Rifampicin-resistant Mutants Research Articles

Mutation Analysis of the rpoB Gene in the Radiation-Resistant Bacterium Deinococcus radiodurans R1 Exposed to Space during the Tanpopo Experiment at the International Space Station.

To investigate microbial viability and DNA damage, dried cell pellets of the radiation-resistant bacterium Deinococcus radiodurans were exposed to various space environmental conditions at the Exposure Facility of the International Space Station (ISS) as part of the Tanpopo mission. Mutation analysis was done by sequencing the rpoB gene encoding RNA polymerase β-subunit of the rifampicin-resistant mutants. Samples included bacteria exposed to the space environment with and without exposure to UV radiation as well as control samples held in the ISS cabin and at ground. The mutation sites of the rpoB gene obtained from the space-exposed and ISS/ground control samples were similar to the rpoB mutation sites previously reported in D. radiodurans. Most mutations were found at or near the rifampicin binding site in the RNA polymerase β-subunit. Mutation sites found in UV-exposed samples were mostly shared with non-exposed and ISS/ground control samples. These results suggest that most mutations found in our experiments were induced during procedures that were applied across all treatments: preparation, transfer from our laboratory to the ISS, return from the ISS, and storage before analysis. Some mutations may be enhanced by specific factors in the space experiments, but the mutations were also found in the spontaneous and control samples. Our experiment suggests that the dried cells of the microorganism D. radiodurans can travel without space-specific deterioration that may induce excess mutations relative to travel at Earth's surface. However, upon arrival at a recipient location, they must still be able to survive and repair the general damage induced during travel.

Biological control of hairy root (Rhizobium rhizogenes) in apple nurseries through Rhizobium radiobacter antagonists (strain K-84 and native strain UHFBA-218)

Hairy root caused by Rhizobium rhizogenes is one of the major hindrances in raising quality nursery apple plants. Based on the survey analysis, carried out in the present study, disease incidence ranging from 3.00 to 21.05% was recorded in different apple nurseries of Himachal Pradesh. The plant pathogen isolated from rhizosphere soil of hairy root-infested apple seedling rootstock, in the present study, identified as Rhizobium rhizogenes strain UHF P-10 (NCBI Gen Bank accession no: MG745303.1) shared 100% homology with R. rhizogenes and belonged to biovar 2. Application of Rhizobium radiobacter antagonists (Strain K-84; native strain UHFBA-218 and their streptomycin and rifampicin-resistant mutants) as seed treatment completely inhibited hairy root disease. The application of strain UHFBA-218 as root dip reduced hairy root incidence to 3.33% whereas, application of K-84 as root dip minimized incidence to 4.99 %. The hairy root incidence in untreated plants during the first and second year of field trials was 18.57% and 20.23%, respectively. Seed and root dip application of both strains of R. radiobacter (K-84; native strain UHFBA-218) along with their antibiotic-resistant counterparts also promoted the growth of apple plants and increased population of antagonists.

Brucellosis re-emergence after a decade of quiescence in Palestine, 2015-2017: A seroprevalence and molecular characterization study.

Brucellosis is an endemic disease in many developing countries and ranked by the World Health Organization among the top seven "neglected zoonoses". Although a Palestinian brucellosis control program was launched in 1998, the disease re-emerged after 2012. Interestingly, a similar re-emerging pattern was reported in the neighbouring Israeli regions. The aim of this work was to characterize the re-emerging strains and delineate their genetic relatedness. During 2015-2017, blood samples from 1324 suspected human cases were analyzed using two serological tests. Seropositive samples were cultured, and their DNAs were analyzed by different genetic markers to determine the involved Brucella species and rule out any possible involvement of the Rev.1 vaccine strain. The rpoB gene was sequenced from nine isolates to screen for rifampicin resistance mutations. Multi locus VNTR analysis (MLVA-16) was used for genotyping the isolates. The molecular analysis showed that all isolates were Brucella melitensis strains unrelated to the Rev.1 vaccine. The rpoB gene sequences showed four single nucleotide variations (SNVs) not associated with rifampicin resistance. MLVA-16 analysis clustered the isolates into 22 unique genotypes that belonged to the East Mediterranean lineage. Altogether, our findingsshow that the re-emergence of brucellosis was due to B. melitensis strains of local origin, the Palestinian and Israeli control programs' weaknesses could be a major factor behind the re-emergence of the disease. However, other socioeconomic and environmental factors must be investigated. Moreover, strengthening brucellosis control programs and enhancing cooperation between all stakeholders is essential to ensure long-term program outcomes to fight brucellosis.

