Capreomycin Resistance Research Articles

Performance evaluation of the Xpert MTB/XDR test for the detection of drug resistance to Mycobacterium tuberculosis among people diagnosed with tuberculosis in South Africa.

Drug-resistant tuberculosis (TB) poses a significant public health concern in South Africa due to its complexity in diagnosis, treatment, and management. This study assessed the diagnostic performance of the Xpert MTB/XDR test for detecting drug resistance in patients with TB by using archived sputum sediments. This study analyzed 322 samples collected from patients diagnosed with TB between 2016 and 2019 across South Africa, previously characterized by phenotypic and genotypic methods. The Xpert MTB/XDR test was evaluated for its ability to detect resistance to isoniazid (INH), ethionamide (ETH), fluoroquinolones (FLQ), and second-line injectable drugs (SLIDs) compared with phenotypic drug susceptibility testing (pDST) and whole-genome sequencing (WGS). Culture, Xpert MTB/RIF Ultra, and Xpert MTB/RIF (G4) tests were performed to determine sensitivity and agreement with this test for TB detection. The sensitivities using a composite reference standard, pDST, and sequencing were >90% for INH, FLQ, amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) resistance, meeting the WHO target product profile criteria for this class. A lower sensitivity of 65.9% (95% CI: 57.1-73.6) for ETH resistance was observed. The Xpert MTB/XDR showed a sensitivity of 98.3% (95% CI: 96.1-99.3) and specificity of 100% (95% CI: 86.7-100) compared with culture, a positive percent agreement (PPA) of 98.8% (95% CI: 93.7-99.8) and negative percent agreement (NPA) of 100.0% (95% CI: 78.5-100.0) compared with G4, and a PPA of 99.5% (95% CI: 97.3-99.9) and NPA of 100.0% (95% CI: 78.5-100.0) compared with Xpert MTB/RIF Ultra for detecting Mycobacterium tuberculosis. The test offers a promising solution for the rapid detection of drug-resistant TB and could significantly enhance control efforts in this setting.

The rv2820c K114N mutation is related with capreomycin tolerance

As one of the factors affecting the treatment outcomes, drug tolerance in mycobacteriosis has not been paid due attention. Genome-wide association studies on 607 Mycobacterium tuberculosis clinical isolates with phenotypic drug susceptibility test data revealed that a K114N mutation on the rv2820c gene was highly enriched in capreomycin-resistant isolates (32/213, 15.02%). However, the mutation was also observed in capreomycin-sensitive isolates (10/394, 2.53%). In most cases (31/42, 73.81%), the rv2820c K114N mutation occurred in isolates with the known capreomycin resistance conferring mutation rrs A1401G. In contrast, the general frequency of the rv2820c K114N mutation was low in 7061 genomes downloaded from the National Center for Biotechnology Information database. To determine the impact of this mutation on the antimycobacterial activity of capreomycin, the intact rv2820c gene and the rv2820c K114N mutant were over-expressed in Mycobacterium smegmatis (Ms), and the results of susceptibility tests showed that the rv2820c K114N mutation did not affect the minimum inhibition concentration (MIC) of capreomycin. Subsequently, the data of time-kill assays showed that, it took only 2 h of capreomycin treatment (40 μg/ml, 5 × MIC) to kill 99.9% bacterial cells of Ms MC2155 pMV261::rv2820cH37Rv, while it took 6 h to achieve that for Ms MC2155 pMV261::rv2820cK114N. Taken together, these data suggested that the rv2820c K114N mutation is related with capreomycin tolerance, which merits further investigation.

