Conferring Rifampicin Resistance Research Articles

Response and adaptation of the transcriptional heat shock response to pressure.

The molecular mechanisms underlying pressure adaptation remain largely unexplored, despite their significance for understanding biological adaptation and improving sterilization methods in the food and beverage industry. The heat shock response leads to a global stabilization of the proteome. Prior research suggested that the heat shock regulon may exhibit a transcriptional response to high-pressure stress. In this study, we investigated the pressure-dependent heat shock response in E. coli strains using plasmid-borne green fluorescent protein (GFP) promoter fusions and fluorescence fluctuation microscopy. We quantitatively confirm that key heat shock genes-rpoH, rpoE, dnaK, and groEL - are transcriptionally upregulated following pressure shock in both piezosensitive Escherichia coli and a more piezotolerant laboratory-evolved strain, AN62. Our quantitative imaging results provide the first single cell resolution measurements for both the heat shock and pressure shock transcriptional responses, revealing not only the magnitude of the responses, but also the biological variance involved. Moreover, our results demonstrate distinct responses in the pressure-adapted strain. Specifically, PgroEL is upregulated more than PdnaK in AN62, while the reverse is true in the parental strain. Furthermore, unlike in the parental strain, the pressure-induced upregulation of PrpoE is highly stochastic in strain AN62, consistent with a strong feedback mechanism and suggesting that RpoE could act as a pressure sensor. Despite its capacity to grow at pressures up to 62 MPa, the AN62 genome shows minimal mutations, with notable single nucleotide substitutions in genes of the transcriptionally important b subunit of RNA polymerase and the Rho terminator. In particular, the mutation in RNAP is one of a cluster of mutations known to confer rifampicin resistance to E. coli via modification of RNAP pausing and termination efficiency. The observed differences in the pressure and heat shock responses between the parental MG1655 strain and the pressure-adapted strain AN62 could arise in part from functional differences in their RNAP molecules.

Rifampicin-resistant RpoB S522L Vibrio vulnificus exhibits disturbed stress response and hypervirulence traits.

Rifampicin resistance, which is genetically linked to mutations in the RNA polymerase β-subunit gene rpoB, has a global impact on bacterial transcription and cell physiology. Previously, we identified a substitution of serine 522 in RpoB (i.e., RpoBS522L ) conferring rifampicin resistance to Vibrio vulnificus, a human food-borne and wound-infecting pathogen associated with a high mortality rate. Transcriptional and physiological analysis of V. vulnificus expressing RpoBS522L showed increased basal transcription of stress-related genes and global virulence regulators. Phenotypically these transcriptional changes manifest as disturbed osmo-stress responses and toxin-associated hypervirulence as shown by reduced hypoosmotic-stress resistance and enhanced cytotoxicity of the RpoBS522L strain. These results suggest that RpoB-linked rifampicin resistance has a significant impact on V. vulnificus survival in the environment and during infection.

Antimicrobial Susceptibility of Staphylococcus aureus Strains and Effect of Phloretin on Biofilm Formation.

Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) are known to be responsible of various infections, including biofilm-associated diseases. The aim of this study was to analyze 19 strains of S. aureus from orthopedic sites in terms of phenotypic antimicrobial susceptibility against 13 selected antibiotics, slime/biofilm formation, molecular analysis of specific antibiotic resistance genes (mecA, cfr, rpoB), and biofilm-associated genes (icaADBC operon). Furthermore, the effect of phloretin on the production of biofilm was evaluated on 8 chosen isolates. The susceptibility test confirmed almost allstrains were resistant to cefoxitin and oxacillin. Most strains possessthe mecA, whereas none of the strains had the cfr gene. Four strains (1, 7, 10, and 24) presented single-nucleotide polymorphisms (SNPs) in rpoB, which confer rifampicin resistance. IcaD was detected in all tested strains, whereas icaR was only found in two strains (24 and 30). Phloretin had a dose-dependent effect on biofilm production. Specifically, 0.5 × MIC determined biofilm inhibition in 5 out of 8 strains (8, 24, 25, 27, 30), whereas an increase in biofilm production was detected with phloretin at the 0.125 × MIC across all tested strains. These data are useful to potentially develop novel compounds against antibiotic-resistant S. aureus.

