Discovery of novel DprE1 inhibitors via computational bioactivity fingerprints and structure-based virtual screening.

Decaprenylphosphoryl-ß-D-ribose oxidase (DprE1) plays important roles in the biosynthesis of mycobacterium cell wall. DprE1 inhibitors have shown great potentials in the development of new regimens for tuberculosis (TB) treatment. In this study, an integrated molecular modeling strategy, which combined computational bioactivity fingerprints and structure-based virtual screening, was employed to identify potential DprE1 inhibitors. Two lead compounds (B2 and H3) that could inhibit DprE1 and thus kill Mycobacterium smegmatis in vitro were identified. Moreover, compound H3 showed potent inhibitory activity against Mycobacterium tuberculosis in vitro (MICMtb = 1.25 µM) and low cytotoxicity against mouse embryo fibroblast NIH-3T3 cells. Our research provided an effective strategy to discover novel anti-TB lead compounds.

Detecting Mycobacterium tuberculosis complex and rifampicin resistance via a new rapid multienzyme isothermal point mutation assay.

Simple, rapid, and accurate detection of the Mycobacterium tuberculosis complex (MTBC) and drug resistance is critical for improving patient care and decreasing the spread of tuberculosis. To this end, we have developed a new simple and rapid molecular method, which combines multienzyme isothermal rapid amplification and a lateral flow strip, to detect MTBC and simultaneously detect rifampin (RIF) resistance. Our findings showed that it has sufficient sensitivity and specificity for discriminating 118 MTBC strains from 51 non-tuberculosis mycobacteria strains and 11 of the most common respiratory tract bacteria. Further, compared to drug susceptibility testing, the assay has a sensitivity, specificity, and accuracy of 54.1%, 100.0%, and 75.2%, respectively, for detection of RIF resistance. Some of the advantages of this assay are that no special instrumentation is required, a constant low temperature of 39 °C is sufficient for the reaction, the turnaround time is less than 20 min from the start of the reaction to read out and the result can be seen with the naked eye and does not require specialized training. These characteristics of the new assay make it particularly useful for detecting MTBC and RIF resistance in resource-limited settings.

Mutations within embCAB Are Associated with Variable Level of Ethambutol Resistance in Mycobacterium tuberculosis Isolates from China.

The EmbCAB proteins have been considered a target for ethambutol (EMB). Mutations in embCAB are known to confer most EMB resistance. However, the knowledge about the effects of embCAB mutations on the EMB resistance level and about the role of mutation-mutation interactions is limited in China. Here, we sequenced embCAB among 125 Mycobacterium tuberculosis isolates from China and quantified their EMB MICs by testing growth at 10 concentrations. Furthermore, a multivariate regression model was established to assess the effects of both individual mutations and multiple mutations. Our results revealed that in China, 82.6% of EMB-resistant isolates (71/86 isolates) harbored at least one mutation within embCAB Most of the mutations were located in the embB and embA upstream region. Several individual mutations and multiple mutations within this region contributed to the different levels of EMB resistance. Their effects were statistically significant. Additionally, there was an association between high-level EMB resistance and multiple mutations.

A Retrospective Study of Culture-confirmed Mycobacterial Infection among Hospitalized HIV-infected Patients in Beijing, China.

A retrospective analysis was performed in two major HIV/AIDS referral hospitals in Beijing to evaluate the prevalence of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacterial (NTM) infections in HIV-infected patients. A total of 627 patients' data were reviewed, and 102 (16.3%) patients were diagnosed with culture-confirmed mycobacterial infection, including 84 with MTB, 16 with NTM, and 2 with both MTB and NTM. The most frequent clinical complication by mycobacterial infection was pulmonary infection (48/102, 47.1%). The overall rates of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) were 11.9% and 3.4%, respectively. This study underlines the urgent need to intensify screening for mycobacteria coinfection with HIV and to prevent the spread of drug-resistant TB among HIV-infected patients.

