Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

Mycobacterium vicinigordonae sp. nov., a slow-growing scotochromogenic species isolated from sputum.

A slow-growing, scotochromogenic mycobacterial strain (24T) was isolated from the sputum of a Chinese male human. Phylogenetic analysis using the 16S rRNA gene assigned strain 24T to the Mycobacterium gordonae complex, which includes Mycobacterium gordonae and Mycobacterium paragordonae. The phenotypic characteristics, unique mycolic acid profile and the results of phylogenetic analysis based on hsp65 and rpoB sequences strongly supported the taxonomic status of strain 24T as a representative of a species distinct from the other members of the M. gordonae complex. The genomic G+C content of strain 24T was 65.40mol%. Genomic comparisons showed that strain 24T and M. gordonae ATCC 14470T had an average nucleotide identity (ANI) value of 81.00 % and a DNA-DNA hybridization (DDH) value of 22.80 %, while the ANI and DDH values between strain 24Tand M. paragordonae 49 061T were 80.98 and 22.80 %, respectively. In terms of phylogenetic, phenotypic and chemotaxonomic features, strain 24T is distinguishable from its closest phylogenetic relatives and represents a novel species of the genus Mycobacterium, therefore the name Mycobacterium vicinigordonae sp. nov. is proposed. The type strain is 24T (=CMCC 93559T=DSM 105979T).

In Vitro Susceptibility Testing of GSK656 against Mycobacterium Species.

In this study, we aimed to assess the in vitro susceptibility to GSK656 among multiple mycobacterial species and to investigate the correlation between leucyl-tRNA synthetase (LeuRS) sequence variations and in vitro susceptibility to GSK656 among mycobacterial species. A total of 187 mycobacterial isolates, comprising 105 Mycobacterium tuberculosis isolates and 82 nontuberculous mycobacteria (NTM) isolates, were randomly selected for the determination of in vitro susceptibility. For M. tuberculosis, 102 of 105 isolates had MICs of ≤0.5 mg/liter, demonstrating a MIC50 of 0.063 mg/liter and a MIC90 of 0.25 mg/liter. An epidemiological cutoff value of 0.5 mg/liter was proposed for identification of GSK656-resistant M. tuberculosis strains. For NTM, the MIC50 and MIC90 values were >8.0 mg/liter for both Mycobacterium intracellulare and Mycobacterium avium In contrast, all Mycobacterium abscessus isolates had MICs of ≤0.25 mg/liter, yielding a MIC90 of 0.063 mg/liter. LeuRS from M. abscessus showed greater sequence similarity to M. tuberculosis LeuRS than to LeuRSs from M. avium and M. intracellulare Sequence alignment revealed 28 residues differing between LeuRSs from M. avium and M. intracellulare and LeuRSs from M. tuberculosis and M. abscessus; among them, 15 residues were in the drug binding domain. Structure modeling revealed that several different residues were close to the tRNA-LeuRS interface or the entrance of the drug-tRNA binding pocket. In conclusion, our data demonstrate significant species diversity in in vitro susceptibility to GSK656 among various mycobacterial species. GSK656 has potent efficacy against M. tuberculosis and M. abscessus, whereas inherent resistance was noted for M. intracellulare and M. avium.

Epidemiology of skeletal tuberculosis in Beijing, China: a 10-year retrospective analysis of data.

