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.

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.

The Homologous Gene of Chromosomal Virulence D (chvD) Presents High Resolution as a Novel Biomarker in Mycobacterium Species Identification.

Objective: To evaluate the resolution of chromosomal virulence D (chvD) as a novel marker for mycobacterial species identification. Methods: A segment of chvD (652 bp) was amplified by PCR from 63 mycobacterial reference strains, 163 nontuberculous mycobacterial clinical isolates, and 16 M. tuberculosis complex (MTBC) clinical isolates. A phylogenetic tree based on the reference strains was constructed by the neighbor-joining and IQ-tree methods. Comparative sequence analysis of the homologous chvD gene efficiently differentiated the species within the genus Mycobacterium. Slowly growing Mycobacterium (SGM) and rapidly growing Mycobacterium (RGM) were separated in the phylogenetic tree based on the chvD gene. Results: The sequence discrepancies were obvious between M. kansasii and M. gastri, M. chelonae and M. abscessus, and M. avium and M. intracellulare, none of which could be achieved by 16S ribosomal RNA (rRNA) homologous gene alignment. Furthermore, chvD manifested larger intraspecies diversity among members of M. intracellulare subspecies. A total of 174 of the 179 (97.21%) clinical isolates, consisting of 12 mycobacterial species, were identified correctly by chvD blast. Four M. abscessus subsp. abscessus were identified as M. abscessus subsp. bolletii by chvD. MTBC isolates were indistinguishable, because they showed 99.84%-100% homology. Conclusion: Homologous chvD is a promising gene marker for identifying mycobacterial species, and could be used for highly accurate species identification among mycobacteria.

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.

Bedaquiline resistance pattern in clofazimine-resistant clinical isolates of tuberculosis patients.

OBJECTIVES: Bedaquiline (BDQ) is a potent drug for treating drug-resistant tuberculosis (TB). Here, we analysed the resistance profiles of BDQ in CFZ-resistant clinical isolates and investigated the clinical risk factors of BDQ and CFZ cross/co-resistance. METHODS: The AlarmarBlue microplate assay was performed to determine the minimum inhibitory concentration (MIC) of the CFZ-resistant Mycobacterium tuberculosis (MTB) clinical isolates to CFZ and BDQ. The clinical characteristics of the respective patients were analysed to explore the possible risk factors of BDQ resistance. The drug-resistance-associated genes including Rv0678, Rv1979c, atpE, pepQ and Rv1453 were sequenced and analysed. RESULTS: A total of 72 clinical CFZ-resistant MTB isolates were collected; among these, half were identified as BDQ-resistant. The MIC value of BDQ closely correlated with CFZ (Spearman's q = 0.766, P < 0.005). Among the isolates with a MIC of CFZ ≥4 mg/L, 92.31% (12/13) were resistant to BDQ. Pre-XDR and exposure to BDQ or CFZ are the major risk factors for concurrent BDQ resistance. Among the 36 cross/co-resistant isolates, 50% (18/36) had mutations in Rv0678, 8.3% (3/36) had mutations in Rv0678+Rv1453, 5.6% (2/36) had mutations in Rv0678+Rv1979c, 2.8% (1/36) had mutations in Rv0678+Rv1979c+Rv1453, 2.8% (1/36) had mutations in atpE+Rv0678+Rv1453, 2.8% (1/36) had mutations in Rv1979c, and 27.7% (10/36) had no variations in the target genes. CONCLUSION: Nearly half of the CFZ-resistant isolates were still sensitive to BDQ, whereas this rate dramatically decreased among patients with pre-XDR TB or those who had been exposed to BDQ or CFZ.

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.

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.

Novel oxazolidinones harbor potent in vitro activity against the clinical isolates of multidrug-resistant Mycobacterium tuberculosis in China.

