rpoB Mutations Research Articles

Regulatory Perturbations of Ribosome Allocation Optimize the Growth Proteome with a Trade-Off in Adaptation Capacity

Bacteria regulate their cellular resource allocation to enable their fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655. Mutants with only 3 copies of the strongest ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied rpoB mutants. Both fast-growing strains showed higher ribosomal content rather than higher ribosomal efficiency. Expression profiles of fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. Mutants showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA Polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments. We show how two different regulatory perturbations (rRNA promoters or rpoB mutations) optimize the proteome for growth with a concomitant fitness cost.

Evaluation of the frequency of mutation genes in multidrug-resistant tuberculosis (MDR-TB) strains in Beijing, China

The aim of this study was to explore the frequency and distribution of gene mutations that are related to isoniazid (INH) and rifampin (RIF)-resistance in the strains of the multidrug-resistant tuberculosis (MDR-TB) Mycobacterium tuberculosis (M.tb) in Beijing, China. In this retrospective study, the genotypes of 173 MDR-TB strains were analysed by spoligotyping. The katG, inhA genes and the promoter region of inhA, in which genetic mutations confer INH resistance; and the rpoB gene, in which genetic mutations confer RIF resistance, were sequenced. The percentage of resistance-associated nucleotide alterations among the strains of different genotypes was also analysed. In total, 90.8% (157/173) of the MDR strains belonged to the Beijing genotype. Population characteristics were not significantly different among the strains of different genotypes. In total, 50.3% (87/173) strains had mutations at codon S315T of katG; 16.8% (29/173) of strains had mutations in the inhA promoter region; of them, 5.5% (15/173) had point mutations at -15 base (C→T) of the inhA promoter region. In total, 86.7% (150/173) strains had mutations at rpoB gene; of them, 40% (69/173) strains had mutations at codon S531L of rpoB. The frequency of mutations was not significantly higher in Beijing genotypic MDR strains than in non-Beijing genotypes. Beijing genotypic MDR-TB strains were spreading in Beijing and present a major challenge to TB control in this region. A high prevalence of katG Ser315Thr, inhA promoter region (-15C→T) and rpoB (S531L) mutations was observed. Molecular diagnostics based on gene mutations was a useful method for rapid detection of MDR-TB in Beijing, China.

Compensatory effects of M. tuberculosis rpoB mutations outside the rifampicin resistance-determining region

ABSTRACT Mycobacterium tuberculosis has been observed to develop resistance to the frontline anti-tuberculosis drug rifampicin, primarily through mutations in the rifampicin resistance-determining region (RRDR) of rpoB. While these mutations have been determined to confer a fitness cost, compensatory mutations in rpoA and rpoC that may enhance the fitness of resistant strains have been demonstrated. Recent genomic studies identified several rpoB non-RRDR mutations that co-occurred with RRDR mutations in clinical isolates without rpoA/rpoC mutations and may confer fitness compensation. In this study, we identified 33 evolutionarily convergent rpoB non-RRDR mutations through phylogenomic analysis of public genomic data for clinical M. tuberculosis isolates. We found that none of these mutations, except V170F and I491F, can cause rifampin resistance in Mycolicibacterium smegmatis. The compensatory effects of five representative mutations across rpoB were evaluated by an in vitro competition assay, through which we observed that each of these mutations can significantly improve the relative fitness of the initial S450L mutant (0.97–1.08 vs 0.87). Furthermore, we observed that the decreased RNAP transcription efficiency introduced by S450L was significantly alleviated by each of the five mutations. Structural analysis indicated that the fitness compensation observed for the non-RRDR mutations might be achieved by modification of the RpoB active centre or by changes in interactions between RNAP subunits. Our results provide experimental evidence supporting that compensatory effects are exerted by several rpoB non-RRDR mutations, which could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant M. tuberculosis strains.

