Rifampicin-resistant Isolates Research Articles

Evaluation of genetic correlation with fluoroquinolones resistance in rifampicin-resistant Mycobacterium tuberculosis isolates

ObjectiveTo detect levofloxacin (LFX) and moxifloxacin (MFX) resistance among rifampicin-resistant tuberculosis (RR-TB) isolates, and predict the resistance level based on specific mutations in gyrA and gyrB genes. MethodsA total of 686 RR-TB isolates were collected from Chinese Drug Resistance Surveillance Program from 2013 to 2020. The minimum inhibitory concentrations (MICs) of 12 anti-TB drugs were acquired using the broth microdilution method, followed by whole genome sequencing (WGS) analysis. ResultsAmong the 686 RR isolates, the most prevalent resistance was to isoniazid (80.5 %) and ethambutol (28.4 %), followed by LFX (26.1 %) and MFX (21.9 %). The resistance rate of LFX (26.1%–99.4 %) was higher than that of MFX (21.9%–83.3 %) across various drug resistance patterns. Of the 180 fluoroquinolones (FQs) resistant isolates, 168 (93.3 %) had mutations in quinolone-resistant determining regions (QRDRs) with 21 mutation types, and Asp94Gly (32.7 %, 55/168) was the predominant mutation. Isolates with mutations in Asp94Asn and Asp94Gly were associated with high levels of resistance to LFX and MFX. Using broth microdilution method as gold standard, the sensitivities of WGS for LFX and MFX were 93.3 % and 98.0 %, and the specificities were 98.6 % and 95.0 %, respectively. ConclusionThe resistance rate of LFX was higher than that of MFX among various drug resistance patterns in RR-TB isolates. The gyrA Asp94Gly was the predominant mutation type underlying FQs resistance. However, no significant difference was observed between mutation patterns in gyrA gene and resistance level of FQs.

Rifampicin resistant Mycobacterium tuberculosis in Vietnam, 2020–2022

ObjectiveWe conducted a descriptive analysis of multi-drug resistant tuberculosis (MDR-TB) in Vietnam’s two largest cities, Hanoi and Ho Chi Minh city. MethodsAll patients with rifampicin resistant tuberculosis were recruited from Hanoi and surrounding provinces between 2020 and 2022. Additional patients were recruited from Ho Chi Minh city over the same time period. Demographic data were recorded from all patients, and samples collected, cultured, whole genome sequenced and analysed for drug resistance mutations. Genomic susceptibility predictions were made on the basis of the World Health Organization’s catalogue of mutations in Mycobacterium tuberculosis associated with drug resistance, version 2. Comparisons were made against phenotypic drug susceptibility test results where these were available. Multivariable logistic regression was used to assess risk factors for previous episodes of tuberculosis. Results233/265 sequenced isolates were of sufficient quality for analysis, 146 (63 %) from Ho Chi Minh City and 87 (37 %) from Hanoi. 198 (85 %) were lineage 2, 20 (9 %) were lineage 4, and 15 (6 %) were lineage 1. 17/211 (8 %) for whom HIV status was known were infected, and 109/214 (51 %) patients had had a previous episode of tuberculosis. The main risk factor for a previous episode was HIV infection (odds ratio 5.1 (95 % confidence interval 1.3–20.0); p = 0.021). Sensitivity for predicting first-line drug resistance from whole genome sequencing data was over 90 %, with the exception of pyrazinamide (85 %). For moxifloxacin and amikacin it was 50 % or less. Among rifampicin-resistant isolates, prevalence of resistance to each non-first-line drug was < 20 %. ConclusionsDrug resistance among most MDR-TB strains in Vietnam’s two largest cities is confined largely to first-line drugs. Living with HIV is the main risk factor among patients with MDR-TB for having had a previous episode of tuberculosis.

Novel and reported compensatory mutations in rpoABC genes found in drug resistant tuberculosis outbreaks.