Characterization of rifampicin-resistant Mycobacterium tuberculosis in Khyber Pakhtunkhwa, Pakistan

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is endemic in Pakistan. Resistance to both firstline rifampicin and isoniazid drugs (multidrug-resistant TB; MDR-TB) is hampering disease control. Rifampicin resistance is attributed to rpoB gene mutations, but rpoA and rpoC loci may also be involved. To characterise underlying rifampicin resistance mutations in the TB endemic province of Khyber Pakhtunkhwa, we sequenced 51 M. tuberculosis isolates collected between 2016 and 2019; predominantly, MDR-TB (n = 44; 86.3%) and lineage 3 (n = 30, 58.8%) strains. We found that known mutations in rpoB (e.g. S405L), katG (e.g. S315T), or inhA promoter loci explain the MDR-TB. There were 24 unique mutations in rpoA, rpoB, and rpoC genes, including four previously unreported. Five instances of within-host resistance diversity were observed, where two were a mixture of MDR-TB strains containing mutations in rpoB, katG, and the inhA promoter region, as well as compensatory mutations in rpoC. Heteroresistance was observed in two isolates with a single lineage. Such complexity may reflect the high transmission nature of the Khyber Pakhtunkhwa setting. Our study reinforces the need to apply sequencing approaches to capture the full-extent of MDR-TB genetic diversity, to understand transmission, and to inform TB control activities in the highly endemic setting of Pakistan.

Predicting rifampicin resistance mutations in bacterial RNA polymerase subunit beta based on majority consensus

BackgroundMutations in an enzyme target are one of the most common mechanisms whereby antibiotic resistance arises. Identification of the resistance mutations in bacteria is essential for understanding the structural basis of antibiotic resistance and design of new drugs. However, the traditionally used experimental approaches to identify resistance mutations were usually labor-intensive and costly.ResultsWe present a machine learning (ML)-based classifier for predicting rifampicin (Rif) resistance mutations in bacterial RNA Polymerase subunit β (RpoB). A total of 186 mutations were gathered from the literature for developing the classifier, using 80% of the data as the training set and the rest as the test set. The features of the mutated RpoB and their binding energies with Rif were calculated through computational methods, and used as the mutation attributes for modeling. Classifiers based on five ML algorithms, i.e. decision tree, k nearest neighbors, naïve Bayes, probabilistic neural network and support vector machine, were first built, and a majority consensus (MC) approach was then used to obtain a new classifier based on the classifications of the five individual ML algorithms. The MC classifier comprehensively improved the predictive performance, with accuracy, F-measure and AUC of 0.78, 0.83 and 0.81for training set whilst 0.84, 0.87 and 0.83 for test set, respectively.ConclusionThe MC classifier provides an alternative methodology for rapid identification of resistance mutations in bacteria, which may help with early detection of antibiotic resistance and new drug discovery.

Complete Whole Genome Sequences of Escherichia coli Surrogate Strains and Comparison of Sequence Methods with Application to the Food Industry.

In 2013, the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) began transitioning to whole genome sequencing (WGS) for foodborne disease outbreak- and recall-associated isolate identification of select bacterial species. While WGS offers greater precision, certain hurdles must be overcome before widespread application within the food industry is plausible. Challenges include diversity of sequencing platform outputs and lack of standardized bioinformatics workflows for data analyses. We sequenced DNA from USDA-FSIS approved, non-pathogenic E. coli surrogates and a derivative group of rifampicin-resistant mutants (rifR) via both Oxford Nanopore MinION and Illumina MiSeq platforms to generate and annotate complete genomes. Genome sequences from each clone were assembled separately so long-read, short-read, and combined sequence assemblies could be directly compared. The combined sequence data approach provides more accurate completed genomes. The genomes from these isolates were verified to lack functional key E. coli elements commonly associated with pathogenesis. Genetic alterations known to confer rifR were also identified. As the food industry adopts WGS within its food safety programs, these data provide completed genomes for commonly used surrogate strains, with a direct comparison of sequence platforms and assembly strategies relevant to research/testing workflows applicable for both processors and regulators.