Skeletal tuberculosis (STB) is one of the most common forms of extrapulmonary tuberculosis; however, limited epidemiological data are available on this public health concern worldwide, especially in developing countries. The aims of this study were to analyze the epidemiological characteristics of STB cases and to identify risk factors associated with drug resistance among STB cases in China. We included STB inpatients at Beijing Chest Hospital from January 2009 through December 2018. The multiple demographic and clinical variables of patients were collected from the hospital's electronic patient record. In total, 3086 STB patients were hospitalized in Beijing Chest Hospital. Of these cases, 1988 (64.4%) were spinal TB cases, 991 (32.1%) were joint TB cases, and 107 (3.5%) were concurrent spinal-joint TB cases. The most frequent localization of the infections in joints included the knee (21.5%), hip (17.9%), and elbow (10.3%). For spinal TB, lumbar, thoracic, and cervical spinal TB were present in 51.7%, 40.6%, and 4.4% of cases, respectively. Positive cultures were reported in only 16.0% of STB cases. When patients aged ≥ 60 years old were used as the control group, youths (< 18 years old) were less likely to have spinal TB (aOR, 0.29; 95% CI, 0.21-0.41). The prevalence of MDR-TB was 12.5% among the STB cases, and more female cases were afflicted with drug-resistant STB than with drug-susceptible STB (aOR, 0.50; 95% CI, 0.27-0.94). In addition, patients aged < 18 years had significantly higher odds of having drug-resistant STB compared with those aged ≥ 60 years (aOR, 20.778; 95% CI, 4.49-96.149). In conclusion, our data demonstrate that spinal TB is the most frequent form of STB in China. The youths are less likely to have spinal TB compared with elderly patients, while the patients aged < 18 years have significantly higher odds of having drug-resistant STB than elderly patients.

External quality control of phenotypic drug susceptibility testing for Mycobacterium tuberculosis in China.

The aim of our study was to evaluate the performance of conventional drug susceptibility testing (DST) among the tuberculosis (TB)-specialized hospitals in China. A total of 40 hospitals participated in the external quality assurance program for assessment of DST results from each hospital. The sensitivity, specificity, and accuracy of DST were analyzed. The mean accuracy was 96.5% for isoniazid (INH), 95.8% for rifampin (RIF), 97.0% for ethambutol (EMB), 96.8% for ofloxacin (OFX), 97.1% for kanamycin (KAN), 96.1% for amikacin (AMK), and 93.6% for capreomycin (CAP), respectively. Of the 40 participating laboratories, 4 (10.0%) and 6 (15%) failed to achieve 90% accuracy for INH and RIF, respectively. In addition, six hospitals (15%) were confirmed as certified to provide reliable DST results for both first-line and second-line drugs. The certified proportion for DST dropped from 73.9% in the non-western region to 59.2% in the western region. The significant difference was observed in the certified proportion for first-line drugs between the western and non-western region (P = 0.013). Our results demonstrate that the quality of phenotypical DST is frequently unsatisfactory, with approximately one-third of participated laboratories failing to produce certified phenotypical DST results. In addition, the uncertified laboratories majorly come from the western region in China.

The effect of culture media dilution on recovery of rapidly growing mycobacteria.

Nontuberculous mycobacteria (NTM) are important environmental opportunistic pathogens of human and animals. Rapidly growing mycobacteria (RGM) are important emerging pathogens causing NTM infections. Unfortunately, the majority of microorganisms in the environment resist cultivation in the laboratory. The aim of the study was to investigate whether the nutrients in the medium have impact on the growth of RGM in vitro. We first assessed the growth rate of rapidly growing mycobacteria strains in broth medium with different dilutions, including M. abscessus, M. chelonae, and M. fortuitum. Our data demonstrate that the majority of M. abscessus, M. chelonae and M. fortuitum strains prefer to grow in nutrient-rich MH medium, whereas a small proportion of RGM strains grew faster in diluted culture medium. Our study identified that dilution culture has a different impact on recovery of various RGM strains.

Epidemiology of Extrapulmonary Tuberculosis among Inpatients, China, 2008-2017.

We investigated the epidemiology of extrapulmonary tuberculosis (TB) among patients admitted to Beijing Chest Hospital, Beijing, China, during January 2008-December 2017. Of 19,279 hospitalized TB patients, 33.4% (6,433) had extrapulmonary TB and 66.6% (12,846) had pulmonary TB. The most frequent forms of extrapulmonary TB observed were skeletal TB (41.1%) and pleural TB (26.0%). Younger, female patients from rural areas were more likely to have extrapulmonary TB. However, patients with diabetes mellitus were less likely to have extrapulmonary TB compared with patients without diabetes. A higher proportion of multidrug-resistant (MDR) TB was observed among patients with extrapulmonary TB than among patients with pulmonary TB. We observed a large increase in MDR TB, from 17.3% to 35.7%, for pleural TB cases. The increasing rate of drug resistance among extrapulmonary TB cases highlights the need for drug susceptibility testing and the formulation of more effective regimens for extrapulmonary TB treatment.