Host-pathogen relationship in retreated tuberculosis with major rifampicin resistance-conferring mutations.

It is assumed that host defense systems eliminating the pathogen and regulating tissue damage make a strong impact on the outcome of tuberculosis (TB) disease and that these processes are affected by rifampicin (RIF) resistance-conferring mutations of Mycobacterium tuberculosis (Mtb). However, the host responses to the pathogen harboring different mutations have not been studied comprehensively in clinical settings. We analyzed clinico-epidemiological factors and blood transcriptomic signatures associated with major rpoB mutations conferring RIF resistance in a cohort study. Demographic data were collected from 295 active pulmonary TB patients with treatment history in Hanoi, Vietnam. When recruited, drug resistance-conferring mutations and lineage-specific variations were identified using whole-genome sequencing of clinical Mtb isolates. Before starting retreatment, total RNA was extracted from the whole blood of HIV-negative patients infected with Mtb that carried either the rpoB H445Y or rpoB S450L mutation, and the total RNA was subjected to RNA sequencing after age-gender matching. The individual RNA expression levels in the blood sample set were also measured using real-time RT-PCR. Logistic and linear regression models were used to assess possible associations. In our cohort, rpoB S450L and rpoB H445Y were major RIF resistance-conferring mutations [32/87 (36.8%) and 15/87 (17.2%), respectively]. H445Y was enriched in the ancient Beijing genotype and was associated with nonsynonymous mutations of Rv1830 that has been reported to regulate antibiotic resilience. H445Y was also more frequently observed in genetically clustered strains and in samples from patients who had received more than one TB treatment episode. According to the RNA sequencing, gene sets involved in the interferon-γ and-α pathways were downregulated in H445Y compared with S450L. The qRT-PCR analysis also confirmed the low expression levels of interferon-inducible genes, including BATF2 and SERPING1, in the H445Y group, particularly in patients with extensive lesions on chest X-ray. Our study results showed that rpoB mutations as well as Mtb sublineage with additional genetic variants may have significant effects on host response. These findings strengthen the rationale for investigation of host-pathogen interactions to develop countermeasures against epidemics of drug-resistant TB.

Molecular Epidemiology of Rifampicin-Resistant Mycobacterium tuberculosis by GeneXpert MTB/RIF Assay in Renal Transplant Recipients: A Single-Center Experience.

There are scarce data on the incidence and resistance pattern of rifampicin-resistant Mycobacterium tuberculosis among kidney transplant recipients. This is a retrospective, single- center study of kidney transplantrecipients suspected of M. tuberculosis infection. The GeneXpert assay we used detected mutations in the rpoB gene that confer rifampicin resistance using 5 overlapping probes (A, B, C, D, and E). The probes can detect mutations in the codons 507 to 511 (probe A), 511 to 518 (probe B), 518 to 523 (probe C), 523 to 529 (probe D), and 529 to 533 (probe E).We also detailed the treatment protocol and outcomes of kidney transplantrecipients infected with rifampicin-resistant M. tuberculosis. In total, 2700 samples were processed during the period from October 2018 to February 2022 with successful results in 2640 samples (97.04%). One hundred and ninety (7.19%) samples were positive for M.tuberculosis, and rifampicin resistance was detected in 12 (0.45%) cases (11 pulmonary, 1 genitourinary). The most common rpoB mutation was located in the region of probe E (75.0%), followed by probe A (16.6%) and in 1 combination probe DE (8.33%). The rpoB mutations were not observed in probe B and probe C. Six patients received bedaquiline-based treatmentfor a short course of 11 months, whereas the other 6 patients required a long course of 18 to 20 months. Three patients died, 2 were lost to follow-up, and 7 were cured. During treatment, 4 patients experienced acute rejection, and 1 graft loss was reported. We report for the first time the incidence and pattern of rifampicin resistance among kidney transplant recipients with tuberculosis infection. Further investigations are required for exploring the molecular and clinical phenotypes.