Preliminary Study on Drug Susceptibility Profile and Resistance Mechanisms to Macrolides of Clinical Isolates of Non-tuberculous Mycobacteria from China.

OBJECTIVE: Macrolide susceptibility and drug resistance mechanisms of clinical non-tuberculous mycobacteria (NTM) isolates were preliminarily investigated for more accurate diagnosis and treatment of the infection in China. METHODS: Four macrolides, including clarithromycin (CLAR), azithromycin (AZM), roxithromycin (ROX), and erythromycin (ERY), were used to test the drug susceptibility of 310 clinical NTM isolates from six provinces of China with the broth microdilution method. Two resistance mechanisms, 23S rRNA and erm, were analyzed with nucleotide sequence analysis. RESULTS: Varied effectiveness of macrolides and species-specific resistance patterns were observed. Most Mycobacterium abscessus subsp. massiliense were susceptible and all M. fortuitum were highly resistant to macrolides. All the drugs, except for erythromycin, exhibited excellent activities against slow-growing mycobacteria, and drug resistance rates were below 22.2%. Only four highly resistant strains harbored 2,058/2,059 substitutions on rrl and none of other mutations were related to macrolide resistance. G2191A and T2221C on rrl were specific for the M. abscessus complex (MABC). Seven sites, G2140A, G2210C, C2217G, T2238C, T2322C, T2404C, and A2406G, were specifically carried by M. avium and M. intracellulare. Three sites, A2192G, T2358G, and A2636G, were observed only in M. fortuitum and one site G2152A was specific for M. gordonae. The genes erm(39) and erm(41) were detected in M. fortuitum and M. abscessus and inducible resistance was observed in relevant sequevar. CONCLUSION: The susceptibility profile of macrolides against NTM was demonstrated. The well-known macrolide resistance mechanisms, 23S rRNA and erm, failed to account for all resistant NTM isolates, and further studies are warranted to investigate macrolide resistance mechanisms in various NTM species.

First Report in China on the Identification and Drug Sensitivity of Mycobacterium elephantis Isolated from the Milk of a Cow with Mastitis.

OBJECTIVE: In this study, milk from a cow with mastitis was analyzed to determine the presence of mycobacterial infection. Milk quality and security problems pertaining to the safe consumption of dairy products were also discussed in this study. METHODS: Milk was preprocessed with 4% NaOH. Then, mycobacteria were isolated from the milk sample on L-J medium. The isolate was identified using multiple loci Polymerase Chain Reaction (PCR) and multi-locus sequence analysis with 16S rRNA, sodA, hsp65, and ITS genes. The drug sensitivity of the isolate to 27 antibiotics was tested through alamar blue assay. RESULTS: Smooth, moist, pale yellow colonies appeared on the L-J medium within a week after inoculation. Based on the results of multiple loci PCR analysis, the isolate was preliminarily identified as non-tuberculous mycobacteria. The 16S rRNA, SodA, hsp65, and ITS gene sequences of the isolate exhibited 99%, 99%, 99%, and 100% similarities, respectively, with those of the published reference strains of Mycobacterium elephantis (M. elephantis). The drug sensitivity results showed that the strain is resistant to isoniazid, p-aminosalicylic acid, and trimesulf but is sensitive to ofloxacin, rifampicin, amikacin, capreomycin, moxifloxacin, kanamycin, levofloxacin, cycloserine, ethambutol, streptomycin, tobramycin, rifabutin, ciprofloxacin, linezolid, cefoxitin, clarithromycin, and minocycline. CONCLUSION: To the best of our knowledge, this study is initially to report the isolation of M. elephantis from the milk of a cow with mastitis in China.

Evaluation of Six Recombinant Proteins for Serological Diagnosis of Lyme Borreliosis in China.

