rpoB Mutations Research Articles

Investigation of the Rv3065, Rv2942, Rv1258c, Rv1410c, and Rv2459 efflux pump genes expression among multidrug-resistant Mycobacterium tuberculosis clinical isolates

BackgroundDifferent resistance mechanisms for multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have been reported. Although mutations in target genes are the main cause of drug resistance, efflux pumps (Eps) also play an important role in this process. Here, we investigated the overexpression of five putative EP genes plus gene mutations in MDR-TB clinical isolates. MethodsA total of 27 M. tuberculosis (Mtb) clinical isolates including, 22 MDR and 5 sensitive isolates were analyzed. Minimum inhibitory concentrations (MIC) were determined in the absence and presence of efflux inhibitor. The expression level of 5 EP genes (Rv3065, Rv2942, Rv1258c, Rv1410c, Rv2459) was investigated by quantitative real time PCR (RT-qPCR). DNA sequencing of rpoB, katG, and inhA promoter was done. ResultsAmong the 22 MDR-TB isolates, 13 (59.1%) showed significant overexpression (>4-fold) for at least one EP gene. The expression levels of 5 genes were significantly higher (P < 0.05) in MDR-TB isolates than sensitive isolates. The Rv3065 (22.7%), and Rv1410c (18.2%) were found to be the most commonly overexpressed EPs. The observed MICs were as follows: RIF (2 to >128 μg/ml) and INH (2–32 μg/ml). After efflux pump inhibitor exposure, 10/22 (45.45%) isolates showed a decrease in MIC of INH, and 17/22 (77.27%) isolates showed a decrease in MIC of RIF. Of the isolates that overexpressed, 4 isolates lacked mutation in inhA, rpoB, and katG genes and 10 ones lacked mutation in inhA and katG. ConclusionThe results showed that overexpression of EP genes in Mtb isolates, besides target gene mutations can contribute to the development of MDR phenotype.

Rarity of rpoB Mutations Outside the Rifampicin Resistance-Determining Region of Mycobacterium tuberculosis Isolates from Patients Responding Poorly to First-Line Tuberculosis Regimens in Beijing, China: A Retrospective Study

BackgroundEarly and accurate diagnosis of rifampicin (RIF)-resistant Mycobacterium tuberculosis (MTB) is essential for controlling community spread of drug-resistant tuberculosis (TB). In order to discover mutations residing outside the rifampicin resistance-determining region (RRDR) of the MTB rpoB gene, we conducted this retrospective study.MethodsWe retrospectively screened patient records to obtain Xpert MTB/RIF assay results for patients who received care at the Beijing Chest Hospital from 2016 to 2019 in order to identify subjects who met study selection criteria. Stored frozen patient isolates were cultured, harvested, and then subjected to drug susceptibility testing. Concurrently, entire rpoB gene DNA of each isolate was amplified and then sequenced to reveal rpoB mutations.ResultsOverall, 104 RIF-susceptible tuberculosis patients who were tested using the Xpert MTB/RIF assay also had poor first-line regimen treatment responses. Isolates obtained from these cases included 101 MTB isolates that possessed wild-type rpoB allelic profiles, as demonstrated using Sanger sequencing. However, sequences from the other three isolates confirmed that rpoB of one isolate harbored a mutation encoding the amino acid substitution Ile491Phe and that rpoB genes of two isolates contained a mutation encoding the amino acid substitution Ser450Leu.ConclusionOur data demonstrated that mutations found outside the RRDR of MTB rpoB are rare in Beijing, China, indicating that World Health Organization-approved molecular diagnostics are generally suitable for diagnosing RIF resistance.

Rapid Molecular Diagnosis of Tuberculosis and Its Resistance to Rifampicin and Isoniazid with Automated MDR/MTB ELITe MGB® Assay.