Genome-Wide Identification of Resveratrol Intrinsic Resistance Determinants in Staphylococcus aureus.

Resveratrol has been extensively studied due to its potential health benefits in multiple diseases, for example, cancer, obesity and cardiovascular diseases. Besides these properties, resveratrol displays inhibitory activity against a wide range of bacterial species; however, the cellular effects of resveratrol in bacteria remain incompletely understood, especially in the human pathogen, Staphylococcus aureus. In this study, we aimed to identify intrinsic resistance genes that aid S. aureus in tolerating the activity of resveratrol. We screened the Nebraska Transposon Mutant Library, consisting of 1920 mutants with inactivation of non-essential genes in S. aureus JE2, for increased susceptibly to resveratrol. On agar plates containing 0.5× the minimum inhibitory concentration (MIC), 17 transposon mutants failed to grow. Of these, four mutants showed a two-fold reduction in MIC, being the clpP protease mutant and three mutants with deficiencies in the electron transport chain (menD, hemB, aroC). The remaining 13 mutants did not show a reduction in MIC, but were confirmed by spot-assays to have increased susceptibility to resveratrol. Several genes were associated with DNA damage repair (recJ, xerC and xseA). Treatment of S. aureus JE2 with sub-inhibitory concentrations of resveratrol did not affect the expression of recJ, xerC and xseA, but increased expression of the SOS–stress response genes lexA and recA, suggesting that resveratrol interferes with DNA integrity in S. aureus. Expression of error-prone DNA polymerases are part of the SOS–stress response and we could show that sub-inhibitory concentrations of resveratrol increased overall mutation frequency as measured by formation of rifampicin resistant mutants. Our data show that DNA repair systems are important determinants aiding S. aureus to overcome the inhibitory activity of resveratrol. Activation of the SOS response by resveratrol could potentially facilitate the development of resistance towards conventional antibiotics in S. aureus.

Culturable endophytic Pseudomonas fluorescens Z1B4 isolated from Zanthoxylum alatum Roxb. with stress-tolerance and plant growth-promoting potential.

Endophytes are an important constituent of sustainable agriculture because of their ability to produce a large number of agriculturally important metabolites. A salt-tolerant fluorescent green pigment-producing endophytic bacterium was isolated on 2.5% NaCl-supplemented nutrient agar from the leaf samples of Zanthoxylum alatum Roxb. The isolate Z1B4 was identified as Pseudomonas fluorescens based on morphological features, fatty acid methyl ester analysis, biochemical tests, and 16S rRNA gene sequencing. P. fluorescens Z1B4 showed positive results for tricalcium phosphate solubilization; 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity; and production of auxins, siderophores, hydrogen cyanide, and ammonia. P. fluorescens Z1B4 also showed strong antagonistic activity against Curvularia lunata (MTCC 283), Fusarium verticillioides (MTCC 3322), and Alternaria alternata (MTCC 1362) and exhibited stress tolerance to a wide range of temperature and pH and concentrations of NaCl and calcium salts. Under natural conditions, following inoculation with the isolate Z1B4, a significant increase in the growth of pea and maize test plants in pots was observed compared to that of uninoculated control plants. The rifampicin-resistant mutant Z1B4Rif was recovered from the roots, shoots, and leaves of the test plants, indicating that the isolated endophytic bacterium can grow well within different plant tissues. The present study indicated that the endophytic bacterium P. fluorescens Z1B4 can be used as a bacterial inoculant in stressed environments for sustainable agriculture.

Mycobacterium tuberculosis Cells Surviving in the Continued Presence of Bactericidal Concentrations of Rifampicin in vitro Develop Negatively Charged Thickened Capsular Outer Layer That Restricts Permeability to the Antibiotic.

