Growth-inhibitory activity of natural and synthetic isothiocyanates against representative human microbial pathogens.

AIMS: The aim of this study was to test the growth inhibition activity of isothiocyanates (ITCs), defence compounds of plants, against common human microbial pathogens. METHODS AND RESULTS: In this study, we have tested the growth-inhibitory activity of a diverse collection of new and previously known representative ITCs of various structural classes against pathogenic bacteria, fungi and moulds by a serial dilution method. Generally, the compounds were more active against Gram-positive bacteria and fungi exhibiting species-specific bacteriostatic or bactericidal effect. The most active compounds inhibited the growth of both drug-susceptible and multi-drug-resistant (MDR) pathogens at micromolar concentrations. In the case of Mycobacterium tuberculosis, some compounds were more active against MDR, rather than against susceptible strains. The average antimicrobial activity for some of the new derivatives was significantly higher than that previously reported for the most active ITC compounds. The structure-activity relationship (SAR) established for various classes of ITC with Bacillus cereus (model organism for B. anthracis) followed a distinct pattern, thereby enabling prediction of new more efficient inhibitors. Remarkably, tested bacteria failed to develop resistance to ITC. While effectively inhibiting microbial growth, ITCs displayed moderate toxicity towards eukaryotic cells. CONCLUSIONS: High antimicrobial activity coupled with moderate toxicity grants further thorough studies of the ITC compounds aimed at elucidation of their cellular targets and inhibitory mechanism. SIGNIFICANCE AND IMPACT OF THE STUDY: This systematic study identified new ITC compounds highly active against common human microbial pathogens at the concentrations comparable with those for currently used antimicrobial drugs (e.g. rifampicin and fluconazole). Tested representative pathogens do not develop resistance to the inhibitors. These properties justify further evaluation of ITC compounds as potential antimicrobial agents for medicinal use and for industrial applications.

[Toxocariasis in the Republic of Altai. Geoinformation mapping simulation].

Toxocariasis is one of the most important zooanthroponotic natural-focal parasitic diseases in the Republic of Altai. The prevalence of their invasion among the inhabitants of the Republic has increased by more than 7 times. The data of the authors' observations ofToxocara infection in animals (cats, dogs), soil contamination with helminth eggs, and prevalence of human toxocariasis in the Republic of Altai, by considering the results of tests for antibodies against its pathogen in the inhabitants of the region, were automatically processed using geoinformation mapping simulation, which yielded a mapping model to rank the region's area by morbidity rates. The use of up-to-date computers and geo information systems makes it possible to systematize information on this invasion and to see major foci of the disease to reveal the reasons for their assignment to the specific type of the region's landscape.

[Analysis of chromosome deletions of TbDl, RD6 and pks15/1 in clinical strains of Mycobacterium tuberculosis].

Deletions are very important sources of the variability among members of the mycobacterial tuberculosis complex (MTC). Deletion analysis of MTC clinical isolates was performed to clarify phylogenetic relationships and help to identify epidemiologically significant groups of the MTC. In this study, the variability of the TbDl, RD6 and pks15/1 chromosome loci in clinical MTC strains and comparison of those results with IS6110-RFLP (restriction fragment length polymorphism), sSNP (synonymous single nucleotide polymorphism), PGG (Principal Genetic Group) typing data were used to determine if these chromosome regions constitute good molecular markers for some of the epidemiologically important groups of the MTC. In the present study, 122, 61 and 294 clinical isolates were tested for the TbDl, RD6 and pks15/1 deletions, respectively. Specific probes were designed and used in RFLP analysis as well as sequencing techniques were applied. We found that all strains with intact TbDl region belonged to the sSNP cluster I, PGG 1 (katG463Leu and gyrA95Thr). The RD6 deletion was not determined to be a strict characteristic feature of any specific genetic group of the tested M.tb strains, but presence of this deletion is presumed for strains of high virulence, and associated with principal genetic groups 2 or 3. The genetic event that led to this deletion likely occurred in the strain that belongs to PGG 1. Identification of strains with an intact pksl5/1 gene cluster provided a potential marker for virulence. An intact pks15/1 gene cluster is required for the biosynthesis of the phenolic glycolipids (PGL-tb), production of which by clinical isolates was correlated with virulence.

Genetic analysis of mycobacterium tuberculosis strains isolated in Ural region, Russian Federation, by MIRU-VNTR genotyping.