Objective: To investigate the in vitro activities of five oxazolidinones in parallel against the reference strains of different mycobacterial species and clinical isolates of Mycobacterium tuberculosis (Mtb), and shed light on the differences in the efficacy of these homolog drugs. Materials and methods: The minimum inhibitory concentrations (MICs) of linezolid, tedizolid, sutezolid, delpazolid, and contezolid against 16 mycobacterial reference strains and 69 M. tuberculosis clinical isolates, including 17 drug-susceptible isolates and 52 multidrug-resistant (MDR) isolates, were determined by microplate alamarBlue assay (MABA). The intracellular killing activities of contezolid and linezolid against Mtb H37Rv were compared. In addition, mutations in the linezolid resistance-related genes (rplC, rplD, and 23S rRNA) of the Mtb clinical isolates were also analyzed. Results: Tedizolid exhibited the strongest inhibitory activities against the reference strains of both rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM), among the tested oxazolidinones. In contrast, sutezolid only manifested potent activity against reference strains of SGM. Linezolid, delpazolid, and contezolid were less active against the non-tuberculous mycobacterial references. For the Mtb clinical isolates, the antimicrobial action was ranked as: sutezolid > tedizolid > contezolid and linezolid > delpazolid, whereas no difference between drug-sensitive and multiple drug-resistant isolates was observed. Notably, contezolid demonstrated obviously superior intracellular antimicrobial activity than linezolid. Few strains harbored mutations in rrl gene or rplD genes, although these strains had drug susceptible profiles to linezolid. Conclusion: Different oxazolidinones can have discrepant antimicrobial activity against different mycobacterial species, or have different manifestations out of cell or in cell. Understanding these differences would be helpful in choosing the appropriate drug in clinical practice.

Case report: A 9-year systematic treatment failure of a pulmonary tuberculosis patient.

Objective: To explore the reasons of failure in a case of pulmonary tuberculosis (PTB) after 9 years systematic treatment. Methods: We extracted the patients' treatment history, drug susceptibility testing (DST), Computed tomography (CT) images, and sequenced the isolated strains by whole gene sequencing (WGS). Results: Although most results of the phenotypical DSTs were consistent with the genotype DST, the occurrence of gene resistance to amikacin (AMK), capreomycin (CAP), moxifloxacin (MFX) was earlier than the phenotypical DST. Based on the continuously reversed results of phenotypical DSTs, CT images in different stages and WGS, it can be confirmed that the patient was infected with two different strains of Mycobacterium tuberculosis (M.TB). Moreover, severe cavities may be another factor leading to treatment failure. Conclusion: Given the suggestive effect of genotype DST is earlier than the phenotypical DST, so genotype DST can play a better guiding role in patients with MDR-TB. Additionally, for patients who have not been cured for a long time, medication should be more cautious and the role of WGS in drug resistance surveillance should be fully utilized.

Doubled Nontuberculous Mycobacteria Isolation as a Consequence of Changes in the Diagnosis Algorithm.

Background: Nontuberculous mycobacteria (NTM) and their associated diseases remain neglected. Through minor modifications in our diagnostic algorithm, we observed an unexpected higher number of cultivable NTM isolates. Therefore, a retrospective study was performed thoroughly to investigate the effect of changed laboratory procedures on NTM isolation in a specialized tuberculosis hospital. Methods: NTM isolation rates and composition of NTM species were compared for the two diagnostic algorithms: (1) by using traditional p-nitrobenzoic acid (PNB) selective medium as a preliminary test to identify NTM isolates among the positive cultures (procedure I) and (2) by using the MPT64 antigen detection method to distinguish between Mycobacterium tuberculosis complex (MTBC) isolates and possible NTM isolates after a positive MGIT960 liquid culture (procedure II). Results: The NTM isolation rate in procedure II was significantly higher than the procedure I (18.08% vs 9.71%; P<0.001). A noticeable increase in the ratio of NTM isolates among the identified mycobacteria was observed over the studied years (ie, from 58.18% in 2019 to 72.93% in 2021), which indicated a more precise prescription of species identification test after prompt information was provided in procedure II. In addition, the consistency of the identified species using multiple specimens from the same patient did not present a significant difference between the procedures. Conclusion: According to our study, NTM infection might be far more underestimated than it is. A diagnostic procedure combining MGIT960 culture and MPT64 antigen detection could timely and easily identify clues of NTM isolates and improve the diagnosis of NTM infections.

