Comparison of growth phase on Salmonella enterica serovar Typhimurium invasion in an epithelial cell line (IPEC J2) and mucosal explants from porcine small intestine

Abstract

Salmonella Typhimurium DT104 is a zoonotic enteropathogen of increasing concern for human health. In this study, the influence of growth phase on invasiveness of a S. Typhimurium DT104 field isolate and two reference strains (SL1344 and ATCC 14028) was compared in IPEC J2 cells and mucosal explants from porcine ileum. Internalized bacteria were quantified by a gentamicin resistance assay. After 90 min of exposure to the apical aspect of epithelial monolayers or luminal surface of explants, internalization of all S. Typhimurium strains in mid logarithmic phase of bacterial growth was comparable. Internalization of stationary phase bacteria was reduced relative to log phase bacteria, with DT104 exhibiting the greatest decrease. Growth phase-related differences in S. Typhimurium invasion are similar in porcine intestinal epithelial cells and mucosal explants, but may be greater in multidrug-resistant strains.

1. Introduction

Salmonella Typhimurium accounts for approximately 17% of world-wide intestinal Salmonella infections reported yearly [1]. The emergence of antibiotic-resistant strains of S. Typhimurium has become a major concern for human health and among resistant strains, definitive type 104 (DT104) is the most common Salmonella phage-type isolated from both humans and animals, including swine [2]. Salmonellae possess several crucial virulence factors that have been mapped to Salmonella pathogenicity islands (SPI). Gene products encoded in SPI-1 form components of the type III secretion system that is required for early Salmonella invasion of the intestinal epithelium [3]. The expression of SPI-1 genes such as hilA, prgH and sipA is dependent upon the phase of bacterial growth [4–6].

In the present investigation, the role of growth phase in the invasiveness of S. Typhimurium DT104 was compared to two S. Typhimurium reference strains in IPEC J2 epithelial cells as well as in isolated sheets of porcine ileal mucosa. Past studies have utilized experimental models for determining Salmonella invasion that have ranged from cultured cells to whole animal models. However, most cell lines used to study intestinal invasion have not been derived from the gastrointestinal tract. IPEC J2 epithelial cells are derived from the swine small intestine and have been used in previous studies of epithelial interactions with salmonellae [7, 8]. Mucosal explants from the porcine ileum, which were mounted in Ussing chambers, have been employed previously to investigate interactions between enteric pathogens and the intestinal epithelium [9–11]. Both mucosal explants and intestinal epithelial cell cultures from the porcine intestine offer technical advantages in investigations of Salmonella invasion in swine, but the results obtained using these models have not been hitherto compared.

2. Materials and Methods

Salmonella enterica serovar Typhimurium (S. Typhimurium) reference strains SL1344 [12] and ATCC 14028 were obtained from Dr. Richard Isaacson (Univ. of Minnesota, Dept. of Veterinary and Biomedical Sciences, St. Paul, MN). A field isolate of Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen (a strain resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline, i.e. R-type ACSSuT; Minnesota Dept. of Health isolate number #E02–000392) was kindly provided by Dr. Jeffrey Bender (Center for Animal Health and Food Safety, University of Minnesota, St. Paul, MN) and identified as definitive type 104 (DT104) by the Centers for Disease Control and Prevention (Atlanta, GA). Bacteria were stored in 4% (vol/vol) glycerol/phosphate-buffered saline (PBS) until time of culture and grown overnight in Luria-Bertani (LB) medium at 37°C in a humidified 5% CO₂ atmosphere.

Explants of the ileal mucosa were obtained from outbred Yorkshire/Landrace-crossed male pigs that were 5–9 weeks old and weighed between 10 and 18 kg; the animals were obtained from a national specific pathogen-free accredited herd. Pigs had continuous access to water and non-medicated feed and were not fasted prior to sacrifice. They were sedated with an intramuscular injection of tiletamine hydrochloride-zolazepam (Telazol; 8 mg/kg, Fort Dodge Laboratories, Fort Dodge, IA), in combination with xylazine (3 mg/kg, Lloyd Laboratories, Shenandoah, IA). They were then euthanized by barbiturate overdose in accordance with approved University of Minnesota Institutional Animal Care and Use Committee protocols. A midline laparotomy was performed to expose the intestine, and a 20 cm segment of the distal ileum extending proximal from the ileo-cecal ligament was isolated. Segments were stripped of smooth muscle coats and the resulting sheets of mucosa with attached submucosa were mounted between two lucite Ussing-type half chambers (2 cm² flux area). Mucosal sheets were bathed on both luminal and contraluminal surfaces in 10 ml of a buffered, physiological saline solution (PSS; composition in mM: 130 NaCl, 6 KCl, 3 CaCl₂, 0.7 MgCl₂, 20 NaHCO₃, 0.29 NaH₂PO₄ and 1.3 Na₂HPO₄) which was maintained at pH 7.4 and 39° C (porcine core temperature). D-Glucose and mannitol (10 mM) were added to the contraluminal and luminal bathing medium, respectively. Explants were continuously oxygenated on both luminal and contraluminal surfaces with 95% O₂-5% CO₂ delivered by gas lift.

