Multiple effects of Escherichia coli Nissle 1917 on growth, biofilm formation and inflammation cytokines profile of Clostridium perfringens type A strain CP4

Abstract

Clostridium perfringens is an important Gram-positive pathogen responsible for food poisoning, necrotic enteritis, gas gangrene, and even death. Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic strain with demonstrated benefits. In this study we evaluated the effects of EcN on growth, toxin production, biofilm formation and inflammatory cytokine responses of C. perfringens. In vitro co-culture experiments demonstrated that EcN inhibited growth, gas production and toxin production (α-toxin and NetB) of C. perfringens in a dose dependent manner. The growth inhibition effect was not observed when C. perfringens was incubated with EcN cell free supernatants (CFSE), suggesting that growth inhibition was caused by nutrition competition during co-incubation. In vitro studies demonstrated that pre-incubation with EcN did not inhibit C. perfringens attachment to Caco-2 cells, but did reduce C. perfringens total number, toxin production and cytotoxicity after 24 h. The similar growth inhibition results were also observed during the formation of C. perfringens biofilm. Finally, pre-incubation of EcN with RAW264.7 cells significantly decreased the production of inflammatory cytokines caused by introduction of C. perfringens. Our results indicate that EcN can inhibit many of the pathological effects of C. perfringens in vitro conditions.

Introduction

Probiotics are live microorganisms which provide beneficial effects when ingested. Although the underlying mechanisms remain poorly understood, a number of studies have demonstrated that probiotics can efficiently inhibit the impact of pathogens in the gut either directly by growth competition or indirectly via production of inhibitory substances such as bacteriocins (Sanders, 2011). Typical probiotics including lactic acid bacteria (LAB), bifidobacteria, certain yeasts and bacilli have been well studied for decades to treat antibiotic-associated diarrhea (D'Souza, et al., 2002), lactose intolerance (Sanders, 2000) and colon cancer (Brady, et al., 2000). The ability of probiotics to improve host immune function (Ouwehand, et al., 2002, Reid, et al., 2003), modulate inflammatory and hypersensitivity responses (Reid, et al., 2003) has also been documented.

As a nonpathogenic representative of the human intestinal flora, Escherichia coli Nissle 1917 (EcN), the active ingredient of the probiotic drug Mutaflor^®, was isolated in 1916 by the German physician Alfred Nissle and has been used as a probiotic agent in human and animal medicine to treat chronic inflammatory and infectious diseases of the human and animal intestine (Kamada, et al., 2005). The EcN strain can reduce bacterial invasion and modulate cytokine expression of Caco-2 cells infected with Crohn’s Disease-Associated E. coli LF82 (Huebner, et al., 2011). EcN has also been engineered to express the autoinducer molecule cholera autoinducer 1 (CAI-1). The recombinant strain significantly inhibited Vibrio cholerae virulence gene expression and colonization in an infant mouse model (Duan & March, 2010). A recent study demonstrates that EcN reduces Salmonella Typhimurium intestinal colonization and expression of proinflammatory cytokines by competing for iron (Deriu, et al., 2013).

C. perfringens is a Gram-positive anaerobic spore-forming bacterium, able to produce more than ten kinds of toxins and enzymes responsible for wound contamination, anaerobic cellulitis and gas gangrene (Songer, 1996). It is also the third most common cause of food poisoning in the United States (Scallan, et al., 2011) mediated primarily by its enterotoxin, CPE (Songer, 1996) and is frequently responsible for non-C. difficile cases of antibiotic associated diarrhea (Modi & Wilcox, 2001). C. perfringens strains are classified into five toxinogenic types (A, B, C, D and E), based on the production of four major toxins (α, β, ι and ε) (Petit, et al., 1999). Type A isolates are the most widespread in the intestines of warm-blooded animals and in the environment, causing enteric disease in a number of domestic animals, including chickens, horses, pigs and sheep (Songer, 1996). The use of probiotics to control C. perfringens infection has drawn more attention recently. A number of probiotic bacteria such as Bacillus subtilis (Teo & Tan, 2005), Lactobacillus salivarius (Kizerwetter-Swida & Binek, 2009) and Lactobacillus fermentum (Allaart, et al., 2011) have been shown to either inhibit C. perfringens growth or repress virulence factor production.