In silico evaluation of WHO-endorsed molecular methods to detect drug resistant tuberculosis

Universal drug susceptibility testing (DST) for tuberculosis is a major goal of the END TB strategy. PCR-based molecular diagnostic tests have been instrumental in increasing DST globally and several assays have now been endorsed by the World Health Organization (WHO) for use in the diagnosis of drug resistance. These endorsed assays, however, each interrogate a limited number of mutations associated with resistance, potentially limiting their sensitivity compared to sequencing-based methods. We applied an in silico method to compare the sensitivity and specificity of WHO-endorsed molecular based diagnostics to the mutation set identified by the WHO mutations catalogue using phenotypic DST as the reference. We found that, in silico, the mutation sets used by probe-based molecular diagnostic tests to identify rifampicin, isoniazid, pyrazinamide, levofloxacin, moxifloxacin, amikacin, capreomycin and kanamycin resistance produced similar sensitivities and specificities to the WHO mutation catalogue. PCR-based diagnostic tests were most sensitive for drugs where mechanisms of resistance are well established and localised to small genetic regions or a few prevalent mutations. Approaches using sequencing technologies can provide advantages for drugs where our knowledge of resistance is limited, or where complex resistance signatures exist.

The evaluation of second line drug susceptibilities and molecular epidemiological profiles of multidrug resistance Mycobacterium tuberculosis isolates isoleted from different region of Turkey

Introduction: This study was planned to determine the second line drug resistance and molecular epidemiological profiles of multidrug resistant Mycobacterium tuberculosis isolates isolated from different geographical regions of Turkey.
 Material and Method: In our study, 63 MDR M. tuberculosis isolates were evaluated for the drug susceptibility sent from different tuberculosis laboratories of Turkey. Secondary antituberculosis drugs resistance was evaluated by indirect proportion method. Epidemiological origins were evaluated by using IS6110-RFLP and spoligotyping methods. 
 Results: Cycloserine, ethionamide, capreomycin, thiacetozone, ofloxacin, kanamsin and paraaminosalicylic acid resistance rates were 15.87%, 19.04%, 7.93%, 6.34%, 11.11%, 12.69% and 6.34.%, respectively. According to spoligotyping results, 11 different patterns were obtained, including 52 isolates consisting of 5 clusters and 11 patterns consisting of a single isolate. When we compared our results with the spoligotype database in the world; 42 of 52 isolates forming 5 clusters were identified as predefined spoligotypes (LAM7-TUR, LAM9, T clade). 10 isolates showed the characteristics of the U spoligotype family. Of the 11 isolates that produced 11 different patterns, 8 were Haarlem and T spoligotypes. It was found that 2 isolates had the characteristics of Orphan and 1 isolate had the characteristics of BOV family. 
 Conclusion: In our study, LAM7-TUR, LAM9, T clade spoligotype families are common in our country and in the world were determined.

Xpert MTB/XDR for rapid detection of drug-resistant tuberculosis beyond rifampicin

Effective control of drug-resistant tuberculosis (DR-TB) requires rapid diagnosis and prompt treatment. 1 WHOWHO operational handbook on tuberculosis. Module 3: diagnosis—rapid diagnostics for tuberculosis detention, 2021 update. World Health Organization, Geneva2021 Google Scholar However, access to diagnostic assays for DR-TB remains a challenge, especially in highly endemic countries. The Xpert MTB/RIF assay and its successor the Xpert MTB/RIF Ultra (Cepheid, Sunnyvale, CA, USA) are used extensively worldwide to detect Mycobacterium tuberculosis and rifampicin resistance but cannot assess resistance to isoniazid and second-line drugs. Tests for second-line-drug resistance are technically demanding and require advanced laboratory infrastructure. Therefore, their use is restricted to central or national tuberculosis laboratories. Almost a decade since the introduction of the GeneXpert technology, the Xpert MTB/XDR assay has been approved for reflex testing of sputum samples after a positive result for M tuberculosis complex on the Xpert MTB/RIF or Ultra assays. Detection of isoniazid, fluoroquinolone, ethionamide, amikacin, kanamycin, and capreomycin resistance by the Xpert MTB/XDR assay: a cross-sectional multicentre diagnostic accuracy studyThe Xpert MTB/XDR assay showed high diagnostic accuracy and met WHO's minimum target product profile criteria for a next-generation drug susceptibility test. The assay has the potential to diagnose drug-resistant tuberculosis rapidly and accurately and enable optimum treatment. Full-Text PDF

Diagnostic performance of whole-genome sequencing for identifying drug-resistant TB in Thailand.