Prevalence and Analysis of Drug Resistance Pattern of MDR-TB in Retreatment Cases at Allied Hospital, Faisalabad, Pakistan

Background: The prevalence of Multi-Drug Resistance-Tuberculosis (MDR-TB) among retreatment cases is very high. It has become a serious public health issue in Pakistan and can affect greatly TB control strategies. So, we conducted the present research work to update a systematic review of the literature available on MDR-TB cases. Objective: To analyze the resistance pattern of multidrug-resistant tuberculosis. To study the prevalence of MDR-TB among new cases, retreatment cases. Study Design: Cross-sectional study. Settings: Pathology department, Allied Hospital, Faisalabad Medical University, Faisalabad, Pakistan. Duration: Six months from January 01, 2020 to June 30, 2020. Methodology: In this study, we included new and retreatment tuberculosis cases. Gene mutation RpoB, DNA band sequence detected by using machine, Cepheid gene Xpert IV – 4 Modules, Sr # 806133. Data was collected and analyzed statistically by using SPSS v 20. Results: 858 cases were included of age 12 years – 100yrs with the mean age (in years) 42.139 ± 16.823.  Two groups of males and females were studied separately. Group I included male patients {456 (56.64%)} and further subdivided into IA [age above 30 years, {354(72.84%)}] and IB [age below 30 years {132 (27.15%)}]. Group II included female patients {372 (43.36%)} and further subdivided into IIA [age above 30 years {239 (64.25%)}] and IIB [age below 30 years {133(35.75%)}]. Out of 858 cases, 347 (40.44%) were New cases, 13 (1.51%) New cases with HIV and 408 (47.55%) were Previously Treated patients (PT). 30 (3.45%) cases were unknown (UK). 28 (3.26%) cases were brought by different lady health workers (LHW) and relapse cases were 32 (3.73%). Overall statistical analysis was found to be highly significant (p=0.000, df= 1, CI= 95%). According to our results, in 517 (60.26%) patients, Mycobacterium Tuberculosis was not detected (MTB-ND). In 214 (24.94%) cases, Mycobacterium Tuberculosis was detected (MTB-D), out of which 13 (6.07%) cases were of First-Line Multi Drug Resistance (MDR). Age of MDR cases was in between 17- 70 years with the mean age (in years) 37.307 ± 17.637. Our results showed MDR more in males (53. 85%) as compared to female patients (46. 15%). Our results showed a significant correlation between status of all cases and their reports (p= -.050). For MTB-ND cases p= 0.000, df =1, CI=95% and for invalid cases p= 0.000, df =5, CI=95%. Conclusion: There is a great need to implement national tuberculosis guidelines fully.  In order to prevent further emergence of drug resistance, we should focus on effective implementation of directly observed therapy short courses.

Tissue specific diversification, virulence and immune response to Mycobacterium bovis BCG in a patient with an IFN-γ R1 deficiency

ABSTRACT Summary: We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits. Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient’s lungs and brain. Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling. Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling. Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.

Genomic surveillance of Neisseria meningitidis serogroup B invasive strains: Diversity of vaccine antigen types, Brazil, 2016-2018.

BackgroundNeisseria meningitidis serogroup B remains a prominent cause of invasive meningococcal disease (IMD) in Brazil. Because two novel protein-based vaccines against serogroup B are available, the main purpose of this study was to provide data on the diversity and distribution of meningococcal vaccine antigen types circulating in Brazil.MethodologyGenetic lineages, vaccine antigen types, and allele types of antimicrobial-associated resistance genes based on whole-genome sequencing of a collection of 145 Neisseria meningitidis serogroup B invasive strains recovered in Brazil from 2016 to 2018 were collected.ResultsA total of 11 clonal complexes (ccs) were identified among the 145 isolates, four of which were predominant, namely, cc461, cc35, cc32, and cc213, accounting for 72.0% of isolates. The most prevalent fHbp peptides were 24 (subfamily A/variant 2), 47 (subfamily A/variant 3), 1 (subfamily B/variant 1) and 45 (subfamily A/variant 3), which were predominantly associated with cc35, cc461, cc32, and cc213, respectively. The NadA peptide was detected in only 26.2% of the isolates. The most frequent NadA peptide 1 was found almost exclusively in cc32. We found seven NHBA peptides that accounted for 74.5% of isolates, and the newly described peptide 1390 was the most prevalent peptide exclusively associated with cc461. Mutated penA alleles were detected in 56.5% of the isolates, whereas no rpoB and gyrA mutant alleles were found.ConclusionDuring the study period, changes in the clonal structure of circulating strains were observed, without a predominance of a single hyperinvasive lineage, indicating that an epidemiologic shift has occurred that led to a diversity of vaccine antigen types in recent years in Brazil.