OBJECTIVE: In this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis (LB) and screened out the appropriate antigens to support the production of a Chinese clinical ELISA (enzyme-linked immunosorbent assay) kit for LB. METHODS: Six recombinant antigens, Fla B.g, OspC B.a, OspC B.g, P39 B.g, P83 B.g, and VlsE B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic (ROC) curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models. RESULTS: Two IgG (VlsE and OspC B.g) and two IgM (OspC B.g and OspC B.a) antigens were left by the logistic regression model screened. VlsE had the highest specificity for syphilis samples in the IgG test (87.7%, P<0.05). OspC B.g had the highest diagnostic value in the IgM test (AUC=0.871). Interactive effects between OspC B.a and Fla B.g could reduce the specificity of the ELISA. CONCLUSION: Three recombinant antigens, OspC B.g, OspC B.a, and VlsE B.a, were useful for ELISAs of LB. Additionally, the interaction between OspC B.a and Fla B.g should be examined in future research.

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients' serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions.

Analysis of embCAB mutations associated with ethambutol resistance in multidrug-resistant mycobacterium tuberculosis isolates from China.

Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(-16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306-497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples.

A set of universal loop-mediated isothermal amplification (LAMP) primers targeting the fla gene was designed to detect Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.l. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.l. in human serum samples.

Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization.

OBJECTIVE: A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). METHODS: 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. RESULTS: The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. CONCLUSION: Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

Molecular Characteristics and Drug Susceptibility of Mycobacterium tuberculosis Isolates from Patients Co-infected with Human Immunodeficiency Virus in Beijing, China.

70 clinical Mycobacterium tuberculosis strains isolated from AIDS patients in two HIV/AIDS referral hospitals in Beijing were used in this study. M. tuberculosis and non-tuberculosis mycobacterium (NTM) were identified by using multi-locus PCR. M. tuberculosis was genotyped by using 15-locus MIRU-VNTR technique and spoligotyping afterwards. Meanwhile, the drug susceptibilities of the strains to the four first-line anti TB drugs (rifampin, isoniazid, streptomycin, and ethambutol) and the four second-line anti-TB drugs (capreomycin, kanamycin, ofloxacin, and ethionanide) were tested with proportional method. In this study, M. tuberculosis and NTM strains isolated from AIDS patients with TB-like symptoms were identified and genotyping analysis indicated that Beijing genotype was the predominant genotype. In addition, the prevalence of drug-resistant TB, especially the prevalence of XDR-TB, was higher than that in TB patients without HIV infection.

Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Hunan, China.

To determine the prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan province, drug susceptibility testing and spoligotyping methods were performed among 171 M. tuberculosis isolates. In addition, the mutated characteristics of 12 loci, including katG, inhA, rpoB, rpsL, nucleotides 388 to 1084 of the rrs gene [rrs(388-1084)], embB, pncA, tlyA, eis, nucleotides 1158 to 1674 of the rrs gene [rrs(1158-1674)], gyrA, and gyrB, among drug-resistant isolates were also analyzed by DNA sequencing. Our results indicated that the prevalences of isoniazid (INH), rifampin (RIF), streptomycin (SM), ethambutol (EMB), pyrazinamide (PZA), capreomycin (CAP), kanamycin (KAN), amikacin (AKM), and ofloxacin (OFX) resistance in Hunan province were 35.7%, 26.9%, 20.5%, 9.9% 15.2%, 2.3%, 1.8%, 1.2%, and 10.5%, respectively. The previously treated patients presented significantly increased risks for developing drug resistance. The majority of M. tuberculosis isolates belonged to the Beijing family. Almost all the drug resistance results demonstrated no association with genotype. The most frequent mutations of drug-resistant isolates were katG codon 315 (katG315), inhA15, rpoB531, rpoB526, rpoB516, rpsL43, rrs514, embB306, pncA96, rrs1401, gyrA94, and gyrA90. These results contribute to the knowledge of the prevalence of drug resistance in Hunan province and also expand the molecular characteristics of drug resistance in China.

Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from China.

