Sixty rifampin (RIF)-resistant and 75 RIF-susceptible Mycobacterium tuberculosis isolates from ShandongProvince, China, were analyzed for rpoB gene mutations and genotyped. Mycobacterial interspersed repetitive unit (MIRU) genotype 223325173533 was overrepresented among RIF-resistant isolates. MIRU combined with IS6110 restriction fragment length polymorphism analysis as the second-line genotyping method may reflect epidemiologic links more reliably than each method alone.Tuberculosis (TB) remains a major public health concern in China, causing 150,000 deaths per year and making China second only to India in TB mortality (22). A significant challenge for TB control in China is the rapid dissemination and high prevalence of drug-resistant Mycobacterium tuberculosis (12). Rifampin (RIF), one of the principal first-line antituberculosis drugs, inhibits DNA-directed RNA synthesis of M. tuberculosis by binding to the  subunit of RNA polymerase (10). Mutations in the rpoB gene, encoding the  subunit of RNA polymerase, have been shown to be strongly associated with RIF-resistant phenotypes in multiple study populations (6,9,14,17). rpoB mutations are more likely segregated in an 81-bp region called the RIF resistance-determining region (RRDR). Because up to 90% of RIF-resistant strains carry RRDR mutations within codons 516, 526, and 531, these mutational "hotspots" are being used to rapidly identify RIF-resistant isolates (8,15).The Beijing family, a dominant M. tuberculosis genotype in China and the rest of Asia, has been associated with increased frequencies of drug resistance in some populations, but not in others (5). More-reliable genotyping strategies are required to refine the Beijing family and identify possible subgroups, which may explain the drug resistance scenario. With few exceptions (7, 13), there have been few investigations into the molecular characterization of RIF-resistant M. tuberculosis isolates from mainland China, reflecting a general lack of understanding of this important topic.In this study, 60 RIF-resistant and 75 RIF-susceptible M. tuberculosis isolates were collected through an M. tuberculosis sentinel surveillance network in Shandong Province, China (21). These isolates were obtained from different patients with no familial ties. Drug susceptibility testing was performed by the proportion method on Löwenstein-Jensen medium using the critical drug concentrations for RIF (40 g/ml), isoniazid (0.2 g/ml), streptomycin (4 g/ml), and ethambutol (EMB; 2 g/ml). The drug resistance profiles of 60 RIF-resistant isolates are shown in Table 1. Seventy-five M. tuberculosis isolates were susceptible to all first-line antituberculosis drugs.Extraction of M. tuberculosis genomic DNA was performed by standard methods (20). A 495-bp region of the rpoB gene including the RRDR was amplified by PCR with forward primer 5Ј GACGACATCGACCACTTC and reverse primer 5Ј GGTCAGGTACACGATCTC. PCR fragments were sequenced with an ABI 377 DNA sequencer (Applied Biosystems, Inc., Foster City, CA). The new alleles were co...