Rifampicin (RIF) is a key first-line drug used to treat tuberculosis, a primarily pulmonary disease caused by Mycobacterium tuberculosis. RIF resistance is caused by mutations in rpoB, at the cost of slower growth and reduced transcription efficiency. Antibiotic resistance to RIF is prevalent despite this fitness cost. Compensatory mutations in rpoABC genes have been shown to alleviate the fitness cost of rpoB:S450L, explaining how RIF resistant strains harbor this mutation can spread so rapidly. Unfortunately, the full set of RIF compensatory mutations is still unknown, particularly those compensating for rarer RIF resistance mutations. We performed an association study on a globally representative set of 4,309 whole genome sequenced clinical M. tuberculosis isolates to identify novel putative compensatory mutations, determine the prevalence of known and previously reported putative compensatory mutations, and determine which RIF resistance markers associate with these compensatory mutations. Of the 1,079 RIF resistant isolates, 638 carried previously reported putative and high-probability compensatory mutations. Our strict criteria identified 46 additional mutations in rpoABC for which no strong prior evidence of their compensatory role exists. Of these, 35 have previously been reported. As such, our independent corroboration adds to the mounting evidence that these 35 also carry a compensatory role. The remaining 11 are novel putative compensatory markers, reported here for the first time. Six of these 11 novel putative compensatory mutations had two or more mutation events. Most compensatory mutations appear to be specifically compensating for the fitness loss due to rpoB:S450L. However, an outbreak of 22 closely related isolates each carried three rpoB mutations, the rare RIFR markers D435G and L452P and the putative compensatory mutation I1106T. This suggests compensation may require specific combinations of rpoABC mutations. Here, we report only mutations that met our very strict criteria. It is highly likely that many additional rpoABC mutations compensate for rare resistance-causing mutations and therefore did not carry the statistical power to be reported here. These findings aid in the identification of RIF resistant M. tuberculosis strains with restored fitness, which pose a greater risk of causing resistant outbreaks.

Mycobacterium tuberculosis Lineage Distribution Using Whole-Genome Sequencing and Bedaquiline, Clofazimine, and Linezolid Phenotypic Profiles among Rifampicin-Resistant Isolates from West Java, Indonesia.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis infection. Indonesia is ranked second in the world for TB cases. New anti-TB drugs from groups A and B, such as bedaquiline, clofazimine, and linezolid, have been shown to be effective in curing drug resistance in TB patients, and Indonesia is already using these drugs to treat patients. However, studies comparing the TB strain types with anti-TB resistance profiles are still relevant to understanding the prevalent strains in the country and their phenotypic characteristics. This study aimed to determine the association between the TB lineage distribution using whole-genome sequencing and bedaquiline, clofazimine, and linezolid phenotypic profile resistance among M. tuberculosisrifampicin-resistant isolates from West Java. M. tuberculosis isolates stock of the Department of Microbiology, Faculty of Medicine, Universitas Indonesia, was tested against bedaquiline, clofazimine, and linezolid using a mycobacteria growth indicator tube liquid culture. All isolates were tested for M. tuberculosis and rifampicin resistance using Xpert MTB/RIF. The DNA genome of M. tuberculosis was freshly extracted from a Löwenstein-Jensen medium culture and then sequenced. The isolates showed phenotypically resistance to bedaquiline, clofazimine, and linezolid at 5%, 0%, and 0%, respectively. We identified gene mutations on phenotypically bedaquiline-resistant strains (2/3), and other mutations also found in phenotypically drug-sensitive strains. Mykrobe analysis showed that most (88.33%) of the isolates could be classified as rifampicin-resistant TB. Using Mykrobe and TB-Profiler to determine the lineage distribution, the isolates were found to belong to lineage 4 (Euro-American; 48.33%), lineage 2 (East Asian/Beijing; 46.67%), and lineage 1 (Indo-Oceanic; 5%). This work underlines the requirement to increase the representation of genotype-phenotype TB data while also highlighting the importance and efficacy of WGS in predicting medication resistance and inferring disease transmission.

A cell-free strategy for host-specific profiling of intracellular antibiotic sensitivity and resistance

Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease-causing microbes. To provide a host-specific tool to study antibiotic susceptibility and resistance, here we develop Klebsiella pneumoniae cell-free gene expression (CFE) systems from laboratory and clinical isolates. Using proteomics, we identify relative differences and unique proteins for these new CFE systems in comparison to an Escherichia coli MG1655 CFE model. Then we profile antimicrobial susceptibility in parallel with whole cells to quantify CFE antibiotic potency. Finally, we apply this native CFE tool to study AMR variants at a proof-of-concept level. Definably we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin—a genotype observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. Overall, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial extract processing requires Biosafety Level 2, the CFE system is non-living, suitable for long-term storage and study in a Biosafety Level 1 lab. We anticipate the K. pneumoniae CFE bioassay is advantageous for host-specific antimicrobial testing, the characterisation of intracellular AMR variants and potentially structure-activity relationship studies.