In Vitro Activity of Dalbavancin against Refractory Multidrug-Resistant (MDR) Staphylococcus aureus Isolates.

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections, always treated with vancomycin and daptomycin, has led to the emergence of vancomycin-intermediate (VISA), heteroresistant vancomycin-intermediate (hVISA) and daptomycin non-susceptible (DNS) S. aureus. Even if glycopeptides and daptomycin remain the keystone for treatment of resistant S. aureus, the need for alternative therapies that target MRSA has now become imperative. The in vitro antibacterial and bactericidal activity of dalbavancin was evaluated against clinically relevant S. aureus showing raised antibiotic resistance levels, from methicillin-susceptible to Multidrug-Resistant (MDR) MRSA, including hVISA, DNS and rifampicin-resistant (RIF-R) strains. A total of 124 S. aureus strains were tested for dalbavancin susceptibility, by the broth microdilution method. Two VISA and 2 hVISA reference strains, as well as a vancomycin-resistant (VRSA) reference strain and a methicillin-susceptible Staphylococcus aureus (MSSA) reference strain, were included as controls. Time–kill curves were assayed to assess bactericidal activity. Dalbavancin demonstrated excellent in vitro antibacterial and bactericidal activity against all S. aureus resistance classes, including hVISA and DNS isolates. The RIF-R strains showed the highest percentage of isolates with non-susceptibility, reflecting the correlation between rpoB mutations and VISA/hVISA emergence. Our observations suggest that dalbavancin can be considered as an effective alternative for the management of severe MRSA infections also sustained by refractory phenotypes.

Positivity Rate of Pyrosequencing to Diagnose Drug-Resistant Tuberculosis Directly from Sputum with Different Bacterial Load

BACKGROUND: Molecular techniques, which detect mutations associated with drug resistance tuberculosis (TB), are promising technologies for rapid diagnosis and monitoring of drug-resistant TB. Pyrosequencing is a potential rapid and robust molecular technique to detect drug resistance but its performance in clinical samples is less investigated. This study aimed to determine the positivity rate of pyrosequencing to diagnose drug-resistant TB directly from sputum samples with different grades of sputum smear microscopy results.METHODS: Thirty-five sputum specimens from drug-resistant TB suspects were submitted for acid-fast bacilli (AFB) microscopy. All specimens were cultured using microscopic observation drug susceptibility (MODS) culture. Pyrosequencing was performed to DNA extracted from sputum of culture-positive patients.RESULTS: MODS culture was positive in 19/35 subjects (54.29%) samples; 16 smear-positive and three smear-negative. Using pyrosequencing, Mycobacterium tuberculosis was identified in all culture-positive samples, including smear-negative samples. A complete resistance profile for 16 (82.35%) samples could be generated. Pyrosequencing failed to show results for eis or gyrA promoter in three samples. Nine of 19 patients were multidrug resistant-TB (MDR-TB), 1/19 was rifampicin-resistance TB (RR-TB), and 4/19 were pre-extensively drug-resistant TB (pre-XDR-TB). Two novel mutations in rpoB and rrs (associated with rifampicin and aminoglycoside, respectively) were found in this study.CONCLUSION: The results of this study demonstrates high positivity rates of pyrosequencing to detect drug-resistant TB directly from sputum samples with different grades of smear microscopy, as the surrogate of bacterial load. The assay can be used as a first prediction test of drug resistance prior to confirmation by phenotypic tests.KEYWORDS: drug-resistant tuberculosis, pyrosequencing, direct sputum