BACKGROUND: Whole-genome sequencing (WGS) is a promising tool for the detection of drug-resistant TB (DR-TB). To date, there have been few comparisons of diagnostic performance of WGS and phenotypic drug susceptibility testing (DST) in DR-TB.METHODS: We compared drug resistance-conferring mutations identified by WGS analysis using TB-Profiler and Mykrobe with phenotypic DST profiles based on the Löwenstein-Jensen proportion method using drug-resistant Mycobacterium tuberculosis (n = 537) isolates from across Thailand. Based on available phenotypic DST results, diagnostic performance was analysed for resistance against isoniazid, rifampicin, ethambutol (EMB), streptomycin, ethionamide (ETH), kanamycin, capreomycin (CPM), para-aminosalicylic acid, ofloxacin and levofloxacin.RESULTS: High agreement between the two methods was observed for most drugs (>91%), except EMB (57%, 95% CI 53-61) and ETH (70%, 95% CI 66-74). Also, low specificity was observed for EMB (49%, 95% CI 44-54) and ETH (66%, 95% CI 61-71). Sensitivity was high for most drugs (range 83-98%), except CPM (77%, 95% CI 59-88).CONCLUSION: Low agreement between WGS and phenotypic tests for drug resistance was found for EMB and ETH. The current genomic database is insufficient for the identification of CPM resistance. Challenges remain for routine usage of WGS-based DST, especially for second-line anti-TB drugs.

Insights into the mutations leading to capreomycin resistance in S-adenosyl-L-methionine binding motif in TlyA from Mycobacterium tuberculosis

Capreomycin is a second line antibiotic used for the treatment of drug resistant Tuberculosis (TB), primary reason of death from a solo infectious organism, Mycobacterium tuberculosis (M.tb). Capreomycin targets the ribosome of bacteria and is known to bind at the interface where the large and small ribosomal subunits interact in M.tb using an S-Adenosyl Methionine (SAM) dependent methyltransferase, TlyA (Rv1794). Besides the methyltransferase activity, TlyA has also been found to show substantial haemolytic activity. The dual activity of TlyA highlights its crucial role in pathogenesis and virulence of M.tb. In the present study, docking and molecular dynamics (MD) simulations were carried out to explore the impact of mutations in a conserved SAM binding motif, 90GASTG94, on the affinity of TlyA enzyme for SAM. Two already reported mutations, A91E and S92L, and the remaining wild type residues, Gly90, Thr93, Gly94 mutated to alanine were taken into consideration resulting in a total of six systems, wild type + SAM, G90A + SAM, A91E + SAM, S92L + SAM, T93A + SAM and G94A + SAM that were subjected to 100 ns MD simulations. Docking scores and MD simulations analyses revealed that in contrast to wild type, mutants reduced the affinity of SAM for TlyA with most prominent effect observed in case of alanine mutants. Mutations also led to the loss of hydrogen bond and hydrophobic interactions and large-scale movement of atoms evident from the principal component analyses indicating their destabilizing impact on TlyA. The present study gives insights into influence of mutations on binding of SAM to TlyA in M.tb and promoting capreomycin resistance. Communicated by Ramaswamy H. Sarma

First molecular-based anti-TB drug resistance survey in Eritrea.

BACKGROUND: In the absence of reliable data on drug-resistant TB in Eritrea, a national survey was conducted in 2018 using molecular-based methods, bypassing the need for culture.METHODS: A cross-sectional study was conducted in all 77 TB microscopy centres in the country. All 629 newly registered sputum smear-positive pulmonary TB patients were enrolled over 12 months. Sputum samples were tested using the Xpert® MTB/RIF assay and targeted next-generation sequencing (Deeplex Myc-TB) to identify resistance and explore the phylogenetics of Mycobacterium tuberculosis complex strains.RESULTS: Drug resistance profiles were obtained for 555 patients (502 new, 53 previously treated). The prevalence of rifampicin-resistant TB (RR-TB) was respectively 2.0% and 7.6% among new and previously treated cases. All RR-TB isolates that were susceptible to isoniazid displayed a phylogenetic marker conferring capreomycin resistance, confirming circulation of a previously described resistant TB sub-lineage in the Horn of Africa. Only one case of fluoroquinolone resistance was detected.CONCLUSION: The prevalence of rifampicin resistance among TB patients is encouragingly low. The scarcity of fluoroquinolone resistance bodes well for the success of the recommended all-oral treatment regimen. Surveillance based on molecular approaches enables a reliable estimation of the burden of resistance and can be used to guide appropriate treatment and care.