Majority of the cells in the bacterial populations exposed to lethal concentrations of antibiotics for prolonged duration succumbs to the antibiotics’ sterilizing activity. The remaining cells survive by diverse mechanisms that include reduced permeability of the antibiotics. However, in the cells surviving in the continued presence of lethal concentrations of antibiotics, it is not known whether any cell surface alterations occur that in turn may reduce permeability of the antibiotics. Here we report the presence of a highly negatively charged, hydrophilic, thickened capsular outer layer (TCOL) on a small proportion of the rifampicin surviving population (RSP) of Mycobacterium tuberculosis (Mtb) cells upon prolonged continuous exposure to bactericidal concentrations of rifampicin in vitro. The TCOL reduced the intracellular entry of 5-carboxyfluorescein-rifampicin (5-FAM-rifampicin), a fluorochrome-conjugated rifampicin permeability probe of negligible bacteriocidal activity but comparable properties. Gentle mechanical removal of the TCOL enabled significant increase in the 5-FAM-rifampicin permeability. Zeta potential measurements of the cells’ surface charge and hexadecane assay for cell surface hydrophobicity showed that the TCOL imparted high negative charge and polar nature to the cells’ surface. Flow cytometry using the MLP and RSP cells, stained with calcofluor white, which specifically binds glucose/mannose units in β (1 → 4) or β (1 → 3) linkages, revealed the presence of lower content of polysaccharides containing such residues in the TCOL. GC-MS analyses of the TCOL and the normal capsular outer layer (NCOL) of MLP cells showed elevated levels of α-D-glucopyranoside, mannose, arabinose, galactose, and their derivatives in the TCOL, indicating the presence of high content of polysaccharides with these residues. We hypothesize that the significantly high thickness and the elevated negative charge of the TCOL might have functioned as a physical barrier restricting the permeability of the relatively non-polar rifampicin. This might have reduced intracellular rifampicin concentration enabling the cells’ survival in the continued presence of high doses of rifampicin. In the context of our earlier report on the de novo emergence of rifampicin-resistant genetic mutants of Mtb from the population surviving under lethal doses of the antibiotic, the present findings attain clinical significance if a subpopulation of the tubercle bacilli in tuberculosis patients possesses TCOL.

Sobrevivência de Pseudomonas marginalis pv. marginalis em bulbilhos de alho

A queima-bacteriana-do-alho causada por Pseudomonas marginalis pv. marginalis é uma das principais doenças foliares da cultura e pouco se conhece de sua dinâmica de sobrevivência no bulbilho. Dentro deste aspecto, o objetivo deste trabalho foi avaliar a sobrevivência desta bactéria no bulbilho com e sem assepsia durante o armazenamento. Bulbilhos de alho do cultivar Chonan foram inoculados com isolado mutante a rifampicina de P. marginalis pv. marginalis para diferenciar de outras bactérias e foram transplantados a campo. Ao final do ciclo da cultura foi avaliada a população presente no bulbilho e mensalmente até o sexto mês de armazenamento. Para quantificação da população bacteriana foram realizadas três repetições composta de dez gramas cada de bulbilhos sem e com assepsia com hipoclorito de sódio por três minutos, seguida do isolamento pela técnica de diluição seriada seguida de plaqueamento em meio de cultura King-B com rifampicina para isolamento seletivo da bactéria. Após incubação de 48 horas a 28 ºC, as colônias foram contadas para analisar mensalmente a sua sobrevivência no bulbilho. Mediante aos resultados obtidos, a bactéria não tem capacidade de sobreviver nos bulbilhos submetidos à assepsia durante o armazenamento, mas consegue sobreviver sem assepsia por até três meses.

Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs.