Comparison of Interpretation between Pyrosequencing and Xpert MTB/RIF Assay in Multidrug-Resistant Tuberculosis

Indonesia is one of the countries with the highest multidrug-resistant tuberculosis cases in the world. Rapid molecular test using the Xpert MTB/RIF assay is one of the detection methods for MDR-TB. Early detection of MDR-TB is crucial for early initiation of treatment. However, Xpert MTB/RIF assay only detects the rpoB gene mutations associated with Rifampicin resistance. Recently, WHO recommends the use of Pyrosequencing, a DNA sequencing method that can detect not only the rpoB gene but also katG and/or inhA gene mutations associated with Isoniazid resistance. The aims of this study were to compare the interpretation between the two methods and to determine the differences in codon mutation position detection of the rpoB gene and mutation detection of the katG and/or inhA gene. This was a cross-sectional comparative observational study on patients ≥18 years old interpreted as RR-TB patients based on Xpert MTB/RIF assay results who had not received MDR-TB drugs at Dr. Hasan Sadikin General Hospital, Bandung, Indonesia. Results showed there were 40 Rifampicin-resistant TB subjects interpreted by Xpert MTB/RIF assay while Pyrosequencing interpreted 30 MDR-TB, 9 RR-TB and one Isoniazid-resistant TB subjects in January - February 2020. The detection of rpoB gene codon mutation position between Xpert MTB/RIF assay and Pyrosequencing methods was not significantly different (p=0.389). Pyrosequencing had detected 27 katG gene mutations, 3 inhA gene mutations, one katG and inhA gene mutation. To conclude, Pyrosequencing can be used for accurate detection of Rifampicin and Isoniazid resistance in MDR-TB.

MDR M. tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era

BackgroundMultidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini.MethodsWe investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017).ResultsMDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2.ConclusionThe high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Mutant RNA polymerase can reduce susceptibility to antibiotics via ppGpp-independent induction of a stringent-like response.

BackgroundMutations in RNA polymerase (RNAP) can reduce susceptibility to ciprofloxacin in Escherichia coli, but the mechanism of transcriptional reprogramming responsible is unknown. Strains carrying ciprofloxacin-resistant (CipR) rpoB mutations have reduced growth fitness and their impact on clinical resistance development is unclear.ObjectivesTo assess the potential for CipRrpoB mutations to contribute to resistance development by estimating the number of distinct alleles. To identify fitness-compensatory mutations that ameliorate the fitness costs of CipRrpoB mutations. To understand how CipRrpoB mutations reprogramme RNAP.Methods E. coli strains carrying five different CipRrpoB alleles were evolved with selection for improved fitness and characterized for acquired mutations, relative fitness and MICCip. The effects of dksA mutations and a ppGpp0 background on growth and susceptibility phenotypes associated with CipRrpoB alleles were determined.ResultsThe number of distinct CipRrpoB mutations was estimated to be >100. Mutations in RNAP genes and in dksA can compensate for the fitness cost of CipRrpoB mutations. Deletion of dksA reduced the MICCip for strains carrying CipRrpoB alleles. A ppGpp0 phenotype had no effect on drug susceptibility.ConclusionsCipRrpoB mutations induce an ppGpp-independent stringent-like response. Approximately half of the reduction in ciprofloxacin susceptibility is caused by an increased affinity of RNAP to DksA while the other half is independent of DksA. Stringent-like response activating mutations might be the most diverse class of mutations reducing susceptibility to antibiotics.

RpoB mutations conferring rifampicin-resistance affect growth, stress response and motility in Vibrio vulnificus.