Capreomycin resistance prediction in two species ofMycobacteriumusing a stacked ensemble method

Predicting bacterial resistance provides valuable information that can assist in clinical decisions. With recent advances in whole genome sequencing technology, the detection of antibiotic resistance (AR) proteins directly from genomic data is becoming feasible. AR genes/proteins can be identified using best-hit methods that work by comparing candidate sequences with known AR genes in public databases. However, these approaches may fail to detect resistance genes with sequences that differ significantly from known sequences. Our goal is to develop a machine learning technique to accurately predict capreomycin resistance in Mycobacteria with low false discovery rates. We present a stacked ensemble learning model as an alternative to traditional DNA sequence alignment-based methods using optimal features generated from the physicochemical, evolutionary and secondary structure properties of protein sequences. We train logistic regression, C5.0 and support vector machine (SVM) algorithms as our base classifiers, and our stacked ensemble predictors combine the results from the base classifiers to achieve higher accuracy. Compared with our most accurate base classifier (SVM), our most accurate stacked ensemble predictor increases training accuracy by 2·43%. Our stacked ensemble predictors achieve test accuracy up to 81·25%. We developed a stacked ensemble model to predict capreomycin resistance for Mycobacteria with an accuracy >80% using protein sequences with sequence similarity ranging between 10% and 70%. This performance cannot be achieved with best-hit methods due to differences in sequence similarity. Today an estimated one-half million cases of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) occur annually worldwide at a great cost. Because capreomycin is a second-line drug used to treat drug-resistant TB, the ability to use a machine learning approach to classify capreomycin-resistant TB in a timely manner is crucial for the successful treatment of MDR or XDR TB.

Assessing capreomycin resistance on tlyA deficient and point mutation (G695A) Mycobacterium tuberculosis strains using multi-omics analysis

Capreomycin (CAP), a cyclic peptide antibiotic, is considered to be an ideal second-line drug for tuberculosis (TB). However, in the past few years, the emergence of more CAP-resistant (CAPr) TB patients has limited its use. Although it has been reported that CAP resistance to Mycobacterium tuberculosis (Mtb) is associated with rrs or tlyA mutation, the exact mechanism of CAPr Mtb strains, especially the mechanism associated with tlyA deficient or mutation, is not fully understood. Herein, we utilized a multi-omics (genome, proteome, and metabolome) approach to assess CAP resistance on tlyA deficient CAPr Mtb strains (CAPr1) and tlyA point mutation CAPr Mtb strains (CAPr2) that we established for the first time in vitro to investigate the CAP-resistant mechanism. Our results showed that the CAPr1 strains (> 40 μg/ml) was more resistant to CAP than the CAPr2 strains (G695A, 10 μg/ml). Furthermore, multi-omics analysis indicated that the CAPr1 strains exhibited greater drug tolerance than the CAPr2 strains may be associated with the weakening of S-adenosyl-L-methionine-dependent methyltransferase (AdoMet-MT) activity and abnormal membrane lipid metabolism such as suppression of fatty acid metabolism, promotion of glycolipid phospholipid and glycerolipid metabolism. As a result, these studies reveal a new mechanism for CAP resistance to tlyA deficient or mutation Mtb strains, and may be helpful in developing new therapeutic approaches to prevent Mtb resistance to CAP.

