NEW APPLICATIONS OF THE MICRO BIOLOGICAL SURVEY METHOD (MRS)

Abstract

Food and water safety is nowadays an extremely central issue concerning public health. In this respect, European and global inspection bodies have enacted many regulations and guidelines with the aim of detecting contaminants and pathogens possibly found in food matrices and water for human consumption, and set limits of acceptance for different microorganisms and matrices under analysis. Traditional microbiological methods of analysis are still the most popular and widely used, due to their reliability, relative simplicity and international consensus by regulatory agencies. They are in fact considered the “gold standards” in food and water diagnostics and thus overall well accepted. These standardized methods are most of the time classical culture methods that rely on specific media to enumerate and isolate viable target organism while suppressing the indigenous background flora. These methods are sensitive, easily adaptable, and can give both qualitative and quantitative information on the number and the nature of microorganisms present in the sample, but display several limitations. Due to their complex operating protocols, they involve a significant amount of time and labor, as well as a high chance of manual error; the processes are highly operator-dependent, and as a result the bacterial count can vary widely when calculated by different operators and different labs resulting in a subjective interpretation of results. Moreover, the costs associated with these methods can be high, not only considering the labor costs, but also the cost of materials necessary to conduct the testing. Most importantly the long time required for obtaining results, which can take up to several days, and the need for equipped laboratories make these methods unpractical in several situations. For these reasons, my PhD project focused on the improvement of the Micro Biological Survey Method (MBS), an original colorimetric method for microbiological analysis. This method measures the catalytic activity of redox enzymes of the main metabolic pathways of bacteria, allowing an unequivocal correlation between the observed enzymatic activity and the number of viable cells present in the samples. This correlation is obtained using ready-to-use disposable analytical vials filled with original reagents for selective counting of microorganisms. Analysis can be automated using a device, called Multi Reader (MR), which can perform simultaneously 8 independent analysis, at different temperatures. The MR is controlled by a computer with a specific software which detects the color change of the vials and calculates the number of microorganisms present in the sample. The method can be applied in many fields and significantly reduces the time required to obtain test results comparable to traditional microbiology testing and its simple interface and operation requires a minimal level of training and operator skills. In particular, I focused my attention on different application fields, such as the use of the MBS method for the quality control of food, to implement the appliance of a self-control plan in the industrial-scale food system using the MBS method, in order to prevent the lack of food quality; its application for the quality control of water, in order to increase water microbiological monitoring of the new and already existing water sources, the optimization of media already used by the MBS method, to make them always faster according to the characteristics of the method, and solve any problem different matrixes could represent towards the method, and really new and original applications of the MBS method in other fields different from the ones already present, to enlarge the flexibility and applicability of the method. Nowadays, the use of the tropical plant Moringa oleifera as an alternative purification tool is becoming very popular in these countries, so the first aim of my research was to verify if this plant could be effective in water purification using the MBS method, developing a protocol of analysis easily applicable in developing countries. The second aim was the development of an enrichment broth for pathogens detection, in particular for Salmonella spp. and Listeria monocytogenes. These broths, in accordance with MBS principles, allow the analysis on 25 g of sample and a significant analysis time reduction, without negatively affecting reliability of results. These characteristics make this new procedure more in accordance with the reference standards, but always following the principles of the MBS method. Concerning the possible use of Moringa oleifera (MO) as an alternative purification tool, after the study on which part of the plant could have the best antimicrobial activity, the analysis on different bacterial strains, in order to evaluate any differences of its action, and the evaluation of its action varying chemical parameters, such as pH, I conducted in field tests, in order to prove that my laboratory evidences could be confirmed on environmental bacteria. After verifying the validity of the system, it was evaluated the possibility that the protocol used in laboratory could be applied to bacteria commonly present in water. For this reason, different sites of rivers in Lazium region were analyzed, testing both chemical-physical parameters of water and microbiological index of total viable count with the MBS method, in order to measure the quality and safety of water. Microbiological analysis were conducted before and after MO treatment, to monitor the action of the treatment, demonstrating the use of the MBS method as a monitoring tool for water analyses, verifying the purifying action of MO on water samples, although its use could not be suitable for human consumption in developing countries due to the fact that MO significantly modifies the organoleptic characteristics of water, making it undesirable to drink. Concerning the study on an enrichment broth for Salmonella spp. detection, which could be applied to the MBS method in order to allow the analysis on 25 g of sample, making the MBS method more in accordance with reference standards, results obtained from this research demonstrated that the MBS enrichment broth promotes a selective growth of Salmonella spp. towards other bacterial species biochemically related to Salmonella. The effectiveness of this broth was also demonstrated in artificially cross contaminated samples, in order to repeat naturally contaminated food samples. Before the introduction of the enrichment broth, analyses using the specific vials developed by the MBS method lasted about 72 hours. After the enrichment broth introduction, a consistent reduction of maximum time of analysis was recorded in the MBS procedure using the MBS enrichment broth, from 72 hours to 48 hours approximatively. This maximum time frame accounts the enrichment phase and the analysis with the reaction vials. In conclusion, this new procedure allows the analysis on 25 g of sample and a significant analysis time reduction, without negatively affecting reliability of results. These characteristics make this new procedure more in accordance with the reference standards, but always following the principles of the MBS method. At last, the same kind of research was conducted on Listeria spp. detection. In this case, the reagent originally developed by the MBS method for Listeria spp. detection was not sensitive and selective, so it had to be optimized. Results obtained from the research demonstrated that the MBS LY was optimized for quantitative analysis with a decrease of false positive results, and the MBS enrichment broth promotes a selective growth of Listeria monocytogenes towards other competitor bacterial species. A reduction of maximum time of analysis was recorded in the MBS quantitative analyses, from 72 hours to 48 hours approximatively. The inoculum of the sample after the selective enrichment phase in the modified MBS LY vials was not so encouraging in results, prompting to further researches, in order to modify again the MBS LY reagent that could be used to perform qualitative analyses according to the method. In conclusion, the optimization of the reagent already present led to a decrease in false positive results ratio, and a consistent analysis time reduction, and the selective broth formulated allows the analysis on 25 g or ml of sample with a significant selective enrichment of Listeria monocytogenes, generally very difficult to detect following reference standards’ procedures, which is faster than the reference broth. It is however necessary to continue the research to modify again the MBS LY reagent that could be suitable for the analysis including an enrichment step, in accordance to the principles of the method in order to make it also in line with qualitative standards.