IPEC J2 cells (passages 50 – 56) were derived from porcine jejunal epithelial cells and were a kind gift from Dr. Bruce Schultz (Dept. of Anatomy and Physiology, Kansas State University, Manhattan, KS). The cells were grown and maintained in 50% Dulbecco’s Modified Eagle Medium and 50% Nutrient Mixture F12 (Ham) (1:1 DMEM/F12; Invitrogen Life Technologies, Carlsbad, CA), 5% fetal bovine serum, 5 μg/ml insulin (Sigma), 5 ng/ml epidermal growth factor (Sigma), 0.1% streptomycin and 0.1% penicillin. IPEC J2 cells were seeded at a density of 5 × 10⁶ per well on Costar Transwell plates (surface area = 4.7 cm²) coated with rat tail collagen (Sigma). The cells were maintained in an atmosphere of 5% CO₂ at 37°C. The culture medium was changed on alternate days after cells adhered to filters, and replaced with antibiotic-free medium 12 h prior to each experiment. Transepithelial electrical resistance of monolayers was determined on a daily basis after seeding (approximately 8 days).

Spectrophotometric evaluations indicated that overnight incubation was sufficient for all bacteria to reach the stationary growth phase. Overnight cultures (300 μl) were inoculated into fresh LB media (30 ml) and incubated for 3–4 hours to obtain mid log phase cultures. Inocula (100 μl) from mid log or stationary phase cultures were added to the luminal medium (total volume = 10 ml) bathing ileal explants. For IPEC J2 cells, inocula (100 μl) from mid log or stationary phase cultures were diluted 1:100 in physiological saline solution comprising the apical bathing media. Two milliliters of the resultant bacterial suspension was added to the apical surface of IPEC J2 cells. All bacterial inoculations are expressed as the final bath density in log colony forming units (CFU)/ml in bathing media.

Salmonella invasion into the ileal mucosa was determined by the method of Elsinghorst [13]. Briefly, mucosal sheets were removed from Ussing chambers after 90 min of luminal exposure to bacteria, washed three times in PBS (pH 7.4) and subsequently incubated at 37° C in a humidified 5% CO₂ atmosphere in a gentamicin solution (100 μg/ml in PBS; Sigma Chemical Co., St. Louis, MO, USA) for 80 min to eliminate extracellular bacteria. Previous experiments indicated that this concentration and exposure time were sufficient to kill S. Typhimurium DT104 under these incubation conditions. Tissues were subsequently homogenized using a Brinkmann Polytron and homogenates were serially diluted and spread-plated on differential and selective media for Salmonella (XLD agar, Becton-Dickinson, Sparks, MD). The resultant black bacterial colonies were counted with a Quebec darkfield colony counter (Leica, Inc., Buffalo, NY).

Bacterial invasion in IPEC J2 cells was determined in six cell monolayers from at least two different passages 8 days after initial seeding. At the time of the experiments, monolayers were bathed on the basolateral side with 2 ml of the identical oxygenated physiological saline solution used to bathe ileal mucosa explants. The apical aspect of the IPEC J2 monolayers was exposed to 2 ml of the resultant bacterial suspension (described above) of S. Typhimurium inoculum for 90 min at 37 °C. After removal of bacteria, monolayers were incubated in PBS containing 100 μg/ml gentamicin for 80 min to eliminate extracellular bacteria. After incubation in gentamicin solution, monolayers were rinsed twice for 5 min in sterile PBS. PBS (200 μl) containing 0.1% Triton-X was added to each transwell filter and after 5 min, it was diluted with 800 μl PBS prior to removing monolayers from each filter. The concentration of Triton X employed did not alter the recovery of viable salmonellae incubated for 90 min in 0.1% (v/v) Triton X in PBS (data not shown). The resulting cell suspension was vortexed, serially diluted and spread-plated on XLD agar.

