Heterogeneity of Mycobacterium tuberculosis strains in Makassar, Indonesia.

SETTING: Patients with suspected pulmonary tuberculosis (TB) visiting government TB diagnostic and treatment centres in Makassar City, South Sulawesi Province, Indonesia, from February to October 2008 were included in the study. OBJECTIVE: To determine the distribution of Mycobacterium tuberculosis genotypes in Makassar. DESIGN: Cross-sectional study. Spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and principal genetic grouping (PGG) were used to genotype the M. tuberculosis clinical isolates. RESULTS: Among 179 isolates derived from pulmonary TB patients, distribution of spoligotypes comprised the East Africa-Indian (30.2%), T (17.9%), H (12.3%) and Beijing (9.5%) lineages. Other lineages found in smaller proportions were the Latin American-Mediterranean, MANU, S and X lineages. Nineteen isolates (10.6%) could not be grouped into any of the reported lineages or shared types. Single nucleotide polymorphism analysis of katG(463) and gyrA(95) grouped these isolates primarily into PGG1 (9/19, 47%). CONCLUSION: Only a few genetically identical clustered isolates were identified within the 9-month study period, and most isolates were genetically diverse. Furthermore, 15 spoligopatterns identified in our study have not been reported previously. To our knowledge, this is the first comprehensive study describing genotypes of M. tuberculosis clinical isolates in Sulawesi.

Molecular analysis of high-level ciprofloxacin resistance in Salmonella enterica serovar Typhi and S. Paratyphi A: need to expand the QRDR region?

Fourteen strains of S. Typhi (n=13) and S. Paratyphi A (n=1) resistant to ciprofloxacin were compared with 30 ciprofloxacin decreased-susceptibility strains on the basis of qnr plasmid analysis, and nucleotide substitutions at gyrA, gyrB, parC and parE. In ciprofloxacin-resistant strains, five S. Typhi and a single S. Paratyphi A showed triple mutations in gyrA (Ser83-->Phe, Asp87-->Asn, Glu133-->Gly) and a novel mutation outside the quinolone resistance determining region (QRDR) (Met52-->Leu). Novel mutations were also discovered in an isolate (minimum inhibitory concentration 8 microg/ml) in gyrA gene Asp76-->Asn and outside the QRDR Leu44-->Ile. Out of 30 isolates with reduced susceptibility, single mutation was found in 12 strains only. Genes encoding qnr plasmid (qnr A, qnr B, AAC1-F) were not detected in ciprofloxacin-resistant or decreased-susceptibility strains. Antimicrobial surveillance coupled with molecular analysis of fluoroquinolone resistance is warranted for reconfirming novel and established molecular patterns of resistance, which is quintessential for reappraisal of enteric fever therapeutics.

Immunological characterization of novel secreted antigens of Mycobacterium tuberculosis.

Proteins secreted by Mycobacterium tuberculosis are targets of host immune responses and as such are investigated for vaccine and immunodiagnostics development. Computer-driven searches of the M. tuberculosis H37Rv genome had previously identified 45 novel secreted proteins. Here, we report the characterization of these antigens in terms of specificity for the M. tuberculosis complex and the ability to induce human immune responses. BLAST homology searches and Southern hybridization identified 10 genes that were either specific for the M. tuberculosis complex or found in only two nontuberculous mycobacterial species of minor medical significance. Selected recombinant proteins were purified from Escherichia coli cells and tested for the ability to elicit antibody responses in tuberculosis patients. Reactivity of the serum panel was ' 36% with at least one of five novel proteins (Rv0203, Rv0603, Rv1271c, Rv1804c and Rv2253), 56% with the 38 kDa lipoprotein, a M. tuberculosis antigen known to be highly seroreactive, and 68% with a combination of Rv0203, Rv1271c and the 38 kDa antigen. Thus, at least five novel secreted proteins induce antibody responses during active disease; some of these proteins may increase the sensitivity of serological assays based on the 38 kDa antigen.

Identification of a W variant outbreak of Mycobacterium tuberculosis via population-based molecular epidemiology.

CONTEXT: Typing of Mycobacterium tuberculosis could provide a more sensitive means of identifying outbreaks than use of conventional surveillance techniques alone. Variants of the New York City W strain of M tuberculosis were identified in New Jersey. OBJECTIVE: To describe the spread of the W family of M tuberculosis strains in New Jersey identified by molecular typing and surveillance data. DESIGN: Population-based cross-sectional study. SETTING AND SUBJECTS: All incident culture-positive tuberculosis cases reported in New Jersey from January 1996 to September 1998, for which the W family was defined by insertion sequence (IS) IS6110 DNA fingerprinting, polymorphic GC-rich repetitive sequence (PGRS) typing, spacer oligotyping (spoligotyping), and variable number tandem repeat (VNTR) analysis. MAIN OUTCOME MEASURE: Identification and characterization of W family clones supplemented by surveillance data. RESULTS: Isolates from 1207 cases were analyzed, of which 68 isolates (6%) belonged to the W family based on IS6110 and spoligotype hybridization patterns. The IS6110 hybridization patterns or fingerprints revealed that43 patients (designated group A) shared a unique banding motif not present in other W family isolates. Strains collected from the remaining 25 patients (designated group B), while related to W, displayed a variety of IS6110 patterns and did not share this motif. The PGRS and VNTR typing confirmed the division of the W family into groups A and B and again showed group A strains to be closely related and group B strains to be more diverse. The demographic characteristics of individuals from groups A and B were specific and defined. Group A patients were more likely than group B patients to be US born (91 % vs 24%, P<.001), black (76% vs 16%, P<.001), human immunodeficiency virus positive (40% vs 0%, P = .007), and residents of urban northeast New Jersey counties (P<.001). Patients with group B strains were primarily non-US born, of Asian descent, and more dispersed throughout New Jersey. No outbreak had been detected using conventional surveillance alone. CONCLUSIONS: The implementation of multiple molecular techniques in conjunction with surveillance data enabled us to identify a previously undetected outbreak in a defined geographical setting. The outbreak isolates comprise members of a distinct branch of the W family phylogenetic lineage. The use of molecular strain typing provides a proactive approach that may be used to initiate, and not just augment, traditional surveillance outbreak investigations.