Reevaluating Rifampicin Breakpoint Concentrations for Mycobacterium tuberculosis Isolates with Disputed rpoB Mutations and Discordant Susceptibility Phenotypes.

In this study, rifampicin resistance breakpoints based on MICs of disrupted rpoB mutants of Mycobacterium tuberculosis (MTB) were explored using the Mycobacteria Growth Indicator Tube (MGIT) system and microplate alamarBlue assay (MABA). Sixty-one MTB isolates with disputed low-level rifampicin resistance-associated rpoB mutations and 40 RIF-susceptible wild-type isolates were included. Among the 61 resistant isolates, 25 (41.0%) had MICs ≥2.0 mg/L via MABA, while 16 (26.2%) were identified as RIF resistant via MGIT. Epidemiological cut-off (ECOFF) values obtained using MABA and MGIT were 0.25 and 0.125 mg/L, respectively. Based on 0.125 mg/L as a tentative critical concentration (CC), MABA RIF resistance-detection sensitivity was 93.4%, prompting the reduction of the MGIT CC to 0.125 mg/L, given that only a single isolate (1.6%) with the borderline mutation would be misclassified as susceptible to RIF based on this CC. Based on DNA sequencing of RRDR as the gold standard, the diagnostic accuracy of MGIT (99.0%) was significantly higher than that of MABA (91.1%). MICs of Leu511Pro mutant isolates were negatively correlated with time to liquid culture positivity (TTP) in our analysis (R = 0.957, P < 0.01). In conclusion, our results demonstrated missed detection of a high proportion of rifampicin-resistant isolates based on the WHO-endorsed CC. Such missed detections would be avoided by reducing the optimal MGIT RIF CC to 0.125 mg/L. In addition, MGIT based on reduced CC outperformed MABA in detecting borderline RIF resistance, with MABA MIC results obtained for isolates with the same mutation correlating with MTB growth rate. IMPORTANCE Tuberculosis (TB) is still one of the world's leading infectious disease killers. The early and accurate diagnosis of RIF resistance is necessary to deliver timely and appropriate treatment for TB patients and improve their clinical outcome. Actually, a proportion of MTB isolates with disputed rpoB mutations present a diagnostic dilemma between Xpert and phenotypical drug susceptibility testing (pDST). Recently, WHO reported a pragmatic approach by lowering critical concentration (CC) to boost sensitivity of resistance detection of pDST. Therefore, a detailed analysis of the association between RIF susceptibility and disrupted mutations within rpoB gene would lay a foundation to assess the diagnostic accuracy of pDST with lowering RIF CC. In this study, we aim to determine the MICs of MTB isolates with disrupted mutations by MGIT and microplate alamarBlue assay (MABA). We also aimed to determine the optimal breakpoints for MTB isolates with these mutations.

Molecular Characteristic of Both Levofloxacin and Moxifloxacin Resistance in Mycobacterium tuberculosis from Individuals Diagnosed with Preextensive Drug-Resistant Tuberculosis.

Aim: Fluoroquinolones (FQs) are the cornerstone in treating drug-resistant tuberculosis (TB); the prevalence of TB among the population is diverse in different regions, understanding the relationship between resistance pattern and molecular characteristic of FQs in preextensive drug-resistant (pre-XDR) clinical isolates is limited in China. Methods: A total of 141 pre-XDR clinical isolates from different individuals stored at the National Clinical Centre were collected from the Beijing Chest Hospital, minimal inhibitory concentrations of levofloxacin (Lfx) and moxifloxacin (Mfx) as well as sequences of quinolone-resistant determining regions in gyrA and gyrB genes were examined. Results: One hundred twelve pre-XDR clinical isolates were resistant to both Lfx and Mfx, molecular analyses showed that 87.50%, 0.89%, and 6.25% of the pre-XDR clinical isolates harbored FQ resistance mutations in gyrA, gyrB, and in both. We found five amino acid mutation positions in gyrA and four in gyrB, The mutation position in gyrA included codons 94, 91, 90, 88, and 74, and in gyrB included codons 504, 500, 512, and 501. Codon 94 of gyrA was the most prevalent mutation (83.04%), containing the Asp amino acid substitution with Gly (50.89%), Asn (15.17%), Ala (8.93%), Tyr (6.25%), and His (1.79%). Conclusions: The mutations of gyrA were most common and the frequency of Asp94Gly was the highest in pre-XDR clinical isolates in Beijing, China. The mutations at codon 94 significantly contributed to the resistance to both Lfx and Mfx in pre-XDR clinical isolates and may cause a high resistance level.

