Mutations Conferring Rifampicin Resistance Research Articles

Missed phenotypic drug resistance in pediatric tuberculosis: A cause of concern in a resource-limited setting

BackgroundMulti-drug resistance (MDR) in pediatric tuberculosis (TB) is a growing global threat. Unavailability of conventional or molecular drug susceptibility test (DST) in resource-limited settings often impede the determination of the extent of first line anti-tubercular drugs deployed in national programs. Materials and methodPulmonary and extra pulmonary specimens were collected from clinically suspected pediatric TB cases, who were microbiologically confirmed. Resistance to first-line anti-TB was detected by 1% proportion method. KatG315 and inhA-15 genes were amplified by PCR and detection of mutations were done by sequencing. Genotypic resistance for rifampicin was detected by Xpert MTB/RIF assay (Cepheid Inc., Sunnyvale, California). ResultsFifty-one cases of pediatric tuberculosis were confirmed microbiologically. Resistance to isoniazid, streptomycin, rifampicin and ethambutol were 5 (14%), 4 (11%), 2 (5.5%) and 2 (5.5%) respectively by 1% proportion method. Genotypic Rifampicin and isoniazid resistance was found in 2 (5.5%) and 7 (14%) samples respectively. ConclusionExisting genotypic methods, detect targeted mutations conferring rifampicin resistance, however isoniazid (INH) resistance often go undetected. Since the resistance to pivotal anti-TB drugs are often encoded by multiple genes which may not be targeted by widely available molecular tests, discrepancies in molecular and culture-based DST reports should be interpreted with caution.

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.

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.

Molecular detection of mutations in rpoB gene involved in rifampin resistance in leprosy patients from Côte d'Ivoire

The control measures recommended for treatment of leprosy with multidrug therapy should limit spread of drug-resistant strains; however, rifampicin resistance continues to be reported in several countries. Resistance to anti-infectious used in multidrug therapy has not yet been molecularly established in Cote d'Ivoire. This preliminary study presents results of evaluation of level rifampicin resistance in Cote d'Ivoire. For each case of clinically confirmed multibacillary leprosy (new or relapsed cases) a dermal juice sample was examined by microscopy after Ziehl-Neelsen staining and a specific PCR targeting RLEP repeat sequence. 48 isolates were used to perform amplification of the rpoB gene involved in rifampicin resistance. The origin of the resistance was then investigated after sequencing and mutations analysis. Thus, 24 isolates (50 %) had mutations, 11 (22. 91 %) of which had mutations conferring rifampicin resistance. The mutations correlated with resistance were as follows: Gln438Val (9. 09 %), Asp441Asn (18. 18 %), Ser456Leu (45. 50 %) and Ser456Phe (27. 27 %) in 9 health districts in Cote d'Ivoire. These mutations were observed in both relapsed and new cases. In conclusion, cases of rifampicin resistance exist in Cote d'Ivoire and an active surveillance should be carried out to monitor the evolution of this resistance. In addition, this information can be used to control the spread of drug-resistant M. leprae strains.

Epidemiological and molecular aspects of rifampicin-resistant Staphylococcus aureus isolated from wounds, blood and respiratory samples

To study the incidence of rifampicin-resistant Staphylococcus aureus in Gipuzkoa, Northern Spain, and to characterize representative resistant isolates and mutations associated with resistance. For rifampicin-resistant isolates, the rpoB gene fragment that includes the most frequent mutations conferring rifampicin resistance in S. aureus was amplified and sequenced. The role of new mutations responsible for rifampicin resistance was confirmed by cloning and complementation in trans. Resistant isolates were characterized by multilocus sequence typing and PFGE. Between 1999 and 2008, 0.59% (96/16 348) of S. aureus clinical isolates studied showed rifampicin resistance. Rifampicin resistance was higher in methicillin-resistant S. aureus (MRSA) than in methicillin-susceptible S. aureus (MSSA) (3.26% versus 0.26%; P < 0.001). Twenty-two randomly selected rifampicin-resistant isolates were studied in depth, 11 showing low-level and 11 showing high-level rifampicin resistance (rifampicin MICs of 2-4 mg/L and ≥8 mg/L, respectively). Overall, 12 different mutations in the rpoB gene were detected, including a newly described N474K mutation followed by the insertion of a glycine residue at position 475. Among the eight different sequence types (STs) found, the most frequent were ST8 and ST863, the latter being associated with respiratory infections. Ten of the 11 low-level rifampicin-resistant isolates were MRSA ST8 and had the same H481N mutation, while the 11 high-level rifampicin-resistant isolates, 6 MSSA and 5 MRSA, belonged to eight different STs and had distinct rpoB mutations. Low-level rifampicin-resistant isolates were mainly clonal while high-level resistant isolates showed a high genetic diversity. Most mutations observed coincided with those found in other studies, but a new mutation conferring rifampicin resistance was detected.