To investigate the molecular characterization of multidrug-resistant tuberculosis (MDR-TB) isolates from China and the association of specific mutations conferring drug resistance with strains of different genotypes, we performed spoligotyping and sequenced nine loci (katG, inhA, the oxyR-ahpC intergenic region, rpoB, tlyA, eis, rrs, gyrA, and gyrB) for 128 MDR-TB isolates. Our results showed that 108 isolates (84.4%) were Beijing family strains, 64 (59.3%) of which were identified as modern Beijing strains. Compared with the phenotypic data, the sensitivity and specificity of DNA sequencing were 89.1% and 100.0%, respectively, for isoniazid (INH) resistance, 93.8% and 100.0% for rifampin (RIF) resistance, 60.0% and 99.4% for capreomycin (CAP) resistance, 84.6% and 99.4% for kanamycin (KAN) resistance, and 90.0% and 100.0% for ofloxacin (OFX) resistance. The most prevalent mutations among the MDR-TB isolates were katG315, inhA15, rpoB531, -526, and -516, rrs1401, eis-10, and gyrA94, -90, and -91. Furthermore, there was no association between specific resistance-conferring mutations and the strain genotype. These findings will be helpful for the establishment of rapid molecular diagnostic methods to be implemented in China.

Fever with thrombocytopenia associated with a novel bunyavirus in China.

BACKGROUND: Heightened surveillance of acute febrile illness in China since 2009 has led to the identification of a severe fever with thrombocytopenia syndrome (SFTS) with an unknown cause. Infection with Anaplasma phagocytophilum has been suggested as a cause, but the pathogen has not been detected in most patients on laboratory testing. METHODS: We obtained blood samples from patients with the case definition of SFTS in six provinces in China. The blood samples were used to isolate the causal pathogen by inoculation of cell culture and for detection of viral RNA on polymerase-chain-reaction assay. The pathogen was characterized on electron microscopy and nucleic acid sequencing. We used enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and neutralization testing to analyze the level of virus-specific antibody in patients' serum samples. RESULTS: We isolated a novel virus, designated SFTS bunyavirus, from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction. RNA sequence analysis revealed that the virus was a newly identified member of the genus phlebovirus in the Bunyaviridae family. Electron-microscopical examination revealed virions with the morphologic characteristics of a bunyavirus. The presence of the virus was confirmed in 171 patients with SFTS from six provinces by detection of viral RNA, specific antibodies to the virus in blood, or both. Serologic assays showed a virus-specific immune response in all 35 pairs of serum samples collected from patients during the acute and convalescent phases of the illness. CONCLUSIONS: A novel phlebovirus was identified in patients with a life-threatening illness associated with fever and thrombocytopenia in China. (Funded by the China Mega-Project for Infectious Diseases and others.).

Establishment of multiple locus variable-number tandem repeat analysis assay for genotyping of Borrelia burgdorferi sensu lato detected in China.

OBJECTIVE: Human Lyme Borreliosis (LB), which is caused by Borrelia burgdorferi sensu lato (B. burgdorferi), has been identified as a major arthropod-borne infectious disease in China. We aimed to develop a multiple locus variable-number tandem repeat (VNTR) analysis (MLVA) assay for the genotyping of Borrelia burgdorferi strains detected in China. METHODS: B. garinii PBi complete 904.246 kb chromosome and two plasmids (cp26 and lp54) were screened by using Tandem Repeats Finder program for getting potential VNTR loci, the potential VNTR loci were analyzed and identified with PCR and the VNTR loci data were analyzed and MLVA clustering tree were constrcted by using the categorical coefficient and the unweighted pair-group method with arithmetic means (UPGMA). RESULTS: We identified 5 new VNTR loci through analyzing 47 potential VNTR loci. We used the MLVA protocol to analyse 101 B. burgdorferi strains detected in China and finally identified 51 unique genotypes in 4 major clusters including B. burgdorferi sensu stricto (B.b.s.s), B. garinii, B. afzelii, and B. valaisiana, consistent with the current MLSA phylogeny studies. The allele numbers of VNTR-1, VNTR-2, VNTR-3, VNTR-4, and VNTR-5 were 7, 3, 9, 7, and 6. The Hunter-Gaston index (HGI) of five VNTR loci were 0.79, 0.22, 0.77, 0.71, and 0.67, respectively. The combined HGI of five VNTR loci was 0.96. Clustering of the strains of Xinjiang, Inner Mongolia and Heilongjiang was confirmed, and this situation was consistent with the close geographical distribution of those provinces. CONCLUSION: The MLVA protocol esytablished in this study is easy and can show strains' phylogenetic relationships to distinguish the strains of Borrelia species. It is useful for further phylogenetic and epidemiological analyses of Borrelia strains.