Distinguishing Relapse From Reinfection With Whole-Genome Sequencing in Recurrent Pulmonary Tuberculosis: A Retrospective Cohort Study in Beijing, China.

Background: Tuberculosis recurrence is still a major problem for the control of tuberculosis, and the cause of the recurrence is still unclear. Methods: We retrospectively recruited 68 pairs of samples of Mycobacterium tuberculosis (MTB) from recurrent TB cases in Beijing Chest Hospital between January 2008 and December 2019. The whole-genome sequencing was conducted to analyze single-nucleotide polymorphism (SNP) and to identify whether recurrent disease was due to relapse or reinfection. The BACTEC MGIT was performed to compare differences in drug susceptibility profiles between two episodes. Results: 62 (91.2%) out of 68 confirmed recurrence were due to relapse, whereas the remaining six (8.8%) were due to reinfection. And there was a strong association between earlier relapse and underlying chronic diseases. In addition, the MTB isolates from non-diabetic patients had a higher mutation rate than those from diabetic patients. A community transmission was also identified in our cohort. Levofloxacin resistance was the most frequently observed drug resistance for 12.9% relapse cases. Conclusion: The relapse of a previous episode in Beijing. The underlying chronic diseases are associated with an earlier TB relapse. MTB isolates were more prone to develop levofloxacin resistance than moxifloxacin resistance after FQ exposure. The patients at high-risk for relapses deserve more careful investigation.

In vitro Antimicrobial Activity Comparison of Linezolid, Tedizolid, Sutezolid and Delpazolid Against Slowly Growing Mycobacteria Isolated in Beijing, China.

BACKGROUND: The antimicrobial activities of some new oxazolidinones against slowly growing mycobacteria (SGM) have never been well evaluated. METHODS: We evaluate the in vitro susceptibility of 20 reference strains and 157 clinical isolates, pertaining different SGM species, against four oxazolidinones, ie, delpazolid, sutezolid, tedizolid and linezolid. In addition, the association of linezolid resistance and mutations in 23srRNA, rplC, rplD were also tested. RESULTS: Sutezolid presented the strongest antimicrobial activity against the clinical isolates of M. intracellulare than the other oxazolidinones, with MIC50 at 2 µg/mL and MIC90 at 4 µg/mL. MICs of sutezolid were usually 4- to 8-fold lower than these of linezolid against M. intracellulare and M. avium. The tested isolates of M. kansasii were susceptible to all of the four oxazolidinones. According to the multiple sequence alignment, novel 23srRNA mutations (A2267C and A2266G) in M. intracellulare and rplD mutations (Thr147Ala) in M. avium were identified in this study which have plausible involvement in rendering resistance against linezolid. CONCLUSION: This study showed that sutezolid harbors the strongest inhibitory activity against M. intracellulare, M. avium and M. kansasii in vitro, which provided important insights on the potential clinical application of oxazolidinones for treating SGM infections.

Comparison of the in vitro activity of linezolid, tedizolid, sutezolid, and delpazolid against rapidly growing mycobacteria isolated in Beijing, China.

BACKGROUND: The natural resistance of rapidly growing mycobacteria (RGM) to multiple antibiotics renders the treatment of the infections caused less successful. The objective of this study was to evaluate the in vitro susceptibilities of four oxazolidinones against different RGM species. METHODS: The microplate alamarBlue assay was performed to identify the minimum inhibitory concentrations (MICs) of four oxazolidinones - delpazolid, sutezolid, tedizolid, and linezolid - for 32 reference strains and 115 clinical strains of different RGM species. The MIC breakpoint concentration was defined as 16 µg/ml for linezolid. Next, the gene fragments associated with oxazolidinone resistance were amplified and sequenced, and mutations were defined in contrast with the sequences of the reference strains. RESULTS: Tedizolid showed the strongest inhibitory activity against the Mycobacterium abscessus isolates. Delpazolid exhibited better antimicrobial activity against the Mycobacterium fortuitum isolates when compared to linezolid, with 4-fold lower MIC values. The protein alignment and structure-based analysis showed that there might be no correlation between oxazolidinone resistance and mutations in the rplC, rplD, and 23S rRNA genes in the tested RGM. CONCLUSIONS: Tedizolid had the strongest inhibitory activity against M. abscessus in vitro, while delpazolid presented the best inhibitory activity against M. fortuitum. This provides important insights into the potential clinical application of oxazolidinones to treat RGM infections.