Change in prevalence and molecular characteristics of isoniazid-resistant tuberculosis over a 10-year period in China.

BACKGROUND: Isoniazid (INH) represents the cornerstone for the treatment of cases infected with Mycobacterium tuberculosis (MTB) strains. Several molecular mechanisms have been shown to be the major causes for INH resistance, while the dynamic change of mutations conferring INH resistance among MTB strains during the past decade is still unknown in China. METHODS: In this study, we carried out a comparative analysis of the INH minimal inhibitory concentration (MIC) distribution, and investigate the dynamic change of molecular characteristics among INH-resistant MTB strains between 2005 and 2015. RESULTS: The proportion of INH resistance (39.0%, 105/269) in 2015 was significantly higher than in 2005 (30.0%, 82/273; P = 0.03). Among 269 isolates collected in 2015, 76 (28.3%, 76/269) exhibited high-level INH-resistance (MIC≥32 mg/L), which was significantly higher than that in 2005 (20.5%, 56/273, P = 0.04). In addition, a significantly higher percentage of INH-resistant isolates carried inhA promoter mutations in 2015 (26.7%) versus that in 2005 (14.6%, P = 0.04), while no significant difference was observed in the rates of isolates containing katG mutations between 2005 (76.8%) and 2015 (70.5%, P = 0.33). Notably, the proportion of MTB isolates with inhA mutations (26.7%, 28/105) for patients who had previous exposure to protionamide (PTH) was higher than that for patients who had no previous exposure to PTH (21.4%, 6/28). CONCLUSIONS: In conclusion, our results demonstrated that the proportion of INH-resistant MTB isolates significantly increased during the last decade, which was mainly attributed to an increase of high-level INH-resistant MTB. In addition, prior exposure to PTH may be associated with the increased frequency of INH-resistant tuberculosis strains with inhA mutations in China.

In Vitro Activity of PBTZ169 against Multiple Mycobacterium Species.

In this study, we demonstrate that PBTZ169 exhibits significant differences in in vitro activity against multiple Mycobacterium species. The amino acid polymorphism at codon 387 of decaprenylphosphoryl-beta-d-ribose oxidase (DprE1) can be used as a surrogate marker for in vitro susceptibility to PBTZ169 in mycobacteria. In addition, the amino acid substitution at codon 154 in DprE1 may be associated with acquired resistance to PBTZ169 in the Mycobacterium fortuitum mutants.

A 10-Year Comparative Analysis Shows that Increasing Prevalence of Rifampin-Resistant Mycobacterium tuberculosis in China Is Associated with the Transmission of Strains Harboring Compensatory Mutations.

In this work, we conducted bacterial population profile studies to assess trends of rifampin (RIF) resistance of Mycobacterium tuberculosis isolates collected across China from 2005 to 2015. Totals of 273 and 269 randomly selected M. tuberculosis isolates from 2005 and 2015, respectively, were analyzed. The rates of RIF resistance (36.4%), isoniazid resistance (39.0%), and levofloxacin resistance (25.7%) in 2015 were significantly higher than those in 2005 (28.2%, 30.0%, and 15.4%, respectively; P < 0.05). Genotypic data revealed 256 (95.2%) Beijing-type isolates in 2015, a rate significantly higher than that in 2005 (86.4%) (P < 0.01). A higher proportion of mutations was identified within the rifampin resistance-determining region (RRDR) of rpoB in isolates from 2015 (99.0%) than in 2005 isolates (85.7%, P < 0.01). In addition, a significantly higher proportion of RIF-resistant isolates carrying compensatory mutations was observed in 2015 (31.6%) than in 2005 (7.8%). Notably, the great majority of these compensatory mutations (91.9%) were observed in isolates that harbored a mutation of codon 531 of the rpoB gene. In conclusion, our data demonstrate that resistance to RIF, isoniazid, and levofloxacin has become significantly more prevalent during the past decade. In addition, the prevalence of the Beijing genotype significantly increased from 2005 to 2015. Notably, a significantly increased frequency of strains with mutations in rpoC or rpoA is observed among those that have codon 531 mutations, which suggests that they may be compensatory and may play a role in facilitating transmission.