Molecular characterisation of Staphylococcus aureus in school-age children in Guangzhou: associations among agr types, virulence genes, sequence types, and antibiotic resistant phenotypes

BackgroundStaphylococcus aureus, one of the most prevalent opportunistic pathogens, mainly colonizes the nasal cavity and is a risk factor for severe infections. Virulence factors and accessory gene regulator (agr) are key to the severity and diversity of staphylococcal infection. In this study, we aimed to characterise S. aureus agr-types and virulence genes and correlated them with genetic background and antibiotic-resistant phenotypes.ResultsAgr types were identified in 704 isolates (98.5%), with only 11 isolates were negative for agr type. Most of our isolates were classified as agr type I, followed by types III, II and IV. The enterotoxin c gene (sec) was detected in 48.6% of isolates, showing the highest prevalence among the five enterotoxin genes detected. The positivity rates for the lukS/F-PV and tsst genes were 4% and 2.2%, respectively, while neither sed nor SasX were detected. ST45, ST59, ST338, ST188, ST6, ST7, ST22, ST25, ST398, and ST944 belonged to agr I group, while ST5 and ST15 belonged to agr II group. ST30 and ST1 were classified into agr III group, and ST121 was assigned into agr IV group. The tsst gene was found exclusively within agr I and III types belonging to ST7 and ST30 isolates, while the lukS/F-PV was predominantly carried by agr I type isolates primarily within CC59 and CC22 clones. Among the methicillin-resistant S. aureus (MRSA) isolates, 89.7% belonged to agr I group, and 97.8% of rifampicin-resistant or intermediate isolates were assigned to agr I group. MRSA isolates harboured more tested virulence genes compared to methicillin-susceptible S. aureus isolates.ConclusionsWe characterized the distributions of agr types and eight major virulence genes of 715 S. aureus isolates, and our findings revealed clear associations between agr types and STs, as well as virulence genes, and drug resistant phenotypes.

Antimicrobial susceptibilities and genomic characteristics of methicillin-resistant Staphylococcus aureus resistant to mupirocin in Stockholm, Sweden.

The aim of this study was to investigate antimicrobial susceptibilities and genomic characteristics of mupirocin-resistant MRSA isolates in Stockholm, Sweden. In total, 44 non-duplicate mupirocin-resistant MRSA isolates detected in Stockholm during 2010-2022 were investigated. Antimicrobial susceptibility testing was performed using broth microdilution method and further tested for high-level mupirocin-resistance (MuH) and rifampicin by Etest®. All isolates were subjected to whole genome sequencing. 41 isolates presented MuH with MICs ≥1024 mg/L whilst three isolates displayed low-level mupirocin resistance (MuL). mupA-gene was detected in all MuH isolates. Point mutations in ileS gene leading to N213D and V588F were identified in the three MuL isolates. Mutation in rpoB (H481N) was detected in a rifampicin-resistant isolate. Among the isolates, 15 multi-locus sequence types (MLST) were identified, with the four most common sequence types (ST22, ST72, ST8, and ST125) accounting for 66% of the isolates. Mupirocin-resistant MRSA in Stockholm was uncommon, with a percentage of <0.5% among MRSA cases during 2010-2022. In the present study, most mupirocin-resistant isolates were MuH and mupA-positive, predominantly linked to ST22 or ST72 isolates. MuL-resistance was associated with a point mutation in the IleS protein. A multidrug-resistant ST1-MRSA-IV strain was resistant to both mupirocin and rifampicin.

