Compensatory Mutations of Rifampin Resistance Are Associated with Transmission of Multidrug-Resistant Mycobacterium tuberculosis Beijing Genotype Strains in China

ABSTRACTMycobacterium tuberculosiscan acquire resistance to rifampin (RIF) through mutations in therpoBgene. This is usually accompanied by a fitness cost, which, however, can be mitigated by secondary mutations in therpoAorrpoCgene. This study aimed to identifyrpoAandrpoCmutations in clinicalM. tuberculosisisolates in northern China in order to clarify their role in the transmission of drug-resistant tuberculosis (TB). The study collection included 332 RIF-resistant and 178 RIF-susceptible isolates. The majority of isolates belonged to the Beijing genotype (95.3%, 486/510 isolates), and no mutation was found inrpoAorrpoCof the non-Beijing genotype strains. Among the Beijing genotype strains, 27.8% (89/320) of RIF-resistant isolates harbored nonsynonymous mutations in therpoA(n= 6) orrpoC(n= 83) gene. The proportion ofrpoCmutations was significantly higher in new cases (P= 0.023) and in strains with therpoBS531L mutation (P< 0.001). In addition, multidrug-resistant (MDR) strains withrpoCmutations were significantly associated with 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat clustering (P= 0.016). In summary, we believe that these findings indirectly suggest an epistatic interaction of particular mutations related to RIF resistance and strain fitness and, consequently, the role of such mutations in the spread of MDRM. tuberculosisstrains.

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Tuberculous Spondylitis in Russia and Prominent Role of Multidrug-Resistant Clone Mycobacterium tuberculosis Beijing B0/W148

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04221-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2349-2357 ◽

Cited By ~ 14

Author(s):

Anna Vyazovaya ◽

Igor Mokrousov ◽

Natalia Solovieva ◽

Alexander Mushkin ◽

Olga Manicheva ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Spinal Tuberculosis ◽

Variable Number Tandem Repeat ◽

Molecular Study ◽

Multidrug Resistant ◽

Variable Number ◽

Beijing Genotype ◽

Tuberculous Spondylitis ◽

Content Type

ABSTRACTExtrapulmonary and, in particular, spinal tuberculosis (TB) constitutes a minor but significant part of the total TB incidence. In spite of this, almost no studies on the genetic diversity and drug resistance ofMycobacterium tuberculosisisolates from spinal TB patients have been published to date. Here, we report results of the first Russian and globally largest molecular study ofM. tuberculosisisolates recovered from patients with tuberculous spondylitis (TBS). The majority of 107 isolates were assigned to the Beijing genotype (n= 80); the other main families were T (n= 11), Ural (n= 7), and LAM (n= 4). Multidrug resistance (MDR) was more frequently found among Beijing (90.5%) and, intriguingly, Ural (71.4%) isolates than other genotypes (5%;P< 0.001). The extremely drug-resistant (XDR) phenotype was exclusively found in the Beijing isolates (n= 7). A notable prevalence of therpoB531andkatG315mutations in Beijing strains that were similarly high in both TBS (this study) and published pulmonary TB (PTB) samples from Russia shows that TBS and PTB Beijing strains follow the same paradigm of acquisition of rifampin (RIF) and isoniazid (INH) resistance. The 24-locus mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) subtyping of 80 Beijing isolates further discriminated them into 24 types (Hunter Gaston index [HGI] = 0.83); types 100-32 and 94-32 represented the largest groups. A genotype of Russian successful clone B0/W148 was identified in 30 of 80 Beijing isolates. In conclusion, this study highlighted a crucial impact of the Beijing genotype and the especially prominent role of its MDR-associated successful clone B0/W148 cluster in the development of spinal MDR-TB in Russian patients.

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First insight into the genotype and molecular epidemiology of MDR-TB isolates from Chongqing Municipality of China using 24-loci MIRU-VNTR technique

10.21203/rs.2.10166/v1 ◽

2019 ◽

Author(s):

Lei Xu ◽

Yan Hu ◽

Tongxin Li ◽

Chun Yang ◽

Yonglin He ◽

...