The MDR/MTB ELITe MGB® kit (ELITech) carried on the ELITe InGenius® platform is a new real-time PCR assay allowing automated extraction and detection of DNA of the Mycobacterium tuberculosis complex (MTB) and mutations in the rpoB and katG genes and inhA promoter region (pro-inhA) associated to resistance to rifampicin and isoniazid, the two markers of multidrug-resistant TB (MDR). We assessed the performances of the test on a collection of strains (n = 54) and a set of clinical samples (n = 242) from routine practice, comparatively to TB diagnosis and genotypic drug susceptibility testing (gDST) as references. Regarding the 242 clinical samples, the sensitivity and specificity of MTB detection by ELITe were 90.9% and 97.5%, respectively. For the detection of resistance-conferring mutations on positive clinical samples, we observed perfect agreement with gDST for katG and pro-inhA (κ = 1.0) and two discordant results for rpoB (κ = 0.82). Considering the 54 cultured strains, very good agreement with gDST was observed for the detection of the 25 distinct mutations in rpoB, katG, and pro-inhA, (κ = 0.95, 0.88, and 0.95, respectively). In conclusion, the automated MDR/MTB ELITe MGB® assay shows great promise and appears to be a valuable tool for rapid detection of pre-MDR- and MDR-TB directly from clinical specimens.

Generation of Tetracycline and Rifamycin Resistant Chlamydia Suis Recombinants.

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the β-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

Adaptive Resistance Mutations at Suprainhibitory Concentrations Independent of SOS Mutagenesis.

Emergence of resistant bacteria during antimicrobial treatment is one of the most critical and universal health threats. It is known that several stress-induced mutagenesis and heteroresistance mechanisms can enhance microbial adaptation to antibiotics. Here, we demonstrate that the pathogen Bartonella can undergo stress-induced mutagenesis despite the fact it lacks error-prone polymerases, the rpoS gene and functional UV-induced mutagenesis. We demonstrate that Bartonella acquire de novo single mutations during rifampicin exposure at suprainhibitory concentrations at a much higher rate than expected from spontaneous fluctuations. This is while exhibiting a minimal heteroresistance capacity. The emerged resistant mutants acquired a single rpoB mutation, whereas no other mutations were found in their whole genome. Interestingly, the emergence of resistance in Bartonella occurred only during gradual exposure to the antibiotic, indicating that Bartonella sense and react to the changing environment. Using a mathematical model, we demonstrated that, to reproduce the experimental results, mutation rates should be transiently increased over 1,000-folds, and a larger population size or greater heteroresistance capacity is required. RNA expression analysis suggests that the increased mutation rate is due to downregulation of key DNA repair genes (mutS, mutY, and recA), associated with DNA breaks caused by massive prophage inductions. These results provide new evidence of the hazard of antibiotic overuse in medicine and agriculture.

In silico mutational analysis in RNA polymerase β subunit (rpoB) gene of rifampicin-resistant in Mycobacterium tuberculosis from Malaysia

Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis (MTB) and remains as a key public health problem worldwide. Most of MTB clinical strains are resistant to rifampicin (RIF), the first-line anti-tuberculosis drug. RIF resistance to MTB is due to mutations that mainly found in RIF resistance-determining region (RRDR) in drug target gene, RNA polymerase β subunit (rpoB). Therefore, the aim of the study is to extend the identification of variants in rpoB gene and to elucidate the effect of variants to the RIF resistance. Four of the strains, MTBR1/09, MTBR2/09, MTBR3/09 and MTB221/11 were subjected to drug susceptibility test (DST). All of the strains sequenced and submitted to the National Center for Biotechnology Information Sequence Read Archive were analyzed to identify the variants in the rpoB gene. The identified new variants were then subjected to docking to examine the drug-protein interactions. DST analysis revealed MTBR1/09, MTBR2/09 and MTBR3/09 were resistant to the RIF drug, while MTB221/11 was a presumptive susceptible strain. Two new variants were observed, the deletion (Phe433_Met434delinsLeu in MTBR1/09) and missense (Lys37Arg in MTBR3/09) variants. Meanwhile, the His445Leu, Ser450Leu, Asp103Asp, Ala1075Ala were reported variants. Docking of RIF to MTBR1/09 and MTBR3/09 mutant models revealed the RIF bound to the RIF binding site at different binding affinity and conformation. Concurrently, the new variants caused the RIF to bind to the different active site and neighboring residues. Findings from DST and docking analyses indicate that new variants potentially disturb the RIF inhibition in RpoB mutant proteins, and thus might be responsible to cause the RIF resistance.

Whole-Genome Sequencing for Tracing the Genetic Diversity of Brucella abortus and Brucella melitensis Isolated from Livestock in Egypt.

