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.

The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex.