Polymorphism of gene cassette promoter variants of class 1 integron harbored in S. Choleraesuis and Typhimurium isolated from Taiwan.

Integrons, mobile genetic units, capture and incorporate antibiotic resistance gene cassette by site-specific recombination. Class 1 integrons are widespread and associated with dispersion of antibiotic resistance among Gram-negative bacteria. The expression of gene cassette in Class 1 can vary, based on the Pc promoter but seldom from another promoter hiding downstream of Pc, called P2. To probe distribution and prevalence of gene cassette promoter variants, we analyzed 169 S. Choleraesuis and 191 S. Typhimurium isolates from humans and animals, finding 95.27% occurrence of integrin among S. Choleraesuis, 83.25% among S. Typhimurium. PCR-RFLP analysis identified four promoters (PcS+P2, PcWTGN-10+P2, PcH1+P2, and PcWTGN-10+P2-GGG) in said integron-positive isolates; major types in S. Choleraesuis and S. Typhimurium were PcS+P2 and PcWTGN-10+P2, respectively. Likewise, ß-galactosidase assay rated promoter strength of variants by transcriptional fusion constructs to show extended -10 promoter (TGn/-10 promoter) in Pc and three-nucleotide insertion (GGG) between -35 and -10 region of P2 improving promoter strength of gene cassette.

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria.

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10(4) colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP(i) and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP(i)-recycling loop was completed using ATP sulfurylase and adenosine 5' phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.

A comparative study of the interaction of Bartonella henselae strains with human endothelial cells.

Bartonella henselae can cause a wide range of clinical outcomes and may lead to severe disease, especially in patients with acquired immunodeficiency syndrome. It is well-known that B. henselae-induced cell proliferation is mediated by anti-apoptotic activity; however, the detailed mechanism is still unclear. In this study, the cellular responses of endothelial cells after infection with four B. henselae strains were compared and protein candidates that may be involved in the interaction between cells and bacteria were determined. The Houston-1 strain elicited the fastest response in terms of stimulating endothelial cell proliferation, and the JK-40 strain had the strongest ability to induce cell proliferation. By Western blot analysis, it was demonstrated that B. henselae-induced cell proliferation involved the mitochondria intrinsic apoptotic pathway. In addition, the adhesion abilities of the U-4 and JK-40 strains were much greater than those of the Houston-1 and JK-47 strains; however, the ability of Houston-1 to invade host cells was high. By two-dimensional gel electrophoresis analysis, it was found that succinyl-CoA synthetase subunit beta, phage-related protein, and ATP synthase subunit alpha might be involved in the invasion process. The expression of superoxide dismutase [Cu-Zn] precursor increased with infection time for all four strains but was significantly higher in the Houston-1 strain, which may increase the competitive advantage of Houston-1 in terms of survival in host cells and render it successful in invading host cells and stimulating cell proliferation. Our data suggest that the interaction of B. henselae and endothelial cells differed between strains, and the results indicated possible candidate proteins that may play a role in the pathogenesis of B. henselae infection.