Integrating informatics tools and portable sequencing technology for rapid detection of resistance to anti-tuberculous drugs

BackgroundMycobacterium tuberculosis resistance to anti-tuberculosis drugs is a major threat to global public health. Whole genome sequencing (WGS) is rapidly gaining traction as a diagnostic tool for clinical tuberculosis settings. To support this informatically, previous work led to the development of the widely used TBProfiler webtool, which predicts resistance to 14 drugs from WGS data. However, for accurate and rapid high throughput of samples in clinical or epidemiological settings, there is a need for a stand-alone tool and the ability to analyse data across multiple WGS platforms, including Oxford Nanopore MinION.ResultsWe present a new command line version of the TBProfiler webserver, which includes hetero-resistance calling and will facilitate the batch processing of samples. The TBProfiler database has been expanded to incorporate 178 new markers across 16 anti-tuberculosis drugs. The predictive performance of the mutation library has been assessed using > 17,000 clinical isolates with WGS and laboratory-based drug susceptibility testing (DST) data. An integrated MinION analysis pipeline was assessed by performing WGS on 34 replicates across 3 multi-drug resistant isolates with known resistance mutations. TBProfiler accuracy varied by individual drug. Assuming DST as the gold standard, sensitivities for detecting multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) were 94% (95%CI 93–95%) and 83% (95%CI 79–87%) with specificities of 98% (95%CI 98–99%) and 96% (95%CI 95–97%) respectively. Using MinION data, only one resistance mutation was missed by TBProfiler, involving an insertion in the tlyA gene coding for capreomycin resistance. When compared to alternative platforms (e.g. Mykrobe predictor TB, the CRyPTIC library), TBProfiler demonstrated superior predictive performance across first- and second-line drugs.ConclusionsThe new version of TBProfiler can rapidly and accurately predict anti-TB drug resistance profiles across large numbers of samples with WGS data. The computing architecture allows for the ability to modify the core bioinformatic pipelines and outputs, including the analysis of WGS data sourced from portable technologies. TBProfiler has the potential to be integrated into the point of care and WGS diagnostic environments, including in resource-poor settings.

Genetic Characterization of Second-Line Drug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis from the Northern Region of India

Rapid detection of drug resistance in Mycobacterium tuberculosis is important for the successful treatment of tuberculosis. Fluoroquinolone and aminoglycoside resistance detection by molecular methods becomes more complex due to cross resistance among them. Thus, we aimed to determine cross-resistance and mutations in resistance genes for these drugs. A total of 336 multidrug-resistant tuberculosis (MDR-TB) cases received in Mycobacteriology laboratory were screened for phenotypic drug sensitivity testing for second-line drugs, i.e., ofloxacin, amikacin, kanamycin, and capreomycin. Molecular characterization of resistance was done by DNA sequencing of gyrA gene for fuoroquinolones (FQ), and multiplex allele-specific polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (RFLP) of rrs gene for aminoglycosides. Of 336 MDR-TB isolates, 12 were extensively drug-resistant tuberculosis and 219 were sensitive to all the drugs tested. Ofoxacin, amikacin, kanamycin, and capreomycin resistance was detected in 101 (30.1%), 23 (6.8%), 27 (8.1%), and 19 (5.6%) cases, respectively. Eight different mutations were detected in gyrA gene in ofloxacin-resistant isolates and A1401G nucleotide change in rrs gene were seen in 55.6% (15/27), 65.2% (15/23), and 68.4% (13/29) for kanamycin-, amikacin-, and capreomycin-resistant isolates, respectively. Information on second-line drug resistance-associated mutations could potentially be used for development of newer rapid diagnostic tests.

Evaluation of Genotype MTBDRplus and MTBDRsl Assays for Rapid Detection of Drug Resistance in Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in Pakistan.

Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries alongside the 6th among countries with the highest burden of drug-resistant TB, including multi-drug resistant (MDR)-TB. Methods for rapid and reliable drug susceptibility testing (DST) are prerequisite for the prompt institution of effective anti-TB treatment. The aim of this study was to evaluate the efficiency of Genotype MTBDRplus and MTBDRsl assays for the detection of MDR and (pre-) extensively drug-resistant (XDR-TB) isolates in Pakistan. The study included 47 pre-XDR and 6 XDR-TB isolates, recovered from 53 patients from Pakistan. Conventional DST was performed using the standard 1% proportion method on the Löwenstein-Jensen medium. For molecular determination of drug resistance, GenoType MTBDRplus and GenoType MTBDRsl assays (Hain Lifescience, Germany) were used. To evaluate discrepancies between conventional and molecular DST results, mutation profiling was performed by amplifying and sequencing seven genetic loci, i.e., katG, inhA, and mabA-inhA promoter, rpoB, gyrA, embB, rrs. The sensitivity of Genotype MTBDRplus was 71.7% for isoniazid (INH) and 79.2% for rifampicin (RIF). Sequence analysis revealed non-synonymous mutations in 93.3 and 27.3% of isolates phenotypically resistant to INH and RIF, respectively, albeit susceptible when tested by GenoType MTBDRplus. GenoType MTBDRsl had a sensitivity of 73.6, 64.7, 20, 25, and 100% for the detection of fluoroquinolones, ethambutol, kanamycin, amikacin, and capreomycin resistance, respectively. Upon sequencing, mutations were detected in 20, 77.8%, and all isolates phenotypically resistant to aminoglycosides, ethambutol, and fluoroquinolones, respectively, yet declared as susceptible with GenoType MTBDRsl. Low sensitivities seriously impede the large-scale application of the Genotype MTBDRplus and MTBDRsl assays. Unless further optimized, the currently available line-probe assays should rather be auxiliary to the conventional, phenotype-based methods in the detection of MDR- and XDR-TB in Pakistan.

Second Line Drugs Resistant Mycobacterium Tuberculosis in Multi-Drug Resistant Tuberculosis Patients

With an increase in Multi-drug resistant tuberculosis (MDR-TB), there is a need of second line drug susceptibility test that helps in early diagnosis and minimize the risk of other powerful drug resistant Mycobacterium tuberculosis. The aim of this study was to determine second line drugs (ofloxacin, kanamycin, capreomycin) resistance pattern in MDR-TB isolates and to determine the prevalence of pre-Extensively drug resistant tuberculosis (pre-XDR-TB) and XDR-TB in MDR-TB patients. The study was conducted from February to September 2015 at National Tuberculosis Centre, Thimi, Bhaktapur. MDR-TB (resistant to isoniazid and rifampicin) patients’ sputum samples were processed by Modified Petroff’s method. Out of 92 samples, 57 were found culture positive. Following the species identification of culture positive MDR-TB isolates, second line drug susceptibility test was performed by conventional proportion method. Of 57 MDR-TB isolates, 22 (38.59%) showed resistance to ofloxacin (Ofx), 9 (15.79%) to capreomycin (Cm) and 9 (15.79%) to kanamycin (Km). One XDR-TB (1.8%) resistant to all drugs was detected. Of the remaining, 21(36.8%) were resistant to ofloxacin only and 8(15.4%) were resistant to two drugs i.e.29 (50.9%) were pre-XDR-TB. The prevalence of pre-XDR-TB and XDR-TB was found to be 50.88% and 1.75% respectively. The resistance pattern of second line anti-tuberculosis drugs showed higher ofloxacin resistance in MDR-TB patients. In a nutshell, MDR-TB cases need urgent and timely susceptibility report for second line anti-tuberculosis drugs to help the clinicians start proper drug combinations to treat MDR-TB patients. Journal of Institute of Science and Technology Volume 22, Issue 2, January 2018, page: 168-174

Impact of gyrB and eis Mutations in Improving Detection of Second-Line-Drug Resistance among Mycobacterium tuberculosis Isolates from Georgia

