Inhibition of Adhesion of Intestinal Pathogens ( Escherichia coli,Vibrio cholerae,Campylobacter jejuni,and Salmonella Typhimurium) by Common Oligosaccharides

Abstract

Inhibition of the binding of pathogenic adhesins to host glycans by suitable oligosaccharides forms the basis of antiadhesion therapies. Experiments were carried out to study the inhibition capability of oligosaccharides on the adhesion of four microorganisms (Escherichia coli, Vibrio cholerae, Campylobacter jejuni, and Salmonella Typhimurium) to HT-29 cells. Results showed that, in the absence of oligosaccharides, all of the four pathogens efficiently adhered to the cells. Cell adhesion with different bacteria was inhibited by distinct oligosaccharides (e.g., the adhesion number relative to control of V. cholerae could be significantly decreased by pectin oligosaccharide and chitooligosaccharide to about 16.1% and 18.9%, respectively). Saturation studies showed that the extent of antiadhesive effect for most of the suitable carbohydrates was dependent on their concentration. The observations from the study suggest that various carbohydrates may have antiadhesive activity and may be useful in future therapeutic study.

Introduction

Bacterial adhesion to host is the prerequisite for the initiation of many bacterial infections (Bavington and Page, 2005; Klemm et al., 2010). Bacteria are protected from expulsion by the mechanical cleaning process of the host by adhering to epithelial cells (Laux et al., 2005). The most common methods of adhesion involve the interaction between host cell surface glycans and protein-assembling adhesins located on surfaces of the pathogens (Sharon and Lis, 2003). Bacteria adhere to specific sites in the body because of the presence of appropriate oligosaccharide receptors at that site. The apparent preference of a particular bacterium for a specific tissue is known as tissue tropism (Alverdy and Stern, 1998). Previous studies have suggested that the selectivity of bacterial adhesins for oligosaccharide receptors is a decisive factor for the susceptibility of particular microbes to the host (Hultgren et al., 1993). Thus, the use of receptor-structure-mimicking oligosaccharides or analogs as antiadhesive agents to competitively inhibit the bacterial colonization is very promising (Shoaf-Sweeney and Hutkins, 2009). Rather than binding to the host cells and initiating bacterial pathogenesis, pathogens can adhere to specific soluble oligosaccharides, and then be expelled from the host (Shoaf et al., 2006).

The theory has been proven in vitro and in vivo with a range of bacterial species. For example, 3-siallyllactose inhibits the attachment of Helicobacter pylori to epithelial cells (Simon et al., 1997). Mannose and globotetraose hinder urinary tract infection caused by Escherichia coli in BALB/c mice (Eden et al., 1982). Many oligosaccharides are derived from natural sources and thus are unlikely to be disruptive to the body. Human milk oligosaccharides provide additional protection for infants against gastrointestinal, urogenital, and respiratory tract infection by inhibiting the adhesion of pathogens to the epithelial surfaces (Newburg, 1997). Chitooligosaccharides are inhibitors of E. coli strains to HT-29 cells (Rhoades et al., 2006). Dietary oligofructose and inulin can protect mice from the infection of enteric pathogens (Buddinton et al., 2002). Considering that oligosaccharides are generally not bactericidal, the strain resistance to oligosaccharides is unlikely to develop as easily as their resistance to antibiotics (Sharon, 2006). The alarming emergence of many antibiotic-resistant bacteria, particularly the spread of Enterobacteriaceae that carries carbapenemases-producing genes (Kumarasamy et al., 2010), makes it possible for the application of antiadhesive oligosaccharides in the therapeutic area to fight infectious diseases.

In this study, oligosaccharides derived from natural sources through enzymatic hydrolysis were applied for the determination of antiadhesion activity to intestinal pathogens. This work aimed to provide an efficient method and theoretical guide for the development of antiadhesive oligosaccharides as dietary supplements in the food and health-product industries.

Materials and Methods

Bacterial strains and culture conditions

Four bacterial strains were used in this study. Salmonella Typhimurium NCMCC 50222 was obtained from the National Center for Medical Culture Collections, China. Vibrio cholerae ATCC 14035 was obtained from the American Type Culture Collection (Manassas, VA, USA). Escherichia coli O157:H7 and Campylobacter jejuni of Penner serotype O 19 were isolated from clinical cases.

Bacteria stock solutions were stored in a Luria–Bertani (LB) broth supplemented with 10% (vol/vol) glycerol at −80°C. A freeze stock was plated onto the appropriate agar and incubated overnight at appropriate conditions prior to each experiment. V. cholerae, E. coli, and Salmonella Typhimurium were grown on nutrient agar plates (Land Bridge Ltd., Beijing, China) at 37°C for 24 h. C. jejuni was grown on Skirrow agar (Land Bridge Ltd.) supplemented with 7% defibrinated sheep's blood (Land Bridge Ltd.) at 42°C under microaerobic conditions (85% N₂, 10% CO₂, and 5% O₂) for 48 h.

For V. cholerae, E. coli, and Salmonella Typhimurium, a single colony was inoculated into 10 mL of the LB broth and incubated overnight without shaking. After the overnight incubation, bacteria cells were harvested by centrifugation and washed twice with phosphate-buffered saline (PBS). C. jejuni was directly harvested from Skirrow agar plates and washed twice with PBS. The harvested bacteria cells of the 4 strains were then resuspended at a concentration of 1×10⁸ colony-forming units (CFU)/mL for the adherence experiments. A sample of this bacterial suspension was subjected to serial dilution, in which 100-μL volumes were plated on organism-specific plates and incubated overnight to determine the CFU per milliliter.