Hedychium coronarium J.Koenig produced extracellular and intracellular anti-tuberculosis activity with morphological alteration of the tubercle bacillus

Hedychium coronarium J.Koenig (Zingiberaceae) is a medium-sized aromatic rhizomatous perennial monocotyledonous plant. The rhizome of H. coronarium is reported in traditional medicine for its use in the treatment of tonsillitis, bronchitis, cough, fever and other respiratory disorders. The present work was carried out to evaluate anti-mycobacterial activity of the ethanolic extract of rhizomes of H. coronarium against respiratory pathogen Mycobacterium tuberculosis (Mtb) with its safety assessment and action on morphology of the bacilli. Anti-mycobacterial activity was assessed using the in-vitro resazurin microtiter plate assay (REMA). The plant extract was further evaluated for its cytotoxicity against human THP-1 macrophages. Ex-vivo intracellular anti-TB activity was also determined using Mtb infected macrophage culture model. Scanning electron microscopy (SEM) was used to examine the morphological changes in Mtb in the presence of plant extract.H. coronarium exhibited promising antimycobacterial activity against drug sensitive Mtb H37Rv and multi drug resistant (MDR) clinical isolates of Mtb with minimum inhibitory concentration (MIC) values ranging from 15.62 to 31.25 µg mL−1. The IC50 (50% inhibitory concentration) of plant extract on THP-1 was found at higher concentration than MIC values against Mtb, indicating that the mammalian cells were not adversely affected at concentrations which are effective against Mtb. The plant extract displayed intracellular killing of bacilli inside the infected macrophages. H. coronarium also produced morphological alteration of the bacilli after 48 and 72 h of treatment. In this study, we conclude that H. coronarium has promising in-vitro and ex-vivo intracellular activity against Mtb and it also displayed activity against rifampicin and isoniazid resistant clinical isolates of Mtb without affecting eukaryotic cells.

Molecular Analysis of Anti-Tuberculosis Drug Resistance of Mycobacterium tuberculosis Isolated in the Republic of Korea.

Rapid and accurate detection of tuberculosis (TB) drug resistance is critical for the successful treatment and control of TB. Here, we investigated resistance to anti-TB drugs and genetic variations in 215 drug-resistant Mycobacterium tuberculosis isolates in Korea. Genetic variations were observed in rpoB Ser531Leu, katG Ser315Thr, and gyrA Asp94Gly; however, the minimum inhibitory concentrations varied, which can be attributed to other resistance mechanisms. Examination of genetic relatedness among drug-resistant isolates revealed that the cluster size of resistant bacteria was less than six strains, suggesting no evidence of a large-scale epidemic caused by a specific strain. However, rpoC mutants of the rifampicin-resistant isolates were composed of five types of clusters, suggesting that these compensatory mutations advance propagation. In the present study, more than 90% of the resistance mechanisms to major anti-TB drugs were identified, and the effect of each mutation on drug resistance was estimated. With the clinical application of recent next-generation sequencing-based susceptibility testing, the present study is expected to improve the clinical utilization of genotype-based drug susceptibility testing for the diagnosis and treatment of patients with drug-resistant TB.

Isoniazid Resistance Level and Associated Resistance Conferring-mutations in Rifampicin Resistant Mycobacterium tuberculosis

Background: Multidrug resistant tuberculosis (MDR-TB) is a global public health problem causing treatment failure. Rifampicin (RIF) resistance has been used as a surrogate marker for MDR-TB diagnosis, but level of isoniazid (INH) resistance and associated resistance conferring mutations for INH in rifampicin-resistant TB cases are little known. Objective: The objective of this study was to determine level of isoniazid resistance and associated resistance conferring mutations in rifampicin resistant Mycobacterium tuberculosis (MTB) in sputum samples. Methods: A total 53 RIF resistant MTB isolates in sputum, detected by Xpert-MTB RIF assay were enrolled in the study. Culture positive samples were tested by BACTEC MGIT 960 system and level of isoniazid resistance was determined, defined as minimum inhibitory concentration of INH of &gt;0.4 µg/mL and 0.1-0.4 µg/mL as high level and low level INH resistance respectively. Distribution of mutation in katG (codon 315) and inhA promoter (-5, -8, -15 and -16) genes by Real-time PCR among the different degrees of INH resistance was investigated. Results: Among the growth positive isolates, 68.8% of the resistant isolates had high level INH resistance, where katG was found to be the prominent mutation, with or without combined with inhA mutation. Positive predictive value (PPV) of katG mutation was 84.6% in detecting a high level of INH resistance. Low level resistance was present in 31.3% isolates, conferring mutation in inhA and katG in equal percentage (40%), but no detectable mutations were found in 20% low level INH resistant MTB isolates. The PPV of inhA mutation was 33.3% in detection low level resistance. Conclusion: Most of the INH resistant isolates conferred high level resistance and were associated with katG mutation. Evaluation of level INH resistance before using high dose INH will help to avoid dose dependent toxicity and to determine an appropriate treatment regimen. Bangladesh Medical Res Counc Bull 2023; 49(2): 120-125

Rifampicin Enhanced Carbapenem Activity with Improved Antibacterial Effects and Eradicates Established Acinetobacter baumannii Biofilms.