In Vitro Activity of Clofazimine against Nontuberculous Mycobacteria Isolated in Beijing, China.

Due to the natural resistance of nontuberculous mycobacteria (NTM) to many antibiotics, the treatment of diseases caused by NTM is often long-term but unsuccessful. The main goal of this study was to evaluate the in vitro susceptibilities to clofazimine of 209 isolates consisting of different NTM species isolated in Beijing, China. Furthermore, 47 reference strains were also tested, including 30 rapidly growing mycobacterium (RGM) species and 17 slowly growing mycobacterium (SGM) species. The potential molecular mechanism contributing to clofazimine resistance of NTM was investigated as well. Clofazimine exhibited excellent activity against both reference strains and clinical isolates of different SGM species, and most of the strains had MICs far below 1 µg/ml. Although the majority of the clinical isolates of Mycobacterium abscessus and Mycobacterium fortuitum had MICs higher than 2 µg/ml, 17 out of the 30 reference strains of different RGM species had MICs below 1 µg/ml in vitro According to the MIC distributions, the tentative epidemiological cutoff (ECOFF) values for Mycobacterium kansasii, Mycobacterium avium, and Mycobacterium intracellulare were defined at 0.5 µg/ml, 1 µg/ml, and 2 µg/ml, respectively. Intriguingly, single-direction cross-resistance between bedaquiline- and clofazimine (Cfz)-resistant isolates was observed among the tested NTM species. This study demonstrates that clofazimine had strong activity against most SGM species in vitro, as well as some RGM species. The data provide important insights into the possible clinical application of Cfz to treat NTM infections.

Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China.

Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

In Vitro Drug Susceptibility of Bedaquiline, Delamanid, Linezolid, Clofazimine, Moxifloxacin, and Gatifloxacin against Extensively Drug-Resistant Tuberculosis in Beijing, China.

Extensively drug-resistant tuberculosis (XDR-TB) is a deadly form of TB that can be incurable due to its extreme drug resistance. In this study, we aimed to explore the in vitro susceptibility to bedaquiline (BDQ), delamanid (DMD), linezolid (LZD), clofazimine (CLO), moxifloxacin (MFX), and gatifloxacin (GAT) of 90 XDR-TB strains isolated from patients in China. We also describe the genetic characteristics of XDR-TB isolates with acquired drug resistance. Resistance to MFX, GAT, LZD, CLO, DMD, and BDQ was found in 82 (91.1%), 76 (84.4%), 5 (5.6%), 5 (5.6%), 4 (4.4%), and 3 (3.3%) isolates among the XDR-TB strains, respectively. The most frequent mutations conferring fluoroquinolone resistance occurred in codon 94 of the gyrA gene (57.8%), and the strains with these mutations (69.2%) were associated with high-level MFX resistance compared to strains with mutations in codon 90 (25.0%) (P < 0.01). All 5 CLO-resistant isolates exhibited ≥4-fold upward shifts in the BDQ MIC, which were attributed to mutations of codons 53 (60.0%) and 157 (20.0%) in the Rv0678 gene. Additionally, mutation in codon 318 of the fbiC gene was identified as the sole mutation related to DMD resistance. In conclusion, our data demonstrate that the XDR-TB strains exhibit a strikingly high proportion of resistance to the current anti-TB drugs, whereas BDQ, DMD, LZD, and CLO exhibit excellent in vitro activity against XDR-TB in the National Clinical Center on TB of China. The extensive cross-resistance between OFX and later-generation fluoroquinolones indicates that MFX and GAT may have difficulty in producing the desired effect for XDR-TB patients.

