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The introduction of novel drugs into treatment must be accompanied by continuous phenotypic susceptibility testing and the analysis of genetic determinants of resistance.

BackgroundThe 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 in vitro and in vivo recombination processes have been developed.ResultsHere, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant E. coli strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-attB 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-attP-site, and second, the λ system is used for excision of inserted vector part, including the plasmid ori-replication and the marker, flanked by λ-attL/R-sites.ConclusionThe 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.

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BackgroundThe steady rise in the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) requires rapid and reliable methods to identify resistant strains. The current molecular methods to detect MTB resistance to second-line drugs either do not cover an extended spectrum of mutations to be identified or are not easily implemented in clinical laboratories. A rapid molecular technique for the detection of resistance to second-line drugs in M. tuberculosis has been developed using hybridisation analysis on microarrays.MethodsThe method allows the identification of mutations within the gyrA and gyrB genes responsible for fluoroquinolones resistance and mutations within the rrs gene and the eis promoter region associated with the resistance to injectable aminoglycosides and a cyclic peptide, capreomycin. The method was tested on 65 M. tuberculosis clinical isolates with different resistance spectra that were characterised by their resistance to ofloxacin, levofloxacin, moxifloxacin, kanamycin and capreomycin. Also, a total of 61 clinical specimens of various origin (e.g., sputum, bronchioalveolar lavage) were tested.ResultsThe sensitivity and specificity of the method in the detection of resistance to fluoroquinolones were 98% and 100%, respectively, 97% and 94% for kanamycin, and 100% and 94% for capreomycin. The analytical sensitivity of the method was approximately 300 genome copies per assay. The diagnostic sensitivity of the assay ranging from 67% to 100%, depending on the smear grade, and the method is preferable for analysis of smear-positive specimens.ConclusionsThe combined use of the developed microarray test and the previously described microarray-based test for the detection of rifampin and isoniazid resistance allows the simultaneous identification of the causative agents of MDR and XDR and the detection of their resistance profiles in a single day.

The pentose-phosphate pathway (PPP) is an important part of central metabolism in many organisms. A pgl(-) mutation that decreases the efficiency of the second stage of PPP has been described and mapped at approx. 17.2 min of the Escherichia coli chromosome more than 30 years ago. Although it has recently been shown that deletion of ORF ybhE leads to earlier detected Pgl(-) phenotype for E. coli mutant strain, 6-phosphogluconolactonase from this organism has not been characterized. In the present, independent investigation we show that the Pgl(-) phenotype of DeltaybhE MG1655 could be complemented by insertion of the well-characterized pgl gene from Pseudomonas putida whose protein product has no visible homology with E. coli YbhE. Moreover, a final confirmation that ybhE actually encodes 6PGL in E. coli was obtained through overexpression of the cloned gene, purification of the protein product, followed by direct determination of its enzymatic activity in vitro.

Federation eIn addition to the obligatory pathogenic species of the Mycobacterium tuberculosis complex and Mycobacterium leprae, the genus Mycobacterium also includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of the gyrB gene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when using gyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice. In addition to the obligatory pathogenic species of the Mycobacterium tuberculosis complex (MTC) and Mycobacterium leprae, the genus Mycobacterium also includes conditionally pathogenic species that in rare cases lead to the development of nontuberculous mycobacterial disorders in humans (1). The main risk factors for such diseases are immunosuppression and chronic obstructive pulmonary disease (2). Because tuberculosis and mycobacteriosis have common clinical signs, the proper identification of the causative agent in a clinical microbiology laboratory is one of the most important tasks for diagnostic verification and treatment with the correct medication (3).The conventional microbiological and biochemical methods used to detect Mycobacterium are labor-intensive and timeconsuming. Efficient mycobacterial species identification using high-performance liquid chromatography of mycolic acids (4) requires expensive equipment and high-level personnel qualifications. Moreover, the spectrum of species that can be identified by microbiological assays is rather narrow, making DNA-based methods preferable (5). A number of molecular genetic techniques have been developed, including in situ hybridization species-specific probes (6), mult...