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the β subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus , even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml−1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

Prediction of rifampicin resistance beyond the RRDR using structure-based machine learning approaches

Rifampicin resistance is a major therapeutic challenge, particularly in tuberculosis, leprosy, P. aeruginosa and S. aureus infections, where it develops via missense mutations in gene rpoB. Previously we have highlighted that these mutations reduce protein affinities within the RNA polymerase complex, subsequently reducing nucleic acid affinity. Here, we have used these insights to develop a computational rifampicin resistance predictor capable of identifying resistant mutations even outside the well-defined rifampicin resistance determining region (RRDR), using clinical M. tuberculosis sequencing information. Our tool successfully identified up to 90.9% of M. tuberculosis rpoB variants correctly, with sensitivity of 92.2%, specificity of 83.6% and MCC of 0.69, outperforming the current gold-standard GeneXpert-MTB/RIF. We show our model can be translated to other clinically relevant organisms: M. leprae, P. aeruginosa and S. aureus, despite weak sequence identity. Our method was implemented as an interactive tool, SUSPECT-RIF (StrUctural Susceptibility PrEdiCTion for RIFampicin), freely available at https://biosig.unimelb.edu.au/suspect_rif/.

Identification of a novel tedizolid resistance mutation in rpoB of MRSA after in vitro serial passage.

Tedizolid is an oxazolidinone antimicrobial with activity against Gram-positive bacteria, including MRSA. Tedizolid resistance is uncommon and tedizolid's capacity to select for cross-resistance to other antimicrobials is incompletely understood. The objective of this study was to further explore the phenotypic and genetic basis of tedizolid resistance in MRSA. We selected for tedizolid resistance in an MRSA laboratory strain, N315, by serial passage until an isolate with an MIC ≥1 log2 dilution above the breakpoint for resistance (≥2 mg/L) was recovered. This isolate was subjected to WGS and susceptibility to a panel of related and unrelated antimicrobials was tested in order to determine cross-resistance. Homology modelling was performed to evaluate the potential impact of the mutation on target protein function. After 10 days of serial passage we recovered a phenotypically stable mutant with a tedizolid MIC of 4 mg/L. WGS revealed only one single nucleotide variant (A1345G) in rpoB, corresponding to amino acid substitution D449N. MICs of linezolid, chloramphenicol, retapamulin and quinupristin/dalfopristin increased by ≥2 log2 dilutions, suggesting the emergence of the so-called 'PhLOPSa' resistance phenotype. Susceptibility to other drugs, including rifampicin, was largely unchanged. Homology models revealed that the mutated residue of RNA polymerase would be unlikely to directly affect oxazolidinone action. To the best of our knowledge, this is the first time that an rpoB mutation has been implicated in resistance to PhLOPSa antimicrobials. The mechanism of resistance remains unclear, but is likely indirect, involving σ-factor binding or other alterations in transcriptional regulation.

Detection of Beijing strains of MDR M. tuberculosis and their association with drug resistance mutations in katG, rpoB, and embB genes

BackgroundMolecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients.MethodsIn this study 381 M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n = 131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene.ResultsThe sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p = 0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p = < 0.0001, < 0.0001 & 0.0014 respectively) was also found.ConclusionsThis rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains.

Transmission of Mycobacterium tuberculosis presenting unusually high discordance between genotypic and phenotypic resistance to rifampicin in an endemic tuberculosis setting

BackgroundSince the implementation of the Xpert MTB/RIF in Sao Paulo, Brazil, numerous Mycobacterium tuberculosis isolates presenting “rifampicin-resistant genotype with rifampicin-susceptible phenotype” were observed. ObjectiveTo evaluate the prevalence, rpoB mutations and transmission of M. tuberculosis resistant to rifampicin on Xpert MTB/RIF but susceptible on BACTEC MGIT system, in Sao Paulo state. MethodsPatients’ isolates with this pattern of rifampicin discordance, collected from 2014 to 2017, had their rpoB predominant rifampicin-resistance-determining region sequenced and were genotyped by IS6110 restriction fragment-length polymorphism. FindingsThe prevalence of rifampicin-discordant M. tuberculosis with genotypic resistance was 55.1% (156/283). Among the sequenced and genotyped isolates, 75.5% (111/147) were in clusters, largely associated with the type of rpoB mutation. Most isolates (98.6%; 72/73) harbouring the predominant mutation, His445Asn, were pooled into the two largest clusters, SP2ga (42/72; 58.3%) and SP5o (12/72; 16.7%). Ranking second, isolates carrying the silent mutation Phe433Phe were mostly (92.3%; 24/26) gathered into four groups of the family SP25. ConclusionThese findings suggest that this unusual high rifampicin discrepancy proportion was greatly influenced by few actively circulating clusters. Further studies on many of the rpoB mutations identified in our setting are needed to elucidate their association with phenotypic rifampicin resistance.