SETTING: The Ural region in Russia is one of the areas most affected by a high incidence of tuberculosis (TB). Molecular epidemiological studies able to trace Mycobacterium tuberculosis transmission are of particular significance. OBJECTIVE: To characterize the population of M. tuberculosis strains circulating in the Ural region, to detect the predominant genotypes and to evaluate their phylogenetic relationship and epidemiological significance. DESIGN: Ninety-two M. tuberculosis clinical samples originating from the Ural region were genotyped using the MIRU-VNTR method. RESULTS: Two major phylogenetically distinct groups of isolates were identified: the W-Beijing family (54.3%) and a previously unreported cluster, named the Ural group (15.2%). Forty-seven different MIRU profiles were identified, including 38 unique (41.3%) and 54 isolates grouped into nine clusters (from 2 to 28 isolates in each cluster). Genetic diversity within the clusters was shown by additional sub-typing of M. tuberculosis isolates in nine additional QUB-VNTR loci. CONCLUSION: W-Beijing family isolates are associated with multiresistance to anti-tuberculosis drugs. It is possible that the strains of this family play a significant role in the spread of multidrug-resistant TB over the Ural region.

Immunological characterization of novel secreted antigens of Mycobacterium tuberculosis.

Proteins secreted by Mycobacterium tuberculosis are targets of host immune responses and as such are investigated for vaccine and immunodiagnostics development. Computer-driven searches of the M. tuberculosis H37Rv genome had previously identified 45 novel secreted proteins. Here, we report the characterization of these antigens in terms of specificity for the M. tuberculosis complex and the ability to induce human immune responses. BLAST homology searches and Southern hybridization identified 10 genes that were either specific for the M. tuberculosis complex or found in only two nontuberculous mycobacterial species of minor medical significance. Selected recombinant proteins were purified from Escherichia coli cells and tested for the ability to elicit antibody responses in tuberculosis patients. Reactivity of the serum panel was ' 36% with at least one of five novel proteins (Rv0203, Rv0603, Rv1271c, Rv1804c and Rv2253), 56% with the 38 kDa lipoprotein, a M. tuberculosis antigen known to be highly seroreactive, and 68% with a combination of Rv0203, Rv1271c and the 38 kDa antigen. Thus, at least five novel secreted proteins induce antibody responses during active disease; some of these proteins may increase the sensitivity of serological assays based on the 38 kDa antigen.

Molecular epidemiology of tuberculosis in Prague and South Moravia, Czech Republic: genetic analysis of Mycobacterium tuberculosis isolates by IS6110-RFLP fingerprinting and spoligotyping.

OBJECTIVES: To genetically characterize and compare Mycobacterium tuberculosis isolates among culture-confirmed TB cases in two regions in the Czech Republic in 1998. METHODS: Consecutive M. tuberculosis isolates from 111 TB patients in Prague and 120 patients in the South Moravia region were genotyped using the standardized IS6110 Southern blot hybridization method and by spoligotyping. RESULTS: Eighty of the Prague patients (72.1%) had isolates with unique RFLP patterns, while 31 (27.9%) had isolates which belonged to 10 clusters. Seventy-eight (64.7%) of the South Moravia strains displayed unique RFLP pattern and 42 (35.3%) were assigned into 15 clusters. The spoligotype profiles previously identified in the U.S. were found in 69 (33%) samples and newly identified Czech spoligotypes in 24 (11.4%) of the total number of examined strains. CONCLUSIONS: The present population-based molecular epidemiological study performed in two regions of the Czech Republic in 1998 demonstrated the distribution of individual genotypes as well as clustered strains of M. tuberculosis isolated from TB patients, and confirmed the similarity between the Czech strain collection and the European Community TB Database, that includes countries with low TB rate. The sporadic import of TB cases from foreign countries and recent transmission events probably do not play significant roles in the epidemiological situation in the Czech Republic.

[Estimation of the efficiency of ERIC-PCR typing of Mycobacterium tuberculosis for evaluation of the prevalence of antibiotic-resistant strains]. / Otsenka éffektivnosti ERIC-PCR-tipirovaniia Mycobacterium tuberculosis pri izuchenii rasprostraneniia ustoichivykh k antibiotikam shtammov.

Twenty-four antibiotic-resistant and sensitive strains of M. tuberculosis isolated from different territories of the Irkutsk region (East Siberia) were studied using PCR genotyping by enterobacterial repetitive intergeneric consensus (ERIC). Evolution relationships are illustrated by phylogenetic trees as a result of analysis by UPGMA and ML approaches. It was found that the studied samples belonged to two genetically different groups, both of which included sensitive and resistant strains. The sensitivity of the method was calculated by the Hunter-Gaston index. Based on these data, a probable pattern of emergence and propagation of antibiotic-resistant forms of tuberculosis in the studied region is discussed.

Multiplex PCR assay to aid in the identification of the highly transmissible Mycobacterium tuberculosis strain CDC1551.