The country of Georgia has a high burden of multi- and extensively drug-resistant tuberculosis (XDR-TB). To evaluate whether mutations in gyrB and eis genes increased the sensitivity of detection of phenotypic resistance to ofloxacin and kanamycin or capreomycin compared to use of the first-generation MTBDRsl assay alone, which tests for mutations in gyrA and rrs genes, a retrospective study of stored Mycobacterium tuberculosis isolates was performed. All isolates underwent DNA sequencing of resistance-determining regions. Among 112 M. tuberculosis isolates with DNA extraction data, targeted sequencing was successfully performed for each gene as follows: for gyrA, 98% sensitivity; for gyrB, 96%; for rrs, 93%; for the eis gene and its promoter, 93%. The specificity and hence the positive predictive value of gyrA and gyrB mutations for detecting ofloxacin resistance were 100%. The addition of gyrB mutations increased the sensitivity of phenotypic ofloxacin resistance detection by 13% (75% to 88%). All rrs resistance-conferring mutations were A1401G, and this mutation had low sensitivity (40% and 18%) and high specificity (95% and 100%) in predicting phenotypic capreomycin and kanamycin resistance, respectively. The eis C-14T mutation increased the sensitivity of phenotypic kanamycin resistance detection by 9% (18% to 27%) and was found solely in kanamycin phenotypic resistance isolates. Our data showed that the inclusion of eis C-14T and gyrB mutations in addition to rrs and gyrA mutations improves the sensitivity of detection of phenotypic ofloxacin and kanamycin resistance, respectively.

Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes.

Mycobacterium abscessus is innately resistant to a variety of drugs thereby limiting therapeutic options. Bacterial resistance to aminoglycosides (AGs) is conferred mainly by AG-modifying enzymes, which often have overlapping activities. Several putative AG-modifying enzymes are encoded in the genome of M. abscessus . The aim of this study was to investigate the molecular basis underlying AG resistance in M. abscessus . M. abscessus deletion mutants deficient in one of three genes potentially involved in AG resistance, aac(2 ' ) , eis1 and eis2 , were generated by targeted gene inactivation, as were combinatorial double and triple deletion mutants. MICs were determined to study susceptibility to a variety of AG drugs and to capreomycin. Deletion of aac(2 ' ) increased susceptibility of M. abscessus to kanamycin B, tobramycin, dibekacin and gentamicin C. Deletion of eis2 increased susceptibility to capreomycin, hygromycin B, amikacin and kanamycin B. Deletion of eis1 did not affect drug susceptibility. Equally low MICs of apramycin, arbekacin, isepamicin and kanamycin A for WT and mutant strains indicate that these drugs are not inactivated by either AAC(2 ' ) or Eis enzymes. M. abscessus expresses two distinct AG resistance determinants, AAC(2 ' ) and Eis2, which confer clinically relevant drug resistance.

Molecular analysis of genetic mutations among cross-resistant second-line injectable drugs reveals a new resistant mutation in Mycobacterium tuberculosis

Mutations causing mono and cross-resistance among amikacin, kanamycin and capreomycin of second-line injectable drugs (SLIDs) namely are not well understood. We investigated 124 isolates of Mycobacterium tuberculosis for mutations within rrs, eis, tlyA and efflux pump (Rv1258c and Rv0194) genes involved in resistance towards SLIDs. The distribution of mutations across these genes were significantly different in strains with mono-resistance or cross-resistance. A new mutation G878A was found in rrs gene, among strains with capreomycin mono-resistant, or in strains with cross-resistance of capreomycin, kanamycin and amikacin. This mutation was associated with the Euro-American X3 lineage (P < 0.0001). Mutations in the two efflux genes Rv1258c and Rv0194 were confined to strains with only capreomycin/amikacin/kanamycin cross-resistance. We further investigated the minimum inhibitory concentration of capreomycin on isolates with new G878A mutation ranging from 8 μg/mL to 64 μg/mL. Inclusion of G878A on new molecular assays could increase the sensitivity of capreomycin resistance detection.

Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables

Objective/backgroundThe in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes. Thus, the aim of our study was to correlate rrs (X52917) and eis (AF144099) promoter mutations, found in M. tuberculosis isolates, with corresponding minimum inhibitory concentrations of amikacin, kanamycin, and capreomycin. MethodsNinety M. tuberculosis clinical isolates were analyzed in this study. The minimum inhibitory concentrations were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions, and 31 isolates with wild-type sequences, as determined by the GenoType MTBDRsl (version 1) assay. ResultsThe rrs A1401G mutation was identified in 48 isolates resistant to the second-line injectables. The eis promoter mutations C-14T (n=3), G-10C (n=3), G-10A (n=3), and C-12T (n=2) were found within 11 isolates with various resistance profiles to the second-line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was amikacin, kanamycin, and capreomycin resistant. The isolates with the rrs A1401G mutation had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of >40mg/L, >20mg/L, and 5–15mg/L, respectively. The isolates with eis promoter mutations had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of 0.25–1.0mg/L, 0.625–10mg/L, and 0.625–2.5mg/L, respectively. ConclusionThis study provides a preliminary basis for the prediction of phenotypic-resistance levels to the second-line injectables based upon the presence of genetic mutations associated with amikacin, kanamycin, and capreomycin resistance. The results suggest that isolates with eis promoter mutations have consistently lower resistance levels to amikacin, kanamycin, and capreomycin than isolates with the rrs A1401G mutation.

Primary Capreomycin Resistance Is Common and Associated With Early Mortality in Patients With Extensively Drug-Resistant Tuberculosis in KwaZulu-Natal, South Africa.

Capreomycin is a key antimycobacterial drug in treatment of extensively drug-resistant tuberculosis (XDR-TB). Drug-susceptibility testing (DST) for capreomycin is not routinely performed in newly diagnosed XDR-TB in South Africa. We performed this study to assess the prevalence, clinical significance, and molecular epidemiology of capreomycin resistance in newly diagnosed patients with XDR-TB in KwaZulu-Natal, South Africa. Retrospective cohort study of consecutive patients with XDR-TB admitted to a TB referral hospital without previous XDR-TB treatment. A subset of isolates had extended DST (including capreomycin), mutational analysis, and IS6110 restriction fragment length polymorphism assays. A total of 216 eligible patients with XDR-TB were identified. The majority were treated with capreomycin (72%), were young (median age: 35.5 years), and were female (56%). One hundred five (76%) were HIV+, and 109 (66%) were on antiretroviral therapy. A subset of 52 patients had full DST. A total of 47/52 (90.4%) patients with XDR-TB were capreomycin resistant. Capreomycin-resistant patients experienced worse mortality and culture conversion than capreomycin susceptible, although this difference was not statistically significant. The A1401G mutation in the rrs gene was associated with capreomycin resistance. The majority of capreomycin-resistant strains were F15/LAM4/KZN lineage (80%), and clustering was common in these isolates (92.5%). Capreomycin resistance is common in patients with XDR-TB in KwaZulu-Natal, is predominantly because of ongoing province-wide transmission of a highly resistant strain, and is associated with high mortality. Capreomycin should be included in routine DST in all patients with XDR-TB. New drug regimens that do not include injectable agents should be operationally tested as empiric treatment in XDR-TB.

Risk factors for acquisition of drug resistance during multidrug-resistant tuberculosis treatment, Arkhangelsk Oblast, Russia, 2005-2010.

Acquired resistance to antituberculosis drugs decreases effective treatment options and the likelihood of treatment success. We identified risk factors for acquisition of drug resistance during treatment for multidrug-resistant tuberculosis (MDR TB) and evaluated the effect on treatment outcomes. Data were collected prospectively from adults from Arkhangelsk Oblast, Russia, who had pulmonary MDR TB during 2005-2008. Acquisition of resistance to capreomycin and of extensively drug-resistant TB were more likely among patients who received <3 effective drugs than among patients who received >3 effective drugs (9.4% vs. 0% and 8.6% vs. 0.8%, respectively). Poor outcomes were more likely among patients with acquired capreomycin resistance (100% vs. 25.9%), acquired ofloxacin resistance (83.6% vs. 22.7%), or acquired extensive drug resistance (100% vs. 24.4%). To prevent acquired drug resistance and poor outcomes, baseline susceptibility to first- and second-line drugs should be determined quickly, and treatment should be adjusted to contain >3 effective drugs.