Primary Clofazimine and Bedaquiline Resistance among Isolates from Patients with Multidrug-Resistant Tuberculosis.

Clofazimine has been repurposed for the treatment of tuberculosis, especially for multidrug-resistant tuberculosis (MDR-TB). To test the susceptibility to clofazimine of Mycobacterium tuberculosis clinical isolates, MICs of clofazimine were determined using the microplate alamarBlue assay (MABA) method for 80 drug-resistant isolates and 10 drug-susceptible isolates for comparison. For five clofazimine-resistant strains isolated from previously treated pre-extensively drug-resistant TB (pre-XDR-TB) and XDR-TB patients without prior exposure to clofazimine or bedaquiline, clofazimine MICs were ≥1.2 µg/ml. Four isolates with cross-resistance to bedaquiline had Rv0678 mutations. The other isolate with no resistance to bedaquiline had an Rv1979c mutation. This study adds to a recent study showing that 6.3% of MDR-TB patients without prior clofazimine or bedaquiline exposure harbored isolates with Rv0678 mutations, which raises concern that preexisting resistance to these drugs may be associated with prior TB treatment. Furthermore, we propose a tentative breakpoint of 1.2 µg/ml for clofazimine resistance using the MABA method. More-widespread surveillance and individualized testing for clofazimine and bedaquiline resistance, together with assessment of their clinical usage, especially among previously treated and MDR-TB patients, are warranted.

Gene cloning, expression and characterization of an exo-chitinase with high ß-glucanase activity from Aeromonas veronii B565.

Objective: We aimed to express and characterize biochemical properties of Chi92, a chitinase from Aeromonas veronii B565, and study its potential application as aquafeed supplement. Methods: The chitinase gene chi92 was cloned from A. veronii strain B565 and expressed in Pichia pastoris GS115. The recombinant chitinase (Chi92) was purified and characterized. Chi92 was supplemented in diets containing P. pastoris powder and fed to zebrafish for 14 days. By comparing with the control group, effect of Chi92 supplementation on growth, feed utilization, microvilli morphology, and disease resistance was investigated. Results: The complete gene sequence encoded a polypeptide with 864 amino acids. Chi92 exhibited optimal activity at pH 6.0 and 40℃, and was resistant to proteases and not substantially inhibited by metal ions. Chi92 had high chitinase activity (69.4 U/mL). The specific activity was 809.2 U/mg and 235.6 U/mg on colloidal chitin and ß-1,3-1,4-glucan, respectively. Thin-layer chromatography and electrospray ionization-coupled mass spectrometry revealed that N-diacetylglucosamine was the dominant product of Chi92 when colloidal chitin was used as substrate. Moreover, Chi92 showed advantages over other chitinases for degradation of yeast cell wall. Supplementation of Chi92 in diet containing yeast product significantly improved the intestine microvilli length and density of zebrafish after two weeks of feeding. Marginally improved growth performance, feed utilization, as well as disease resistance were also observed in the Chi92 supplement group. Conclusion: The pH stability, resistance against metal ions/chemical reagents/proteases, and high yeast cell wall degradation activity of Chi92 suggest its potential use as feed additive enzyme for warm water aquaculture.

High-yield production of a chitinase from Aeromonas veronii B565 as a potential feed supplement for warm-water aquaculture.

