Characterisation of Escherichia coli isolates from the blood of haematological adult patients with bacteraemia: translocation from gut to blood requires the cooperation of multiple virulence factors.

The aim of the study was to investigate whether there are unique pathotypes of Escherichia coli capable of transmission from the gastrointestinal tract to the vascular bed. The study included E. coli strains isolated from clinical materials collected from 115 patients suffering from haematologic malignancies diagnosed with bacteraemia. The genotyping techniques established that 89 E. coli isolates from the blood had the same genotype as the E. coli from the patient's bowel. The presence of 21 genes encoding virulence factors typical of various E. coli pathotypes and their relationship with the phylogenetic group was established. One-dimensional analysis showed that the focG gene occurred more frequently in the control bowel group, while the ampicillin-resistant afa/dr E. coli were associated with bacteraemia. Blood isolates with the highest occurrence of virulence factors belonged to pathogenic group B2 and non-pathogenic group A. The co-occurrence of multiple genes encoding papC, sfa, usp and cnf1 virulence factors probably predisposes E. coli to translocation from the gastrointestinal tract to the vascular bed in the group of patients with haematologic malignancies. Based on clustering analysis, dominance of the most virulent strains assigned to the cluster with seven virulence factors encoded by the following genes, papC, sfaD/E, cnf1, usp, agn43, hlyA and iutA, was found. The obtained results enforce the previously proposed concept of bowel-blood translocation and further expand our hypothesis by defining the unique virulence characteristics of E. coli isolates, which predispose them to bowel colonisation or translocation and bacteraemia in this group of patients.

A subset of two adherence systems, acute pro-inflammatory pap genes and invasion coding dra, fim, or sfa, increases the risk of Escherichia coli translocation to the bloodstream.

An analysis of the phylogenetic distribution and virulence genes of Escherichia coli isolates which predispose this bacteria to translocate from the urinary tract to the bloodstream is presented. One-dimensional analysis indicated that the occurrence of P fimbriae and α-hemolysin coding genes is more frequent among the E. coli which cause bacteremia. However, a two-dimensional analysis revealed that a combination of genes coding two adherence factors, namely, P + Dr, P + S, S + Dr, S + fim, and hemolysin + one adherence factor, were associated with bacteremia and, therefore, with the risk of translocation to the vascular system. The frequent and previously unrecognized co-existence of pro-inflammatory P fimbriae with the invasion promoting Dr adhesin in the same E. coli isolate may represent high-risk and potentially lethal pathogens.

Leukemia and risk of recurrent Escherichia coli bacteremia: genotyping implicates E. coli translocation from the colon to the bloodstream.

In patients with leukemia, the portal(s) and reasons for the persistence of an Escherichia coli recurrent bacteremia remain unclear. Adult Hematology Clinic (AHC) databases at the State Clinical Hospital in Gdansk were reviewed to evaluate the frequency of E. coli bacteremia between 2002 and 2005. Blood and bowel E. coli strains were obtained and the genetic relatedness of the strains was analyzed. The rate of E. coli bacteremia per 1,000 admissions at the AHC was higher (85.0) than in the other clinics of the hospital (2.9), p < 0.001. A higher mortality was observed in patients with a history of E. coli versus non-E. coli bacteremia [30/95 (31 %) vs. 53/430 (12 %), p < 0.001]; 72.8 % of patients with leukemia had an unknown source of bacteremia. In 2005, 6 out of 25 (24 %) patients with leukemia had ≥2 episodes of E. coli-positive blood cultures. These gastrointestinal E. coli isolates were replaced within 3-8 weeks with a new E. coli H genotype. A recurrent episode of bacteremia was usually caused by an infection with a transient E. coli H genotype identical to that found in the subject's bowel. Consistent with the definition of bowel/blood translocation, the bowel appeared to be a portal for E. coli in these subjects and, hence, a clear source for their recurring bacteremia.

Retrospective analysis of the genetic diversity of Klebsiella oxytoca isolated in Poland over a 50-year period.