Biofilm formation which can enhance persistence and increase resistance to stress, has been demonstrated to be involved in an estimated 80% of all infections (Biel, 2010). After colonization, the cells are able to communicate via quorum sensing using products such as N-acyl homoserine lactone to form biofilm, consisting of polysaccharide, extracellular DNA, environment material such as minerals and blood components (Murga, et al., 2001, Sutherland, 2001, Moscoso, et al., 2006, Nadell, et al., 2008). It has been reported that all sequenced C. perfringens strains can form biofilms that protect them from oxidative and antibiotic stress (Varga, et al., 2008). Since the probiotics EcN can outcompete a number of different pathogenic E. coli during biofilm formation (Hancock, et al., 2010), it would be meaningful to determine the interaction between EcN and C. perfringens during biofilm formation.

The aim of this study was to investigate the impact of EcN on C. perfringens type A strain. Our results show that EcN inhibits both growth and toxin production by C. perfringens in vitro, as well as biofilm formation, indicating that the use of EcN may be a useful, alternative approach to controlling the pathogenic effects of C. perfringens type A infection.

Materials and Methods

Bacterial strains and growth conditions

Strains and plasmids used in this study are listed in Table 1. C. perfringens strain CP4 (CP4) is a virulent type A strain isolated from a necrotic enteritis (NE) case in Ontario (Thompson, et al., 2006). Plasmid pJIR750 (Bannam & Rood, 1993) was used to transform CP4 as described (Scott & Rood, 1989) to yield CP4(pJIR750) and provided a chloramphenicol resistance (Cm⁺) marker to distinguish CP4 from EcN whenever co-culture experiments were performed. Plasmid pWSK129 (Wang & Kushner, 1991) was used to transform EcN yielding EcN(pWSK129) with kanamycin resistance (Kan⁺) marker whenever needed. Stability of both plasmids was evaluated as described before (Kang, et al., 2002, Brenneman, et al., 2012) with the exception that strains were grown in the absence of chloramphenicol or kanamycin. Plasmids pWSK129 and pJIR750 were found to be stable in the absence of antibiotics for more than 50 generations. Strains containing each plasmid grew at a similar rate in the absence of antibiotics as wild type strains did. All experiments were performed under anaerobic conditions using the BD GasPak EZ Anaerobe Container System.

Table 1.

Strains and plasmids used in this study

Strains and plasmid	Relevant serotype or genotype	Source or reference
E. coli
Nissle 1917 (EcN)	O6:H1:K5	Ardeypharm GmbH, Herdecke Germany
C. perfringens CP4	Type A, field isolate, α-toxin, NetB toxin positive	J.F. Prescott, Guelph, Canada
pJIR750	Chloramphenicol resistant, shuttle vector	Julian Rood, Monash University, Australia
pWSK129	pSC101 replicon, kanamycin-resistance	Lab stock (Wang & Kushner, 1991)

All strains were routinely cultured at 37°C in trypticase-peptone-glucose (TPG) broth consisting of 5% (w/v) Bacto-Tryptone (Difco Laboratories, Detroit), 0.5% (w/v) proteose peptone (Difco), 0.4% (w/v) glucose and 0.1% (w/v) sodium thioglycolate (Leslie, et al., 1989) unless otherwise specified. CP4 and EcN strains grow well in TPG broth under anaerobic condition. Tryptone sulfite cycloserine agar (TSC) plates (Merck) with 5 µg/ml chloramphenicol and Luria-Bertani (LB) agar plates with 50 µg/ml kanamycin were used for the enumeration of CP4(pJIR750) and EcN(pWSK129) from mixed cultures separately. Tryptic Soy Broth (TSB) with 10 mM lactose was used in the C. perfringens biofilm study. All centrifugation were performed at room temperature unless otherwise specified.