Chitin, present in crustacean shells, insects, and fungi, is the second most plentiful natural organic fiber after wood. To effectively use chitin in a cost-saving and environmentally friendly way in aquaculture, crustacean shells (e.g., shrimp-shell meal) are supplemented into aquafeed after degradation by chemical methods. Herein, we describe a chitinase from Aeromonas veronii B565, designated ChiB565, which potently degrades shrimp-shell chitin and resists proteolysis. We isolated recombinant ChiB565 of the expected molecular mass in large yield from Pichia pastoris. ChiB565 is optimally active at pH 5.0 and 50 °C and stable between pH 4.5 and 9.0 at 50 °C and below. Compared with the commercial chitinase C-6137, which cannot degrade shrimp-shell chitin, ChiB565 hydrolyzes shrimp-shell chitin in addition to colloidal chitin, powdered chitin, and ß-1,3-1,4-glucan. The optimal enzyme concentration and reaction time for in vitro degradation of 0.1 g of powdered shrimp shell are 30 U of ChiB565 and 3 h, respectively. A synergistic protein-release effect occurred when ChiB565 and trypsin were incubated in vitro with shrimp shells. Tilapia were fed an experimental diet containing 5% (w/w) shrimp bran and 16.2 U/kg ChiB565, which significantly improved growth and feed conversion compared with a control diet lacking ChiB565. Dietary ChiB565 enhanced nitrogen digestibility and downregulated intestinal IL-1ß expression. The immunologically relevant protective effects of dietary ChiB565 were also observed for 2 to 3 days following exposure to pathogenic Aeromonas hydrophila.

Biological interpretation of the sporadic sputum smear-positive-culture-negative outcome for patients with tuberculosis undertaking treatments.

Introduction: The objective of the study was to identify the causes of smear-positive-culture-negative (S+/C-) outcomes of patients with tuberculosis during the treatment course. Methods: A laboratory-based retrospective study was performed at the Beijing Chest Hospital in China. Within the study period, all patients with pulmonary tuberculosis (PTB) who undertook anti-TB treatments and yielded smear positive outcomes with simultaneous culture outcomes on sputa were considered. Patients were classified into three groups: (I) performed LJ medium culture only; (II) performed BACTEC MGIT960 liquid culture only; and (III) performed both LJ culture and MGIT960 culture. The S+/C- rates of each group were analyzed. The clinical medical records regarding patient category, follow-up bacteriologic examination data, and treatment response were investigated. Results: In total, 1,200 eligible patients were enrolled, and the overall S+/C- rate was 17.5% (210/1,200). Group I had obviously higher S+/C- rate (37%) than group II (18.5%) and group III (9.5%). When solid and liquid cultures were considered independently, the S+/C- outcome was observed more frequently in the solid culture group than in the liquid culture group (30.4%, 345/1,135 vs. 11.5%, 100/873; p < 0.001, χ2 = 102.64). Among the 102 S+/C- patients who had follow-up cultures performed, 35 (34.3%) had positive culture outcomes. Whereas among the 67 patients with follow-up information for more than 3 months but without supportive bacteriological evidence, 45 (67.2%, 45/67) had unfavorable prognosis (including relapse and unimproved conditions), and only 22 (32.8%, 22/67) patients had improved conditions. Compared with new cases, retreated cases produced S+/C- outcomes more frequently and had more chances to be cultivated bacilli successfully afterward. Conclusions: Among our patients, the sporadic smear positive and culture negative outcomes for sputa are more likely associated with the technical failures of culture than with dead bacilli, and this is especially noteworthy for LJ medium culture.

In vitro activity of tubercidin against Mycobacterium tuberculosis and nontuberculosis Mycobacteria.

Tubercidin is an adenosine analogue that has been shown to exhibit good activity against some tumours and parasites. In this study, the in vitro activity of tubercidin was evaluated against Mycobacterium tuberculosis (Mtb) and nontuberculosis Mycobacteria (NTM). For determining the MICs of tubercidin, 23 fully drug-sensitive (DS) Mtb strains, 33 multi-drug resistance tuberculosis (MDR-TB) strains, 29 pre-extensively drug-resistant tuberculosis (pre-XDR-TB) strains, 21 extensively drug-resistant tuberculosis (XDR-TB) strains, 17 rapidly growing mycobacteria (RGM) and nine slowly growing mycobacteria (SGM) references strains were tested by microplate-based Alamar Blue assay (MABA) method. The results indicate that tubercidin has high in vitro activity against some drug-resistance Mtb strains and NTM reference strains, which warrants further investigation on the actions of tubercidin and its derivatives as potential drugs for mycobacterial infections.