Mutations in rpoB That Confer Rifampicin Resistance Can Alter Levels of Peptidoglycan Precursors and Affect β-Lactam Susceptibility.

Bacteria can adapt to stressful conditions through mutations affecting the RNA polymerase core subunits that lead to beneficial changes in transcription. In response to selection with rifampicin (RIF), mutations arise in the RIF resistance-determining region (RRDR) of rpoB that reduce antibiotic binding. These changes can also alter transcription and thereby have pleiotropic effects on bacterial fitness. Here, we studied the evolution of resistance in Bacillus subtilis to the synergistic combination of RIF and the β-lactam cefuroxime (CEF). Two independent evolution experiments led to the recovery of a single rpoB allele (S487L) that was able to confer resistance to RIF and CEF through a single mutation. Two other common RRDR mutations made the cells 32 times more sensitive to CEF (H482Y) or led to only modest CEF resistance (Q469R). The diverse effects of these three mutations on CEF resistance are correlated with differences in the expression of peptidoglycan (PG) synthesis genes and in the levels of two metabolites crucial in regulating PG synthesis, glucosamine-6-phosphate (GlcN-6-P) and UDP-N-acetylglucosamine (UDP-GlcNAc). We conclude that RRDR mutations can have widely varying effects on pathways important for cell wall biosynthesis, and this may restrict the spectrum of mutations that arise during combination therapy. IMPORTANCE Rifampicin (RIF) is one of the most valued drugs in the treatment of tuberculosis. TB treatment relies on a combination therapy and for multidrug-resistant strains may include β-lactams. Mutations in rpoB present a common route for emergence of resistance to RIF. In this study, using B. subtilis as a model, we evaluate the emergence of resistance for the synergistic combination of RIF and the β-lactam cefuroxime (CEF). One clinically relevant rpoB mutation conferred resistance to both RIF and CEF, whereas one other increased CEF sensitivity. We were able to link these CEF sensitivity phenotypes to accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc), which feedback regulates GlmS activity and thereby peptidoglycan synthesis. Further, we found that higher CEF concentrations precluded the emergence of high RIF resistance. Collectively, these results suggest that multidrug treatment regimens may limit the available pathways for the evolution of antibiotic resistance.

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.

Bovine Mastitis in Egypt: Bacterial Etiology and Evaluation of Diagnostic Biomarkers

In current study a total of 153 tuberculous lesion samples collected from cattle after PM examination (69 from El-Menufia, 47 from El-Sharkia and 37 from El-Qaliobia governorates) were subjected to bacteriological examination. Eighty-three samples showed the AF bacilli in direct smears stained by ZN stain at a percentage of 54.2% while by the culture method, M. bovis was isolated from 100 (65.4%) samples. Sixty-five isolates, confirmed to be M. bovis by conventional PCR targeting Mpb70, were tested for the sensitivity to anti-tuberculous drugs and 25 isolates showed resistance to at least one tested drug. The overall resistance to EMB was 3.1% and resistance to STR, INH and RIF were 20, 30.8 and 32.3% respectively. MDR was detected in 14 (21.5%) isolates. For DNA sequencing, PCR targeting rpoB gene, conferring rifampicin resistance, was performed on selected 6 isolates and identified three mutations designated as H526Y (4/6), S531L (1/6) and D516V (1/6) and the sequences were submitted to the NCBI GenBank with the accession numbers (OM448574, OM448575, OM448576, OM448577, OM448578 and OM448579). The obtained results are epidemiologically significant and augment the necessity of further investigation on ante-mortem identification of M. bovis infection in cattle to minimize public risk.

