Optimization and applications of the microbiological survey method MBS

Abstract

Traditional microbiological analyses used in food safety, environmental monitoring and clinical diagnosis, were developed at the end of XIX century. Despite advances in technology and the introduction over the past decade of a plethora of commercial assays and kits for the detection and enumeration of a wide range of microorganisms, traditional culture based methods are still the most used, thanks to their relatively technical and scientific simplicity and their acceptance by regulatory agencies (ISO, FDA, USEPA, EU). These methods are however labor intensive, time consuming and present several shortfalls, such as subjectivity in the interpretation of results, the possible interference of matrices and, above all, the prolonged time (from 2 to 7 days) needed to give definitive results, depending on the ability of the organisms to multiply into visible colonies. These reasons have led to the development and refinement of alternative microbiological methods of analysis. These alternative methods are quicker and easier to perform than the corresponding reference methods; they minimize manipulation, provide results in less time, and reduce costs. They also normally involve some form of automation, and often capture data electronically. In particular alternative microbiological method technologies aim to provide more sensitive, accurate, precise, and reproducible test results when compared with conventional, growth-based methods. Rapid methods generally include immuno-based and DNA-based assays. However the variable reliability, cost, novelty and the need of highly equipped laboratories of some of these methods still limit their adoption. In this context, an alternative method called Micro Biological Survey (MBS) has been developed by “Roma Tre” University in order to meet the need to provide reliable results thus diminishing the time of analysis, facilitating procedures and interpretation of data, increasing the detection sensitivity, limiting costs and allowing analysis also in the absence of a laboratory. The MBS method is a colorimetric system for detection and selective counting of bacteria in food, water and environmental samples. In contrast with traditional methods that measure the capability of cells to grow in discrete colonies, the MBS method measures the catalytic activity of redox enzymes in the main metabolic pathways of bacteria allowing an unequivocal correlation between enzymatic activity and bacterial concentration in the samples. The time required for a color change is inversely related to the logarithm of bacterial concentration in the sample: like an enzymatic reaction, the greater the number of bacteria, the faster the color change. The MBS analysis is performed in disposable ready to use reaction vials that contain the specific reagent for the analysis to perform. Samples can be inoculated without any preliminary treatment. Analyses can be carried out by untrained personnel and anywhere they are necessary, without the need for any instrumentation other than a thermostatic device. The method was initially conceived for food analysis and it has proven to be a reliable system to evaluate microbiological quality and assess rapidly contamination of different food samples. Further studies have demonstrated that the simple analytical procedure, the reduced labor and automation, positively affect also the analytical performance of the MBS method that displays greater reproducibility and repeatability compared to traditional methods. The MBS method represents a promising and attractive alternative both to traditional and alternative methods until now developed. Its features make it useful in many contexts and are the subject of this thesis. In particular my research was aimed to study alternative applications of the MBS method. The first aim was to verify the effectiveness of the MBS method as an alternative method for microbiological analysis of drinking water, with the purpose to develop a new point of use test to assess water quality in developing countries. The second aim was to investigate the possibility to use the MBS method in clinical settings and in particular for the detection of bacteria in urine and a preliminary evaluation of their susceptibility/resistance to antibiotics, with the final goal to develop a new Point of care test (POCT) for diagnosis and management of Urinary Tract Infections (UTIs). In both cases the need to obtain rapid, accurate, reliable results in a simple, errorless approach, was a central issue to facilitate investigations, allow fast critical decisions and corrective actions. In the context of the analysis of potable water I investigated the possibility to apply the MBS method as an alternative method for drinking water analysis demonstrating its sensitivity, specificity, linearity and accuracy. Further experiments were carried to demonstrate that the simple evaluation of total coliforms in 1ml of water samples, instead of 100ml as required by law, could be effective to roughly assess water quality, particularly in developing countries, in the absence of specific facilities and instrumentations. I therefore applied the MBS method on field and in particular in the evaluation of the microbiological quality of water in Douala, Cameroon. Sixty one water points were selected within 20 quarters of the Douala V City council. The water points were divided into dug wells and drilled wells and all of them were used as drinking water sources by the resident population. Data were collected in three different phases in order to study the effects of inter-annual meteorological variation of rainfall and temperature on groundwater characteristics. A strict correlation between the well type and the level of contamination was observed: 70% of the examined dug wells resulted at least once contaminated by total coliforms with a high average contamination; on the other hand, 100% of drilled wells resulted non-contaminated, underlining the safeness of these water sources and the importance of their use in both rural and city areas. The MBS method has therefore proven to be a valid and accurate method to frequently evaluate the microbiological quality of many water sources. Its simple procedure and interpretation of results can be suitable for use in rural areas, by local personnel, operating without a microbiological laboratory. Concerning the use of the MBS method as a Point of care test for diagnosis and management of Urinary Tract Infections the first step was the development and preliminary in vitro validation of new MBS reagents for the detection of bacteria in urine and for the evaluation of their susceptibility/resistance to a panel of antibiotics. These were tested in a preliminary clinical study, performed in collaboration with the “Azienda ospedaliera Sant’Andrea” of Rome, that demonstrated the great potential of the MBS POCT as a diagnostic tool for a rapid and accurate detection of bacteria causing UTIs. A comparative analysis between the results obtained with the MBS method and results of urine cultures, conducted by the hospital laboratory, was performed using the Receiver Operating Characteristics (ROC) analysis. Results demonstrated that the MBS POCT was able to reveal the presence of a significant bacterial load in urine, hence diagnose a clinical UTI, in only 5 hours (Area Under the Curve=0,93). More importantly, the MBS POCT showed much higher accuracy (90.2%), sensitivity (91.2%) and specificity (89,8%) compared to urine dipsticks, widely used for a presumptive diagnosis of UTI. In a relatively short time compared to standard methods, the MBS method was able to give an accurate indication of UTI and a preliminary evaluation of the antibiotic susceptibility of the infecting bacteria, ensuring a prompt diagnosis and guiding the antibiotic choice long before the conventional antibiotic susceptibility test is performed. Different issues linked to the specific composition of the reagent, the operating procedures and the manufacturing process were also optimized in order to further improve the overall performance of the MBS POCT and meet the essential premarketing requirements mandatory for all in vitro diagnostic devices. Together the results obtained demonstrate that the MBS method is an effective, reliable and precise alternative to traditional microbiological testing, proven in laboratory settings and on field trials. It has also been demonstrated that the solid principles of the MBS method make it extremely flexible and adaptable to completely different needs, allowing simplified, rapid, reliable solutions and achieving health-effective and productive results.