Conventional molecular tests for detecting Mycobacterium tuberculosis complex (MTBC) drug resistance on clinical samples cover a limited set of mutations. Whole-genome sequencing (WGS) typically requires culture.Here, we evaluated the Deeplex Myc-TB targeted deep-sequencing assay for prediction of resistance to 13 anti-tuberculous drugs/drug classes, directly applicable on sputum.With MTBC DNA tests, the limit of detection was 100–1000 genome copies for fixed resistance mutations. Deeplex Myc-TB captured in silico 97.1–99.3% of resistance phenotypes correctly predicted by WGS from 3651 MTBC genomes. On 429 isolates, the assay predicted 92.2% of 2369 first- and second-line phenotypes, with a sensitivity of 95.3% and a specificity of 97.4%. 56 out of 69 (81.2%) residual discrepancies with phenotypic results involved pyrazinamide, ethambutol and ethionamide, and low-level rifampicin or isoniazid resistance mutations, all notoriously prone to phenotypic testing variability. Only two out of 91 (2.2%) resistance phenotypes undetected by Deeplex Myc-TB had known resistance-associated mutations by WGS analysis outside Deeplex Myc-TB targets. Phenotype predictions from Deeplex Myc-TB analysis directly on 109 sputa from a Djibouti survey matched those of MTBSeq/PhyResSE/Mykrobe, fed with WGS data from subsequent cultures, with a sensitivity of 93.5/98.5/93.1% and a specificity of 98.5/97.2/95.3%, respectively. Most residual discordances involved gene deletions/indels and 3–12% heteroresistant calls undetected by WGS analysis or natural pyrazinamide resistance of globally rare “Mycobacterium canettii” strains then unreported by Deeplex Myc-TB. On 1494 arduous sputa from a Democratic Republic of the Congo survey, 14 902 out of 19 422 (76.7%) possible susceptible or resistance phenotypes could be predicted culture-free.Deeplex Myc-TB may enable fast, tailored tuberculosis treatment.

Diabetes and multidrug-resistance gene mutation: tuberculosis in Zunyi, Southwest China.

The aim of this study was to clarify the characteristics of gene mutation related to multidrug-resistance (MDR) of tuberculosis (TB) and diabetes in Zunyi. A total 763 patients with TB were screened for TB-DNA, TB-RNA, and acid-fast staining (all were positive). They were divided into the tuberculosis (TB) group and the diabetes mellitus-tuberculosis (DM-TB) group. We compared and analyzed the MDR gene rpoB, KatG, and inhA characteristics of gene mutations in the two groups by polymerase chain reaction (PCR)-reverse dot hybridization, and collected relevant clinical data to explore its correlation with the occurrence of multidrug resistance. Multidrug resistance occurred in 32 of the 525 patients in the TB group, and extensive drug resistance occurred in 15 of the 207 patients in the DM-TB group. In the DM-TB group, the mutation rates of ropBS531L and ropB531 (both 53.33%) were lower than those of the TB group (both 59.38%) in rifampicin resistance mutations. Most of the mutations were at the KatG315N site, conferring isoniazid resistance. The mutation sites of multidrug-resistant patients in Zunyi are mainly ropB531 and ropBS531L mutations, which are prone to co-occurrence; patients with MDR-TB alone are prone to mutations at the KatG315N site, while patients with diabetes and MDR-TB are more likely to have inhA15M site mutations.

Controlling the hypermutation: Exploitation of the MutS protein in Pseudomonas aeruginosa

Pseudomonas aeruginosa infections commonly develop in individuals with cystic fibrosis (CF), and its adaptation in such an unfavourable condition is always found to be related to hypermutation. In fact, most of the hypermutation is due to the defects in mutS gene which involves in the mismatch repair mechanism, causing the acceleration of mutation rate and adaptive evolution. In order to rheostatically express the MutS protein and achieve “hypomutation” (in which the rate of mutation is lower than that of wild type strain), an exogenous mutS gene with rhamnose-inducible promoter was cloned into MPAO1 mutS::Tn mutant strain. Present findings demonstrate that this system is tightly-controlled and stable, with less rifampicin-resistant mutant frequency and more fluorescence intensity from a GFP-tagged MutS expressing cells were observed when the concentration of the inducer increases. Interestingly, the results from Western blot analysis show that less MutS protein is required to suppress hypermutation in the wild type strain, as compared to our construct that behaves similar to the wild type but obviously needs more MutS expression to achieve such state. This indicates that the exogenous MutS might be lacking of other important protein to work efficiently in mismatch recognition. Therefore, based on our cDNA analysis, we found that fdxA gene next to the mutS gene is in the same operon, which could suggest that they might be functionally related in the DNA repair machinery.

Nutritional conditions and oxygen concentration affect spontaneous occurrence of homologous recombination events but not spontaneous mutagenesis in Escherichia coli.