Nosocomial transmission of a drug-sensitive W-variant Mycobacterium tuberculosis strain among patients with acquired immunodeficiency syndrome in Tennessee.

OBJECTIVE: To use DNA fingerprinting to characterize nosocomial spread of Mycobacterium tuberculosis following hospitalization of a patient with acquired immunodeficiency syndrome and active pulmonary tuberculosis, for whom respiratory isolation was not initiated promptly. DESIGN: Epidemiological investigation. SETTING: A tertiary-care medical center in Tennessee. PARTICIPANTS: Patients and healthcare workers potentially exposed to the infectious patient in 1992. RESULTS: Of 172 healthcare workers exposed, 35 (20%) were judged to have acquired tuberculous infection. Risk of acquisition was greatest for nurses and medical receptionists. Active tuberculosis later developed in one healthcare worker and one hospitalized patient. Nosocomial transmission was supported by epidemiological evidence and DNA fingerprinting. The outbreak strain of Mycobacterium tuberculosis differed from other isolates at this hospital, but its DNA hybridization pattern was highly similar to that of the multidrug-resistant outbreak strain W that has been prevalent in New York City, suggesting a common strain ancestry. However, the Tennessee isolates were susceptible to all first-line antituberculous agents. CONCLUSIONS: This report suggests the possibility that a molecular characteristic(s) shared by these successful outbreak strains is associated with increased transmissibility or pathogenicity and emphasizes the need for continued vigilance for tuberculosis in the nosocomial setting.

Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region.

SETTING: Five IS6110 chromosomal insertion sites were characterized in the multidrug resistant Mycobacterium tuberculosis 'W' strain. OBJECTIVE: To use insertion site probes to study the phylogenetic distribution of IS6110 in the M. tuberculosis genome. DESIGN: A total of 722 M. tuberculosis isolates, previously genotyped using the standard IS6110 Southern blot hybridization methodology, were re-hybridized with the Region A insertion site probe and representative strains were further hybridized with the Region B and C probes. Strains were grouped on the basis of having IS6110 insertions in these different regions and their relatedness was further compared by sequencing the IS6110 insertion sites. RESULTS: The insertion site probes revealed that the collection of Chinese isolates previously grouped as the Beijing strain family shared IS6110 insertions in common with the W and other genotypic group 1 strains. Unexpectedly, we found that IS6110 integrated at least 10 independent times between the dnaA and dnaN genes encoding deoxyribonucleic acid replication proteins. CONCLUSIONS: IS6110 insertion site mapping is able to identify genetic relatedness among a collection of M. tuberculosis clinical strains representing the breadth of species diversity. The mapping data indicate that IS6110 insertion sites are not always random.

Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosis clone family.

OBJECTIVE: To determine whether isolates of Mycobacterium tuberculosis from New York and elsewhere that are resistant to four or more primary antimicrobial agents and responsible for widespread disease in the 1990s represent a newly emerged clone or a heterogeneous array of unrelated organisms. SETTING: New York City area and selected locations in the United States. PATIENTS: M tuberculosis isolates from 1953 patients in New York and multidrug-resistant isolates from six patients from other US communities. DESIGN: Convenience sample of all M tuberculosis strains (M tuberculosis isolates resistant to rifampin, streptomycin, isoniazid, and ethambutol, and sometimes ethionamide, kanamycin, capreomycin, or ciprofloxacin) submitted to the Public Health Research Institute Tuberculosis Center since 1991 and samples submitted to the Centers for Disease Control and Prevention from throughout the United States. The samples submitted were representative of the New York City strains of M tuberculosis. MAIN OUTCOME MEASURE: Characterization of resistant M tuberculosis strains studied by IS6110 and polymorphic GC-rich repetitive sequence (PGRS) hybridization patterns, multiplex polymerase chain reaction (PCR) analysis, and automated DNA sequencing of genes containing mutations associated with resistance to rifampin (rpoB), isoniazid (katG and inhA locus), and streptomycin (strA and rrs). RESULTS: Multidrug-resistant M tuberculosis isolates were recovered from 253 New York City patients and had the same or closely allied IS6110 and PGRS patterns, multiplex PCR type, and gene mutations associated with resistance to rifampin, isoniazid, and streptomycin. Isolates with these same molecular characteristics were recovered from patients in Florida and Nevada, health care workers in Atlanta, Ga, and Miami, Fla, and an individual who recently moved from New York City to Denver, Colo, and caused disease or skin test conversion in at least 12 people in a nursing home environment. CONCLUSIONS: The results document the molecular origin and spread of progeny of a closely related family of multidrug-resistant M tuberculosis strains that have recently shared a common ancestor and undergone clonal expansion. The multidrug-resistant phenotype in these organisms arose by sequential acquisition of resistance-conferring mutations in several genes, most likely as a consequence of antibiotic selection of randomly occurring mutants in concert with inadequately treated infections. Dissemination of these difficult-to-treat bacteria throughout New York City and to at least four additional US cities has adverse implications for tuberculosis control in the 21st century.