Highly Invasive Listeria monocytogenes Strains Have Growth and Invasion Advantages in Strain Competition

Abstract

Multiple Listeria monocytogenes strains can be present in the same food sample; moreover, infection with more than one L. monocytogenes strain can also occur. In this study we investigated the impact of strain competition on the growth and in vitro virulence potential of L. monocytogenes. We identified two strong competitor strains, whose growth was not (or only slightly) influenced by the presence of other strains and two weak competitor strains, which were outcompeted by other strains. Cell contact was essential for growth inhibition. In vitro virulence assays using human intestinal epithelial Caco2 cells showed a correlation between the invasion efficiency and growth inhibition: the strong growth competitor strains showed high invasiveness. Moreover, invasion efficiency of the highly invasive strain was further increased in certain combinations by the presence of a low invasive strain. In all tested combinations, the less invasive strain was outcompeted by the higher invasive strain. Studying the effect of cell contact on in vitro virulence competition revealed a complex pattern in which the observed effects depended only partially on cell-contact suggesting that competition occurs at two different levels: i) during co-cultivation prior to infection, which might influence the expression of virulence factors, and ii) during infection, when bacterial cells compete for the host cell. In conclusion, we show that growth of L. monocytogenes can be inhibited by strains of the same species leading potentially to biased recovery during enrichment procedures. Furthermore, the presence of more than one L. monocytogenes strain in food can lead to increased infection rates due to synergistic effects on the virulence potential.

Listeria monocytogenes Strains Underrepresented during Selective Enrichment with an ISO Method Might Dominate during Passage through Simulated Gastric Fluid and In Vitro Infection of Caco-2 Cells

“…We showed previously that coenrichment of L. monocytogenes strains-also used in this study-following the ISO method might favor the recovery of certain strains, resulting in a biased outcome (24). In addition, we have demonstrated previously that growth competition could occur between L. monocytogenes strains during their cocultivation in TSB-Y at 10°C (23). The 10°C temperature was initially chosen as one at which at we could observe equal levels of growth of all single cultures of L. monocytogenes strains, thus ensuring that the observed inhibition would not be the result of differences in the individual growth potentials of strains under the conditions tested.…”

“…The L. monocytogenes strains used in this study are listed in Table 1. The selection of strains was performed according to two previous studies investigating the growth, virulence, and enrichment competition of L. monocytogenes strains (23,24). Strains selected for resistance to rifampin (Rifambicin; AppliChem) or streptomycin (Streptomycin Sulfate Biochemica; AppliChem) according to the method described by Blackburn and Davies (25) were used for enabling selective enumeration of each strain in coculture.…”

“…Inoculation of TSB-Y was performed as previously described for single or mixed listerial cultures (23). Briefly, the activated 18-h cultures (corresponding to approximately 10 9 CFU/ml) of L. monocytogenes strains were washed with Ringer solution (Lab M; LAB100Z), resuspended in 10 ml TSB-Y, and serially diluted in TSB-Y to obtain a final inoculum of approximately 10 3 CFU/ml.…”

“…Invasion efficiency and intracellular proliferation were assessed for L. monocytogenes strains in Caco-2 cell monolayers, as previously described (23). Briefly, Caco-2 cells were seeded into 24-well tissue culture plates (Greiner Bio-One) in MEM supplemented with 15% (vol/vol) FBS until confluence was reached.…”

“…The growth of single and mixed cultures of L. monocytogenes strains at 10°C was evaluated in our previous study (23). The population of each strain (grown individually or in coculture in TSB-Y) after 2, 4, 6, and 8 days of incubation is given in Table 2 as Log 10 (N0) [where Log 10 (N0) represents the measured population (log CFU/milliliter) of L. monocytogenes strains in TSB-Y and the initial inoculum used for enrichments or exposure to SGF on each day].…”