Dependence of Xpert MTB/RIF Accuracy for Detecting Rifampin Resistance in Bronchoalveolar Lavage Fluid on Bacterial Load: A Retrospective Study in Beijing, China.

INTRODUCTION: We assessed the effect of Mycobacterium tuberculosis (MTB) bacterial load on Xpert MTB/RIF accuracy for detection of rifampicin (RIF)-resistant MTB in bronchoalveolar lavage fluid (BALF) specimens obtained at a national tuberculosis (TB) specialized hospital in Beijing, China. METHODS: A retrospective study was conducted at Beijing Chest Hospital. Patients with symptoms suggestive of pulmonary TB who provided BALF specimens for routine MTB detection between June 2019 and July 2020 were enrolled in the study. Chi-square test and Student's t-test were used to compare results across groups stratified according to BALF bacterial load. RESULTS: In total, 1125 patients with positive Xpert results who were enrolled in final analysis, 263 provided BALF specimens that tested positive for RIF-resistant MTB via Xpert MTB/RIF. The RIF-resistance rate of specimens with very low MTB bacterial load was 30.9%, a resistance rate significantly greater than rates obtained for groups with high (25.0%), medium (17.3%) and low (19.2%) MTB loads (P<0.01). Notably, false-positive results obtained for the very low bacterial load group led to markedly reduced positive predictive value of Xpert MTB/RIF to provide correct RIF-resistance predictions for that group (67.1%, 95% CI: 56.1%-78.1%5) relative to the predictive value obtained for all other groups combined (about 90%, P<0.05). Sanger sequencing data obtained for 20 (32.8%) MTB isolates deemed RIF-resistant via Xpert (Probe E) lacked rpoB RRDR mutations. Meanwhile, of another group of 23 isolates deemed RIF-susceptible via DST but RIF-resistant via Xpert MTB/RIF, 20 isolate sequences (87.0%) lacked rpoB RRDR mutations, while sequences of the remaining 3 isolates harbored single rpoB RRDR mutations predicted to cause amino acid substitutions. CONCLUSION: Xpert MTB/RIF assay performed alarmingly poorly when used to detect RIF-resistant MTB in BALF specimens with very low bacterial loads. A high rate of Xpert probe E hybridization failure was the main driver of false-positive RIF-resistant results.

Rarity of rpoB Mutations Outside the Rifampicin Resistance-Determining Region of Mycobacterium tuberculosis Isolates from Patients Responding Poorly to First-Line Tuberculosis Regimens in Beijing, China: A Retrospective Study.

BACKGROUND: Early and accurate diagnosis of rifampicin (RIF)-resistant Mycobacterium tuberculosis (MTB) is essential for controlling community spread of drug-resistant tuberculosis (TB). In order to discover mutations residing outside the rifampicin resistance-determining region (RRDR) of the MTB rpoB gene, we conducted this retrospective study. METHODS: We retrospectively screened patient records to obtain Xpert MTB/RIF assay results for patients who received care at the Beijing Chest Hospital from 2016 to 2019 in order to identify subjects who met study selection criteria. Stored frozen patient isolates were cultured, harvested, and then subjected to drug susceptibility testing. Concurrently, entire rpoB gene DNA of each isolate was amplified and then sequenced to reveal rpoB mutations. RESULTS: Overall, 104 RIF-susceptible tuberculosis patients who were tested using the Xpert MTB/RIF assay also had poor first-line regimen treatment responses. Isolates obtained from these cases included 101 MTB isolates that possessed wild-type rpoB allelic profiles, as demonstrated using Sanger sequencing. However, sequences from the other three isolates confirmed that rpoB of one isolate harbored a mutation encoding the amino acid substitution Ile491Phe and that rpoB genes of two isolates contained a mutation encoding the amino acid substitution Ser450Leu. CONCLUSION: Our data demonstrated that mutations found outside the RRDR of MTB rpoB are rare in Beijing, China, indicating that World Health Organization-approved molecular diagnostics are generally suitable for diagnosing RIF resistance.