Effects of environmental factors for growth of Escherichia coli on spontaneous mutagenesis and homologous recombination events are described. By analyzing rifampicin-resistant (Rifr) mutation frequencies in an E. coli strain lacking MutM and MutY repair enzymes, which suppress base substitution mutations caused by 8-oxoguanine (7,8 dihydro-8-oxoguanine; 8-oxoG) in DNA, we examined levels of oxidative DNA damage produced in normally growing cells. The level of 8-oxoG DNA damage was about 9- and 63-fold higher in cells grown in M9-glucose and M9-glycerol media, respectively, than in those grown in LB medium. We also found that about 14-fold more 8-oxoG DNA damage was produced in cells grown in about 0.1% oxygen than in those grown in the normal atmosphere. However, Rifr mutation frequency in wild-type cells was unchanged in such different growth conditions, suggesting that the capacity of repair mechanisms is sufficient to suppress mutations caused by 8-oxoG even at very high levels in cells growing in the particular conditions. On the other hand, the frequency of spontaneous homologous recombination events in wild-type E. coli cells varied with different growth conditions. When cells were grown in M9-glucose and M9-glycerol media, the spontaneous recombination frequency increased to about 4.3- and 7.3-fold, respectively, higher than that in LB medium. Likewise, the spontaneous recombination frequency was about 3.5-fold higher in cells growing in the hypoxic condition than in cells growing in the atmosphere. When cells were grown in anaerobic conditions, the recombination frequency decreased to half of that in the atmosphere. These data indicated that spontaneous homologous recombination is highly responsive to environmental factors such as nutrition and oxygen concentration.

GeneXpert MTB/RIF Based Detection of Rifampicin Resistance and Common Mutations in rpoB Gene of Mycobacterium Tuberculosis in Tribal Population of District Anuppur, Madhya Pradesh, India

Introduction: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) continues to be one of the most significant causes of death in the developing countries. Development of Multi Drug Resistance (MDR) and Extremely Drug Resistance (XDR) strains of MTB has been recognised as a major threat. Rapid diagnosis along with drug sensitivity analysis is the prerequisite for effective treatment of TB, especially in rural and remote location settings. Aim: The goal of this study was to investigate the Rifampicin Resistance (RR) using GeneXpert MTB/Rifampicin (RIF) in tribal patients suffering from Pulmonary Tuberculosis (PTB) in District Anuppur, Madhya Pradesh, India. Materials and Methods: Sputum samples were obtained from 413 patients with symptoms of PTB, who visited District Hospital, Anuppur from April 2017- April 2018. Based on clinical symptoms and chest X-ray, GeneXpert MTB/RIF assay was performed for the confirmation of TB and detection of RR. The data was analysed and expressed in percentage. Results: Out of 413 samples, 104 (25.18%) were diagnosed with PTB. Out of 104 TB positive samples, RR was detected in 7(6.73%) samples. The most common mutations conferring RR were located in the region of Probe B (71.42%), followed by Probe C (14.28%) and Probe E (14.28%), while no mutations were found in the region of Probe A and Probe D. Conclusion: Possibly, this is the first report of RR and probe mutational analysis from this tribal region of India. High rate of mutation at Probe B locus may be the chief reason for RR development. Gene sequencing may be carried for understanding the higher rates of mutations at probe B locus.

Ultrasensitive Real-Time Rolling Circle Amplification Detection Enhanced by Nicking-Induced Tandem-Acting Polymerases.

Padlock probe ligation-based rolling circle amplification (RCA) can distinguish single-nucleotide variants, which is promising for the detection of drug-resistance mutations in, e.g., Mycobacterium tuberculosis (Mtb). However, the clinical application of conventional linear RCA is restricted by its unsatisfactory picomolar-level limit of detection (LOD). Herein, we demonstrate the mechanism of a nicking-enhanced RCA (NickRCA) strategy that allows several polymerases to act simultaneously on the same looped template, generating single-stranded amplicon monomers. Limiting factors of NickRCA are investigated and controlled for higher amplification efficiency. Thereafter, we describe a NickRCA-based magnetic nanoparticle (MNP) dimer formation strategy combined with a real-time optomagnetic sensor monitoring MNP dimers. The proposed methodology is applied for the detection of a common Mtb rifampicin-resistance mutation, rpoB 531 (TCG/TTG). Without additional operation steps, an LOD of 15 fM target DNA is achieved with a total assay time of ca. 100 min. Moreover, the proposed biosensor holds the advantages of single-nucleotide mutation discrimination and the robustness to quantify targets in 10% serum samples. NickRCA produces short single-stranded monomers instead of the DNA coils produced in conventional RCA, which makes it more convenient for downstream operation, immobilization or detection, thus being applicable with different molecular tools and biosensors.

