A close-up on the epidemiology and transmission of multidrug-resistant tuberculosis in Poland.

Multidrug-resistant tuberculosis (MDR-TB) poses a serious challenge to the global control of the disease. The purpose of this study was to characterize MDR-TB patients from Poland and to determine the extent of MDR-TB disease attributable to recent transmission. The study included all 46 patients diagnosed with MDR-TB in Poland in 2004 and followed up for 6 years (until 2011). For each patient, sociodemographic and clinical characteristics, treatment outcomes, and bacteriological data were collected by the review of medical and laboratory records. Mycobacterium tuberculosis isolates from all patients were characterized using spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, IS6110 restriction fragment length polymorphism (RFLP) analysis, and sequencing analysis of drug resistance-associated loci (katG, mabA-inhA, rpoß, rpsL, and embB). The majority of patients were male (86.9%), 40-64 years of age (60.8%), with a history of TB treatment (84.8%), and producing smear-positive sputa (86.9%). Twenty-two (47.8%) patients suffered from concomitant diseases and 28 (60.8%) were alcohol abusers. Treatment outcome assessment revealed that 8 (17.4%) patients were cured or completed therapy, while 15 (32.6%) died of TB, 11 (23.9%) defaulted, 8 (17.4%) failed, and 1 (2.2%) was transferred and lost to follow-up. Upon genotyping, 10 (21.7%) isolates were allocated in four clusters. These were further subdivided by mutational profiling. Overall, in 6 (13%) patients, MDR-TB was a result of recent transmission. For 4 (8.7%) of these patients, a direct epidemiological link was established. The study shows that the transmission of MDR-TB occurs at a low rate in Poland. Of urgent need is the implementation of a policy of enforced treatment of MDR-TB patients in Poland.

Interaction of lectin pathway of complement-activating pattern recognition molecules with mycobacteria.

We have demonstrated that mannose-binding lectin (MBL) recognizes various slow-growing, pathogenic mycobacteria [Mycobacterium tuberculosis (MTB), M. bovis, M. kansasii, M. gordonae] as well as non-pathogenic M. smegmatis. Recognition resulted in activation of the lectin pathway (LP) of complement and an enhancement of phagocytosis (shown for M. tuberculosis). Although MBL may be considered the main factor activating the LP upon recognition of mycobacteria, involvement of ficolins has also to be considered. Interaction of ficolin-3 with M. tuberculosis, M. bovis and M. kansasii, and ficolin-1 with M. tuberculosis and M. bovis was shown for the first time. Binding of recombinant MBL or ficolin-3 to MTB H37 Rv led to the agglutination of bacteria and promoted their phagocytosis, but little effect was apparent with ficolin-1 or ficolin-2. Data from Western blots suggest mannosylated lipoarabinomannan (ManLAM) to be one of the main cell components of slow-growing mycobacteria, involved in LP activation. However, the LP was also activated by other cell fractions. Results presented here supplement considerably the data concerning the ability of complement-activating lectins to interact with mycobacteria. Ficolins (especially ficolin-3) might influence host response to infection and thus have clinical significance, at least as disease modifiers.

2,4-Disubstituted pyridine derivatives are effective against intracellular and biofilm-forming tubercle bacilli.

It was recently reported that 4-substituted picolinohydrazonamides carrying hydrophilic cyclic amines, such as morpholine and pyrrolidine, at the end of their thiosemicarbazide chain have potent antimycobacterial activity in vitro at concentrations below 1 µg/ml. Here, two selected compounds, 2,4-disubstituted pyridine derivatives 11 and 15, revealed significant bactericidal activity against Mycobacterium tuberculosis localized intracellularly within human macrophages, as well as against biofilm-forming tubercle bacilli. Mutants were selected that were resistant to the investigated compounds at an efficiency similar to that identified in the presence of the first line antituberculosis drug rifampicin. The resistant mutants were viable in the presence of the tested compounds exclusively on solid media. Genome-wide sequencing of the mutants selected in the presence of compound 11 revealed the accumulation of nonsynonymous mutations in the mmpR5 gene encoding a transcriptional repressor of the MmpS5-MmpL5 efflux pump, whose upregulation has been associated with bedaquiline resistance. The depletion of MmpR5 in wild-type M. tuberculosis using CRISPR-Cas9 technology increased the resistance of this strain to compound 11. Mass spectrometry-based proteomics (LC-MS/MS) of wild-type tubercle bacilli growing in subinhibitory concentrations of compounds 11 or 15 revealed 15 overproduced proteins not detectable in the control cells, including virulence-related proteins.

