Persistent Listeria monocytogenes in fermented meat sausage production facilities in Portugal represent diverse geno- and phenotypes

Listeria monocytogenes is the causative agent of listeriosis, a foodborne disease responsible for life-threatening infections, and a great concern for the food industry. Persistence of Listeria monocytogenes in food-associated environments represents a key factor responsible for contamination of foods with this pathogen. While it has been suggested that persistent strains are better adapted and possess specific characteristics that influences survival to environmental stresses of the processing facilities, up till now, the reason why strains persist in food processing plants haven’t been disclosed.
During the period between 2003 and 2007, a total of 1723 L. monocytogenes isolates were recovered from samples of fermented meat sausages produced in the northern region of Portugal, representing 11 different processors were characterized by different phenotypic and molecular subtyping methods, namely by Random Amplified Polymorphic DNA (RAPD), PCR-based molecular serotyping and epidemic clone characterization, and cadmium, arsenic, and tetracycline resistance typing. Use of multiple subtyping methods revealed considerable subtype diversity, including presence of epidemic clone I and II, among L. monocytogenes isolates from Portuguese fermented meat sausages.
To allow for further characterization of representative isolates, results obtained with the four typing techniques were combined to generate a single typing profile for each isolate from a given processor. 240 representative isolates were further characterized by pulsed-field gel electrophoresis (PFGE) typing. Among a total of 32 L. monocytogenes PFGE types identified, seven PFGE types were found in isolates from more than one plant, which could be explained by the geographic proximity of the studied plants (which were all located in two cities approx. 40 km apart) or by possible common sources of raw materials. PFGE data also indicated persistence of specific L. monocytogenes strains in 7 different processing facilities, with time of persistence ranging from at least 14 to at least 32 months.
A total of 50 L. monocytogenes were characterized for invasion efficiency in Caco-2 cells; these isolates were selected to include one isolate for each PFGE profile obtained from a given sample. The average invasion efficiencies for these isolates ranged from 0.49 to 402.64%. These reduced invasion phenotypes appear to be due to different mechanisms, including (i) different premature stop codon (PMSC) mutations in inlA, which encodes a protein (internalin A) that promotes L. monocytogenes internalization into human epithelial cells, thus playing an important role in L. monocytogenes virulence, (ii) reduced transcription of inlA, and (iii) reduced swarming such as the cell line Caco-2.
In this study we aimed to study the possible relation between lysogeny and persistence of L. monocytogenes as temperate phages have been recognized as important agents of horizontal gene transfer, playing an important role in bacterial evolution. Among 41 isolates representing sporadic and persistent PFGE types, 22 isolates represented lysogens. Neither strains with reduced invasion nor lysogens were overrepresented found among persistent isolates. While susceptibility of isolates to phages also did not correlate with persistence, it appeared to be associated with molecular serotype. Our data show that (i) RAPD may not be suitable for analysis of large L. monocytogenes isolate sets, (ii) while a large diversity of L. monocytogenes subtypes is found in Portuguese fermented meat sausages, persistence of L. monocytogenes in this food chain is common, and (iii) persistent L. monocytogenes are diverse and do not appear to be characterised by unique genetic or phenotypic characteristics.