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.

Differentiation of Clavibacter michiganensis subsp. sepedonicus using PCR melting profile and variable number of tandem repeat methods.

The potato phytopathogen Clavibacter michiganensis subsp. sepedonicus (Cms) is a causative agent of bacterial ring rot, which is a serious threat to crops. In EU member countries, Cms is subject to quarantine and has to be combated. The knowledge about the transmission of C. michiganensis strains is limited due to a lack of methods which could be used for epidemiological analysis. In this study, PCR melting profile (PCR MP) and variable number tandem repeat methods were used in Cms epidemiological analysis for the first time. PCR MP was based on the melting temperature analysis of BamHI restriction fragments of chromosomal DNA. Respectively, for the variable number tandem repeat (VNTR) method, six loci were identified and used in the differentiation of Cms isolates. PCR MP was used for 93 Cms isolated in Poland. Both PCR MP and VNTR methods were used for the differentiation of 47 Cms strains in this collection. Both these methods were found to be useful for the epidemiological analysis of Cms. SIGNIFICANCE AND IMPACT OF THE STUDY: The potato phytopathogen, Clavibacter michiganensis subsp. sepedonicus (Cms), is a serious threat to crops and lead to significant economic losses. The only way to control and eliminate the disease caused by this pathogen is the use of certified seed potato and strict quarantine of infected fields. Here, for the first time, two molecular typing methods (PCR melting profile (PCR MP) and variable number tandem repeat (VNTR)) were evaluated in respect of their potential in differentiation of Cms isolates. As a result, we obtained characteristic profiles of DNA fragments (PCR MP) and numeric patterns (VNTR), which enable the intraspecies genotyping of Cms strains confirming the effectiveness of PCR MP and VNTR methods in differentiation of Cms strains.

Cyanophages Infection of Microcystis Bloom in Lowland Dam Reservoir of Sulejów, Poland.

An increased incidence of cyanobacterial blooms, which are largely composed of toxigenic cyanobacteria from the Microcystis genus, leads to a disruption of aquatic ecosystems worldwide. Therefore, a better understanding of the impact of environmental parameters on the development and collapse of blooms is important. The objectives of the present study were as follows: (1) to investigate the presence and identity of Microcystis-specific cyanophages capable of cyanobacterial cell lysis in a lowland dam reservoir in Central Europe; (2) to investigate Microcystis sensitivity to phage infections with regard to toxic genotypes; and (3) to identify key abiotic parameters influencing phage infections during the summer seasons between 2009 and 2013. Sequencing analysis of selected g91 gene amplification products confirmed that the identified cyanophages belonged to the family Myoviridae (95 % homology). Cyanophages and Microcystis hosts, including toxic genotypes, were positively correlated in 4 of the 5 years analyzed (r = 0.67-0.82). The average percentage of infected Microcystis cells varied between 0.1 and 32 %, and no particular sensitivity of the phages to toxigenic genotypes was recorded. The highest number of cyanophages (>10(4) gene copy number per microliter) was observed in the period preceded by the following: an increase of the water retention time, growth of the water temperature, optimum nutrient concentrations, and the predomination of Microcystis bloom.

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.

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.

Mycobacterium tuberculosis CwsA interacts with CrgA and Wag31, and the CrgA-CwsA complex is involved in peptidoglycan synthesis and cell shape determination.

Bacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein from Mycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog in M. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [(14)C]N-acetylglucosamine cell wall precursor. The M. smegmatis MSMEG_0023 crgA double mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA for cell wall synthesis and cell shape protein A, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria.

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.

Characterization of CrgA, a new partner of the Mycobacterium tuberculosis peptidoglycan polymerization complexes.

The role(s) in cell division of the Mycobacterium tuberculosis Rv0011c gene product, a homolog of the Streptomyces CrgA protein that is responsible for coordinating growth and cytokinesis in sporogenic aerial hyphae, is largely unknown. We show that an enhanced cyan fluorescent protein-M. tuberculosis CrgA (ECFP-CrgA(MT)) fusion protein is localized to the cell membrane, midcell, and cell pole regions in Mycobacterium smegmatis. Furthermore, the ECFP-CrgA(MT) fusion protein colocalized with FtsZ-enhanced yellow fluorescent protein (EYFP) in M. smegmatis. Bacterial two-hybrid assays indicated strong interactions of M. tuberculosis CrgA with FtsZ, FtsQ, and the class B penicillin-binding proteins, FtsI (PBPB) and PBPA. The midcell localization of CrgA(MT) was severely compromised under conditions of FtsZ depletion, which indicated that CrgA localizes to the midcell region after assembly of the FtsZ ring. M. tuberculosis cells with reduced CrgA levels were elongated and grew more slowly than wild-type cells, which indicated defects in cell division, whereas CrgA overproduction did not show growth defects. A M. smegmatis ΔcrgA strain exhibited a bulged cell morphology, elongated cells with a chain-like phenotype, cells with polar bulbous structures, and a modest growth defect. FtsZ and FtsI levels were not affected in cells producing altered levels of CrgA. Septal and membrane localization of GFP-FtsI was enhanced by CrgA overproduction and was diminished in a ΔcrgA strain, which indicates that one role of CrgA is to promote and/or stabilize FtsI localization. Overall, these data indicate that CrgA is a novel member of the cell division complex in mycobacteria and possibly facilitates septum formation.

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.

Detection of Mycobacterium tuberculosis in clinical samples using insertion sequences IS6110 and IS990.