SETTING: Mycobacterium tuberculosis strain CDC1551 outbreak area in Tennessee and Kentucky and selected locations in the USA. OBJECTIVE: Develop a PCR assay to distinguish the highly transmissible CDC1551 from strains which have similar 4-band IS6110 fingerprints. DESIGN: Compare the IS6110 insertion sites in CDC1551 with those in 10 isolates which have similar 4-band IS6110 fingerprints. Utilize unique characteristics of insertion sites in CDC1551 to design a multiplex PCR to identify this strain. RESULTS: A multiplex PCR was developed which targets an IS6110 insertion conserved in most IS6110 low copy number strains and a deletion within the direct repeat region adjacent to an IS6110 insertion. Of 139 isolates with similar 4-band fingerprints, the CDC1551 PCR pattern was generated by only the 14 outbreak associated isolates. Of 154 isolates with different fingerprints, only four generated the CDC1551 pattern and these could be distinguished from CDC1551 by their IS6110 fingerprint. CONCLUSIONS: The multiplex PCR used in conjunction with the IS6110 fingerprint should be a useful tool to aid in the continued surveillance of the outbreak area and follow the spread of this highly transmissible strain of M. tuberculosis.

The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus.

Tst, the gene for toxic shock syndrome toxin-1 (TSST-1), is part of a 15.2 kb genetic element in Staphylococcus aureus that is absent in TSST-1-negative strains. The prototype, in RN4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a Dichelobacter nodosus VapE homologue and a putative integrase. It is readily transferred to a recA recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same orientation. It is excised and circularized by staphylococcal phages phi13 and 80alpha and replicates during the growth of the latter, which transduces it at very high frequency. Because of its site and orientation specificity and because it lacks other identifiable phage-like genes, we consider it to be a pathogenicity island (PI) rather than a transposon or a defective phage. The tst element in RN4282, near tyrB, is designated SaPI1. That in RN3984 in the trp region is only partially homologous to SaPI1 and is excised by phage 80 but not by 80alpha. It is designated SaPI2. These PIs are the first in any gram-positive species and the first for which mobility has been demonstrated. Their mobility may be responsible for the spread of TSST-1 production among S. aureus strains.

Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region.

SETTING: Five IS6110 chromosomal insertion sites were characterized in the multidrug resistant Mycobacterium tuberculosis 'W' strain. OBJECTIVE: To use insertion site probes to study the phylogenetic distribution of IS6110 in the M. tuberculosis genome. DESIGN: A total of 722 M. tuberculosis isolates, previously genotyped using the standard IS6110 Southern blot hybridization methodology, were re-hybridized with the Region A insertion site probe and representative strains were further hybridized with the Region B and C probes. Strains were grouped on the basis of having IS6110 insertions in these different regions and their relatedness was further compared by sequencing the IS6110 insertion sites. RESULTS: The insertion site probes revealed that the collection of Chinese isolates previously grouped as the Beijing strain family shared IS6110 insertions in common with the W and other genotypic group 1 strains. Unexpectedly, we found that IS6110 integrated at least 10 independent times between the dnaA and dnaN genes encoding deoxyribonucleic acid replication proteins. CONCLUSIONS: IS6110 insertion site mapping is able to identify genetic relatedness among a collection of M. tuberculosis clinical strains representing the breadth of species diversity. The mapping data indicate that IS6110 insertion sites are not always random.

[Plasmid genes participating in the synthesis of microcin C51]. / Plazmidnye geny, uchastvuiushchie v sinteze mikrotsina C51.

Microcin C51 is an antibiotic with a wide application range produced by Escherichia coli cells. Using insertions of transposon Tn5 a set of mutations was induced in the recombinant plasmid pAST, which determines synthesis of microcin C51. The mutations were physically mapped. Complementation analysis was performed for insertions and deletions in the Mic+ plasmids pAST and pUHAB that lead to the absence of microcin or immunity to it. The analysis showed that at least three plasmid genes take part in microcin production and two genes determine immunity of producer cells to microcin. Functioning of the ompR gene product was necessary for synthesis of microcin C51.

Cloning and mapping of the genetic determinants for microcin C51 production and immunity.

Microcin C51 is a small peptide antibiotic produced by Escherichia coli cells harbouring the 38 kb low copy number plasmid pC51, which codes for microcin production and immunity. The genetic determinants for microcin synthesis and immunity were cloned into the vectors pBR325, pUC19 and pACYC184. Physical and phenotypic analysis of deletion derivatives and mutant plasmids bearing insertions of transposon Tn5 showed that a DNA fragment of about 5 kb is required for microcin C51 synthesis and expression of complete immunity to microcin. Partial immunity can be provided by a 2 kb DNA fragment. Mutant plasmids were tested for their ability to complement Mic- mutations. Results of these experiments indicate that at least three plasmid genes are required for microcin production. The host OmpR function is also necessary for microcin C51 synthesis.

