Prevalence and molecular diversity of pHS-2 plasmids,marker for arthritogenicity,among clinical Escherichia coli Shigella isolates

Reactive arthritis can occur after numerous bacterial infections, including bacillary dysentery caused by Escherichia coli Shigella. A major risk factor for the disease is the HLA B27 phenotype in the human host. By comparison between plasmid profiles of arthritogenic vs. nonarthritogenic Shigella strains, the pHS-2 plasmid has been previously associated with the arthritogenic capacity of Shigella isolates. However, the prevalence of this plasmid in the various Shigella biotypes and serotypes is largely unknown. On this background, 188 clinical isolates from intestinal disease representing all 46 Shigella serogroups were studied for the presence of the pHS-2 plasmid, using PCR, dot blot and Southern blot techniques and by analysis of restriction fragment length polymorphisms. The pHS-2 plasmid was found in nine of 14 E. coli Flexneri serogroups, in E. coli Dysenteriae 1 and in E. coli Boydii 16. In addition, we show marked variability of this plasmid in E. coli Flexneri 3A and 4A strains. Major biological diversity of the pHS-2 plasmid was found to be strictly related to Shigella serogroups. The prevalence pattern of the pHS-2 plasmid matches published data on arthritogenic Shigella isolates, providing additional indirect evidence for the potential validity of this plasmid as a marker for arthritogenicity.     

Mercury resistance among clinical isolates of Escherichia coli

The use of organomercurials in liquid detergents and disinfectants promoted resistance to mercury among bacteria. Dental amalgam and industries using mercury are the main source of human exposure to mercury vapor. Release of mercury from dental amalgam contributes to the enrichment of the intestinal flora with mercury resistance plasmids which may be associated with antibiotic resistance. The aim of our study was to evaluate the frequency of E. coli strains resistant to mercury and other antimicrobial agents currently used in therapy. The bacterial mercury and ampicillin, cephalexin, cefotaxime, gentamicin, tetracycline and chloramphenicol resistance was tested against 363 E. coli strains obtained from faeces and urine between 1999-2000. According to the guidelines suggested by NCCLS (1998), minimum inhibitory concentrations (MICs) were determined on Mueller-Hinton agar, using the dilution technique with an inoculum of about 10(5) CFU. The MICs were read after 18 h incubation at 37 degrees C as the lowest concentration that inhibited the development of visible growth. Plasmids in enterobacteria may carry genes encoding resistance to both mercury and antibiotics. Among the tested E. coli strains, mercury resistance rose to 29.2%. Mercury resistance in E. coli is significantly linked to multiresistance to antimicrobial agents. Between 91.5-23.6 of mercury chloride resistant isolates were also resistant to the tested antibiotics. The increased use of non antibiotic antimicrobial agents is a possible selection factor for antibiotic-resistant strains in clinical and domestic environments.     

Unexpected fluctuations of trace element levels in cell culture medium in vitro: caveat emptor

Copper is an essential trace element micronutrient in human and animal nutrition. Trace amounts present even in ultrapure water, serum and other cell culture medium components are sufficient to support the health requirements of most cell types in culture. Analysis of a variety of different types of basal media from a number of different suppliers revealed large fluctuations in the levels of copper, and also of other micronutrients including zinc, iron, selenium and cobalt. Investigations on proliferating Caco-2 cells revealed reductions in growth with increasing copper concentrations within the range seen in the commercial media and changes in expression of apoptosis- and autophagy-related proteins were noted. Even at concentrations of 1 μM CuSO₄ where there was no significant change in cell growth, there was a significant decrease in procaspase-3 expression. These results stress the importance of batch testing of basal media when undertaking trace metal research since the baseline levels may vary. Batch variation of serum is well established but our results suggest that batch variation of the media may also be important.     

Construction,Purification, and Characterization of a Homodimeric Granulocyte Colony-Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) has found widespread clinical application, and modified forms with improved biopharmaceutical properties have been marketed as well. PEGylation, the covalent modification of G-CSF with polyethylene glycol (PEG), has a beneficial effect on drug properties, but there are concerns connected to the immunogenicity of PEGylated compounds and bioaccumulation of the synthetic polymer. To overcome challenges connected with chemical modifications, we developed fusion proteins composed of two G-CSF molecules connected via different peptide linkers. Three different homodimeric G-CSF proteins were purified, and their in vitro and in vivo activities were determined. A G-CSF dimer, GCSF-Lα, was constructed using an alpha-helix-forming peptide linker, and it demonstrated an extended half-life in serum with a stronger neutrophil response as compared to the monomeric G-CSF protein. The GCSF-Lα protein, therefore, might be selected for further studies as a potential drug candidate.