Molecular characterization and drug-resistance of Mycobacterium tuberculosis strains in Xuzhou, China.

To understand the genetic diversity and drug resistance status of Mycobacterium tuberculosis (M. tuberculosis) circulating in Xuzhou of China, the spacer-oligonucleotide typing (Spoligotyping) and multi-loci VNTRs (variable number tandem repeats) analysis (MLVA) were utilized for the genotyping of the isolates. Drug susceptibility test (DST) was performed by the proportion method on the Lowenstein-Jensen (L-J) medium using isoniazid, rifampicin, ethambutol, and streptomycin. By Spoligotyping, 287 M. tuberculosis isolates were differentiated into 14 clusters. Then with 15-loci MLVA, these strains could be divided into 32 clusters, 228 genotypes. Of 15 VNTRs, 6 loci had the highly discriminatory powers, 6 loci presented moderate discrimination and 3 loci demonstrated less polymorphism. The DST results showed that 46 strains were resistant to at least one first-line anti-tuberculosis agent. There was a difference in the isoniazid resistance between Beijing and non-Beijing genotype strains. We concluded that the combination of Spoligotyping and 15 VNTR loci as the genotyping in our study was applicable for this region, the drug resistant isolates were identified, and the Beijing family was the most prevalent genotype in the rural counties of Xuzhou.

Optimization of pulse-field gel electrophoresis for Borrelia burgdorferi subtyping.

OBJECTIVE: To optimize the performance of Pulsed-Field Gel Electrophoresis (PFGE) for the comparison of inter-laboratory results and information exchange of Borrelia burgdorferi subtyping. METHODS: A panel of 34 strains of B. burgdorferi were used to optimize PFGE for subtyping. In order to optimize the electrophoretic parameters (EPs), all 34 strains of B. burgdorferi were analyzed using four EPs, yielding different Simpson diversity index (D) values and the epidemiological concordance was also evaluated. RESULTS: The EP of a switch time of 1 s to 25 s for 13 h and 1 s to 10 s for 6 h produced the highest D value and was declared to be optimal for MluI and SmaI PFGE of B. burgdorferi. MluI and SmaI were selected as the first and second restriction enzymes for PFGE subtyping of B. burgdorferi according to discrimination and consistency with epidemiological data. CONCLUSION: PFGE can be used as a valuable test for routine genospecies identification of B. burgdorferi.

A study of the technique of western blot for diagnosis of lyme disease caused by Borrelia afzelii in China.

OBJECTIVE: To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. METHODS: FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. RESULTS: Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively. CONCLUSION: Establishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China.

Novel species including Mycobacterium fukienense sp. is found from tuberculosis patients in Fujian Province, China, using phylogenetic analysis of Mycobacterium chelonae/abscessus complex.

OBJECTIVE: To identify the novel species 'Mycobacterium fukienense' sp. nov of Mycobacterium chelonae/abscessus complex from tuberculosis patients in Fujian Province, China. METHODS: Five of 27 clinical Mycobacterium isolates (Cls) were previously identified as M. chelonae/abscessus complex by sequencing the hsp65, rpoB, 16S-23S rRNA internal transcribed spacer region (its), recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobacterium. Clinical Mycobacterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed. RESULTS: The 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 16S-23S rRNA internal transcribed spacer region (its), sodA, and recA genes as compared with the M. abscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences. CONCLUSION: The novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. chelonea/abscessus complex.