Fractional recovery was calculated as a fraction of the total number of salmonellae recovered in gentamicin-treated cells or tissues relative to the total number of bacteria added respectively to the apical or luminal bathing medium in each experiment; these data were subsequently transformed to log₁₀ values and expressed as mean ± SE. Comparisons of means were made by a two-tailed, paired or unpaired t tests.

3. Results and Discussion

The ability of various S. Typhimurium strains in different growth phases to invade IPEC J2 cells and the porcine ileal mucosa was examined over 90 min and the results obtained in each model were generally comparable. In IPEC J2 cells, log fractional recovery (Table 1) of the S. Typhimurium ATCC 14028 reference strain and the field isolate were significantly greater (P < 0.0009 and 0.0001, respectively) for bacteria in the mid log phase of growth than in stationary phase. Similarly in the porcine ileal mucosa, internalization and fractional recovery of reference strain SL1344 or the DT104 field isolate was significantly higher (P < 0.04 and 0.0003, respectively) for bacteria in the mid log growth phase than in stationary phase (Table 1). Growth phase is an important determinant in the ability of S. Typhimurium to invade epithelial cells, as it influences the expression of virulence genes required for invasion [6, 14].

Table 1.

Log fractional recovery (mean ± SEM) of intracellular S. Typhimurium from IPEC J2 epithelial monolayers or porcine ileal mucosa after 90 min apical/luminal exposure to bacteria in either the mid log or stationary growth phase.

S. Typhimurium strain	Mean log10 fractional recovery ± SEM
	Mid log	Stationary
	IPEC J2 cells †
SL1344	−2.96 ± 0.15	−3.22 ± 0.09
ATCC 14028	−3.14 ± 0.09	−3.56 ± 0.07***
DT104	−3.04 ± 0.12	−4.06 ± 0.15***
	Porcine ileal mucosa
SL1344a	−2.29 ± 0.12	−3.19 ± 0.36‡
ATCC 14028b	−4.14 ± 0.54	−3.04 ± 0.36
DT104c	−2.86 ± 0.26	−4.49 ± 0.22‡‡‡

^†Six cell monolayers were used for each condition.

^aMeans represent results in 22 and 10 ileal mucosa explants from 12 and 5 pigs for mid log and stationary phase, respectively.

^bMeans represent results in 12 and 14 ileal mucosa explants from 6 and 7 pigs for mid log and stationary phase, respectively.

^cMeans represent results in 28 and 19 ileal mucosa explants from 16 and 13 pigs for mid log and stationary phase, respectively.

^**P < 0.01

^***P < 0.001 vs. mid log growth phase in IPEC J2 cells, paired t test.

^‡P < 0.05

^‡‡‡P < 0.001 vs. mid log growth phase in ileal mucosa explants, unpaired t test.

Although the fractional recovery of internalized S. Typhimurium strain ATCC 14028 in ileal explants appeared to be higher in mid log growth phase than in stationary phase, this difference did not attain statistical significance owing to relatively low invasiveness under the mid log condition and high variability in recovery from gentamicin-treated tissues. This may have obscured an effect of growth phase on internalization of this organism in the ileal mucosa. Alternatively, a decreased effect of growth phase on invasiveness of ATCC 14028 may be due to genes expressed on the Gifsy-3 prophage, which is not present in SL1344. Gifsy-3 encodes the SPI-1 effector protein, SspH1, which can down-regulate the expression of interleukin 8 in infected epithelial cells [15, 16]. Unidentified functions for SspH1 may alter the invasiveness of ATCC 14028 in mucosal sheets relative to the other bacterial strains examined.

In both IPEC J2 cells and mucosal explants, the greatest effect of growth phase on the invasiveness of salmonellae was observed with the DT104 field isolate. This appears to be due to the relatively low invasiveness of this strain in the stationary phase in both IPEC J2 cells and ileal explants. A previous study has shown that some DT104 strains manifest a hypoinvasive phenotype [17]. The cellular mechanisms underlying apparent growth phase-associated differences in DT104 invasiveness are undefined and this phenomenon clearly warrants further investigation.

To our knowledge, this is the first study to compare S. Typhimurium invasion in cultured epithelial cell monolayers and mucosal explants from the same host species. Such comparisons of bacterial interactions in isolated intestinal epithelial cells and intact intestinal mucosae are of potential importance as a functional approach to analyzing the biological processes and consequences of early intestinal infection by enteric pathogens.