Biofilm-mediated infections are critical to public health and a leading cause of resistance among pathogens, amounting to a prolonged hospital stay and increased mortality rate in the intensive care unit. In this study, the antibacterial and antibiofilm activities of rifampicin or carbapenem monotherapies were compared with rifampicin and carbapenem combination therapies against rifampicin-resistant and carbapenem-resistant Acinetobacter baumannii isolates. Among 29 CRAB isolates, 24/29 (83%) were resistant to rifampicin, with MIC values between 2-256 µg/mL. Checkerboard assays disclosed that combination therapies at FICIs between 1/8 and 1/4 improved the activity of carbapenems at subinhibitory concentrations. Time-kill kinetics indicated a 2- to 4-log reduction at 1/2 MIC rifampicin + 1/4 MIC carbapenem and 1/4 MIC rifampicin + 1/4 MIC carbapenem against the isolates, with the MIC values ranging from 2-8 µg/mL. The MTT assay revealed a dose-dependent decrease of the cell viability of established bacterial biofilm at 4 MIC rifampicin + 2 MIC carbapenems, with a percentage reduction of 44-75%, compared with monotherapies at 16 MIC. Scanning electron microscopy further confirmed bacterial cell membrane disruption, suggesting a synergism between carbapenem and rifampicin against a representative isolate. The findings demonstrated that the combination of rifampicin with carbapenems could improve antibacterial activities and eradicate established Acinetobacter baumannii biofilm.

Epidemiology of Xanthomonas euvesicatoria in Tokat Province

This study was carried out to determine the life span of Xanthomonas euvesicatoria (Xeu) in seeds, soil and plant residues and the first inoculum source that cause the onset of the disease. The study was carried out in the province of Tokat. Rifampicin-resistant Xeu isolate was inoculated into soil, seeds and plant residues. As a result of the studies, it was determined that Xeu remained viable in inoculated seeds until the 220th day (7 months), and lived in the soil for 105 days in summer and 14 days in winter. In plant residues, it was determined that the the life span in summer period was 60 days at 0 cm depth, 30 days at 20 cm depth; 30 days at 0 cm depth and 15 days at 20 cm depth in that of winter period. According to the results of this study, it was determined that the Xeu did not survive for a long time in plant residues and soil in Tokat province and thus was not carried to the next year, on the other hand the seed was the most important source of inoculum.

Clinical and microbiological characteristics of rifampicin-resistant MRSA bacteraemia.

The clinical significance of rifampicin resistance in Staphylococcus aureus infections has not been demonstrated. Here, we evaluated the clinical characteristics of rifampicin-resistant S. aureus infection. Data were collected from adult patients who were hospitalized with MRSA bacteraemia between March 2007 and May 2020 at a tertiary hospital in South Korea. The clinical characteristics and treatment outcomes of patients infected with rifampicin-resistant MRSA were compared with those of rifampicin-susceptible isolates. All-cause death and recurrence of MRSA infection were assessed for 90 days. Of the 961 patients with MRSA bacteraemia, 61 (6.3%) were infected by rifampicin-resistant isolates. The type of infection focus and duration of bacteraemia did not significantly differ between the two groups. Rifampicin-resistant MRSA isolates were more likely to have multidrug resistance and a higher vancomycin MIC relative to the rifampicin-susceptible isolates. The 90-day recurrence rate was higher in the patients infected with rifampicin-resistant MRSA compared with those with rifampicin-susceptible MRSA (18.0% versus 6.2%, P < 0.001), whereas the 90-day mortality was comparable between the two groups (27.9% versus 29.2%, P = 0.94). After adjusting for potential confounding factors, rifampicin resistance was significantly associated with 90-day recurrence (subdistributional HR: 2.31; 95% CI: 1.05-5.10; P = 0.04). Rifampicin-resistant MRSA isolates showed distinct microbiological features in terms of multidrug resistance and a high vancomycin MIC. Although the management of MRSA bacteraemia was not significantly different between the two groups, recurrence was significantly more common in the rifampicin-resistant group. Rifampicin resistance may play a significant role in infection recurrence.

Profiles of Isoniazid and Rifampicin drug resistance conferring mutations in Mycobacterium tuberculosis among new and previously treated pulmonary tuberculosis cases from Kisumu County, Kenya

Mycobacterium tuberculosis infection is one of the leading causes of mortality and morbidity in developing countries and drug resistance remains a challenge. Drug resistance is hastened by point mutations in the bacilli genome and their frequencies vary geographically hence the need to understand regional specific patterns for early detection of mutant strains. In this cross sectional study, Sputum samples from pulmonary tuberculosis clinical suspects were collected to detect mutations in Mycobacterium tuberculosis that confers resistance to Isoniazid and rifampicin anti-anti tuberculosis drugs. Detection of mutations was done using GenoType MTBDRplus. Out of a sample of 256 from Tuberculosis clinical suspected cases, 145 were Mycobacterium tuberculosis bacilli confirmed out of which 32 (22%) were new Tuberculosis (TB) cases and 113(78%) retreatment. The total for isoniazid resistance was 9(6.2%), out of which 2(6.3%) were in new cases and 7(6.3%) in retreatment cases. Multi Drug Resistance (MDR) was 2(1.8%) and all in retreatment cases. Rifampicin resistance was 7(4.8%), 1 (3.1%) in new case and 6(5.3%) in retreatment. The MDR among Rifampicin Resistance(RR) was 28.6%. Low Isoniazid resilient strains had changes in the katG gene resulting to nucleotide change from A G C to A C C. Four rifampicin resistant isolates showed mutations at the rpoB gene with nucleotide change from C A C to T A C and a single isolate displayed mutation at the rpoB gene with nucleotide change from T C G to T T G. Same nucleotide change A G C to A C C from different patients in the same facility might be an indication of local transmission of drug resistance and greater variability of mutations observed in HIV positive and retreatment cases are possibilities of mutations acquired during treatment courses by repeated administration of the same anti-TB drugs.

Whole genome sequencing of Bulgarian rifampicin resistant Mycobacterium tuberculosis strains.

The transmission of drug-resistant tuberculosis is one of the greatest challenges facing the global tuberculosis control. The aim of the study was to investigate the resent transmission of rifampicin resistant tuberculosis in Bulgaria and to describe the mutations related to the antimicrobials' resistance using whole genome sequencing. As part of an ECDC funded pilot study for evaluation of the systematic use of whole genome sequencing (WGS) of Mycobacterium tuberculosis (MTB) surveillance (EUSeqMyTB), Bulgaria provided 65 rifampicin resistant isolates over a three years' timeframe (2017-2019) representing 87.5% of the notified rifampicin resistant cases. Drug resistance prediction and relatedness analysis of the resistant isolates was performed in collaboration with San Raffaele Scientific Institute, Milan, Italy. Almost all of the isolates were identified as Euro-American lineage (96.9%); 18.5% of the isolates were found to be resistant to fluoroquinolones, but no mutations conferring resistance to bedaquiline or linezolid could be identified. Less than half (43.3%) of the isolates were clustered (<5 SNPs distance) into a total of seven national SNP-based clusters, while a total of six isolates were found to be part of different cross-border clusters. All clustered cases originated from Bulgaria. WGS has proven to be a reliable tool for surveillance and tracing of recent transmission of tuberculosis and has the potential for resistance prediction for most of the antituberculosis drugs.

Evaluation of whole-genome sequence to predict drug resistance of nine anti-tuberculosis drugs and characterize resistance genes in clinical rifampicin-resistant Mycobacterium tuberculosis isolates from Ningbo, China.