Analysis of Xpert MTB/RIF results in retested patients with very low initial bacterial loads: A retrospective study in China.

BACKGROUND: The Xpert MTB/RIF (Xpert) assay has been widely used to diagnose suspected active tuberculosis (TB) and rifampicin-resistant TB cases. Despite its excellent performance record, false-positive Xpert rifampicin (RIF) resistance results are obtained for specimens with extremely low bacterial loads. OBJECTIVE: We aimed to study the feasibility of repeat Xpert testing as a strategy for reducing the odds of obtaining false-positive results when testing paucibacillary TB patients. METHODS: We enrolled previously tested TB patients with very low initial bacterial loads from May 2016 to February 2022 for Xpert retesting. A total of 251 TB patients were retested using the Xpert assay. RESULTS: RIF resistance was noted in 65 (25.9 %) patients when tested by Xpert at initial diagnosis. Only 107 (42.6 %) of 251 patients tested positive for MTB when retested via Xpert. The majority (98.6 %) of RIF-susceptible cases were still susceptible to RIF when retested. Initial Xpert testing yielded 35 positive results for MTB in the RIF-resistant group, of whom 25 (71.4 %) still exhibited RIF resistance when retested. All culture-positive MTB isolates in the RIF-susceptible group were also RIF-susceptible by phenotypic DST. In the RIF-resistant group, 10 of 14 culture-positive MTB isolates exhibited RIF resistance, of which 4 isolates were deemed RIF-susceptible by phenotypic DST. The proportion of double mutations within the MTB rpoB RRDR sequence, as detected by hybridization of Xpert D and E probes, was significantly higher in the RIF-susceptible group than in the RIF-susceptible group. CONCLUSIONS: Our results demonstrated that initial RIF-susceptible results were more accurate than RIF-resistant results. Additionally, patients with double mutations that delayed probe D/E hybridization were more likely to have false-positive Xpert results. Our findings emphasize that repeat Xpert MTB/RIF testing is necessary for TB patients with extremely low bacterial loads who are at high risk for RIF-resistant TB.

Rv1453 is associated with clofazimine resistance in Mycobacterium tuberculosis.

Clofazimine (CFZ) has been repurposed for treating tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB). However, the mechanisms of resistance to clofazimine are poorly understood. We previously reported a mutation located in the intergenic region of Rv1453 that was linked to resistance to CFZ and demonstrated that an Rv1453 knockout resulted in an increased minimum inhibitory concentration (MIC) of CFZ. The current study aims to go back and describe in detail how the mutation was identified and further explore its association with CFZ resistance by testing additional 30 isolates. We investigated MICs of clofazimine against 100 clinical strains isolated from MDR-TB patients by microplate alamarBlue assay. Whole-genome sequencing (WGS) was performed on 11 clofazimine-resistant and 7 clofazimine-susceptible strains, including H37Rv. Among the 11 clofazimine-resistant mutants subjected to WGS, the rate of mutation in the intergenic region of the Rv1453 gene was 55% (6/11) in clofazimine-resistant strains. Among another 30 clofazimine-resistant clinical isolates, 27 had mutations in the intergenic region of the Rv1453 gene. A mutation in the Rv1453 gene associated with clofazimine resistance was identified, which shed light on the mechanisms of action and resistance of clofazimine. IMPORTANCE Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, especially the emergence of multidrug-resistant tuberculosis (MDR-TB) brings great distress to humans. Clofazimine (CFZ) plays an important role in the treatment of MDR-TB. To understand the underlying mechanism of clofazimine resistance better, in this study, we review and detail the findings of the mutation of intergenic region of Rv1453 and find additional evidence that this mutation is related to clofazimine resistance in 30 additional isolates. The significance of our research is to contribute to a comprehensive understanding of clofazimine-resistant mechanisms, which is critical for reducing the emergence of resistance and for anti-TB drug discovery.