Genotypic Distribution and a Potential Diagnostic Assay of Multidrug-Resistant Tuberculosis in Northern Thailand.

IntroductionKnowledge of the prevalence and distribution of multidrug-resistant tuberculosis (MDR-TB) genotypes in northern Thailand is still limited. An accurate, rapid, and cost-effective diagnostic of MDR-TB is crucial to improve treatment and control of increased MDR-TB.Materials and MethodsThe molecular diagnostic assays named “RIF-RD” and “INH-RD” were designed to detect rifampicin (RIF) and isoniazid (INH) resistance based on real-time PCR and high-resolution melting curve analysis. Applying the ∆Tm cutoff values, the RIF-RD and INH-RD were evaluated against the standard drug susceptibility testing (DST) using 107 and 103 clinical Mycobacterium tuberculosis (Mtb) isolates from northern Thailand. DNA sequence analysis of partial rpoB, katG, and inhA promoter of 73 Mtb isolates, which included 30 MDR-TB, was performed to elucidate the mutations involved with RIF and INH resistance.ResultsWhen compared with the phenotypic DST, RIF-RD targeting rpoB showed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 83.9, 98.6, 96.9, and 92.0%, respectively. The multiplex reaction of the INH-RD targeted both katG and inhA promoter showed high sensitivity, specificity, PPV, and NPV of 97.1, 94.2, 89.2, and 98.5%, respectively. Six patterns of rpoB mutation, predominately at codons 531 (50%) and 526 (40%) along with a rare S522L (3.33%) and D516V (3.33%), were detected. A single pattern of katG mutation (S315T) (63.3%) and four patterns of inhA promoter mutation, predominately −15 (C>T), were found. Approximately, 17% of MDR-TB strains possessed double mutations within the katG and inhA promoter.ConclusionUp to 86.7% and 96.7% of MDR-TB could be accurately detected by RIF-RD and INH-RD, emphasizing its usefulness as a low unit price assay for rapid screening of MDR-TB, with confirmation of INH resistance in low and middle-income countries. The MDR-TB genotypes provided will be beneficial for TB control and the development of drug-resistant TB diagnostic technology in the future.

Diagnostic Evaluation of Non-Interpretable Results Associated with rpoB Gene in Genotype MTBDRplus Ver 2.0.

BackgroundLine probe assay (LPA) is standard diagnostic tool to detect multidrug resistant tuberculosis. Non-interpretable (NI) results in LPA (complete missing or light wild-type 3 and 8 bands with no mutation band in rpoB gene region) poses a diagnostic challenge.MethodsSputum samples obtained between October 2016 and July 2017 at the Intermediate Reference Laboratory, All India Institute of Medical Sciences Hospital, New Delhi, India were screened. Smear-positive and smear-negative culture-positive specimens were subjected to LPA Genotype MTBDRplus Ver 2.0. Smear-negative with culture-negative and culture contamination were excluded. LPA NI samples were subjected to phenotypic drug susceptibility testing (pDST) using MGIT-960 and sequencing.ResultsA total of 1,614 sputum specimens were screened and 1,340 were included for the study (smear-positive [n=1,188] and smear-negative culture-positive [n=152]). LPA demonstrated 1,306 (97.5%) valid results with TUB (Mycobacterium tuberculosis) band, 24 (1.8%) NI, three (0.2%) valid results without TUB band, and seven (0.5%) invalid results. Among the NI results, 22 isolates (91.7%) were found to be rifampicin (RIF) resistant and two (8.3%) were RIF sensitive in the pDST. Sequencing revealed that rpoB mutations were noted in all 22 cases with RIF resistance, whereas the remaining two cases had wild-type strains. Of the 22 cases with rpoB mutations, the most frequent mutation was S531W (n=10, 45.5%), followed by S531F (n=6, 27.2%), L530P (n=2, 9.1%), A532V (n=2, 9.1%), and L533P (n=2, 9.1%).ConclusionThe present study showed that the results of the Genotype MTBDRplus assay were NI in a small proportion of isolates. pDST and rpoB sequencing were useful in elucidating the cause and clinical meaning of the NI results.

