Strains and media
The clinical isolate, Listeria monocytogenes ScottA, carrying erythromycin resistance, and labeled with either Cyan Fluorescent Protein (CFP) or Yellow Fluorescent Protein (YFP) as previously described  was used in all experiments. Bacteria were cultivated on BHI-agar (Oxoid) or in liquid BHI (Oxoid) buffered with 100 mM 3-(N-Morpholino)-propanesulfonic acid (MOPS), pH = 6. When appropriate, Erythromycin (Sigma) was used at a final concentration of 10 ug/mL and Nalidixic acid at a final concentration of 100 ug/mL.
Preparation of L. monocytogenes for infection studies
A fluorescent single colony of L. monocytogenes was inoculated into 10 mL MOPS buffered BHI media supplemented with Erythromycin and incubated at 37°C for 8 hours. The resulting cultures, for which the optical densies (OD600) ranged between 1,5 and 2, were subsequently diluted between 104 and 105 fold into 2 L Bluecap flasks containing 450 mL MOPS buffered BHI medium supplemented with Erythromycin.
To obtain oxygen-restricted cultures of Listeria monocytogenes ScottA, atmospheric air above the diluted cultures were exchanged with sterile Nitrogen and the lid of the Bluecap flasks was tightened and sealed prior to incubation. To obtain non-restricted cultures, the Bluecap flasks were incubated with the lid loosely tightened allowing free exchange of atmospheric air. All cultures were incubated at 37°C in a rotary shaker set at 200 rpm and samples for in vitro invasion studies and in vivo infection studies were taken after approximately 20 hours of incubation, when they reached the optical densities reported below.
Samples for in vitro invasion studies were taken at OD600 = 0.5 (exponential phase), OD600 = 1.0 (exponential phase), and from cultures that had been in stationary phase for approximately 10 hours (referred to as over night cultures). The samples were diluted directly into 37°C MEM medium (Gibco) to a concentration of 107 bacteria/mL and immediately used for challenge of Caco-2 cells as described below.
Samples for oral challenge of guinea pigs were cultivated until OD600 = 1, and 400 mL culture per group of 6 animals was harvested by centrifugation (5 minutes, 7000 g, 25°C). The pellet was resuspended in 4 mL of double cream (38 % fat, pH 7). Aliqouts of 0.5 mL, containing either Listeria monocultures, or a mixture of two cultures grown at oxygen restricted and unrestricted conditions, respectively, were subsequently administered to guinea pigs as described below. Samples of the inoculums were diluted and spread onto BHI-agar supplemented with Erythromycin to estimate the numbers of Listeria present in the double cream suspensions. Microscopy revealed that the bacteria were present in the hydric phase.
Caco-2 cell infection experiments
Enterocyte-like Caco-2 cells were cultivated and prepared as previously described . Bacteria were cultivated as described above to the desired optical density and diluted in 37°C MEM medium (Gibco) to a concentration of 107 bacteria/mL immediately before the invasion assays. One ml of bacterial culture was applied to each well, resulting in a multiplicity of infection of approximately 25 bacteria per Caco-2 cell. Following 1 hour of invasion and 2 hours of gentamycin treatment to kill extracellularly located bacteria, Caco-2 cells were lysed and the numbers of Listeria present in each well was estimated as described in the section 'Enumeration of L. monocytogenes in samples'. Sampling was done in triplicate, and the experiments were performed twice.
Male and female Hartley guinea pigs (Charles River Laboratories; Germany) with a weight of 275 g (± 10 g) were used. After seven days of acclimatization in pens (custom made, 90 × 130 × 61 cm), animals were randomized and housed individually in Polycarbonate cages, Eurostandard Type III H (425 × 266 × 185 mm) with Tapvei bedding (peeled Aspen hardwood, Tapvei Kaavi, Finland) in negatively pressurized isolators. Fecal samples from the guinea pigs were tested by plating on Palcam agar (Oxoid) to verify the absence of Listeria prior to dosage.
A total of 48 guinea pigs were dosed with L. monocytogenes ScottA. Two groups of 12 animals were dosed with monocultures of CFP-labelled bacteria, cultivated either with or without oxygen-restriction. Two other groups of 12 animals were dosed with a 1:1 mixture of oxygen-restricted and un-restricted bacteria carrying the two different fluorescent labels CFP and YFP. In one of these groups, it were the oxygen-restricted Listeria, which carried the CFP label, while in the other group the labels were reversed so that the unrestricted bacteria were labelled with CFP.
All animals were dosed with 0.5 ml double cream containing 38 % milk fat and approximately 5 × 1010
L. monocytogenes at Day 0 and again at Day 1. The number of cells in the inoculum was approximately the same in the mono- and mixed cultures.
Dosage was done directly in the oral cavity, between the incisors and the molars. Following dosage the animals had access to food and water ad libitum throughout the duration of the study. Fresh fecal samples were collected every day, avoiding bedding and traces of urin from the cages. Six animals from each of the groups were euthanized on Days 4 and 7, respectively, for investigation of intestinal segments, liver and spleen. Samples of jejunal content from the middle part of the small intestine were squeezed out with tweezers, and samples from spleen and liver of approximately 0.6 g were homogenized prior to investigation as described below.
Animal experiments were carried out under the supervision of the Danish National Agency for Protection of Experimental Animals
The recently developed guinea-pig model [BB Roldgaard, JB Andersen, TR Licht and BB Christensen, submitted] is much less stressful to the animals than previously published models , since it involves no intubation, injection or anesthetizing. Additionally, the approach of treating each animal with a mixture of cultures reduces the number animals needed.
Estimation of L. monocytogenes in samples
Samples of lysed Caco-2 cells and homogenized organs were cultivated on BHI-agar supplemented with Erythromycin. Samples from faeces and jejunum were cultivated on BHI-agar supplemented with Erythromycin and Nalidixic acid. After 48–72 hours of incubation at 37°C, BHI plates were placed on a UV table (excitation at 302 nm), and fluorescent colonies (either CFP or YFP) were enumerated.
Statistical analysis was performed using the software package JMP (SAS institute, Copenhagen, Denmark). Pearsons Chi2 test was used to compare the numbers of infected animals after dosage with either oxygen-restricted or un-restricted L. monocytogenes. The same test was used to compare the results obtained with either mono infection or mixed infection, and with either YFP or CFP labelling. A significance level of 0.05 was used in all cases.