Population genetics analyses and determination of the phylogenetic relationships between strains have proven to be extremely useful approaches, enabling deeper insights into the epidemiological pattern of bacterial species. There is no longitudinal data describing the molecular epidemiology of Klebsiella oxytoca strains that are opportunistic pathogens responsible for an increasing number of multi-resistant infections in hospitals. The aim of the present study was to assess the genetic diversity of K. oxytoca strains over a 50-year period using internal transcribed spacer polymerase chain reaction (ITS-PCR) and PCR MP (ang. PCR melting profiles) genotyping methods on a large collection of strains isolated from the patients of several hospitals in Poland. The phylogenetic analysis based on ITS-PCR exhibited six distinct branches. Two main groups, KoX and KoY, with four and two sub-groups within KoX and KoY, respectively, have been identified. Typing by the PCR MP method showed a higher level of genetic diversity. However, all K. oxytoca strains were also divided into six genotype groups (KoA, KoB, KoC, KoD, KoE and KoF). In conclusion, we found that the ITS-PCR and PCR MP methods are useful for the phylogenetic delineation of genetic groups in K. oxytoca.

The effects of nucleoside analogues on promoter methylation of selected tumor suppressor genes in MCF-7 and MDA-MB-231 breast cancer cell lines.

The effects of 2-chloro-2'-deoxyadenosine, 9-beta-D-arabinofuranosyl-2-fluoroadenine, and 5-aza-2'-deoxycytidine on promoter methylation of the selected tumor suppressor genes (i.e., ERalpha, BRCA1, RARbeta2, E-cadherin, PTEN, and APC) were estimated using methylation-sensitive restriction analysis. The studies were carried out in hormone-responsive, low-invasive cell line MCF-7 and hormone-insensitive, highly invasive cell line MDA-MB-231. The results demonstrate an implication of the tested adenosine analogues and 5-aza-dCyd in regulation of DNA methylation process. Moreover, the effects of nucleoside analogues on PTEN promoter methylation suggest distinct mechanism of regulation of the epigenetic DNA modification in low-invasive compared to highly invasive breast cancer cells.

Studies on the methylation status of CpG sequences located in promoters of selected tumour suppressor genes in breast cancer cells.

In the tested samples of sporadic breast cancer (100 cases), hypermethylation of CpG sequences located in ERalpha promoter was observed in 62 cases. It correlated with: (i) deficiency of ERalpha protein in 45%, (ii) hypermethylation of BRCA1 promoter in 95%, and (iii) nonmethylated E-cadherin promoter in 90%. Fifty-eight percent of the patients with nonmethylated E-cadherin promoter (56 cases) did not show metastasis to lymphatic nodes. The analysis of the methylation level of the promoter of ERalpha, BRCA1, and E-cadherin, frequently connected with their activity, shows that it can be an important parameter in the diagnosis and therapeutic strategies in sporadic breast cancer.

Alteration of DNA methylation status in K562 and MCF-7 cancer cell lines by nucleoside analogues.

The effects of 2-chloro-2'-deoxyadenosine, beta-D-arabinofuranosyl-2-fluoroadenine, and 5-aza-2'-deoxycytidine on promoter methylation of the selected tumor suppressor genes (i.e., ERalpha, BRCA1, E-cadherin, PTEN, and APC) were estimated using methylation-sensitive restriction analysis (MSRA) in K562 cells (human erythroleukemic cell line) and MCF-7 cells (human breast cancer cell line). In both cell lines all tested drugs completely reduced methylation of PTEN and APC promoters. The results indicate that the tested nucleoside analogues, which are known inhibitors of DNA synthesis, also are implicated in indirect (or direct in the case of 5-aza-dCyd) regulation of post-replicative DNA modifications (i.e., DNA methylation).

Systematic position of copepods of the genus Achtheres (Crustacea: Copepoda: Siphonostomatoida) parasitizing perch, Perca fluviatilis L., and zander, Sander lucioperca (L.).