Keyword(s):

Variable Number Tandem Repeat ◽

Melting Curve ◽

Multidrug Resistant ◽

Variable Number ◽

Beijing Genotype ◽

Molecular Characteristics ◽

Chongqing Municipality ◽

Resistant Tuberculosis ◽

Mdr Tb ◽

High Discriminatory Power

Abstract Background: Multidrug-resistant tuberculosis (MDR-TB) has become a great threat to TB control around the world. In 2017, there were 889,000 new tuberculosis cases in China, and 31.1% were MDR/RR-TB (TB resistant to rifampicin). This study aims to explore the the molecular characteristics and factors associated with infection among different genotype MDR strains in Chongqing, China. Methods: All the 230 MDR-TB isolates were genotyped using 24-loci mycobacterial interspersed repetitive unite variable number tandem repeat (MIRU-VNTR) method and multiplex real-time PCR melting curve assay of Rv2952 gene and mutT2 gene. Polymorphism and clustering analysis of each locus was carried out by BioNumerics Version 5.0. Results: By genotyping, 83.0% (191/230) of the MDR-TB isolates were Beijing strains, among which 41.9% from the ancient Beijing genotype and 58.1% from the modern Beijing genotype. Based on 24-loci, the 230 MDR isolates were classified into 208 genotypes, among which 38 isolates belonged to 16 clusters. The clustering rate was 16.5%. The percentages of SM-resistant and EMB-resistant in Beijing genotype were significantly higher than those in non-Beijing genotype (P < 0.01). The Beijing genotype had a significantly high risk to be clustered than non-Beijing genotype (p < 0.01). The percentages of SM-resistant isolates in clustered group were significantly higher than non-clustered group (P < 0.01). According to 24-loci, the HGDI was 0.9988, and five loci (Qub11b, Qub26, Mtub21, MIRU26, Mtub04) have high discriminatory power (HGDI > 0.6), while 7 loci (MIRU4, MIRU23, Mtub34, MIRU20, Mtub29, MIRU2, MIRU24) showed negligible diversity (HGDI < 0.1). The percentage of clustered rate showed no difference between 24-loci and 15-loci (p = 0.19). Conclusion: Among MDR isolates in Chongqing China, Beijing genotype is more likely to be drug resistant and clustered, and SM-resistant isolates are more likely to be clustered that may related to the MDR epidemic. Although 24-loci had a high resolution of genotype, some new loci should be found to replace the poor diversity loci in 24-loci and additional analysis of specific sublineage of Beijing genotype is needed in order to better understand the relations between the molecular characteristics of strains and MDR-TB epidemic.

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Prevalence and Molecular Characterization of Fluoroquinolone-Resistant Mycobacterium tuberculosis Isolates in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01228-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 364-369 ◽

Cited By ~ 45

Author(s):

Zhijian Zhang ◽

Jie Lu ◽

Yufeng Wang ◽

Yu Pang ◽

Yanlin Zhao

Keyword(s):