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).

Low Rate of Acquired Linezolid Resistance in Multidrug-Resistant Tuberculosis Treated With Bedaquiline-Linezolid Combination.

In this retrospective study in China, we aimed to: (1) determine the prevalence of linezolid (LZD) resistance among multidrug-resistant tuberculosis (MDR-TB)-infected patients; (2) monitor for dynamic LZD susceptibility changes during anti-TB treatment; and (3) explore molecular mechanisms conferring LZD resistance. A total of 277 MDR-TB patients receiving bedaquiline (BDQ)-containing regimens in 13 TB specialized hospitals across China were enrolled in the study. LZD and BDQ susceptibility rates were determined using the minimum inhibitory concentration (MIC) method, then DNA sequences of patient isolates were analyzed using Sanger sequencing to detect mutations conferring LZD resistance. Of 277 patients in our cohort, 115 (115/277, 41.5%) with prior LZD exposure yielded 19 (19/277, 6.9%) isolates exhibiting LZD resistance. The LZD resistance rate of LZD-exposed group isolates significantly exceeded the corresponding rate for non-exposed group isolates (P = 0.047). Genetic mutations were observed in 10 (52.6%, 10/19) LZD-resistant isolates, of which a Cys154Arg (36.8%, 7/19) substitution within ribosomal protein L3 was most prevalent. Analysis of sequential positive cultures obtained from 81 LZD-treated patients indicated that cultured organisms obtained from most patients (85.2%, 69/81) retained original LZD MIC values; however, organisms cultured later from two patients exhibited significantly increased MIC values that were attributed to the rplC substitution T460C. Overall, LZD resistance was detected in 6.9% of patients of an MDR-TB cohort in China. Low rate of acquired LZD resistance was noted in MDR-TB treated with BDQ-LZD combination.

Comparative in vitro susceptibility of a novel fluoroquinolone antibiotic candidate WFQ-228, levofloxacin, and moxifloxacin against Mycobacterium tuberculosis.

OBJECTIVE: WFQ-228 is a novel developed fluoroquinolone (FQ) displaying potent antimicrobial activity against various clinical isolates of pathogens, including FQ-resistant isolates. The aim was to comparatively analyze in vitro susceptibilities of WFQ-228, levofloxacin (LFX), and moxifloxacin (MFX) against Mycobacterium tuberculosis (MTB) isolates, especially with gyrA mutations. METHODS: We selected a panel of 75 MTB isolates, consisting of 25 FQ-susceptible and 50 FQ-resistant isolates determined by conventional drug susceptibility testing. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of FQs to MTB isolates were assessed. RESULTS: MFX exhibited the most potent activity against FQ-susceptible MTB, demonstrating a MIC50 of 0.031 mg/L, which was lower than that of LFX and WFQ-228. Against FQ-resistant MTB isolates, the MIC50 of WFQ-228 was higher than that of MFX but lower than that of LFX. For WFQ-228, there was a significant overlap existing in the MIC distributions between the probable susceptible (PS) and probable resistant (PR) groups. Six out of 50 PR isolates were classified as susceptible based on a proposed critical concentration (CC) of 0.5 mg/L, yielding a poor sensitivity of 88.0%. These discordant isolates had GyrA substitution in Ala90Val, Ser91Pro, and Asp94Tyr. Additionally, MFX exhibited bactericidal activity against MTB isolates without gyrA mutations, which was significantly higher than that of isolates with gyrA mutations. CONCLUSION: WFQ-228 is more efficacious than LFX in isolates with specific mutations conferring low-level FQ resistance. The bactericidal effect is noted more frequently in FQ-susceptible isolates than FQ-resistant isolates for MFX.