Cloning and expression in Escherichia coli of Thermotoga neapolitana genes coding for enzymes of carbohydrate substrate degradation.

A genomic library of thermophilic anaerobic eubacterium Thermatoga neapolitana was constructed in E. coli using the pTZ19R plasmid vector. Some groups of recombinant clones with different cellulase activities were revealed: clones carrying genes for an 1,4-beta-glucanases, 1,3-beta-glucanases, beta-xylanases, beta-glucosidases and beta-xylosidases. One clone possessing avicelase activity was obtained. Some clones were selected with amylolytic activities toward amylose, amylopectin and pullulan.

[Mapping of plasmid genes responsible for microcin R51 synthesis and immunity to it]. / Kartirovanie plazmidnykh genov, opredeliaiushchikh sintez mikrotsina R51 i immunitet k nemu.

Microcin R51 is plasmid-determined low-molecular-weight peptide antibiotic produced by Escherichia coli. The spectrum of its action includes many different species of gram negative and some gram positive bacteria. Microcinogenic strains are immune to the action of the microcin they synthesize. As shown earlier, genes responsible for MccR51 production and immunity are located in a continuous 11.1 kb DNA fragment. These genes were cloned in pUC19 and pACYC184 plasmid vectors. Deletion derivatives and Tn5 insertion mutant plasmids which determined no microcin synthesis and immunity were obtained. Analysis of clones' phenotypes and physical mapping of mutant plasmids demonstrated that the 5 kb DNA fragment was indispensable for microcin production. The region of about 4.6 kb confers complete immunity of the producing strains, while partial immunity is provided by 1.8-1.9 kb DNA fragment.

[Cloning and expression of the gene for thermostable pullulanase from Clostridium thermohydrosulfuricum in Escherichia coli]. / Klonirovanie i ékspressiia gena termostabil'noi pullulanazy Clostridium thermohydrosulfuricum v kletkakh Escherichia coli.

Using a pUC19-based genomic library of the anaerobic thermophilic bacterium C. thermohydrosulfuricum a DNA fragment that confers pullulanase activity to E. coli cells has been identified. Subcloning and restriction mapping procedures was carried out and the primary structure of the 5'-region of the pullulanase gene (pul) was determined. The pul enzyme was shown to be a protein with molecular weight of approximately 60,000. It was found that both pullulanase and glucoamylase activities resides in pullulanase. The intracellular distribution of pullulanase was studied. An E. coli strain that produces large amounts of thermostable pullulanase has been constructed.

[Microcin R51 and a plasmid determining its synthesis]. / Mikrotsin R51 i plazmida, opredeliaiushchaia ego sintez.

A new microcin produced by an Citrobacter R51 strain has been detected. This antibiotic has been shown to inhibit the growth of a number of Gram-negative as well as Gram-positive strains of bacteria on the minimal medium plates. The properties of partially purified microcin were characterized. Constitutive synthesis of microcin is determined by a conjugative plasmid. The genes of microcin synthesis and immunity were cloned on a plasmid and plasmid vehicles. A physical map of the 12 kb fragment coding for the production of microcin R51 and immunity to this antibiotic is presented.

[The nature of typhimuricin--a substance produced by Salmonella typhimurium LT2 and relation of its synthesis to the cryptic plasmid]. / O prirode tifimuritsina--veshchestva, obrazuemogo Salmonella pyphimurium LT2, i sviazi ego sinteza s kripticheskoi plazmidoi.

The effect of typhimuricin on Escherichia coli K12 cells and the properties of this substance produced by Salmonella typhimurium LT2 have been studied. The obtained data permit one to conclude that typhimuricin is similar to the aminoacid L-valine or the chemical very similar to it in properties. The synthesis of typhimuricin is not controlled by the cryptic plasmid present in Salmonella typhimurium LT2 cells. The experiments aimed at isolation of enterobacterial strains producing low molecular mass antibiotics--microcines have been done. It was found that the capability to produce macrocines is not widespread among the isolates of enterobacteria.

[Microcins: their nature and genetic determination]. / Mikrotsiny: priroda i geneticheskoe determinirovanie.

A number of gram-negative bacteria are capable of synthesizing the low molecular mass antibiotics microcins, the substances with molecular masses up to 10 000 D possessing a broad range of antibiotic activity. The synthesis is not lethal for the producing cells and is not induced by DNA-damaging agents. The investigated microcins are either of oligopeptide nature or are analogues of methionine. Five types of microcins have been described. The A- and C-types of microcins inhibit protein synthesis, microcins B suppress DNA replication, microcins D and F affect cellular energy potential. Microcin synthesis and immunity to microcins are shown to be plasmid mediated. The role of microcins in ecology of enterobacteria and the ranking among bacterial antibiotics are discussed.