Brucellosis is a highly contagious zoonosis that occurs worldwide. Whole-genome sequencing (WGS) has become a widely accepted molecular typing method for outbreak tracing and genomic epidemiology of brucellosis. Twenty-nine Brucella spp. (eight B. abortus biovar 1 and 21 B. melitensis biovar 3) were isolated from lymph nodes, milk, and fetal abomasal contents of infected cattle, buffaloes, sheep, and goats originating from nine districts in Egypt. The isolates were identified by microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differentiation and genotyping were confirmed using multiplex PCR. Illumina MiSeq® was used to sequence the 29 Brucella isolates. Using MLST typing, ST11 and ST1 were identified among B. melitensis and B. abortus, respectively. Brucella abortus and B. melitensis isolates were divided into two main clusters (clusters 1 and 2) containing two and nine distinct genotypes by core-genome SNP analysis, respectively. The genotypes were irregularly distributed over time and space in the study area. Both Egyptian B. abortus and B. melitensis isolates proved to be genomically unique upon comparison with publicly available sequencing from strains of neighboring Mediterranean, African, and Asian countries. The antimicrobial resistance mechanism caused by mutations in rpoB, gyrA, and gyrB genes associated with rifampicin and ciprofloxacin resistance were identified. To the best of our knowledge, this is the first study investigating the epidemiology of Brucella isolates from livestock belonging to different localities in Egypt based on whole genome analysis.

Investigation of Mutations in the Rifampin-Resistance-Determining Region of the rpoB Gene of Brucella melitensis by Gene Analysis

Background: RNA polymerase beta subunit (rpoB) gene analysis in bacterial communities is known as a method for determining rifampin sensitivity and genetic diversity among Brucella spp. Detection of antibiotic resistance among Brucella isolates can be a critical approach to control brucellosis. However, rpoB gene analysis of Brucella melitensis for assessing rifampicin resistance has not yet been performed in Iran, which is considered an endemic area for brucellosis. Objectives: The aim of this study was to analyze the whole sequence of rpoB genes of different B. melitensis isolates from humans to identify the single-nucleotide polymorphisms (SNPs) and mutations related to rifampin resistance and to analyze the genetic diversity of these bacteria in Iran. Methods: Between 2017 and 2019, a total of 156 blood samples along with 12 synovial fluid specimens were collected from brucellosis patients in different Iranian provinces and subjected to bacterial culture in Brucella selective media. Brucella identification was carried out using classical biotyping and molecular examinations. Polymerase chain reaction (PCR)-based amplification of the rpoB gene was performed by specific rpoB primers for whole gene sequencing. The antimicrobial susceptibility of Brucella isolates was assessed using disk diffusion susceptibility tests and minimal inhibitory concentration (MIC) methods. The presence of rifampin-binding sites and SNPs were investigated through rpoB whole gene sequencing. Results: Clinical B. melitensis isolates were obtained from blood (13) and synovial fluid (1) samples of patients from different regions of Iran. The results of MIC and disk diffusion susceptibility tests showed that all the isolates were sensitive to rifampin except for one isolate showing intermediate rifampin resistance based on the standards defined for slow-growing bacteria by the Clinical and Laboratory Standards Institute (CLSI). Gene analysis for identifying the mutations related to rifampin resistance and investigating genetic diversity showed that none of the B. melitensis isolates had missense mutations, confirming the susceptibility of all the studied isolates to rifampin. Conclusions: The present study revealed that rpoB gene analysis could be used for the efficient and precise identifying of the mutations related to rifampin resistance, investigating rifampin binding sites, and genotyping Brucella species. Furthermore, the identification of B. melitensis isolates with intermediate resistance to rifampicin highlighted the importance of periodically carrying out antibiotic susceptibility testing. The molecular detection of rpoB mutations in different Brucella isolates may help to prevent the spread of rifampin-resistant Brucella spp. among humans and livestock.