Mycobacterium Tuberculosis ◽

Molecular Characterization ◽

Multidrug Resistant ◽

Beijing Genotype ◽

Content Type ◽

Treatment Regimens ◽

Resistant Tuberculosis ◽

Detection Of Mutations ◽

High Level

ABSTRACTChina is one of the countries with the highest burdens of multidrug-resistant (MDR) and fluoroquinolone (FQ)-resistant tuberculosis (TB) globally. Nevertheless, knowledge about the prevalence and molecular characterization of FQ-resistantMycobacterium tuberculosisisolates from this region remains scant. In this study, 138M. tuberculosisisolates determined by the agar proportion susceptibility method to be resistant to ofloxacin (OFX) were enrolled from a national drug resistance survey of China. All these strains were tested for susceptibility to ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, and sparfloxacin using liquid Middlebrook 7H9 medium. The entiregyrAandgyrBgenes conferring FQ resistance were sequenced, and spoligotyping was performed to distinguish different genotypes. Overall, the prevalence of resistance in China was highest for ofloxacin (3.76%), intermediate for levofloxacin (3.18%) and moxifloxacin (3.12%), and lowest for sparfloxacin (1.91%) and gatifloxacin (1.33%). Mutations in thegyrAgene were observed in 89 (64.5%) out of the 138 OFX-resistantM. tuberculosisstrains. Positions 94 and 90 were the most frequent sites of mutation conferring FQ resistance on these strains, accounting for high-level FQ resistance. Furthermore, the Beijing genotype showed no association with high-level FQ resistance or distribution in hot spots in the quinolone resistance-determining region (QRDR) ofgyrA. Our findings provide essential implications for the feasibility of genotypic tests relying on detection of mutations in the QRDR ofgyrAand the shorter first-line treatment regimens based on FQs in China.

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Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

Journal of Clinical Microbiology ◽

10.1128/jcm.02012-15 ◽

2015 ◽

Vol 53 (12) ◽

pp. 3805-3811 ◽

Cited By ~ 10

Author(s):

Elis R. Dalla Costa ◽

Sidra E. G. Vasconcelos ◽

Leonardo S. Esteves ◽

Harrison M. Gomes ◽

Lia L. Gomes ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Latin American ◽

Variable Number Tandem Repeat ◽

Multidrug Resistant ◽

Variable Number ◽

Compensatory Mutation ◽

Single Nucleotide ◽

Content Type ◽

A Site ◽

South Brazil

We recently detected the spoligotype patterns of strains ofMycobacterium pinnipedii, a species of theMycobacterium tuberculosiscomplex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9,pks15/1, RDRio, andfbpC, the insertion of IS6110at a site specific to theM. tuberculosisLatin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in factM. tuberculosisand more specifically belong to the LAM genotype. Most of these isolates (n= 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of therpoA,rpoB,rpoC,katG, andinhAgenes. Seven isolates (77.8%) carried the S315T mutation inkatG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) ininhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 ofrpoB. No putative compensatory mutation was found in eitherrpoAorrpoC. This is the first report of anM. tuberculosisLAM family strain with a convergentM. pinnipediispoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in theM. tuberculosiscomplex on the basis of single genetic markers.

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Prediction of Local Transmission of Mycobacterium tuberculosis Isolates of a Predominantly Beijing Lineage by Use of a Variable-Number Tandem-Repeat Typing Method Incorporating a Consensus Set of Hypervariable Loci

Journal of Clinical Microbiology ◽

10.1128/jcm.01016-17 ◽

2017 ◽

Vol 56 (1) ◽

Author(s):

Yoshiro Murase ◽

Kiyohiko Izumi ◽

Akihiro Ohkado ◽

Akio Aono ◽

Kinuyo Chikamatsu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Tandem Repeat ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Trend Test ◽