Bacteriological and Molecular studies on Mycobacterium Bovis in Cattle, with Special Reference to its Antimicrobial Resistance

In current study a total of 153 tuberculous lesion samples collected from cattle after PM examination (69 from El-Menufia, 47 from El-Sharkia and 37 from El-Qaliobia governorates) were subjected to bacteriological examination. Eighty-three samples showed the AF bacilli in direct smears stained by ZN stain at a percentage of 54.2% while by the culture method, M. bovis was isolated from 100 (65.4%) samples. Sixty-five isolates, confirmed to be M. bovis by conventional PCR targeting Mpb70, were tested for the sensitivity to anti-tuberculous drugs and 25 isolates showed resistance to at least one tested drug. The overall resistance to EMB was 3.1% and resistance to STR, INH and RIF were 20, 30.8 and 32.3% respectively. MDR was detected in 14 (21.5%) isolates. For DNA sequencing, PCR targeting rpoB gene, conferring rifampicin resistance, was performed on selected 6 isolates and identified three mutations designated as H526Y (4/6), S531L (1/6) and D516V (1/6) and the sequences were submitted to the NCBI GenBank with the accession numbers (OM448574, OM448575, OM448576, OM448577, OM448578 and OM448579). The obtained results are epidemiologically significant and augment the necessity of further investigation on ante-mortem identification of M. bovis infection in cattle to minimize public risk.

Mycobacterium abscessus HelR interacts with RNA polymerase to confer intrinsic rifamycin resistance

Rifampicin (RIF), the frontline drug against M.tuberculosis, is completely ineffective against M.abscessus, partially due to the presence of an ADP-ribosyltransferase (Arr) that inactivates RIF. Using RNA-seq, we show that exposure of M.abscessus to sublethal doses of RIF and Rifabutin (RBT), a close analog of RIF, results in an ∼25-fold upregulation of Mab_helR in laboratory and clinical isolates. An isogenic deletion in Mab_helR results in RIF/RBT hypersensitivity, and overexpression of Mab_helR confers RIF tolerance in M.tuberculosis. We demonstrate an increased HelR-RNAP association in RIF-exposed bacteria and a MabHelR-mediated dissociation of RNAP from stalled initiation complexes invitro. Finally, we show that the tip of the PCh-loop of Mab_helR, present in proximity to RIF, is critical for conferring RIF resistance but dispensable for dissociation of stalled RNAP complexes, suggesting that HelR-mediated RIF resistance requires a step in addition to displacement of RIF-stalled RNAP.

Mutations compensating for the fitness cost of rifampicin resistance in Escherichia coli exert pleiotropic effect on RNA polymerase catalysis.

The spread of drug-resistant bacteria represents one of the most significant medical problems of our time. Bacterial fitness loss associated with drug resistance can be counteracted by acquisition of secondary mutations, thereby enhancing the virulence of such bacteria. Antibiotic rifampicin (Rif) targets cellular RNA polymerase (RNAP). It is potent broad spectrum drug used for treatment of bacterial infections. We have investigated the compensatory mechanism of the secondary mutations alleviating Rif resistance (Rifr) on biochemical, structural and fitness indices. We find that substitutions in RNAP genes compensating for the growth defect caused by βQ513P and βT563P Rifr mutations significantly enhanced bacterial relative growth rate. By assaying RNAP purified from these strains, we show that compensatory mutations directly stimulated basal transcriptional machinery (2–9-fold) significantly improving promoter clearance step of the transcription pathway as well as elongation rate. Molecular modeling suggests that compensatory mutations affect transcript retention, substrate loading, and nucleotidyl transfer catalysis. Strikingly, one of the identified compensatory substitutions represents mutation conferring rifampicin resistance on its own. This finding reveals an evolutionary process that creates more virulent species by simultaneously improving the fitness and augmenting bacterial drug resistance.

Mycobacterium Abscessus HelR Interacts with RNA Polymerase to Confer Intrinsic Rifamycin Resistance

