| Listeria monocytogenes is a Gram-positive foodborne pathogen and often isolated from food processing industry. Its ability to attach and survive on various food processing surfaces can cause cross-contamination of food products and outbreaks of listeriosis resulting in a deadly outcome. The objective of this work was to identify genes related to the biofilm formation of L. monocytogenes.;Among the 1920 screened mutants, 194 (10.1%) were determined as weak, 1612 (84%) as normal, 106 (5.5%) as strong and eight (0.4%) as hyper-strong biofilm formers, relative to the wild type. Of the selected four mutants, three were weak (12A3, 17F6, 21F10) and one strong (16G3) biofilm formers. In 12A3 and 17F6, Tn917 was inserted in different sites of the same gene, lmo1598 (highly similar to the tyrosyl-tRNA synthetase, tyrS). The interrupted site of Tn917 in 21F10 resided in lmo1528, an ORF of unknown function, located between two protein-export membrane proteins SecDF and SecYajC. In mutant 16G3, the site of Tn917 is inserted in a tetracycline resistance protein (lmo0839). Significant differences in growth rates were found between the four selected mutants and Lm568 at all three temperatures. All mutants and Lm568 formed biofilm at 20°C and 37°C but not at 4°C. Significantly higher populations of biofilm cells (p<0.05) were observed at 20°C compared to at 37°C, except for mutant 16G3. Mutant 21F10 was not motile and produced irregular shaped colonies at 20°C.;In conclusion, the results presented in this thesis will help us to understand more about the genetic mechanisms of biofilm formation in L. monocytogenes and lead to the determination of new strategies for elimination of L. monocytogenes and listeriosis.;A library of Tn917 transposon mutants in a L. monocytogenes 568 (Lm568, serotype 1/2a, food isolate) background was obtained from Agriculture and Agri-Food Canada (Kentville, Nova Scotia), and a total of 1920 mutants were screened for their biofilm formation abilities on stainless steel coupons under static conditions following repeated transfers in BHI for six days at 37°C. Coupons were stained with 0.01% (w/v) acridine orange and examined under an ultraviolet transilluminator. The mutants were grouped into four biofilm phenotypes (weak, normal, strong and hyper-strong) according to the intensity of the observed fluorescence as compared to the parent strain Lm568. Southern hybridization followed by inverse PCR and DNA sequencing was used to determine the Tn917 insertion sites in the four mutants with altered biofilm formation. Biofilm formation, growth rate and motility of the four mutants and Lm568 were compared at 4 (ten days), 20 (six days) and 37°C (six days). Analysis of biofilm cells was carried out by spread plating on BHI agar (30°C, two days) and LIVE/DEAD fluorescence microscopy. Growth rates were modeled using a modified Gompertz function and motility was determined in semi-soft BHI agar (0.25% w/v). |
