness of comprehensive variable number of tandem repeat (VNTR) analysis in areas with a low incidence of tuberculosis].

PURPOSE: To ascertain the effectiveness of variable number of tandem repeat (VNTR) analysis in areas with a low incidence of tuberculosis (TB), we examined the combination of comprehensive VNTR analyses and field epidemiological investigation results in Yamagata Prefecture, Japan, where estimated incidence of new TB cases per 100,000 population was 11.3 in 2011. METHODS: We collected Mycobacterium tuberculosis isolates from 184 (69.2%) of 266 pulmonary TB patients across the whole of Yamagata Prefecture between 2009 and 2011. Next, 24 loci [JATA (12), QUB-11a, ETR A, QUB-18, QUB3232, v3820, v4120, MIRU04, MIRU16, MIRU40, ETR C, Mtub30, Mtub39] in VNTR genotypes were determined. The relationships among TB patients derived from the respective clusters were surveyed using field epidemiological investigation results provided by the Public Health Center. RESULTS: Seventeen clusters were formed by 49 (26.6%) of the 184 isolates. We found 3 hospital infection cases, 3 family infection cases, and 1 nursing home infection case forming 6 clusters. Among these cases, two relationships among patients were revealed after additional epidemiological investigation at the Public Health Center. The VNTR pattern of the largest cluster, which was formed by 12 isolates, was identical with that of an incipient patient of a TB mass infection that occurred in 2007. DISCUSSION: In areas with a low incidence of TB, a combination of comprehensive VNTR analysis and field epidemiological investigation is useful to find unknown transmission routes, identify for new risk groups, and trace mass infections.

[Clustering analysis of Mycobacterium tuberculosis using the JATA(12)-VNTR system for molecular epidemiological surveillance in broad areas of Japan].

OBJECTIVES: Japan Anti-Tuberculosis Association (JATA) (12)-variable numbers of tandem repeats (VNTR) is a standard method for genotyping of clinical isolates of Mycobacterium tuberculosis in Japan. As a model study for nationwide surveillance, this study aimed to describe the tendency and frequency of genotypes of M. tuberculosis in a large number of clinical samples. METHODS: Clinical isolates of M. tuberculosis (n = 1,778) were obtained from patients with tuberculosis in 3 areas, i.e., Osaka City, Osaka Prefecture, and Kobe City, during 2007 and 2008. The samples were analyzed using JATA (12)-VNTR. All genotypes were subjected to clustering analysis. RESULTS AND DISCUSSION: In total, 1,086 (61.1%) isolates showed clustering. The most common clusters were composed of 3 members. Such clusters were considered to reflect either actual transmission or low discriminatory power of JATA (12)-VNTR. Several prevalent JATA(12)-VNTR genotypes formed large clusters and were discussed in relation with epidemiological findings of other studies. The findings of this study will aid in the construction of an effective genotyping-based surveillance system of M. tuberculosis, through improvement of interpretation of VNTR types, observation of certain particular strains in an area, and efficient detection of unidentified outbreaks.