Tissue culture cells and growth conditions

Human epithelial colorectal adenocarcinoma cells (Caco-2) cells and mouse macrophage cells (RAW264.7) were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (pen/strep) and maintained in a humidified environment with an atmosphere of 5% CO₂ at 37°C.

Co-culture experiments with C. perfringens and EcN

CP4(pJIR750) and EcN(pWSK129) strains were cultured individually under anaerobic conditions in 3 ml TPG broth with chloramphenicol or kanamycin respectively overnight to reach stationary phase. Both cultures were inoculated into fresh 3 ml TPG broth (1:20) with appropriate antibiotics and grown to an optical density at 600 nm (OD₆₀₀) of 0.8. The cultures were then centrifuged at 16,000×g for 5 min and the pellet was washed once with phosphate-buffered saline (PBS) and resuspended in the same volume of fresh TPG broth. A preliminary assay we found that at equal OD₆₀₀ values (0.8), the EcN culture contained about twice as many CFU as the CP4 culture. For all co-culture experiments, a total final volume of 3.2 ml was used and 0.1 ml of resuspended CP4(pJIR750) was inoculated into each tube. Then we added 0.05 ml, 0.1 ml or 0.2 ml of resuspended EcN(pWSK129) to tubes designated Co-1 (Cp:EcN≈1:1), Co-2 (Cp:EcN≈1:2) and Co-4 (Cp:EcN≈1:4) to achieve the indicated ratios of Cp to EcN. Negative control cultures contained CP4(pJIR750) only (C.p.) and EcN(pWSK129) only. The EcN controls EcN1, EcN2 and EcN4 were inoculated with 0.05 ml, 0.1 ml and 0.2 ml EcN, respectively. Fresh TPG medium was added to all tubes to achieve the final volume of 3.2 ml. All experiments were performed in triplicate. Cultures were incubated anaerobically for 20 h and in some experiments, OD₆₀₀ and pH measurements were taken every two hours until 8 h and CFU of CP4(pJIR750) and EcN(pWSK129) were determined on TSC agar plates (5 µg/ml Cm) and LB agar plates (50 µg/ml Kan), respectively. Supernatants obtained from 20 h cultures were collected by centrifugation (16,000×g, 10 min) and filtered through a 0.22-µm-pore-size filter (Millipore), followed by western blot as described below.

The effect of EcN supernatants on C. perfringens growth and toxin production

EcN was incubated anaerobically in TPG broth overnight at 37°C. After centrifugation at 16,000×g for 10 min, the supernatant was filtered (0.22 µm, Millipore) to remove any resident cells yielding the EcN cell free supernatant (CFS_E). Amicon Ultra-15 Centrifugal Filter Units (10,000 NMWL, Millipore) were used to prepare 5X and 10X concentrated EcN supernatants, designated 5CFS_E and 10CFS_E. All CFS_Es were used immediately or stored at −20°C until use.

Fresh CP4 cultures were prepared as mentioned above. Then, 0.1 ml of resuspended CP4 cells were inoculated into either 3 ml TPG medium (Cp-neg), 2.4 ml TPG plus 0.6 ml 5CFS_E (Cp-5CFS_E) or 2.4 ml TPG plus 0.6 ml 10CFS_E (Cp-10CFS_E). All experiments were performed in triplicate. Cultures were incubated anaerobically at 37°C. OD₆₀₀ values were determined at 2 h intervals for 8 h, at which time, culture supernatants collected and subjected to western blot analysis.

Caco-2 cell attachment and cytotoxicity assay

Caco-2 cell attachment experiments were performed as previously described (Martin & Smyth, 2010) with some modification. Caco-2 cells were seeded into 24-well plates at 5×10⁵ cells/well and cultured for 4 days. EcN cultures were collected by centrifugation at 16,000×g for 10 min and resuspended in DMEM containing 5% FBS to a final concentration of 5 × 10⁷ CFU/ml. Caco-2 cells were washed once with PBS and replaced in 1 ml fresh DMEM with 5% FBS. Then, 100 µl of EcN (5 × 10⁶ CFU) was added at a multiplicity of infection (MOI) ≈10 and incubated for either 2 h (Cp+, EcNpre2h) or 6 h (Cp+, EcNpre6h). Caco-2 cells without EcN were used as the negative control (Cp+, EcN−). Overnight cultures of CP4(pJIR750) grown in TPG broth (5 µg/ml Cm) were collected by centrifugation and resuspended with DMEM containing 5% FBS to 5 × 10⁷ CFU/ml. 100 µl of resuspended CP4(pJIR750) were inoculated into each well (MOI≈10) and incubated at 37°C under anaerobic conditions.

