Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure

<p>Abstract</p> <p>Background</p> <p>The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on <it>in vitro </it>and <it>in vivo </it>recombination processes have been developed.</p> <p>Results</p> <p>Here, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant <it>E. coli </it>strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-<it>attB </it>sites into desired points of the chromosome followed by two site-specific recombination processes: first, the φ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with φ80-<it>attP</it>-site, and second, the λ system is used for excision of inserted vector part, including the plasmid <it>ori</it>-replication and the marker, flanked by λ-<it>attL/R</it>-sites.</p> <p>Conclusion</p> <p>The developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry.</p

Technical Report on critical concentrations for drug susceptibility testing of isoniazid and the rifamycins (rifampicin, rifabutin and rifapentine)

The critical concentrations for culture-based phenotypic drug susceptibility testing (DST) to first-line anti-TB drugs have been revised by the the World Health Organization (WHO). Critical concentrations for rifampicin have been lowered while those for isoniazid have been maintained at the present level. This update helps address the discordance observed between phenotypic and molecular methods to detect rifampicin resistance and improves the accuracy of DST. As a result patients with TB will have a more accurate diagnosis. This document is the outcome of a Technical Expert Group meeting convened by WHO in 2020 to assess the results of a systematic review of published literature on critical concentrations for DST of the most important first-line anti-TB drugs, isoniazid and the rifamycins (rifampicin, rifabutin and rifapentine). These critical concentrations had not been revised since 2008. New evidence over the past decade showed that critical concentrations used for phenotypic methods to detect rifampicin resistance may incorrectly classify strains with certain mutations. The following media were considered: Löwenstein-Jensen (LJ), Middlebrook 7H10 (7H10), Middlebrook 7H11 (7H11) and BACTEC™ Mycobacterial Growth Indicator Tube™ 960 (MGIT). Guidance has been provided to resolve discordance between genotypic and phenotypic results for these drugs and areas for further research have been highlighted. DST methods continue to have a very important role to identify resistance not detected by molecular assays and to support the interpretation of molecular assays results. However, they require sophisticated laboratory infrastructure, qualified staff and strict quality assurance procedures.Peer reviewe

Updating the approaches to define susceptibility and resistance to anti-tuberculosis agents: implications for diagnosis and treatment

11 páginas, 2 figuras, 1 tablaInappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance.I. Comas was supported by PID2019-104477RB-I00 from the Spanish Science Ministry and by ERC (CoG 101001038)Peer reviewe

The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis.

Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation

Variability of Mycobacterium avium Complex Isolates Drug Susceptibility Testing by Broth Microdilution

Non-tuberculous mycobacteria are widely distributed in environments and are capable of infecting humans, particularly those with a compromised immune system. The most prevalent species that cause nontuberculous mycobacterial lung diseases are slow-growing bacteria from the Mycobacterium avium complex (MAC), mainly M. avium or M. intracellulare. The key treatment of MAC infections includes macrolides, ethambutol, and rifampicin; however, the therapy outcomes are unsatisfactory. Phenotypic drug susceptibility testing is a conditional recommendation prior to treatment, and critical concentrations for clarithromycin, amikacin, moxifloxacin, and linezolid have been established. In this review, data from studies on the determination of MIC of clinical isolates using the broth microdilution method were summarized. A significant variation in the MIC distributions from different studies was found. The main reasons could impact the findings: insufficient reproducibility of the phenotypic testing and variation in species lineages identified in different laboratories, which could have various intrinsic susceptibility to drugs. For most of the drugs analyzed, the MICs are too high, which could undermine the treatment efficiency. Further improvement of treatment outcomes demands the validation of microbiological resistance criteria together with the identification of molecular mechanisms of resistance

Molecular Determinants of Ethionamide Resistance in Clinical Isolates of <i>Mycobacterium tuberculosis</i>

Background: Ethionamide and prothionamide are now included in group C of the WHO recommended drugs for the treatment of tuberculosis resistant to rifampicin and multidrug-resistant tuberculosis. The clinical relevance of ethionamide and prothionamide has increased with the wide spread of resistant tuberculosis. Methods: We retrospectively analyzed 349 clinical isolates obtained between 2016 and 2020. The susceptibility to ethionamide was tested using both the BactecTM MGITTM 960 system and the SensititreTM MYCOTB plate. Results: The MIC of ethionamide increases with the total resistance of the isolates in a row from susceptible to XDR strains. A significant part of the isolates have a MIC below the breakpoint: 25%, 36%, and 50% for XDR, pre-XDR, and MDR strains. Sensitivity and specificity of detection of mutations were 96% and 86% using MGIT resistance as a reference. Conclusions: Phenotypic methods for testing ethionamide are imperfectly correlated, and the isolates with MIC of 5 mg/L have the intermediate resistance. A significant proportion of resistant TB cases are susceptible and eligible for ethionamide treatment. Resistance could be explained using only analysis of loci ethA, PfabG1, and inhA for most isolates in the Moscow region. The promoter mutation PfabG1 c(-15)t predicts resistance to ethionamide with high specificity but low sensitivity

A Comparison of the Sensititre MycoTB Plate, the Bactec MGIT 960, and a Microarray-Based Molecular Assay for the Detection of Drug Resistance in Clinical <i>Mycobacterium tuberculosis</i> Isolates in Moscow, Russia

<div><p>Background</p><p>The goal of this study was to compare the consistency of three assays for the determination of the drug resistance of <i>Mycobacterium tuberculosis</i> (MTB) strains with various resistance profiles isolated from the Moscow region.</p><p>Methods</p><p>A total of 144 MTB clinical isolates with a strong bias toward drug resistance were examined using Bactec MGIT 960, Sensititre MycoTB, and a microarray-based molecular assay TB-TEST to detect substitutions in the <i>rpoB</i>, <i>katG</i>, <i>inhA</i>, <i>ahpC</i>, <i>gyrA</i>, <i>gyrB</i>, <i>rrs</i>, <i>eis</i>, and <i>embB</i> genes that are associated with resistance to rifampin, isoniazid, fluoroquinolones, second-line injectable drugs and ethambutol.</p><p>Results</p><p>The average correlation for the identification of resistant and susceptible isolates using the three methods was approximately 94%. An association of mutations detected with variable resistance levels was shown. We propose a change in the breakpoint minimal inhibitory concentration for kanamycin to less than 5 μg/ml in the Sensititre MycoTB system. A pairwise comparison of the minimal inhibitory concentrations (MICs) of two different drugs revealed an increased correlation in the first-line drug group and a partial correlation in the second-line drug group, reflecting the history of the preferential simultaneous use of drugs from these groups. An increased correlation with the MICs was also observed for drugs sharing common resistance mechanisms.</p><p>Conclusions</p><p>The quantitative measures of phenotypic drug resistance produced by the Sensititre MycoTB and the timely detection of mutations using the TB-TEST assay provide guidance for clinicians for the choice of the appropriate drug regimen.</p></div