[In silico modeling of izoniazid-steroid conjugates interactions with cytochromes P450 of mycobacteria and their bioconversion in vitro by the cells]. / In silico modelirovanie vzaimodeistviia kon"iugatov izoniazid-steroid s tsitokhromami P450 mikobakterii i ikh prevrashchenie in vitro v kletkakh dannykh mikroorganizmov.

Molecular docking of four hydrazones of isoniazid with steroids (dehydroepiandrosterone, pregnenolone, 16α,17α-epoxypregnenolone, cholestenone) - IDHEA, IPRE, IEP5, ICHN, to mycobacterial cytochromes P450 was performed. The in silico study has shown than these hydrazones can be effectively bound to CYP121, CYP124, CYP125, CYP126A1, CYP130, and CYP51 with binding energy ranged from -9 kcal/mol to -12 kcal/mol. Calculations also demonstrated enhancement of passive lipid bilayer permeability with respect to isoniazid. In vitro IDHEA, IPRE, IEPR were found to undergo bioconversion into their 3-keto-4-en derivatives. This suggests their ability to penetrate into M. tuberculosis H37Rv cells. The results of this study are important in the context of understanding of specificity of binding of synthetic steroid derivatives to mycobacterial CYPs and indicate the possibility of using the steroid compounds studied by us as new ligands for these enzymes.

Molecular characterisation of streptomycin-resistant Mycobacterium tuberculosis strains isolated in Poland.

Primary drug resistance of Mycobacterium tuberculosis strains in Poland increased two-fold between 1997 and 2000. Among 3705 drug-resistant strains investigated in 2000, 169 were resistant to streptomycin alone or in combination with isoniazid, rifampicin and/or ethambutol. The molecular basis of streptomycin resistance for 88 (52%) of these strains in comparison with 15 susceptible controls was determined. The most prevalent mutation was the single substitution Lys43Arg in the rpsL gene, found in 30.7% of the strains analysed. However, as many as 51% of the strains investigated carried no mutation in the rpsL or rrs genes. The multiple mutations present in two Beijing family strains were also identified.

Molecular epidemiology of drug-resistant Mycobacterium tuberculosis strains isolated from patients with pulmonary tuberculosis in Poland: a 1-year study.

OBJECTIVE: To characterise drug-resistant Mycobacterium tuberculosis strains isolated in Poland and to estimate the amount of recent transmission in the population. DESIGN: M. tuberculosis strains isolated from 251 patients with resistant pulmonary tuberculosis in Poland in 2000 were analysed by spoligotyping and IS6110 DNA fingerprinting. Part of the strains was also characterised by sequencing of the rpoB, katG and/or the regulatory region of the inhA gene. RESULTS: Using combined spoligotyping/IS6110-RFLP defined clusters, 29% of the strains were clustered, suggesting possible recent transmission. In some cases, transmission links among strains in clusters could be confirmed by epidemiological data and in addition, for most of the strains, by analysis of the mutations associated with resistance to rifampicin and/or isoniazid. Younger age, sex, immigration and history of previous treatment were not associated with clustering, whereas multidrug-resistant disease was more likely to cluster. Strains of the Beijing family could also be found in Poland, although with a much lower frequency than in the neighbouring countries. CONCLUSION: Transmission of drug-resistant M. tuberculosis strains was demonstrated, which might contribute to the emergence of drug-resistant tuberculosis in Poland.

Development of a new ligation-mediated PCR method for the differentiation of Mycobacterium tuberculosis strains.