SettingControlling drug-resistant tuberculosis in Ningbo, China.ObjectiveWhole-genome sequencing (WGS) has not been employed to comprehensively study Mycobacterium tuberculosis isolates, especially rifampicin-resistant tuberculosis, in Ningbo, China. Here, we aim to characterize genes involved in drug resistance in RR-TB and create a prognostic tool for successfully predicting drug resistance in patients with TB.DesignDrug resistance was predicted by WGS in a “TB-Profiler” web service after phenotypic drug susceptibility tests (DSTs) against nine anti-TB drugs among 59 clinical isolates. A comparison of consistency, sensitivity, specificity, and positive and negative predictive values between WGS and DST were carried out for each drug.ResultsThe sensitivities and specificities for WGS were 95.92 and 90% for isoniazid (INH), 100 and 64.1% for ethambutol (EMB), 97.37 and 100% for streptomycin (SM), 75 and 100% for amikacin (AM), 80 and 96.3%for capreomycin (CAP), 100 and 97.22% for levofloxacin (LFX), 93.33 and 90.91% for prothionamide (PTO), and 70 and 97.96% for para-aminosalicylic acid (PAS). Around 53 (89.83%) and 6 (10.17%) of the isolates belonged to lineage two (East-Asian) and lineage four (Euro-American), respectively.ConclusionWhole-genome sequencing is a reliable method for predicting resistance to INH, RIF, EMB, SM, AM, CAP, LFX, PTO, and PAS with high consistency, sensitivity, and specificity. There was no transmission that occurred among the patients with RR-TB in Ningbo, China.

The RNA Polymerase Inhibitor Corallopyronin A Has a Lower Frequency of Resistance Than Rifampicin in Staphylococcus aureus.

Corallopyronin A (CorA) is active against Gram-positive bacteria and targets the switch region of RNA polymerase. Because of the high frequency of mutation (FoM) leading to rifampicin resistance, we determined the CorA FoM in S. aureus using fluctuation analysis at 4 × minimum inhibitory concentration (MIC). Resistant mutants were characterized. S. aureus strains HG001, Mu50, N315, and USA300 had an MIC of 0.25 mg/L. The median FoM for CorA resistance was 1.5 × 10−8, 4.5-fold lower than the median FoM of 6.7 × 10−8 for rifampicin, and was reflected in a 4-fold lower mutation rate for CorA than rifampicin (6 × 10−9 for CorA vs. 2.5 × 10−8 for rifampicin). In CorA-resistant/rifampicin-sensitive strains, the majority of amino acid exchanges were S1127L in RpoB or K334N in RpoC. S. aureus Mu50, a rifampicin-resistant clinical isolate, yielded two further exchanges targeting amino acids L1131 and E1048 of the RpoB subunit. The plating of >1011 cells on agar containing a combination of 4 × MIC of rifampicin and 4 × MIC of CorA did not yield any growth. In conclusion, with proper usage, e.g., in combination therapy and good antibiotic stewardship, CorA is a potential antibiotic for treating S. aureus infections.

Whole-Genome Sequencing for Resistance Level Prediction in Multidrug-Resistant Tuberculosis.

ABSTRACTDefining the precise relationship between resistance mutations and quantitative phenotypic drug susceptibility testing will increase the value of whole-genome sequencing (WGS) for predicting tuberculosis drug resistance. However, a large number of WGS data sets currently lack corresponding quantitative phenotypic data—the MICs. Using MYCOTBI plates, we determined the MICs to nine antituberculosis drugs for 154 clinical multidrug-resistant tuberculosis isolates from the Shenzhen Center for Chronic Disease Control in Shenzhen, China. Comparing MICs with predicted drug-resistance profiles inferred by WGS showed that WGS could predict the levels of resistance to isoniazid, rifampicin, streptomycin, fluoroquinolones, and aminoglycosides. We also found some mutations that may not be associated with drug resistance, such as EmbB D328G, mutations in the gid gene, and C−12T in the eis promoter. However, some strains carrying the same mutations showed different levels of resistance to the corresponding drugs. The MICs of different strains with the RpsL K88R, fabG1 C−15T mutations and some with mutations in embB and rpoB, had MICs to the corresponding drugs that varied by 8-fold or more. This variation is unexplained but could be influenced by the bacterial genetic background. Additionally, we found that 32.3% of rifampicin-resistant isolates were rifabutin-susceptible, particularly those with rpoB mutations H445D, H445L, H445S, D435V, D435F, L452P, S441Q, and S441V. Studying the influence of bacterial genetic background on the MIC and the relationship between rifampicin-resistant mutations and rifabutin resistance levels should improve the ability of WGS to guide the selection of medical treatment regimens.IMPORTANCE Whole-genome sequencing (WGS) has excellent potential in drug-resistance prediction. The MICs are essential indications of adding a particular antituberculosis drug dosage or changing the entire treatment regimen. However, the relationship between many known drug-resistant mutations and MICs is unclear, especially for rarer ones. The results showed that WGS could predict resistance levels to isoniazid, rifampicin, streptomycin, fluoroquinolones, and aminoglycosides. However, some mutations may not be associated with drug resistance, and some others may confer various MICs to strains carrying them. Also, 32.3% of rifampicin (RIF)-resistant strains were classified as sensitive to rifabutin (RFB), and some mutations in the rpoB gene may be associated with this phenotype. Our data on the MIC distribution of strains with some rarer mutations add to the accumulated data on the resistance level associated with such mutations to help guide further research and draw meaningful conclusions.