For bacteria attachment experiments, the culture medium was removed after 1 h incubation with CP4(pJIR750) and the cells were washed twice with sterile PBS. 200 µl 0.05% trypsin was added each well and incubated for 15 min at room temperature. An additional 800 µl PBS was added to each well and mixed completely by pipetting. A series of ten-fold dilutions were plated onto TSC plates with 5 µg/ml chloramphenicol. The bacterial colonies obtained from each well were counted and recorded after 24 h anaerobic incubation at 37°C.

For cell cytotoxicity experiments, Caco-2 cells were incubated with either CP4(pJIR750) alone (MOI≈10) or together with EcN pre-incubation (MOI≈10) for 2 h or 6 h as described above. After incubation at 37°C for 20 h, the supernatants from each well were collected by centrifugation at 16,000×g for 10 min and passed through a 0.22 µm filter (Millipore). Lactate dehydrogenase (LDH) enzyme activity in each well was determined using the lactose dehydrogenase activity kit (Biovision) following the manufacturers protocol. Equal volumes of residual supernatants from each well were precipitated with 10% TCA and subjected to western blot assay. Additional triplicate wells were included in each group and the CFU number of CP4 were determined at the end of the assay.

SDS-PAGE and western blot assay

Total proteins in supernatants were precipitated by adding 10% (v/v) trichloroacetic acid (TCA) and incubated at 4°C overnight. Then the pellets were collected by centrifugation at 16,000×g for 10 min at 4°C and washed twice with cold acetone containing 0.1% β-mercaptoethanol. The pellets were then dissolved in 100 µl SDS loading buffer. Equal volumes of each sample were separated by discontinuous 12% SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. Membranes were blocked with 5% skim milk in 100 mM Tris containing 0.9% NaCl and 0.1% Tween 20 (pH7.4). Filters were incubated first with rabbit polyclonal antibodies specific for α-toxin C terminal (PlcC) (Zekarias, et al., 2008) and NetB (laboratory stock, made by Hua Mo). Filters were washed and the secondary antibody, an alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Southern Biotechnology, Birmingham, AL) was added. Immunoreactive bands were detected by the addition of nitroblue tetrazolium (NBT)-5-bromo-4-chloro-3-indolylphosphate (Sigma). The reaction was stopped after 2 to 5 min by washing the membranes several times with large volumes of deionized water. The band intensities were analyzed by Image J software (Wayne Rasband National Institutes of Health, Bethesda, MD, USA) and the relative expression rate was calculated and normalized to control cultures.

Biofilm assays

C. perfringens biofilm assays were performed as described by Varga et al. (Varga, et al., 2008) with some modifications. Overnight cultures of CP4(pJIR750) and EcN(pWSK129) were washed once in PBS and resuspended to an OD₆₀₀ of 0.1 in TSB medium supplemented with filter-sterilized lactose (10 mM). Then 100 µl of resuspended CP4(pJIR750) was inoculated into 96-well polystyrene tissue culture plates. Additional 100 µl or 200 µl of resuspended EcN(pWSK129) culture was inoculated into each well, designated Cp+EcN1 and Cp+EcN2, respectively. Wells with 100 µl CP4(pJIR750) or 100 µl EcN(pWSK129) were also included and named Cp-control and EcN-control individually as negative controls. Extra TSB medium (10 mM lactose) was added into wells to achieve a final volume of 300 µl whenever needed. Cultures were incubated anaerobically at 30°C for 5 days in a sealed container to prevent evaporation. Each plate also contained another series of wells to which 300 µl of TSB medium was added to serve as negative controls. Biofilm formation, as well as bacteria colony numbers in biofilm and floating supernatant were determined.

