Effect of chlorine on cultivability of Shiga toxin producing Escherichia coli (STEC) and ß-lactamase genes carrying E. coli and Pseudomonas aeruginosa.

The worldwide spread of toxin-producing and multi-drug resistant bacteria in water, food and the environment is considered a major threat to human health. Drinking water quality is controlled by inspection of fecal indicators presence whereby viable contaminants will be efficiently reduced by chlorination which is a common process for disinfection. However, the all-out efficiency is arguable, because bacterial regrowth has been documented after disinfection. In this study, we investigated the stability of Shiga toxin producing Escherichia coli (STEC) and ß-lactamase expressing E. coli and Pseudomonas aeruginosa isolates, both equipped with multiple or single ß-lactamase resistance genes. The aim of the study was to analyze the efficiency of chlorine (Cl2) disinfection against shigatoxigenic or ß-lactamase producing bacteria. Cl2 reacts with the bacterial cells after first contact. Counts of antibiotic resistant E. coli were lower after short than upon extended Cl2 treatment. P. aeruginosa counts decreased moderately upon 15-60 min treatment with 1.2 mg Cl2/l, while cells adapted to tap water were not cultivable anymore. We assume that the bacterial physiology changed to a temporary non-cultivatable state at first Cl2 contact followed by resuscitation of some cells at later stages. STEC viability went down continuously at low Cl2 concentrations and these toxigenic E. coli isolates exhibited slightly increased stability to Cl2 treatment compared with non-toxigenic E. coli. Controlling the efficiency of disinfection, realistic counts of cultivatable cells are achieved after extended Cl2 action.

Development and applications of simultaneous immunochemical staining and serial detection of overlapping proteins in blotting procedures.

Immunoblotting techniques are widely used for specific protein identifications and characterizations. The specific bindings of antibodies to epitopes in a protein sequence permits determination of antigens and gives detailed information about protein compositions and expression levels in complex suspensions. However the techniques are mostly restricted to one specific antibody determination. Overlaying proteins are detected using numerous repeated gel runs. For multiple but specific protein determinations on one immunoblot, here we describe the detection of several antigens by simultaneous incubation of antibodies originated from different species followed by sequential addition of secondary antibodies labelled with horseradish peroxidase (HRP) and binding to analogous primary antibodies. Particular signals were visualized step by step using a HRP chemiluminescence substrate while enzymatic HRP reactions were meanwhile inactivated irreversibly by hydrogen peroxide incubation. We demonstrate flexible applications of multiple antigen detections using the Western blotting technique with determination of the CNS protein markers neuron specific enolase, glial fibrillary acid protein and the physiological prion protein (PrP(C)) in brains and in meats as food contaminations and with glycotyping of PrP(C) using antibodies binding to different epitopes. We showed the use of the dot blotting technique with serial determination of different antigens in complex protein suspensions. The method is easy to handle and is flexible and applicable in the fields of diagnostics and public health for detection of overlaying proteins on one immunoblot.

Immunological one-step determination of the central nervous system indicator proteins, neuron-specific enolase and glial fibrillary acidic protein, in meat products.

The determination of specific marker proteins is important in the prevention of infections and transmission of disease. Several diagnostic assays have been developed but these are mostly restricted to the detection of single antigens. Thus there is a need for multiplex detection assays for the simultaneous analysis of several disease indicators. Consumer protection against the transmission of prion diseases is ensured by the removal of specified risk material from cattle meat during slaughtering and this is regulated by law. The investigation reported here describes a one-step determination on immunoblots of the simultaneous detection of two indicator proteins, neuron-specific enolase and glial fibrillary acidic protein, in tissues of the central nervous system. Although the two proteins run in polyacrylamide gels with similar molecular masses the indicators are differentiated by immunological reactions followed by visualizing the different coloured specific protein bands which develop. The enolase exhibits a brown band, whereas the glial fibrillary acidic protein is red. Immunoblotting has proved to be a suitable assay for multiplex analysis of marker proteins possessing similar molecular weights and is therefore a suitable tool for application in food, veterinary and medical facilities.