Bacterial RNA polymerase is a target of the potent and broad-spectrum rifamycin group of antibiotics. Mutations within rpoB and inactivation by a diverse group of enzymes constitute the most widespread mechanisms of resistance. Rifampicin (RIF) constitutes the frontline therapy against M. tuberculosis as well as most slow-growing non-tuberculous mycobacteria (NTM) but is completely ineffective against M. abscessus. This high level of RIF resistance is despite the absence of mutations in the rifampicin resistance determining region of Mab_rpoB and is partially attributed to the presence of an ADP-ribosyltransferase (Arr) activity that inactivates RIF. Rifabutin (RBT), a close analogue of RIF, has recently been shown to be effective against M. abscessus in vitro and in a mouse model and comprises a promising therapeutic against M. abscessus infections. Using RNA sequencing we show that exposure of M. abscessus to sublethal doses of RIF and RBT results in ~25-fold upregulation of Mab_ helR in laboratory and clinical isolates; an isogenic deletion mutant of Mab_ helR is hypersensitive to RIF and RBT, and over-expression of Mab_ helR confers RIF tolerance in M. tuberculosis implying that Mab_helR constitutes a significant determinant of inducible RIF and RBT resistance. We demonstrate a preferential association of MabHelR with RNA polymerase in vivo in bacteria exposed to RIF and show that purified MabHelR can rescue transcription inhibition in the presence of RIF in in vitro transcription assays. Furthermore, MabHelR can dissociate RNAP from RIF-stalled initiation complexes in vitro, a species we envisage accumulates upon RIF exposure. Lastly, we show that the tip of the PCh-loop of Mab_ helR, in particular residues E496 and D497 that are in proximity to RIF, is critical for conferring RIF resistance without being required for dissociation of antibiotic-stalled RNAP complexes. This suggests that HelR may be additionally involved in displacing RIF bound to RNAP and function as an RNAP protection protein.

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.

Relationship between rifampicin resistance and RpoB substitutions of Rhodococcus equi strains isolated in France

ObjectivesStudy of the rifampicin resistance of Rhodococcus equi strains isolated from French horses over a 20-year period. MethodsRifampicin susceptibility was tested by disk diffusion (DD) and broth macrodilution methods, and rpoB gene sequencing and MLST were performed on 40 R. equi strains, 50.0% of which were non-susceptible to rifampicin. ResultsConsistency of results was observed between rifampicin susceptibility testing and rpoB sequencing. Strains non-susceptible to rifampicin by DD had a substitution at one of the sites (Asp516, His526 and Ser531) frequently encountered and conferring rifampicin resistance. High-level resistance was correlated with His526Asp or Ser531Leu substitutions; low-level resistance was correlated with Asp516Tyr substitution, a novel substitution for R. equi. Strains susceptible to rifampicin by DD showed no substitution in the three sites, except for two strains carrying, respectively, the His526Asn and Asp516Val substitutions (previously correlated with low-level rifampicin resistance). Both strains were isolated from an animal from which ten other strains were also isolated and found to be rifampicin-non-susceptible by DD. MLST showed the presence of 10 STs (including the novel ST43), but no association was observed with rifampicin resistance. ConclusionsThis study confirms that certain substitutions in RpoB are more likely to confer high- or low-level rifampicin resistance, describes a new substitution conferring rifampicin resistance in R. equi and suggests non-clonal dissemination of rifampicin-resistant strains in France. Standard DD may miss strains with a low-level rifampicin-resistant substitution; further studies are needed to remedy the absence of R. equi-specific clinical breakpoints.

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.

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.

Does Xpert\xae MTB/RIF assay give rifampicin resistance results without identified mutation? Review of cases from Addis Ababa, Ethiopia