Typing Method ◽

Content Type ◽

Tuberculosis Transmission ◽

High Prevalence ◽

Hypervariable Loci

ABSTRACT Strain genotyping based on the variable-number tandem repeat (VNTR) is widely applied for identifying the transmission of Mycobacterium tuberculosis. A consensus set of four hypervariable loci (1982, 3232, 3820, and 4120) has been proposed to improve the discrimination of Beijing lineage strains. Herein, we evaluated the utility of these four hypervariable loci for tracing local tuberculosis transmission in 981 cases over a 14-month period in Japan (2010 to 2011). We used six different VNTR systems, with or without the four hypervariable loci. Patient ages and weighted standard distances (a measure of the dispersion of genotype-clustered cases) were used as proxies for estimating local tuberculosis transmission. The highest levels of isolate discrimination were achieved with VNTR systems that incorporated the four hypervariable loci (i.e., the Japan Anti-Tuberculosis Association [JATA]18-VNTR, mycobacterial interspersed repetitive unit [MIRU]28-VNTR, and 24Beijing-VNTR). The clustering rates by JATA12-VNTR, MIRU15-VNTR, JATA15-VNTR, JATA18-VNTR, MIRU28-VNTR, and 24Beijing-VNTR systems were 52.2%, 51.0%, 39.0%, 24.1%, 23.1%, and 22.0%, respectively. As the discriminative power increased, the median weighted standard distances of the clusters tended to decrease (from 311 to 80 km, P < 0.001, Jonckheere-Terpstra trend test). Concurrently, the median ages of patients in the clusters tended to decrease (from 68 to 60 years, P < 0.001, Jonckheere-Terpstra trend test). These findings suggest that strain typing using the four hypervariable loci improves the prediction of active local tuberculosis transmission. The four-locus set can therefore contribute to the targeted control of tuberculosis in settings with high prevalence of Beijing lineage strains.

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Sulfamethoxazole Susceptibility of Mycobacterium tuberculosis Isolates from HIV-Infected Ugandan Adults with Tuberculosis Taking Trimethoprim-Sulfamethoxazole Prophylaxis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01101-15 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5844-5846 ◽

Cited By ~ 3

Author(s):

Sam Ogwang ◽

Caryn E. Good ◽

Brenda Okware ◽

Mary Nsereko ◽

Michael R. Jacobs ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

Sub Saharan Africa ◽

Content Type ◽

Pulmonary Tb ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Sub Saharan

ABSTRACTAdditional drugs are needed for the treatment of multidrug-resistant tuberculosis (TB). Sulfamethoxazole has been shown to havein vitroactivity againstMycobacterium tuberculosis; however, there is concern about resistance given the widespread use of trimethoprim-sulfamethoxazole prophylaxis among HIV-infected patients in sub-Saharan Africa. Thirty-eight of 40Mycobacterium tuberculosisisolates (95%) from pretreatment sputum samples from Ugandan adults with pulmonary TB, including HIV-infected patients taking trimethoprim-sulfamethoxazole prophylaxis, were susceptible with MICs of ≤38.4 μg/ml.

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Bazedoxifene Suppresses Intracellular Mycobacterium tuberculosis Growth by Enhancing Autophagy

mSphere ◽

10.1128/msphere.00124-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 3

Author(s):

Qi Ouyang ◽

Kehong Zhang ◽

Dachuan Lin ◽

Carl G. Feng ◽

Yi Cai ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Antimycobacterial Activity ◽

Multidrug Resistant ◽

Drug Resistant ◽

New Host ◽

Drug Resistant Tuberculosis ◽

Content Type ◽

Receptor Modulator ◽

Resistant Tuberculosis ◽

Negative Side Effects

ABSTRACT Tuberculosis (TB) is still the leading killer caused by Mycobacterium tuberculosis infection. There is a clear need for new treatment strategy against TB. It has been reported that tamoxifen, known as a selective estrogen receptor modulator (SERM), exhibits antimycobacterial activity and inhibits M. tuberculosis growth in macrophages. However, it remains unknown whether such antimicrobial activity is a general property of all SERMs and how it works. In this study, we identified that bazedoxifene (BZA), a newer SERM, inhibits intracellular M. tuberculosis growth in macrophages. BZA treatment increases autophagosome formation and LC3B-II protein expression in M. tuberculosis-infected macrophages. We further demonstrated that the enhancement of autophagy by BZA is dependent on increased reactive oxygen species (ROS) production and associated with phosphorylation of Akt/mTOR signaling. In summary, our data reveal a previously unappreciated antimicrobial function of BZA and suggest that future investigation focusing on the mechanism of action of SERMs in macrophages may lead to new host-directed therapies against TB. IMPORTANCE Since current strategies for the treatment of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have low efficacy and highly negative side effects, research on new treatments including novel drugs is essential for curing drug-resistant tuberculosis. Host-directed therapy (HDT) has become a promising idea to modulate host cell responses to enhance protective immunity against pathogens. Bazedoxifene (BZA), which belongs to a new generation of SERMs, shows the ability to inhibit the growth of M. tuberculosis in macrophages and is associated with autophagy. Our findings reveal a previously unrecognized antibacterial function of BZA. We propose that the mechanism of SERMs action in macrophages may provide a new potential measure for host-directed therapies against TB.