Biofilm formation was determined using the distribution between planktonic (free-floating) and sessile (biofilm) cells as described (Varga, et al., 2008) with minor modifications. After 5 days, the supernatant from each well was transferred to a new 96-well plate and the OD₆₀₀ values were measured. The biofilm remaining in the original wells was then gently washed twice with PBS and incubated with 300 µl of 0.1% crystal violet for 30 min at room temperature. The crystal violet was then removed from the wells carefully and the resident wells were gently washed twice with PBS. Then, 300 µl of methanol was added into each well and incubated for 30 min at room temperature to extract bound crystal violet. After transferring the extracted crystal violet to a new 96-well plate, the A₅₇₀ values of the methanol-extracted dye were measured in a SpectraMax M2 Multi-Mode Microplate Reader (Molecular Devices, LLC). Wells containing TSB medium were used to subtract nonspecific staining background. The ratio of the A₅₇₀/OD₆₀₀ was used as a relative measure of biofilm production. The number of CP4(pJIR750) and EcN(pWSK129) in both biofilm and planktonic phase were also determined on selective plates.

Cytokines reaction in presence of RAW264.7 cells

Murine macrophage RAW264.7 cells were seeded into 24 wells cell culture plates at 5×10⁵ cells/well and cultured at 37oC for 24 h. After washing twice with PBS, 1 ml DMEM containing 5% FBS was added each well. Overnight EcN cultures were collected by centrifugation (16,000×g for 5 min) and resuspended with the same volume of TPG. Then 100 µl of EcN was inoculated into each well (MOI≈100) and incubated for 3 h before the infection of CP4. CP4 cells from an overnight culture were collected and resuspended in TPG and diluted appropriately. 100 µl of CP4 (MOI≈10 or MOI≈100) was added to each well. Cells incubated with EcN alone (MOI≈100), CP4 alone (MOI≈10 or MOI≈100) and mock-infected cells were used as controls. After a 20 h incubation, the supernatants from each well were collected and centrifuged (16,000×g, 10 min, 4°C) to remove suspended bacteria. The supernatants were analyzed directly by Bio-Plex Multiplex Cytokine Assay (Bio-Rad) or stored at −20°C prior to cytokine analysis. Granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) were measured.

Statistical analysis

All statistics were carried out using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA). Data were expressed as means ± standard errors (n=2 for cytokines assay; n=3 for the rest of assays) and evaluated with two-tailed t-test. Differences were considered significant at P < 0.05.

Results

EcN represses growth of C. perfringens during co-culture

In a preliminary co-culture experiments with C. perfringens strain CP4, we found that EcN interestingly decreased gas production by CP4. At an inoculation ratio of 1:1 (Co-1), gas production by CP4(pJIR750) decrease slightly compare to the negative control (C.p.). When the ratio of CP4(pJIR750) to EcN(pWSK129) was increased to 1:2 (Co-2) and 1:4 (Co-4), gas production was dramatically reduced (Fig. 1A). These results indicate that EcN(pWSK129) inhibits CP4(pJIR750) gas production in a dose dependent manner. The major pathogenic effects of C. perfringens are primarily due to the toxins they produce. CP4 is a poultry isolate that causes necrotic enteritis, and is known to produce and secrete two toxins important for that disease, α-toxin and NetB toxin (Chalmers, et al., 2008, Keyburn, et al., 2008). Compared to the C.p. control, the amount of α-toxin and NetB toxin present in culture supernatants after 20 h incubation was reduced as the amount of EcN(pWSK129) present in the initial inoculation increased from 1:1 to 1:2 and 1:4 (Fig. 1B). We note that the band on the western blot below the α-toxin, is a cross-reactive band present in the EcN(pWSK129) culture supernatant and not a degradation product of α-toxin, since the band could also be observed in EcN(pWSK129) supernatant control (indicated by an arrow in Fig. 1B). The intensity of western bands were calculated using Image J software and the relative expression ratio of toxins were described as the percentage normalized to control cultures (Fig. 1C). Presence of EcN(pWSK129) significantly repressed the production of both alpha-toxin and NetB toxin in a dose dependent manner. We also determined the bacteria number of CP4(pJIR750) and EcN(pWSK129) in each group after 20 h incubation. The results indicated that the final CP4(pJIR750) number decreased dramatically as the inoculation ratio of EcN(pWSK129) increased from 1:1 to 1:2 and 1:4 (Fig. 1D). Besides, there appeared to be a growth competition effect between EcN(pWSK129) and CP4(pJIR750) since the presence of CP4(pJIR750) also inhibited the growth of EcN(pWSK129), while this kind of inhibition effects were decreased as the original inoculation dose of EcN(pWSK129) increased from 1:1 to 1:4 (Fig. 1E).