To detect Mycobacterium tuberculosis in clinical samples, we used the M. tuberculosis-complex specific insertion sequence IS990 as the target in a simple DIG-PCR ELISA assay, as this element is present as a single copy in all strains of M. tuberculosis we have examined to date. The IS990 test was compared with a similar PCR that utilizes IS6110 as target. For detection of PCR product, digoxigenin-11-dUTP (DIG-dUTP) was incorporated into the product. After amplification, the PCR product was hybridized with biotinylated capture probe, which was complementary to the inner part of the amplicon. The hybrid was captured onto streptavidin-coated microtiter plate and DIG-labeled PCR product was detected using a peroxidase-conjugated antibody to DIG. We evaluated DIG-PCR ELISA for the detection of M. tuberculosis DNA in 265 respiratory and non-respiratory specimens taken from patients with known and suspected tuberculosis disease or from controls. The sensitivity and specificity of both IS990-based test and IS6110-based test was 96.5% and 95.3% respectively, comparable to the sensitivity and specificity of the IS6110-based test. The results demonstrate that the IS990 PCR ELISA test is a rapid and sensitive tool for the detection and identification of M. tuberculosis in clinical samples, and may have advantages to the more widely used IS6110-based tests, particularly in areas where IS6110-negative strains are found.

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.

Specificity of insertion sequence-based PCR assays for mycobacterium tuberculosis complex.

OBJECTIVE: To determine the specificity of different insertion sequence-targeted polymerase chain reaction (PCR) tests for Mycobacterium tuberculosis complex. DESIGN: One M. bovis BCG strain, two M. tuberculosis strains and ten species of mycobacteria other than tuberculosis (MOTT) were tested by three PCR assays based on the repetitive elements IS6110, IS1081 and IS990 under variable amplification conditions (different temperatures of primer annealing and numbers of reaction cycles). RESULTS: DNA amplifications based on the three insertion sequences yielded fragments of expected sizes only in DNA from M. tuberculosis complex strains when the tests were conducted at high stringency (65 degrees C). At the annealing temperature of 60 degrees C the PCR assay with IS6110-specific primers yielded a 245 bp fragment also in nine MOTT strains tested. This could result from previously reported homology between non-tuberculous mycobacteria and a central region of IS6110. Amplification assays based on IS1081 and IS990 gave false-positive results in some MOTT isolates only under very low stringency (55 degrees C), which could be due to non-specific priming of the target DNA at that temperature. CONCLUSION: Repetitive elements IS1081 and IS990 may represent a more reliable alternative to the more widely used IS6110 PCR target for tuberculosis diagnosis.

IS1607, a single-copy insertion sequence-related element of the Mycobacterium tuberculosis complex.

The species specificity of IS1607, a new Mycobacterium tuberculosis complex insertion sequence-related element, was investigated. IS1607 was present as a single copy in M. tuberculosis, M. bovis and M. bovis BCG strains. This sequence also existed in M. africanum and M. microti, but was not present in 69 strains of 19 atypical mycobacterial species analyzed by using polymerase chain reaction (PCR) or Southern hybridization assay. IS1607 appears to be specific for the M. tuberculosis complex.

DNA fingerprinting as an indicator of active transmission of multidrug-resistant tuberculosis in Poland.

OBJECTIVES: To use genetic fingerprinting to investigate the epidemiology of tuberculosis (TB) caused by Mycobacterium tuberculosis in Poland, a country with a relatively high incidence of tuberculosis, to improve TB control. DESIGN: One hundred M. tuberculosis isolates from 98 patients in the Institute of Tuberculosis and Lung Diseases in Warsaw from 1993 to 1995 and 85 isolates obtained from 50 patients in the Hospital of Lung Diseases in Lodz in 1996 were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the insertion sequence IS6110 as a probe. RESULTS: IS6110-associated banding patterns of the M. tuberculosis isolates originating from different localities varied considerably, but isolates from Lodz had a higher degree of similarity. Strains with identical RFLP types were identified in patients of the same family or patients living in the same area, indicating active transmission. Of strains isolated in Warsaw, 45% were resistant to at least one drug, and 35% were resistant to two or more drugs and were classified as multidrug-resistant (MDR). Some drug-resistant isolates were found to have identical banding patterns and originated from epidemiologically linked cases. CONCLUSIONS: Active transmission of TB, including MDR TB, is occurring in Poland. Active measures must be taken to prevent the spread of drug-resistant TB in Poland and potentially, the rest of Europe.

IS990, a new species-specific insertion-sequence-related element of the Mycobacterium tuberculosis complex.

The structure and distribution of 1S990, a new Mycobacterium tuberculosis DNA sequence with homology to characterized insertion sequences (ISs), were investigated. IS990 was related to IS elements of the IS3 family and was present as a single copy in all 21 investigated M. tuberculosis strains, two Mycobacterium bovis strains and two M. bovis BCG strains. The sequence appears to be specific for the M. tuberculosis complex. The element carries two frameshift mutations and appears to be defective.

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.

Cloning, sequencing and characterisation of a downstream region of ksdDI operon of Arthrobacter simplex.

An 8-kb region downstream of the ketosteroid dehydrogenase (ksdD)-ketosteroid isomerase (ksdI) genes of Arthrobacter simplex was cloned. The nucleotide sequence of the first 3-kb segment downstream of ksdD-ksdI operon was determined. Three open reading frames (ORFs) preceded by Shine-Dalgarno (SD) sequences have been found. Homology search revealed that the putative product encoded by ORF3 has high level of similarity with the 3-oxosteroid dehydrogenases from A. simplex and P. testosteroni (90% identity in their putative active sites). The role of ORF3 product as a FAD-containing dehydrogenase in steroid degradation pathway is discussed.