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Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolated in Nepal

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06418-11 ◽

2012 ◽

Vol 56 (6) ◽

pp. 2831-2836 ◽

Cited By ~ 38

Author(s):

Ajay Poudel ◽

Chie Nakajima ◽

Yukari Fukushima ◽

Haruka Suzuki ◽

Basu Dev Pandey ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Promoter Region ◽

Multidrug Resistant ◽

Content Type ◽

Molecular Tests ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Mdr Tb ◽

Substitution Mutations ◽

Drug Resistant Strains

ABSTRACTDespite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance inMycobacterium tuberculosisis required. In the present study, we investigated the prevalence of mutations inrpoBandkatGgenes and theinhApromoter region in 158M. tuberculosisisolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) ofrpoBwere identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in thekatGgene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in theinhApromoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance inM. tuberculosisin Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.

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Molecular Characterization of Ofloxacin-Resistant Mycobacterium tuberculosis Strains from Russia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00036-08 ◽

2008 ◽

Vol 52 (8) ◽

pp. 2937-2939 ◽

Cited By ~ 66

Author(s):

Igor Mokrousov ◽

Tatiana Otten ◽

Olga Manicheva ◽

Yulia Potapova ◽

Boris Vishnevsky ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Molecular Characterization ◽

Local Population ◽

Multidrug Resistant ◽

Beijing Genotype ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis

ABSTRACT In this work, we studied the variation in the gyrA and gyrB genes in ofloxacin- and multidrug-resistant Mycobacterium tuberculosis strains circulating in northwest Russia. Comparison with spoligotyping data suggested that similar to the spread of multidrug-resistant tuberculosis, the spread of fluoroquinolone-resistant tuberculosis in Russia may be due, at least partly, to the prevalence of the Beijing genotype in a local population of M. tuberculosis.

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Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00395-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4457-4463 ◽

Cited By ~ 57

Author(s):

Benoit Lechartier ◽

Stewart T. Cole

Keyword(s):

Mycobacterium Tuberculosis ◽

Bacterial Load ◽

Multidrug Resistant ◽

Content Type ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Drug Resistant Strains ◽

Transfer Chain

ABSTRACTClofazimine (CZM) is an antileprosy drug that was recently repurposed for treatment of multidrug-resistant tuberculosis. InMycobacterium tuberculosis, CZM appears to act as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), to release reactive oxygen species upon reoxidation by O2. CZM presumably competes with menaquinone (MK-4), a key cofactor in the mycobacterial electron transfer chain, for its reduction by NDH-2. We studied the effect of MK-4 supplementation on the activity of CZM againstM. tuberculosisand found direct competition between CZM and MK-4 for the cidal effect of CZM, against nonreplicating and actively growing bacteria, as MK-4 supplementation blocked the drug's activity against nonreplicating bacteria. We demonstrated that CZM, like bedaquiline, is synergisticin vitrowith benzothiazinones such as 2-piperazino-benzothiazinone 169 (PBTZ169), and this synergy also occurs against nonreplicating bacteria. The synergy between CZM and PBTZ169 was lost in an MK-4-rich medium, indicating that MK-4 is the probable link between their activities. The efficacy of the dual combination of CZM and PBTZ169 was testedin vivo, where a great reduction in bacterial load was obtained in a murine model of chronic tuberculosis. Taken together, these data confirm the potential of CZM in association with PBTZ169 as the basis for a new regimen against drug-resistant strains ofM. tuberculosis.

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