Figure 1.

To gain some insight into the mechanism driving the above results, we used the cultures containing equal inoculation dose of EcN(pWSK129) and CP4(pJIR750) (Co-1), as well as negative control cultures C.p. and EcN1, to explore the inhibition effect in more detail. Samples were taken at 2 h, 4 h, 6 h, 8 h and 20 h to measure the OD₆₀₀, CFU and pH values. The OD₆₀₀ values of both control cultures were similar, although the final values of EcN1 were a little higher than C.p. culture (Fig. 1F). However, the actual CFU numbers of EcN(pWSK129) increased much faster compared to CP4(pJIR750) (Fig. 1G). As we mentioned before, the growth competition effects were observed for both strains, since the CFU numbers of each bacteria in co-culture condition were lower than the individual control groups (Fig. 1G). While the pH in all cultures declined steadily during the first 6 h of growth, although the pH decreased faster and reached a lower minimum pH, around 5.7, when EcN was present (Fig. 1H). We also noticed that the pH declined much lower as long as the inoculation dose of EcN increased (data not shown). The lower pH in the mixed culture may be at least partially responsible for lower titer achieved by CP4 in the presence of EcN (Fig. 1G). All the results above indicate that the presence of EcN(pWSK129) could inhibit the growth of CP4(pJIR750) in a dose dependent manner.

EcN supernatants do not inhibit the growth of C. perfringens

The growth inhibition observed when EcN is present could be due to a direct competition for nutrients or secretion of inhibitory compounds by EcN (e.g. bacteriocins), or some combination of the two. To evaluate the possibility that EcN is secreting an inhibitory compound, we prepared cell-free supernatants from EcN cultures and concentrated them either 5-fold (5CFS_E) or 10-fold (10CFS_E) (See Materials and Methods). The results showed that the addition of either CFS_E had no apparent effect on the growth of CP4 and toxins production (data not shown). Thus, we conclude that secretion of inhibitory compounds by EcN is not a factor in the observed effects on CP4 growth and toxin production. Therefore, the direct competition during co-culture process should be responsible for the inhibition effect.

EcN represses C. perfringens growth in the presence of Caco-2 cells

Adherence of enteropathogens to intestinal epithelium cells is an important first step in establishing an infection (Reis & Horn, 2010). Some probiotics have shown the ability to compete with pathogens for adherence to host cells (Lu & Walker, 2001, Vine, et al., 2004). Thus, we examined the effects of EcN on CP4 adherence to Caco-2 epithelial cells. The results showed that the preincubation of Caco-2 cells with EcN(pWSK129) (MOI≈10) for 2 h or 6 h had no obvious inhibition effect on C. perfringens cell attachment ability (Fig. 2A). However, the total number of C. perfringens after 20 h was decreased in cells with EcN pretreatment for 2 h and 6 h compared to wells in which EcN was not present (P < 0.01) (Fig. 2B). The production of both α-toxin and NetB toxin were decreased in the presence of EcN (P < 0.05) (Fig. 2C and D), consistent with co-culture experiments described previously. Finally, presence of EcN increased the intactness of Caco-2 cells about 6-fold (P < 0.0001) shown by decreased level of cellular LDH (Fig. 2E). The results demonstrated that the inhibition effects of EcN on C. perfringens during co-culture experiments were also observed in the presence of host cells.

Figure 2.