Detection of genomic mutations in katG and rpoB genes among multidrug-resistant Mycobacterium tuberculosis isolates from Tehran, Iran

Multidrug-resistant (MDR) Mycobacterium tuberculosis strains, defined as resistant to at least isoniazid and rifampin, have emerged as a major worldwide health threat. Spontaneous point mutations in various genes of M. tuberculosis cause resistance to isoniazid, with the most frequent gene target being katG; and resistance to rifampin is usually due to mutation in the rpoB gene. The current study was aimed to detect the point mutations in the katG and rpoB regions related to isoniazid and rifampin resistance. A total of 203 respiratory specimens were collected from patients suspected of having tuberculosis respiratory infections referred to hospitals of Tehran, Iran, during 2018–2019. The isolation and identification of M. tuberculosis isolates were performed according to the WHO protocol. Drug susceptibility testing was carried out by proportional method. PCR analysis and sequencing were used to detect mutations in the selected katG and rpoB regions. Forty-four M. tuberculosis strains were isolated, of which 12 (27.3%) and 10 (22.7%) were resistant to isoniazid and rifampin, respectively. Ten isolates were resistant to both isoniazid and rifampin and were considered as MDR isolates. Of the ten MDR isolates, six (60%) carried mutations in both rpoB and katG. The most common mutations among isoniazid- and rifampin-resistant isolates were in codon 315 of the katG gene (70%) and codon 441 of the rpoB gene (50%), respectively. The results of this study indicated that MDR-TB continues to be a serious public health problem in Iran.

Vancomycin heteroresistance in Staphylococcus haemolyticus: elusive phenotype.

Aim:To determine the presence of vancomycin heteroresistance in Staphylococcus haemolyticus.Materials & methods:A total of 48 rifampicin-resistant S. haemolyticus isolates from bloodstream infections were included. Vancomycin heteroresistance was determined using the population analysis profile-area under curve (PAP-AUC) method. All the isolates were screened for the presence of mecA gene, mutations in the rpoB gene, staphylococcal cassette chromosome mec and multilocus sequence types.Results:Fifteen isolates were identified as heteroresistant vancomycin-intermediate S. haemolyticus using PAP-AUC method. Dual rpoB mutations (D471E and I527M) contributed for the rifampicin resistance. The sequence types of heteroresistant vancomycin-intermediate S. haemolyticus were highly diverse.Conclusion:These findings illustrate the potential of S. haemolyticus to develop heteroresistance, which emphasizes the need for routine surveillance of S. haemolyticus isolated from intensive care units for infection control practices.

Resistance Profiles and Biological Characteristics of Rifampicin-Resistant Staphylococcus aureus Small-Colony Variants.

BackgroundStaphylococcus aureus (S. aureus) is a major contributor to nosocomial and community-acquired infections. S. aureus small colony variants (SCVs) which changed in relevant phenotype have made more limited and difficult for therapeutic options against S. aureus infections increasingly. Rifampicin is considered as the “last-resort” antibiotic against S. aureus. Our study investigated resistance profiles and biological characteristics of rifampicin-resistant S. aureus SCVs.MethodsWe collected S. aureus SCVs that were selected from 41 rifampicin-resistant clinical isolates. Then, biological characteristics, resistance spectrum, and rifampicin resistance mechanisms of tested S. aureus SCVs and corresponding parental strains were investigated by classic microbiological methods, agar dilution method, polymerase chain reaction (PCR). Moreover, the fitness cost of S. aureus SCVs, including growth, biofilm formation ability, and virulence profile, was also determined by bacterial growth curve assay, biofilm formation assay, and Galleria mellonella infection model.ResultsThere were three S. aureus SCVs (JP310 SCVs, JP1450 SCVs, JP1486 SCVs) that were selected from 41 rifampicin-resistant S. aureus. S. aureus SCVs colonies were tiny, with decreased pigmentation, and the hemolysis circle was not obvious compared with corresponding parental strains. And SCVs could not be restored to normal-colony phenotype after hemin, menaquinone, or thymidine supplementation. Different rpoB mutations occurred in JP1486 SCVs. Antimicrobial susceptibility testing revealed MICs of SCVs were higher than corresponding parental strains. Besides, the growth ability and virulence of SCVs were lower, and biofilm formation ability of which increased compared with parental strains.ConclusionS. aureus SCVs share the rifampicin resistance mechanisms with parental strains, although there were some differences in the position of rpoB mutations. Moreover, we found that the biological characteristics of SCVs were significantly different from corresponding parental strains. In contrast, decreased susceptibility to other antibiotics of SCVs was observed during phenotype switch. Furthermore, SCVs incur the fitness cost.