Parasitic copepods of the genus Achtheres commonly infect perch, Perca fluviatilis, and zander, Sander lucioperca, in Europe. The aim of this study was to verify the specific identity of these copepods. The parasites were examined morphologically, biometrically and genetically. Statistical processing of the biometrical data was based on both empirical measurements and transformed data related to total length and genital trunk width. Principal component analysis was applied to both sets of data. DNA of both parasite 'forms' was amplified using two sets of primers (EU5.8S+EU18S and 18SF1+28SR) and the products were subjected to restriction fragment length polymorphism (RFLP). Morphological differences were found in the overall shape of the copepod bodies as well as in the details of the armament of some appendages. The morphometric study emphasized the importance of second maxillae and genital process as the variables most distinctly distinguishing the two 'forms'. The two 'forms' of Achtheres differed in the DNA sequence amplified by one set of primers. RFLP revealed even more extensive differences between these two copepods. We concluded that the copepods parasitizing perch should be referred to as Achtheres percarum von Nordmann, 1832, whereas a long-forgotten name, A. sandrae Gadd, 1901, should be applied to the copepods from zander.

Comparative studies of the Acinetobacter genus and the species identification method based on the recA sequences.

The recA gene is indispensable for a maintaining and diversification of the bacterial genetic material. Given its important role in ensuring cell viability, it is not surprising that the RecA protein is both ubiquitous and well conserved among a range of prokaryotes. Previously, we reported Acinetobacter genomic species identification method based on PCR amplification of an internal fragment of the recA gene with subsequent restriction analysis (RFLP) with HinfI and MboI enzymes. In present study, the PCR products containing the internal fragment of the recA gene, for 25 Acinetobacter strains belonging to all genomic species, were sequenced. Based on the nucleotide sequences the restriction maps and phylogenetic tree were prepared. The restriction maps revealed that Tsp509I restriction enzyme is the most discriminating for RFLP. To verify the computer analysis, the amplified DNAs from all reference genomic species available (43 strains) and 34 clinical strains were digested with each of the three restriction endonucleases mentioned. The results of digestion confirmed the computer analysis. The reconstructed phylogenetic tree showed linkages between genomic species 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3, 'between 1 and 3', TU13 and 'close to TU13'; genomic species 4, 6, BJ13, BJ14, BJ15, BJ16 and BJ17; genomic species 7 (Acinetobacter johnsonii) and TU14; genomic species 10 and 11; genomic species 8 (Acinetobacter Iwoffii), 9, 12 (Acinetobacter radioresistens) and TU15; and genomic species 5 (Acinetobacter junii). It is interesting that one branch in the phylogenetic tree contains haemolytic species-genomic species 4 (A. haemolyticus), BJ13, BJ14, BJ15, BJ16 and BJ17. The proposed genotypic method clearly revealed that the RFLP profiles obtained with Tsp509I enzyme might be useful for species identification of Acinetobacter strains. In this context, recA/RFLP genotypic method should be seen as an ideal preliminary screening method for large numbers of isolates, with the ultimate confirmatory role reserved for DNA hybridization analysis.

Thermal reactions of anti- and syn-dispiro[5.0.5.2]tetradeca-1,8-dienes: stereomutation and fragmentation to 3-methylcyclohexenes. Entropy-dictated product ratios from diradical intermediates?

A series of cyclobutanes substituted 1,2- by polyenes of increasing radical-stabilizing power has been investigated to test the proposition that stabilization energies obtained independently from apposite, cis,trans geometric isomerizations can be successfully transferred to another system, in this paper, cyclobutanes. The first member of the series, 3-methylenecyclohexene (1), is photodimerized to anti- and syn-dispiro[5.0.5.2]tetradeca-1,8-dienes (anti-2 and syn-2), which undergo stereomutation (stereochemical interconversion) and cycloreversion (fragmentation) to 1 when heated in the range 72.1-118.2 degrees C: anti-2 --> syn-2, DeltaH() = 30.3 kcal mol(-)(1), DeltaS() = 0.2 cal mol(-)(1) K(-)(1); anti-2 --> 1, DeltaH() = 32.8 kcal mol(-)(1), DeltaS() = +8.0 cal mol(-)(1) K(-)(1). Agreement with an enthalpy of activation predicted by assuming full allylic stabilization in a hypothetical diradical intermediate is good. An example of further activation by a radical-stabilizing group is manifested by the approximately 20 000-fold acceleration in rate shown by the system 1-phenyl-3-methylenecyclohexene (3) and anti- and syn-2,9-diphenyldispiro[5.0.5.2]tetradeca-1,8-dienes (anti-4 and syn-4), measured, however, only at 43.6 degrees C. In both systems 2 and 4, volumes of activation for stereochemical interconversion and cycloreversion have been determined and found to be essentially identical within experimental uncertainties, DeltaV() = +10.2 +/- 1.0 and +12.6 +/- 1.4 cm(3) mol(-)(1), respectively (weighted means). These strongly positive values are consistent with the rate-determining step being the first bond-breaking, while the near identity of the volumes of activation argues against the indispensable second bond-breaking being a determining factor in fragmentation. These results are consistent with the theoretically based construct of Charles Doubleday for the paradigm, cyclobutane, in which the ratio between two channels of exit from a "generalized common biradical" is not controlled by enthalpy and entropy, as in the transition state model, but by entropy alone.