Presence of EcN outcompete CP4 growth during biofilm formation

The ability to form biofilms is an important strategy for bacterial persistence in host and free-living environments, including C. perfringens (Varga, et al., 2008). EcN is a good biofilm former and able to outcompete enteropathogenic, enterotoxigenic and enterohaemorrhagic E. coli strains during biofilm formation (Hancock, et al., 2010). Thus, we were interested in determining whether EcN could decrease biofilm formation of C. perfringens. To address this question, we set up a biofilm assay. CP4(pJIR750) cells were inoculated into 96 well plates with and without the addition of EcN(pWSK129) at the ratio (CP4/EcN) of 1:1 (Cp+EcN1) or 1:2 (Cp+EcN2). After 5 days, biofilm formation was observed in all wells according to ratio of A₅₇₀/OD₆₀₀, especially in wells containing CP4(pJIR750) alone (Fig. 3A), consistent with previous reports for other C. perfringens strains (Varga, et al., 2008). Both mixed cultures (Cp+EcN1 and Cp+EcN2) had significantly less biofilm formation compared to CP4 alone or EcN alone (Fig. 3A), indicating that there were some interference effects between C. perfringens and EcN during biofilm formation. We then determined individual bacteria numbers in biofilm and floating supernatant, separately. The existence of EcN decreased the colony number of CP4 in both biofilm (Fig. 3B) and supernatant (Fig. 3C), especially when the starting inoculation ratio was 1:2 (Cp+EcN2). At the same time, the number of EcN in both biofilm and floating supernatant were not obviously affected by CP4 (Fig. 3D and E). We noticed that high dose inoculation (Cp+EcN2) did not cause more EcN cells in biofilm, instead, significantly more EcN cells were observed in supernatants (Fig. 3E). The ratio of CP4 cells in the presence of EcN were also calculated using the mean number of CP4 in Cp-control culture as a standard, which demonstrated that the CP4 number decreased about 60% in biofilm and 70% in supernatant in the presence of EcN (Cp+EcN2) (Fig. 3F). Therefore, we concluded that the presence of EcN significantly outcompeted growth of CP4 during biofilm formation.

Figure 3.

EcN modulates cytokine expression in RAW264.7 cells infected with CP4

Tissue damage and inflammation response are caused by toxins secreted by C. perfringens (Songer, 1997, Wallace, et al., 1999, Stevens, 2000, Oda, 2012). Mice injected with α-toxin secreted dramatically increased proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IL-1β, IL-6 and interferon gamma (IFN-γ) which could be important factors responsible for lethality and systemic hemolysis (Oda, et al., 2008). Since the co-incubation experiments mentioned above showed that EcN could inhibit CP4 growth and reduced toxins production, therefore it would be interesting to measure the inflammatory cytokines profile caused by C. perfringens with or without the presence of EcN.

Compared to mock-infected cells, RAW264.7 cells infected with CP4 (MOI≈10) caused release of significantly increased levels of inflammatory molecules, including 4906 ± 256.0 (n=2) pg/ml IL-1β (P < 0.01) (Fig. 4A), 408.4± 59.90 pg/ml IL-6 (P < 0.05) (Fig. 4B), 57021 ±375.2 pg/ml G-CSF (P < 0.0001) (Fig. 4C) and 2207 ± 43.52 pg/ml GM-CSF (P < 0.001) (Fig. 4D). Pre-incubation with EcN (MOI≈100) significantly decreased the level of cytokine synthesis compared to CP4 infection alone. Similar results were observed when RAW cells were incubated with CP4 plus EcN at an MOI≈100. The level of cytokine secretion was lower when RAW cells were incubated with a high dose of CP4 (MOI≈100) than at a lower dose (MOI≈10) (Fig.4A, B, C, and D), probably due to early cell death caused by the higher dose of CP4.

Figure 4.

Discussion

The relationship between commensals and pathogens have drawn more and more attention recently (Kamada, et al., 2012). In particular, probiotics may provide an alternative to antibiotics for fighting against infectious diseases. In our study, we observed direct competition effects between EcN and C. perfringens type A strain CP4. The presence of EcN in co-culture experiments inhibited growth of CP4 in a dose dependent manner, resulted in reduced gas production, total CP4 numbers and toxin production (α-toxin and NetB toxin).