Mycobacterium tuberculosis Complex Mutations in Drug Resistant Clinical Isolates from Southwest Mexico

Mutations in target genes have been described in Mycobacterium tuberculosis Complex (MTBc) drug resistant isolates worldwide. In Mexico, not enough information has been reported in this concern. The aim of this study was to characterize mutations related to resistance to first line drugs in MTBc isolates from Oaxaca, Mexico. MTBc isolates were identified in clinical samples from Tuberculosis (TB) patients. Susceptibility to isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide was tested through nitrate reductase assay. PCR based analysis and sequencing were employed to characterize mutations in katG, inhA, rpoB, embB, rrs, rpsL and pncA genes. Mutations in katG and the promoter of the mabA-inhA operon were found in isoniazid resistant isolates. Sequence analysis of Rifampin Resistance-Determining Region in the rpoB gene showed novel mutations along this region besides mutations at codons 516, 526 and 531. Polymorphisms at codon 306 embB gene were found in ethambutol resistant isolates. Frequent mutations associated to resistance to streptomycin were characterized in rrs and/or rpsL genes. pncA analysis showed variable number of mutations in resistant and susceptible pyrazinamide isolates. Most frequent mutations related to resistance to first line antituberculous drugs were identified in phenotypically resistant MTBc isolates. New mutations were characterized in rpoB, rrs and rpsL genes.

Low-Level Rifampin Resistance and rpoB Mutations in Mycobacterium tuberculosis: an Analysis of Whole-Genome Sequencing and Drug Susceptibility Test Data in New York.

Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multidrug-resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of rpoB mutations that do not confer high levels of RIF resistance, as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF MICs compared to fully susceptible strains but remain phenotypically susceptible by mycobacterial growth indicator tube (MGIT) testing and have been associated with poor patient outcomes. Here, we assess RIF resistance prediction by whole-genome sequencing (WGS) among a set of 1,779 prospectively tested strains by both prevalence of rpoB gene mutation and phenotype as part of routine clinical testing during a 2.5-year period. During this time, 139 strains were found to have nonsynonymous rpoB mutations, 53 of which were associated with RIF resistance, including both low-level and high-level resistance. Resistance to RIF (1.0 μg/ml in MGIT) was identified in 43 (81.1%) isolates. The remaining 10 (18.9%) strains were susceptible by MGIT but were confirmed to be low-level RIF resistant by MIC testing. Full rpoB gene sequencing overcame the limitations of critical concentration phenotyping, probe-based genotyping, and partial gene sequencing methods. Universal clinical WGS with concurrent phenotypic testing provided a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF resistance in New York.

On the Consequences of Poorly Defined Breakpoints for Rifampin Susceptibility Testing of Mycobacterium tuberculosis Complex.

In a recent report of a systematic review of critical concentrations (CCs), the World Health Organization (WHO) lowered the rifampin (RIF) CC for antimicrobial susceptibility testing (AST) of the Mycobacterium tuberculosis complex using Middlebrook 7H10 medium and the Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system from 1 to 0.5 μg/ml. The previous RIF CC for 7H10 had been in use for over half a century. Because it had served as the de facto reference standard, it contributed to the endorsement of inappropriately high CCs for other AST methods, including the U.S. Food and Drug Administration (FDA)-approved MGIT system. Moreover, this resulted in confusion about the interpretation of seven borderline resistance mutations in rpoB (i.e., L430P, D435Y, H445L, H445N, H445S, L452P, and I491F). In this issue of the Journal of Clinical Microbiology, Shea et al. (J Clin Microbiol 59:e01885-20, 2021, https://doi.org/10.1128/JCM.01885-20) provide evidence that the CC endorsed by the Clinical and Laboratory Standards Institute for the Sensititre MYCOTB system, which is not FDA approved but is CE-IVD marked in the European Union, is likely also too high. These findings underscore the importance of calibrating AST methods against a rigorously defined reference standard, as recently proposed by the European Committee on Antimicrobial Susceptibility Testing, as well as the value of routine next-generation sequencing for investigating discordant AST results.

In vitro Tolerability of Biofilm-Forming Trimethoprim-/Sulfamethoxazole-Resistant Small Colony Variants of Staphylococcus aureus Against Various Antimicrobial Agents.