Rifampicin Resistance Phenotyping of Brucella melitensis by rpoB Gene Analysis in Clinical Isolates

Rifampicin resistance of Brucella melitensis by rpoB gene analysis has not yet been performed in Turkey, where brucellosis is endemic. In this study, we investigated the efficacy of E-test and single nucleotide polymorphism (SNP) analysis of the B. melitensis rpoB gene, for the detection of mutations conferring rifampicin resistance, by sequencing 21 human B. melitensis strains from the Southeast and Marmara regions of Turkey. On CLSI slow-growing bacteria standards, all isolates were sensitive to rifampicin except for 6 which showed intermediate resistance to rifampicin. MIC50 and MIC90 values were 1 μg/ml and 1.5 μg/ml respectively (range 0.50 -1.5 μg/ml). The rifampicin-resistant phenotype was investigated at Cd 154 (GTT/TTT), Cd 526 (GAC/TAC, GAC/AAC, GAC/GGC), Cd 536 (CAC/CTC, CAC/TAC), Cd 539 (CGC/AGC), Cd 541 (TCG/TTG) and Cd 574 (CCG/CTG) of the rpoB gene in B. melitensis 16M and B115 strains, and in clinical isolates. No missense mutations were found in any of the B. melitensis isolates, which indicates that all isolates were rifampicin-susceptible. In conclusion, SNP analysis was useful as a molecular tool for rifampin resistance testing. Although resistance to rifampicin was not detected in our strains of B. melitensis; the presence of strains with intermediate resistance to rifampicin indicates that susceptibility testing should be performed periodically.

Distribution of rpoB mutations among multidrug-resistant Mycobacterium tuberculosis (MDRTB) strains from Thailand and development of a rapid method for mutation detection

Since rifampicin resistance is a surrogate marker for multidrug-resistant Mycobacterium tuberculosis (MDRTB), the present study aimed to investigate rpoB mutations conferring rifampicin resistance in M. tuberculosis strains from Thailand, and to develop a rapid, inexpensive and simple PCR-based method for rapid detection of MDRTB. Overall, 267 M. tuberculosis isolates, including 143 MDRTB isolates, were investigated. Isolates of the Beijing strain predominated among the MDRTB isolates (79.1%), but accounted for only 45.5% of the susceptible isolates. Mutations in the rpoB gene were found most commonly at codons 531, 526 and 516 (58%, 25.2% and 9.1%, respectively). A multiplex allele-specific PCR was developed and tested with 216 clinical isolates. In comparison with the proportion method, the method showed 94.2% sensitivity and 100% specificity, and had a 100% positive predictive value and a 95% negative predictive value, which suggested that this method could be useful for screening for MDRTB, particularly in resource-limited countries.

Development and evaluation of a novel multiple-primer PCR amplification refractory mutation system for the rapid detection of mutations conferring rifampicin resistance in codon 425 of the rpoB gene of Mycobacterium leprae

Rifampicin-resistant Mycobacterium leprae is regularly reported and drug resistance is a major threat for the elimination of leprosy. There is an urgent need for a simple method that can detect rifampicin resistance in clinical isolates. This study developed a multiple-primer PCR amplification refractory mutation system, a simple, reliable and economical method for clinical specimens that allowed the rapid detection of mutations in the nucleotides of the codon for Ser425 of the M. leprae rpoB gene, mutation of which to Leu, Met or Phe is associated with rifampicin resistance. The approach involved a multiple-primer PCR in which both mutant-specific and normal sets of primers were included in the reaction. The mutant-specific primer was complementary to the corresponding sequence of the wild-type gene except for one additional deliberate mismatch at the fourth nucleotide from the 3'-OH terminus. A single mismatch has little influence on the yield of PCR products, but if there are two mismatches as a result of mutation at the position being tested, the mutant-specific primer will not function in PCR under appropriate conditions, leading to no yield of PCR product from the mutant allele. The assay was evaluated successfully using a panel of plasmids and M. leprae reference strains carrying the wild-type or known rpoB mutations. The assay was subsequently applied to M. leprae DNA extracts from skin biopsies taken from patients. In all biopsy samples, the wild-type allele was detected for Ser425. The PCR results correlated with rifampicin susceptibility, as also measured by the traditional in vivo mouse footpad technique.

Evaluation of a rapid screening test for rifampicin resistance in re-treatment tuberculosis patients in the Eastern Cape.