EcN has been demonstrated to inhibit pathogens adhesion and invasion ability. The mechanisms behind this are still not completely clear, but it is generally accepted that probiotics outcompete pathogens for nutrients and space, often producing inhibitory substances, such as lactic acid and other organic acids, hydrogen peroxide, and bacteriocins (Mital & Garg, 1995, Bogovic-Matijasic, et al., 1998, Salminen, et al., 2010, Dobson, et al., 2012). In this study, pre-incubation of epithelial cells Caco-2 with EcN did not inhibit binding of C. perfringens, as was shown for Salmonella (Schierack, et al., 2011) and adherent-invasive E. coli (AIEC) strains (Boudeau, et al., 2003), possibly due to a difference in cell receptors targeted by different pathogens. However, preincubation with EcN dramatically reduced the overall numbers of CP4 and the level of toxins production (Fig. 2) in consistent with previous co-culture experiments.

It has been reported that all sequenced C. perfringens can form biofilms that protect them from oxidative and antibiotic stress (Varga, et al., 2008). To be consistent, we observed obvious biofilm formation using C. perfringens CP4 strain (Fig. 3A). EcN has been demonstrated to outcompete several pathogenic bacteria such as enterotoxigenic E. coli and enteropathogenic E. coli strains in biofilm formation (Hancock, et al., 2010). In our studies, the presence of EcN severely decreased the C. perfringens CP4 biofilm production (Fig. 3A), as well as number of C. perfringens in biofilm (Fig. 3B) and floating supernatant (Fig. 3C). The mechanism underlined is possibly due to the unique yersiniabactin system of EcN, the ability to produce several adhesions and fimbriae, or the combination (Hancock, et al., 2010). Our results demonstrated that the competition between EcN and C. perfringens observed in vitro culture condition also existed during the biofilm formation.

Cytokines are a key element in the inflammatory response that characterizes sepsis and septic shock (Oda, et al., 2008). Pro-inflammatory cytokines induced by C. perfringens and its toxins include IL-8, TNF-α, IL-1 and IL-6 (Zhou, et al., 2009, Forder, et al., 2012, Oda, 2012, Lee, et al., 2013, Tuovinen, et al., 2013) are likely to be responsible for C. perfringens-induced septic shock and death. Chickens infected with C. perfringens have been shown to produce increased levels of proinflammatory cytokines such as IL-1β and IL-6 (Sugiarto & Yu, 2004), responsible for unregulation of several β-defensins (van Dijk, et al., 2007). GM-CSF and G-CSF are induced in in vitro studies by the lipoteichoic acid component of the cell membranes of Gram-positive bacteria (Saba, et al., 2002, Seo, et al., 2008, Chou & Lu, 2011). Thus, the observed increased levels of cytokines by RAW cells in our study induced by either the C. perfringens cell membrane and/or secreted toxins. It has been shown that probiotics can ameliorate the inflammation by inhibiting proinflammatory cytokine production (Hegazy & El-Bedewy, 2010, Mencarelli, et al., 2011, Ganguli, et al., 2013). As a classic probiotic bacteria, EcN has been shown to modulate the cytokine profile in Caco-2 cells infected with Crohn’s Disease-Associated E. coli LF82 (Huebner, et al., 2011) and to decrease IL-10 and TNF-α levels induced by Salmonella Typhimurium in gnotobiotic pigs (Splichalova, et al., 2011). The administration of EcN also results in reductions of intestinal inflammation in S. Typhimurium-infected C57BL/6 mice (Deriu, et al., 2013). In this study, the inhibitory effects of EcN on C. perfringens growth and toxin production were likely responsible for the observed decrease in secretion of inflammatory cytokines.

Taken together, our results indicate that EcN can repress the growth of C. perfringens and decrease toxin production both in vitro condition in a dose dependent manner. EcN also inhibits the pro-inflammation response caused by C. perfringens. All these results indicate that EcN could possibly provide another strategy to control C. perfringens related infection.