Characterization of mutations in the rpoB gene conferring rifampicin resistance in Mycobacterium tuberculosis complex isolated from lymph nodes of slaughtered cattle from South Africa.

Tuberculosis (TB) is an ongoing public health care, with the state of affairs exacerbated by the growth of anti-TB drug-resistant forms in South Africa. Not much attention is given to zoonotic TB. Thus, this study aimed to determine the presence of rpoB mutations among Mycobacterium tuberculosis complex (MTBC) isolates of lymph nodes from slaughtered cattle. A count of 14,950 carcasses from selected abattoirs were examined for nodular lesions and enlarged lymph nodes; 376 lymph nodes were cultured for MTBC. Positive isolates were tested for drug sensitivity against three anti-TB drugs, rifampicin, isoniazid, and ethambutol, using the Lowenstein-Jensen proportion method. Rifampicin-resistant isolates were sequenced, and spoligotyping was performed for lineage classification. A total of 162 isolates were confirmed as MTBC and 42 isolates were resistant to rifampicin. All rifampicin-resistant isolates carried the H526D rpoB mutation, and almost all of them carried an additional nonsynonymous nucleotide substitution in the hot spot region, in three other codons (510, 516 and 522). In total, 5 different mutations at four codons are reported, including one isolate showing 3 of them which has never been reported in South Africa. In addition, we report 4 different spoligo patterns, with 34 isolates known and 8 unknown spoligotype international types. From the known clades, 5 (11.9%) isolates were identified as Bov_4 caprae lineage, 29 (69%) Beijing, and 8 (19.1%) remaining unknown clades. The detection of MTBC-resistant patterns from cattle lymph nodes (Eastern Cape, South Africa) necessitates the investigation of other possible routes of MTBC transmission.

Fluorescein diacetate and rapid molecular testing for the early identification of rifampicin resistance in Mali.

BACKGROUND: Non-conversion on auramine smear microscopy indicates a lack of treatment response, possibly associated with initial rifampicin-resistant tuberculosis (RR-TB). However, dead bacteria still stain positive and may be detected. Fluorescein diacetate smear microscopy (FDA) shows live mycobacteria only. Therefore, we studied the potential of 2-month (2M) FDA for the identification of initial RR-TB.METHODS: Between 2015 and 2018, we enrolled new smear-positive pulmonary TB patients from five local centres in Bamako, Mali. After baseline screening, sputum samples were collected at 1M, 2M, 5M and 18M. We used rpoB sequencing to identify initial RR-TB.RESULTS: Of 1359 patients enrolled, 1019 (75%) had rpoB sequencing results. Twenty-six (2.6%, 95%CI: 1.7-3.7) had mutations conferring rifampicin resistance. Most frequent rpoB mutations were located at the codons Asp435Val (42.4%) and Ser450Leu (34.7%). Among patients with initial RR-TB, 72.2% were FDA-negative at 2M (P = 0.2). The positive and negative predictive value of 5M FDA for culture-based failure was respectively 20.0% and 94.7%.CONCLUSION: FDA did not identify the majority of patients with initial RR-TB or culture-based failure. As the full spectrum of mutations identified on sequencing was identified using Xpert, our data support its rapid universal implementation in Mali.