BACKGROUND: There is a need for rapid, inexpensive methods for analysing a limited number of Mycobacterium tuberculosis strains. The ligation-mediated polymerase chain reaction (LM-PCR) method appears to be sufficiently discriminative and reproducible to be considered as a molecular tool for the initial evaluation of hospital outbreaks, laboratory cross-contamination, and family or small community transmission. OBJECTIVE: To develop a new LM-PCR method based on PCR amplification of the 5'-flanking region of insertion sequence (IS) 6110 consisting of SalI/PvuII digestion of chromosomal DNA, ligation of a SalI linker and differentiation of IS6110-carrying restriction fragments by suppression subtractive hybridisation. DESIGN: The fast ligation amplification polymorphism (FLAP) method was applied in the analysis of 62 M. tuberculosis clinical isolates and compared with IS6110-restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) analyses of the same strains. RESULTS: The sensitivity of FLAP was estimated at 0.25 ng/l. FLAP yielded 32 patterns among the 62 M. tuberculosis strains compared to respectively 28 and 36 patterns obtained using MIRU-VNTR and IS6110-RFLP. Its Hunter-Gaston discriminatory index value (0.973) is similar to that of MIRU-VNTR (0.966) and IS6110-RFLP (0.971). The specificity of the FLAP patterns was also confirmed. CONCLUSION: FLAP proved highly discriminating, sensitive and specific and could be a valuable molecular tool, especially for analysing a limited number of M. tuberculosis strains.

Epidemiological analysis of Mycobacterium tuberculosis strains isolated in Lodz, Poland.

BACKGROUND: Mycobacterium tuberculosis is one of the most dangerous human pathogens. Molecular typing of M. tuberculosis has allowed better control of tuberculosis and, among other benefits, identification of genetic lineages among strains. OBJECTIVE: To test the potential of polymerase chain reaction (PCR) based methods for the epidemiological study of M. tuberculosis strains isolated from patients residing in a single city. DESIGN: We performed spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing and insertion sequence (IS) 6110 restriction fragment length polymorphism (RFLP) analyses of 234 clinical strains of M. tuberculosis collected over 2 years from the Polish city of Lodz. RESULTS: Spoligotyping analysis revealed 84 spoligotypes with a shared international type and 50 unique spoligotypes. Subtyping via 15- and 19-loci MIRU-VNTR analyses revealed 154 patterns with 117 unique profiles, and 159 patterns with 126 unique profiles, respectively. Spoligotyping combined with MIRU-VNTR 15- and 19 loci analyses revealed 132 and 146 unique profiles, respectively. Overall, 96 strains clustered via MIRU-VNTR typing were used in IS6110-RFLP analysis. Complete congruence of patterns revealed by PCR-based methods was noted for 40 strains, of which 36 were isolated from epidemiologically linked patients. CONCLUSION: The combination of 15-loci MIRU-VNTR typing with spoligotyping is useful for primary analysis of M. tuberculosis strains; however, additional use of MIRU 23 should be considered. Strains clustered by PCR-based methods should be further analysed by IS6110-RFLP typing.

Characterisation of a new host-vector system for fast-growing mycobacteria.

In this paper we describe the development of a host-vector system for genetic studies of fast-growing mycobacteria able to biotransform sterols. A wild strain Mycobacterium smegmatis SN38 and a biotechnological mutant Mycobacterium vaccae B3805 were transformed by electroporation with the pSMT3 E. coli-Mycobacterium shuttle plasmid harbouring the hygromycin resistance gene. Both, the pSMT3 plasmid and its derivative pSMT3-ksdD carrying the 3-ketosteroid-delta 1-dehydrogenase gene (ksdD) from Arthrobacter simplex were stably maintained in M. vaccae B3805. The presence of the pSMT3 vector did not affect biotransformation activities of the host strain. We consider the M. vaccae B3805 strain and the pSMT3 plasmid to be a good host-vector system for cloning in mycobacteria genes coding enzymes involved in steroid degradation pathway.

The DNA probe and PCR assay as useful tools to control an acid fast bacteria-dependent biotechnological process.

Specific DNA probe has been developed for fast-growing, mycobacterial mutants able to selectively biotransform side chain of plant sterols. The PCR assay, using primers complementary to the sequence of the probe, was shown to distinguish biotechnological mutants from other fast-growing mycobacteria. Moreover, the species identification of biotechnological strains was done using PCR-restriction analysis based on amplification and digestion of the inner part of hsp65 gene (PRA-assay) as well as 16S rRNA sequencing.

Methods of introduction of foreign DNA into mycobacteria.

Two methods: triparental conjugation and electrotransformation were used for introduction of plasmid DNA into mycobacterial cells. The introduction of shuttle plasmid pMY10 into M. fortuitum mutant caused the activation of its chromosomal cryptic KmR gene. The used of integration vector pUS 903 allowed to obtain a collection of mutants interesting for studies of genetic determination of steroid biotransformation and drug-resistance in mycobacteria.