Trimethoprim-/sulfamethoxazole-resistant small colony variants (SCVs) of Staphylococcus aureus, which are selected by use of trimethoprim/sulfamethoxazole, are involved in intractable biofilm-forming infection. This study aimed to determine the biofilm formation ability in trimethoprim-/sulfamethoxazole-resistant SCVs of S. aureus and investigate the bactericidal activity of differential antimicrobial agents to its biofilm-forming S. aureus. Between 32 S. aureus wild type (WT) and 32 SCVs selected from its WT, the amount of formed biofilm was compared. Vancomycin, daptomycin, rifampicin, and minocycline were exposed to biofilm-forming S. aureus to determine viable bacterial counts and its susceptibility. The biofilm-producing quantify of SCVs was approximately twice that formed by its WT. Vancomycin and daptomycin reduce 4 logs the bacterial counts of biofilm-forming WT at 24 hours, but did not affect SCVs. Rifampicin and minocycline considerably decreased both WT and SCVs; however, both bacterial counts recovered to an initial number 48 hours later. These survival strains showed resistance to each drug, and rpoB mutation or tet38 mRNA overexpression was confirmed.

Abattoir survey of bovine tuberculosis in tanta, centre of the Nile delta, with in silico analysis of gene mutations and protein-protein interactions of the involved mycobacteria.

Bovine tuberculosis is a transboundary disease of high economic and public health burden worldwide. In this study, post-mortem examination of 750 cattle and buffalo in Tanta abattoir, Centre of the Nile Delta, revealed visible TB in 4% of animals and a true prevalence of 6.85% (95% CI: 5.3%-8.9%). Mycobacterial culture, histopathology and RT-PCR targeting all members of M.tuberculosis complex were performed, upon which 85%, 80% and 100% of each tested lesions were confirmed as TB, respectively. Mpb70-targeting PCR was conducted on ten RT-PCR positive samples for sequencing and identified nine Mycobacterium (M.) bovis strains and, interestingly, one M. tuberculosis (Mtb) strain from a buffalo. Bioinformatics tools were used for prediction of mutations, nucleotide polymorphisms, lineages, drug resistance and protein-protein interactions (PPI) of the sequenced strains. The Mtb strain was resistant to rifampicin, isoniazid and streptomycin, and to the best of our knowledge, this is the first report of multidrug resistant (MDR)-Mtb originating from buffaloes. Seven M.bovis strains were resistant to ethambutol and ethionamide. Such resistances were associated with KatG, rpoB, rpsL, embB and ethA genes mutations. Other mutations and nucleotide polymorphisms were also predicted, some are reported for the first time and require experimental work for validation. PPI revealed more interactions than what would be expected for a random set of proteins of similar size and had dense interactions between nodes that are biologically connected, as a group. Two M.bovis strains belonged to BOV AFRI lineage (Spoligotypes BOV 1; BOV 2) and eight strains belonged to East-Asian (Beijing) lineage. In conclusion, visible TB was prevalent in the study area, RT-PCR is the best to confirm the disease, MDR-Mtb is associated with buffalo TB, and mycobacteria of different lineages carry many resistance genes to chemotherapeutic agents used in treatment of human TB constituting a major public health risk.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017–2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

Rifampin-resistance-associated mutations in the rifampin-resistance-determining region of the rpoB gene of Mycobacterium tuberculosis clinical isolates in Shanghai, PR China.