Patients with multidrug-resistant (MDR) tuberculosis (TB) are at high risk of treatment failure. It is anticipated that early identification of MDR-TB and appropriate treatment will improve patient outcome and disease control. We evaluated the rapid detection of rifampicin resistance in previously treated TB patients, directly from acidfast bacilli (AFB)-positive sputum using a phage-based test, FASTPlaque-Response (Biotec Laboratories Ltd, Ipswich, UK). The ability of rifampicin resistance to predict MDR-TB was also determined. A prospective study was done comparing performance of the rapid phage test with conventional culture and drug susceptibility testing (DST) in AFB-positive TB patients. Five primary health clinics and one TB referral centre in the Port Elizabeth Metropolitan area, Eastern Cape. Sensitivity, specificity and overall accuracy of the phage test were determined compared with gold standard culture and DST. Discrepant results were resolved by molecular detection of mutations conferring rifampicin resistance. The proportion of rifampicin-resistant strains that were MDR was also determined. Previously treated patients were at a high risk of MDRTB (35.7%). Sensitivity, specificity and overall accuracy of FASTPlaque-Response for rifampicin resistance determination were 95.4% (95% confidence interval (CI): 91.0 - 99.8%), 97.2% (95% CI: 94.5 - 99.9%) and 96.5% (95% CI: 94.1 - 98.9%) respectively compared with conventional DST (unresolved), calculated for specimens that had both FASTPlaque-Response and conventional DST results available. FASTPlaque-Response results were available in 2 days instead of 28 - 85 days with conventional DST. However, only 70.6% of FASTPlaque-Response results were interpretable compared with 86.3% of conventional DST results. The majority (95.5%) of rifampicinresistant strains were MDR-TB. Rapid detection of rifampicin resistance using FASTPlaque-Response could contribute to improved management of patients at risk of MDR-TB, such as previously treated patients. However, improvement in control of specimen-related contamination is needed to ensure that a higher proportion of FASTPlaque-Response results are interpretable. Where indicated, early modification of therapy could improve patient prognosis and reduce disease transmission.

Use of a high-density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis

A Mycobacterium high-density DNA probe array designed to detect rpoB mutations conferring rifampicin resistance in Mycobacterium tuberculosis was evaluated. The rpoB hybridisation patterns produced by 41 susceptible (RifS) and 59 rifampicin-resistant (RifR) clinical isolates of M. tuberculosis were compared with the results of conventional dideoxynucleotide sequencing of the rpoB gene. For all the RifR isolates, the rpoB hybridisation patterns correlated with the rpoB sequencing results. Among the 59 isolates, 11 distinct amino-acid changes were detected by the DNA probe array. Of these, 36 (61%) corresponded to replacement of the serine residue found in position 531 (S531L in 34 isolates and S531W in two isolates), 16 (27%) affected histidine 526 (five H526D, five H526Y, four H526L, one H526N and one H526R), four (6.8%) replaced aspartate 516 with a valine, and one (1.7%) replaced glutamine 513 with a leucine. Deletion of the asparagine residue at position 519 was detected in one isolate susceptible to rifampicin, but yielding c. 0.1% resistant colonies on rifampicin-containing medium. No mutation was detected in the rpoB region from one isolate yielding c. 5% of resistant colonies on rifampicin-containing medium. Finally, a D516Y substitution was detected in association with an unexpected mutation, G523W, not tiled on the DNA probe array, but which could be detected by analysing the hybridisation pattern obtained with the wild-type probes covering codon 523. In conclusion, the Mycobacterium probe array is a promising approach to rapid detection of mutations involved in rifampicin resistance in M. tuberculosis.

Mutations in ORF D13L and Other Genetic Loci Alter the Rifampicin Phenotype of Vaccinia Virus

Phenotypic analysis of vaccinia virus temperature-sensitive mutants identified several virus isolates that were either resistant or hypersensitive to rifampicin. Temperature-sensitive mutants 1085, 7743, 1085R, and 7743R were found to be rifampicin resistant (Rif R). Each virus contained at least one mutation in ORF D13L which resulted in a change of amino acid sequence. Analysis of each point mutation by marker rescue demonstrated that mutations gly to asp at residue 80, pro to phe at 458, lys to asn at 484, and ile to val at 485 conferred the resistance phenotype, while the mutation changing ser to leu at 176 did not affect the rifampicin phenotype. Temperature-sensitive mutants C6, C17, and C43 were found to be rifampicin hypersensitive. Only C43, however, was found to contain a lesion in ORF D13L. This mutation, ser to asn at 169 was confirmed by marker rescue to confer the hypersensitive phenotype. The results presented here along with those from previous reports show that known mutations conferring rifampicin resistance cluster at the C or N-terminus of D 13L while a mutation mapping at residue 169 confers rifampicin hypersensitivity. Further, the finding that the hypersensitive mutants C6 and C17 contain a wild-type D 13L ORF suggests a possible role of other vital functions in the interaction of rifampicin with vaccinia virus.