BackgroundXpert® MTB/RIF assay is currently used in Ethiopia for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and mutations that confer Rifampicin resistance. Rifampicin resistance is determined based on any mutation in the 81 bp of rpoB gene using five overlapping probes represented as Probe A (codons 507–511), Probe B (codons 512–518), Probe C (codons 518–523), Probe D (codons 523–529) and Probe E (codons 529–533). In this review, we assessed the frequency of missed probe types for Rifampicin Resistance results.MethodsData were reviewed from specimens received and tested using Xpert® MTB/RIF assay at Ethiopian National Tuberculosis Reference Laboratory, in Addis Ababa from 15 July 2016 to 31 December 2018 retrospectively. All archived data were reviewed carefully to describe missed probe types and the quantity of DNA in the sample.ResultsA total of 100 specimens were reported as MTB Detected Rifampicin Resistance Detected by Xpert® MTB/RIF assay. More than half (55%) of these results were reported from male patients. The median age was 28.0 years (5 months to 88 years). Majorities (62%) of the cases were detected from sputum. Among the total of 38 extrapulmonary samples, lymph node aspirates were accounted for 50% (19/38). The most common mutations (81.0%) were found in the Probe E region followed by Probe D (10.0%), and Probe B (3.0%). Mutations in Probe A and Probe C regions were not observed. However, six (6.0%) Rifampicin resistance cases were found without any missed probe type. The delta Ct max is ≥4.3. No specimen yielded Rifampicin resistance associated with more than one probe failure or mutation combinations.ConclusionMutations associated with Probe E (codons 529–533) region were identified as the commonest rpoB gene mutations. The Rifampicin resistance results found without any identified missing probe needs further study. The lower DNA amount was observed in extrapulmonary specimens compared with sputum.

Global spread of three multidrug-resistant lineages of Staphylococcus epidermidis.

Staphylococcus epidermidis is a conspicuous member of the human microbiome, widely present on healthy skin. Here we show that S. epidermidis has also evolved to become a formidable nosocomial pathogen. Using genomics, we reveal that three multidrug-resistant, hospital-adapted lineages of S. epidermidis (two ST2 and one ST23) have emerged in recent decades and spread globally. These lineages are resistant to rifampicin through acquisition of specific rpoB mutations that have become fixed in the populations. Analysis of isolates from 96 institutions in 24 countries identified dual D471E and I527M RpoB substitutions to be the most common cause of rifampicin resistance in S. epidermidis, accounting for 86.6% of mutations. Furthermore, we reveal that the D471E and I527M combination occurs almost exclusively in isolates from the ST2 and ST23 lineages. By breaching lineage-specific DNA methylation restriction modification barriers and then performing site-specific mutagenesis, we show that these rpoB mutations not only confer rifampicin resistance, but also reduce susceptibility to the last-line glycopeptide antibiotics, vancomycin and teicoplanin. Our study has uncovered the previously unrecognized international spread of a near pan-drug-resistant opportunistic pathogen, identifiable by a rifampicin-resistant phenotype. It is possible that hospital practices, such as antibiotic monotherapy utilizing rifampicin-impregnated medical devices, have driven the evolution of this organism, once trivialized as a contaminant, towards potentially incurable infections.

The occurrence and frequency of genomic mutations that mediate Isoniazid and Rifampicin resistance in Mycobacterium tuberculosis isolates from untreated pulmonary Tuberculosis cases in urban Blantyre, Malawi.

The emergence and spread of drug-resistant Tuberculosis (TB) is a major public health threat. TB resistance originates in the course of treatment due to genomic mutations in Mycobacterium tuberculosis (MTB). An increase in new cases with drug-resistant TB could be an indicator of high levels of circulating resistant strains. This study was conducted to determine the occurrence and frequency of genomic mutations that mediate Isoniazid (INH) and Rifampicin (RIF) resistance among isolates from untreated TB cases in urban Blantyre, Malawi. A cross-sectional retrospective study was conducted on a panel of 141(n=141) MTB clinical isolates recovered between June 2010 and January 2012 from >2+ Ziehl-Neelsen smear positive new pulmonary-TB patients with no history of treatment. Frozen isolates were revived using the BACTEC MGIT detection system. DNA was extracted using GenoLyse DNA extraction kit and detection of genomic mutations was carried out using the GenoType MTBDRplus Ver 2.0 assay. Out of the 141 isolates studied, 3 (2.1%) were found carrying mutations in the katG gene that confer resistance to Isoniazid (INH). No mutations were detected in the inhA promoter region gene that confer weak INH resistance or in the rpoB gene that confer Rifampicin resistance. All katG mutant genes had a S315T1 single point mutation, a genomic alteration that mediates high INH resistance. The katG mutant gene conferring resistance to INH was the only genomic mutation observed among the isolates studied and the frequency of occurrence was low. Our findings suggest low levels of circulating drug-resistant MTB strains in urban Blantyre, Malawi.