Introduction. Resistance to rifampin (RIF) in Mycobacterium tuberculosis infection is associated with mutations in the rpoB gene coding for the β-subunit of RNA polymerase. The contribution of various rpoB mutations to the development and level of RIF resistance remains elusive.Hypothesis/Gap Statement. Various rpoB mutations may be associated with differential levels of RIF resistance.Aim. This study aimed to investigate the relationship between specific rpoB mutations and the MICs of RIF and rifabutin (RFB) against M. tuberculosis.Methodology. Of the 195 clinical isolates, 105 and 90 isolates were randomly selected from isolates resistant to RIF and sensitive to RIF, respectively. The MICs of 12 agents for M. tuberculosis isolates were determined using commercial Sensititre M. tuberculosis MIC plates and the broth microdilution method. Strains were screened for rpoB mutations by DNA extraction, rpoB gene amplification and DNA sequence analysis.Results. One hundred isolates (95.24 %) were found to have mutations in the RIF-resistance-determining region (RRDR) of the rpoB gene. Three rpoB mutations were identified in 90 RIF-susceptible isolates. Out of 105 isolates, 86 (81.90 %) were cross-resistant to both RIF and RFB. The most frequent mutation occurred at codons 450 and 445. We also found a novel nine-nucleotide (ATCATGCAT) deletion (between positions 1543 and 1551) in the rpoB gene in two strains (1.90 %) with resistance to RIF, but susceptibility to RFB. In addition, the mutation frequency at codon 450 was significantly higher in RIF-resistant/RFB-resistant (RIFR/RFBR) strains than in RIFR/RFBS strains (75.58 % versus 21.05 %, P<0.01), whereas the mutation frequency at codon 435 was significantly lower in RIFR/RFBR strains than in RIFR/RFBS strains (1.16 % versus 26.32 %, P<0.01).Conclusion. Our data support previous findings, which reported that various rpoB mutations are associated with differential levels of RIF resistance. The specific mutations in the rpoB gene in RIFR/RFBR isolates differed from those in the RIFR/RFBS isolates. A novel deletion mutation in the RRDR might be associated with resistance to RIF, but not to RFB. Further clinical studies are required to investigate the efficacy of RFB in the treatment of infections caused by M. tuberculosis strains harbouring these mutations.

The Value of the inhA Mutation Detection in Predicting Ethionamide Resistance Using Melting Curve Technology.

ObjectiveThis study aims to analyze the correlation between gene inhA mutations by melting curve technology and phenotypic drug susceptibility (DST) results of ethionamide (ETH), and evaluate whether gene inhA mutations detection can serve as a molecular marker in predicting ETH resistance.MethodsA retrospective analysis was conducted on 382 strains of Mycobacterium tuberculosis (MTB) with the anti-tuberculosis drugs isoniazid (INH), rifampicin (RIF), ETH, and others. Phenotypic drug susceptibility and the results of inhA and katG genotypes (mutation and no mutation) were obtained using the melting curve technology MeltPro TB assay.ResultsOf the 382 clinical strains of MTB tested, 118 (30.9%) were resistant to INH, and 28 (7.3%) were resistant to ETH. Among the 28 phenotypically ETH-resistant strains, inhA mutations accounted for 42.9% (12/28). These ETH-resistant strains comprise 35.3% (12/34) of the 34 inhA mutant strains. Of 8 single inhA mutation strains (without katG or rpoB mutation), 4(50%) were resistant to INH; however, all of these 8 strains were sensitive to ETH.ConclusionThe inhA mutation test may not be a reliable predictor of ETH resistance. Mutant inhA strains are not necessarily resistant to ETH. The strains with single inhA mutation (without katG or rpoB mutation) may be effective for ETH treatment. The use of ETH in clinical medicine should be guided by gene (other than inhA alone) detection and phenotypic drug susceptibility testing.

Novel tigecycline resistance mechanisms in Acinetobacter baumannii mediated by mutations in adeS, rpoB and rrf

ABSTRACT Acinetobacter baumannii is an important pathogen in hospital acquired infections. Although tigecycline currently remains a potent antibiotic for treating infections caused by multidrug resistant A. baumannii (MDRAB) strains, reports of tigecycline resistant isolates have substantially increased. The resistance mechanisms to tigecycline in A. baumannii are far more complicated and diverse than what has been described in the literature so far. Here, we characterize in vitro-selected MDRAB strains obtained by increasing concentrations of tigecycline. We have identified mutations in adeS, rrf and rpoB that result in reduced susceptibility to tigecycline. Using in situ complementation experiments, we confirm that mutations in rrf, rpoB, and two types of mutations in adeS correlate with tigecycline resistance. By Western blot and polysome profile analysis, we demonstrate that the rrf mutation results in decreased expression of RRF, which affects the process of ribosome recycling ultimately leading to increased tigecycline tolerance. A transcriptional analysis shows that the mutated rpoB gene plays a role in regulating the expression of the SAM-dependent methyltransferase (trm) and transcriptional regulators, to confer moderate tigecycline resistance. This study provides direct in vitro evidence that mutations in the adeS, rpoB and rrf are associated with tigecycline resistance in A. baumannii.