Innovative strategies to detect antibiotic resistance in food sector

Antibiotic resistance is a rapidly growing global issue that poses significant threats to animal and human health. This Ph.D. thesis aimed to determine the presence of antibiotic-resistant bacteria in raw food materials and to develop a rapid and low-cost method for detecting antibiotic resistance genes. The research focuses on two key bacterial species, Staphylococcus aureus and Escherichia coli and addresses the same topics in parallel for both microorganisms, as major contributors to antibiotic resistance, selected due to their presence in hospitals and agri-food chain. The bacteria were isolated from raw cow milk, raw pork and beef meat that can act as vehicles for transmission of resistant bacteria to humans. Currently, the most widely used method for identifying antibiotic resistance is the gold standard antibiotic susceptibility test which involves isolation of the microorganism and exposure to specific antibiotics. Based on this technique, S. aureus isolates showed high levels of resistance to ampicillin, cefoxitin and tetracycline, while E. coli isolates to ampicillin. Since the bacterial cell surface is the primary interface with the surrounding environment and human host cells, and alterations in surface properties can impact both antibiotic susceptibility and virulence, the relation between antibiotic resistance in S. aureus isolates, bacterial cell surface characteristics and the interaction with human Caco-2 intestinal epithelial cell line were investigated. Multi-drug-resistant S. aureus did not show toxicity against Caco-2, but modified their surface, such as reducing permeability to block antibiotic entry or altering their surface charge. These findings underscore the need for further research to fully understand the risks associated with foodborne S. aureus, particularly its capability to evade antibiotic treatment and adapt its surface properties. Although effective, these methods required several days to provide results and were costly. To improve the detection of resistant genes, molecular techniques were employed to identify the most prevalent genes responsible for methicillin-resistance (mecA gene), tetracycline-resistance (tetK gene) and β-lactam-resistance (blaTEM gene). PCR and multiplex PCR were applied to isolates’ DNAs for the detection of target genes providing results within a few hours. These methods require specialized laboratories and expert operators and often the time before obtaining results is still high, in fact, the first hours from the infection are the critical ones for identifying the presence of resistance and choosing the appropriate therapy. Biosensors offered a promising solution to reduce costs and speed up the analysis. These innovative, small-size devices combine a recognition element for detecting the target with an electronic component for signal transduction. Biosensors are versatile, compact, fast, easy to use and ideal for self-testing systems and in situ applications, providing a faster and more accessible approach to detect antibiotic resistance. In this project, DNA sequences of mecA, tetK and blaTEM genes were used as templates to design single-stranded DNA probes, which worked as the recognition element for the biosensor. The probes were evaluated in silico using AmplifX, OligoAnalyzer and BLAST (Basic Local Alignment Search Tool) programs for their specificity further confirmed through dot blot testing. These newly designed probes were successfully applied in an electrochemical genosensor, allowing the detection of mecA, tetK and blaTEM genes in 20 minutes on the isolates’ DNAs. Optimization of both dot blot and biosensor protocols was crucial for improving their practical application, in fact, lastly, these methods were directly applied to the whole DNA extracted from food matrices with promising results on their effectiveness.