Cell Envelope Thickening: A Mechanism of Drug Resistance in Mycobacterium Tuberculosis

Emergence of multi-drug-resistant (MDR) strains of Mycobacterium tuberculosis (Mtb) is recognized as an alarming threat to public health and it limits the number of compounds available for treatment of global tuberculosis (TB) epidemic. MDR is defined as resistance to at least two first line drugs, e.g. Rifampicin and Isoniazid, used commonly for the treatment of this disease. Multi-drug resistance may be due to presence of a thick, hydrophobic, impermeable, and waxy cell envelope (cell wall and cell membrane combined together). The present study investigated the cell envelope thickness in Rifampicin and Isoniazid resistant and sensitive Mtb isolates by transmission electron microscopy (TEM). Electron microscopic examination exhibited evident differences in cell envelope thickness between the sensitive (approx. 24 - 27 nm) and resistant (approx. 34 - 42 nm) Mtb isolates. Thus, this study demonstrated that thickening of cell envelope in resistant isolates might be one of the primary defense mechanisms adopted by Mtb against antibacterial drugs and the observed resistance towards Rifampicin and Isoniazid, the major first line drugs used against TB, might be attributed to this enhanced cell envelope thickness in drug resistant strains.

Whole genome sequencing-based drug resistance predictions of multidrug-resistant Mycobacterium tuberculosis isolates from Tanzania.

BackgroundRifampicin- or multidrug-resistant (RR/MDR) Mycobacterium tuberculosis complex (MTBC) strains account for considerable morbidity and mortality globally. WGS-based prediction of drug resistance may guide clinical decisions, especially for the design of RR/MDR-TB therapies.MethodsWe compared WGS-based drug resistance-predictive mutations for 42 MTBC isolates from MDR-TB patients in Tanzania with the MICs of 14 antibiotics measured in the Sensititre™ MycoTB assay. An isolate was phenotypically categorized as resistant if it had an MIC above the epidemiological-cut-off (ECOFF) value, or as susceptible if it had an MIC below or equal to the ECOFF.ResultsOverall, genotypically non-wild-type MTBC isolates with high-level resistance mutations (gNWT-R) correlated with isolates with MIC values above the ECOFF. For instance, the median MIC value (mg/L) for rifampicin-gNWT-R strains was >4.0 (IQR 4.0–4.0) compared with 0.5 (IQR 0.38–0.50) in genotypically wild-type (gWT-S, P < 0.001); isoniazid-gNWT-R >4.0 (IQR 2.0–4.0) compared with 0.25 (IQR 0.12–1.00) among gWT-S (P = 0.001); ethionamide-gNWT-R 15.0 (IQR 10.0–20.0) compared with 2.50 (IQR; 2.50–5.00) among gWT-S (P < 0.001). WGS correctly predicted resistance in 95% (36/38) and 100% (38/38) of the rifampicin-resistant isolates with ECOFFs >0.5 and >0.125 mg/L, respectively. No known resistance-conferring mutations were present in genes associated with resistance to fluoroquinolones, aminoglycosides, capreomycin, bedaquiline, delamanid, linezolid, clofazimine, cycloserine, or p-amino salicylic acid.ConclusionsWGS-based drug resistance prediction worked well to rule-in phenotypic drug resistance and the absence of second-line drug resistance-mediating mutations has the potential to guide the design of RR/MDR-TB regimens in the future.