In vitro interaction of the IHF-like proteins Acinetobacter junii and Proteus vulgaris with ihf sites.

The ability of the IHF-like proteins of Acinetobacter junii and Proteus vulgaris to interact with the H' attP and pR' ihf sites of lambda DNA was investigated. IHF from A. junii and P. vulgaris was found to bind the examined ihf sites in a way similar to IHF from Escherichia coli as shown by gel mobility shift DNA binding assays and footprinting analysis. The three IHF proteins bound to the pR' ihf site that overlaps the-35 region of that promoter and in vitro repression of transcription by each IHF was observed. These results confirm that IHF-like proteins from Gram-negative bacteria can recognize the same specific DNA sequences and appear to be important in regulation of transcription.

Purification of IHF-like protein from gram-negative bacteria in one chromatographic step.

We describe a fast and very efficient method of purification which yields highly purified integration host factor-like proteins in one chromatographic step. IHF-like proteins from Acinetobacter junii or Proteus vulgaris are each an alpha beta heterodimer (subunits of 10 and 11 kDa) similar to the IHF of Escherichia coli when analyzed by polyacrylamide gel electrophoresis. The purified IHF are able to bind to the same ihf sites as IHF of E. coli. The results presented confirm that IHF is conserved during evolution in gram-negative bacteria.

Integration host factor, a histone-like Escherichia coli protein, binds to at least four sites of the DNA fragment containing the recA gene.

The integration host factor (IHF) is a sequence-specific, histone-like, multifunctional DNA-binding and -bending protein of Escherichia coli. The characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements. Less is known concerning the role of IHF in E. coli, although it has been implicated in a number of processes in this organism including DNA replication, site-specific recombination, and gene expression. In this paper we report data concerning the binding of IHF protein to the recA gene region. IHF binds to at least four sites of the DNA fragment containing the recA gene, as shown by gel mobility shift assays. On the basis of the ihf consensus sequences published, we have been able to identify two sequences of putative ihf sites (ihf 1 and ihf2) into the 1390 bp long sequence containing the recA gene, but only the ihf2 site was able to bind IHF, as measured by gel mobility shift experiments. The nonfunctional ihf1 sequence was found to overlap the -35 region of the recA promoter and the functional ihf2 sequence was found within the recA gene structure at nt +780 to +807 (both with three mismatches according to the consensus sequence of Kur et al., 1989). This confirms our earlier results that the IHF-DNA interaction does not depend on any very rigid sequence, but also on the suitable sequences of the neighbouring regions, together with the proper DNA conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of climatic conditions on the heat balance of the human body.

The structure of heat exchange between the human body and its surroundings has been studied according to M.I. Budyko's model. Comparative measurements were carried out in the Polish Lakeland (maritime, temperate warm climate), in Central Mongolia (continental, temperate cool climate), and in the Kara Kum desert (dry subtropical climate). The results deal with the summer and early autumn seasons. The calculations indicate that the quantitative apportionment of various forms of heat exchange depend on specific weather conditions, which are typical for the distinguished climatic zones.

Practical aspects of selection of streptomyces mutants.

The methods of industrial strains selection on the basis of some regulatory mechanisms are presented. The selection of the producing S. erythreus mutants exhibiting higher activity for transformation of erythromycin C to erythromycin A is one of the examples for the practical use of the presented method. Some new techniques including isotopic methods are presented.