Fitness of Spontaneous Rifampicin-Resistant Staphylococcus aureus Isolates in a Biofilm Environment.

Biofilms of S. aureus accumulate cells resistant to the antibiotic rifampicin. We show here that the accumulation of rifampicin resistant mutants (RifR) in biofilms is not equable but rather is a local event, suggesting that the growth of a few locally emerged mutants is responsible for this. Competition assays demonstrated that, compared to wild-type bacteria, the isolated RifR mutants have a growth advantage in biofilms, but not in planktonic culture. To gain insight into the mechanism of the growth advantage, we tested the involvement of the two-component systems (TCS) that sense and respond to environmental changes. We found that a deletion of SrrAB or NreBC has a drastic effect on the growth advantage of RifR mutants, suggesting the importance of oxygen/respiration responses. All six of the RifR isolates tested showed increased resistance to at least one of the common stresses found in the biofilm environment (i.e., oxidative, nitric acid, and organic acid stress). The RifR mutants also had a growth advantage in a biofilm flow model, which highlights the physiological relevance of our findings.

Cryptic Resistance Mutations Associated With Misdiagnoses of Multidrug-Resistant Tuberculosis.

Understanding why some multidrug-resistant tuberculosis cases are not detected by rapid phenotypic and genotypic routine clinical tests is essential to improve diagnostic assays and advance toward personalized tuberculosis treatment. Here, we combine whole-genome sequencing with single-colony phenotyping to identify a multidrug-resistant strain that had infected a patient for 9 years. Our investigation revealed the failure of rapid testing and genome-based prediction tools to identify the multidrug-resistant strain. The false-negative findings were caused by uncommon rifampicin and isoniazid resistance mutations. Although whole-genome sequencing data helped to personalize treatment, the patient developed extensively drug-resistant tuberculosis, highlighting the importance of coupling new diagnostic methods with appropriate treatment regimens.

Bacillus subtilis RarA acts at the interplay between replication and repair-by-recombination

Bacterial RarA is thought to play crucial roles in the cellular response to blocked replication forks. We show that lack of Bacillus subtilis RarA renders cells very sensitive to H2O2, but not to methyl methane sulfonate or 4-nitroquinoline-1-oxide. RarA is epistatic to RecA in response to DNA damage. Inactivation of rarA partially suppressed the DNA repair defect of mutants lacking translesion synthesis polymerases. RarA may contribute to error-prone DNA repair as judged by the reduced frequency of rifampicin-resistant mutants in ΔrarA and in ΔpolY1 ΔrarA cells. The absence of RarA strongly reduced the viability of dnaD23ts and dnaB37ts cells upon partial thermal inactivation, suggesting that ΔrarA cells are deficient in replication fork assembly. A ΔrarA mutation also partially reduced the viability of dnaC30ts and dnaX51ts cells and slightly improved the viability of dnaG40ts cells at semi-permissive temperature. These results suggest that RarA links re-initiation of DNA replication with repair-by-recombination by controlling the access of the replication machinery to a collapsed replication fork.

Adaptive Evolution of Escherichia coli K-12 MG1655 Grown on Ethanol and Glycerol

The FEt and FGl derivatives of the E. coli K-12 MG1655 and YA1461 strains with improved ethanol and glycerol utilization, respectively, under aerobic conditions were obtained by laboratory adaptive evolution. These fitness strains have a shorter lag-phase and higher growth rate on glucose, xylose, and their mixture as compared with the parental strains. DNA sequencing and cross-transduction analysis showed that the change in the growth rate of the rifampicin-resistant fitness mutants FGl and YA1461 RifRII are caused by mutations in the rpoB gene encoding the RNA polymerase β-subunit. These mutations affect conservative amino acid segments, which leads to the amino acid substitutions of RpoBD483G and RpoBH526N, respectively.