HT-29 cell cultures

The HT-29 human colon epithelial cells were obtained from the Qingdao University Medical College (Qingdao, China). Cells were cultured in 25-cm² tissue culture flasks (Corning Costar, Corning, USA), which contained Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (Gibco, Invitrogen Ltd., Paisley, UK) supplemented with 5% (vol/vol) fetal bovine serum (Gibco, Invitrogen Ltd.). The cells were incubated at 37°C in 5% CO₂ in a humidified atmosphere, and subcultured every 3 to 4 d. For experimental assays, the confluent cell monolayers were washed with PBS (pH of 7.2±0.2, 0.1 M) and trypsinized with trypsin/0.05% (wt/vol) EDTA. After harvesting from the monolayers, cells were seeded in 24-well microtiter plates (Corning Costar) with approximately 10⁵ cells per well and then incubated for 72 h. The cell monolayers were washed twice with PBS prior to the assay.

Carbohydrates

Monosaccharides, including d-mannose, d-galactose, d-fructose, d-fucose, l-fucose, and d-glucose, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oligosaccharides (Table 1), including pectin oligosaccharide (POS), alginate oligosaccharide (AOS), chitooligosaccharide (COS), fucoidan oligosaccharide (FOS), guar gum oligosaccharide (GOS), konjak gum oligosaccharide (KOS), and tara gum oligosaccharide (TOS), were prepared via enzymolysis using the corresponding microbial depolymerase. AOS, COS, and FOS were prepared according to the methods of Wang et al. (2007a) and Zhang et al. (2004). POS was prepared according to the method of Wang et al. (2007b). GOS, KOS, and TOS were prepared according to the method of Mou et al. (2011). Oligosaccharides with molecular weights <2000 Da were obtained by ultrafiltration, which was performed in membrane-separating devices (Shiyuan Biological Engineering Equipment Co., Ltd., Shanghai, China). Samples were dissolved in PBS and sterilized by passing through a 0.22-μm syringe filter. The oligosaccharide solutions were further diluted in sterile PBS as required.

Table 1.

Composition and Structure of Oligosaccharides Used in This Study

Samples	Structures	Sources
POS	α-(1, 4)-d-galacturonic acid	Orange peel
AOS	β-(1, 4)-d-mannuronate, α-(1, 4)-l-guluronate	Algae colloid
COS	β-(1, 4)-d-glucosamine, N-acetyl-d-glucosamine	Crab shell
FOS	β-(1, 4)-mannose, GlcUA, Gal, α-(1, 3)-fucose	Marine algae
GOS	β-(1, 4)-mannose, α-(1, 6)-galactose	Guar gum
KOS	β-(1, 4)-mannose, α-(1, 6)-glucose	Konjak gum
TOS	β-(1, 4)-mannose, α-(1, 6)-galactose	Tara gum

POS, pectin oligosaccharide; AOS, alginate oligosaccharide; COS, chitooligosaccharide; FOS, fucoidan oligosaccharide; GOS, guar gum oligosaccharide; KOS, konjak gum oligosaccharide; TOS, tara gum oligosaccharide.

Adhesion assay

For inhibiting the bacterial adhesion, 0.5 mL of carbohydrates-containing PBS (oligosaccharides and monosaccharides) was added to each well. Subsequently, 0.5 mL of the bacterial suspension was added in PBS at a density of 1×10⁸ CFU/mL. The actual numbers of bacteria were evaluated via serial dilution and plate counting. Moreover, PBS without carbohydrates was used as the control sample. The microtiter plates were incubated at 37°C for 1 h to allow the adhesion of bacteria to the cells at aerobic conditions for V. cholerae, Salmonella Typhimurium, and E. coli, and at microaerobic conditions for C. jejuni. Preliminary experiments revealed that 1 h of incubation was adequate to measure the initial adherence, which was then used throughout the study (Vo et al., 2007; Ganan et al., 2010).

After incubation, cells were washed three times with PBS to remove the unattached bacteria. The monolayers were lysed with 0.5 mL of 0.1% (vol/vol) Triton X-100 for 20 min. The adhered bacteria were evaluated via serial dilution and plate counting. The results are shown as the percentage of bacteria adhered relative to an adherence control (ARC) (Rhoades et al., 2006; Ganan et al., 2010).

The inhibition of bacterial adhesion to HT-29 monolayers was also assessed via Gram staining. HT-29 cells were seeded on each 10×10-mm glass coverslip in 24-well plates at a density of 10⁵ cells per well. Adhesion inhibition assay was performed as described in the previous section. Some attached bacteria detached during incubation and washing, and those that were resistant to washing were stabilized by fixing with methanol for 15 min. The coverslips were allowed to dry and Gram-stained and then examined microscopically.

Statistical analysis

All experiments were performed in triplicate. Results are expressed as mean values, and SDs are provided where appropriate. P-values were calculated using paired t-test. Differences were considered significant at p<0.05.

Results

Adherence of HT-29 cells by bacteria

Initial experiments determined the relative adhesion to host cells by pathogens that occupy different anatomical niches. A bacterial inoculum of 10⁸ CFU/mL was used. The percentages of the four bacteria species (V. cholerae, E. coli, Salmonella Typhimurium, and C. jejuni) capable of adhering to HT-29 cells were compared with the initial inoculum. The results of plate counting indicated that different pathogens possessed distinct adhesive abilities, with adhesion percentages of 1.213% (V. cholerae), 0.653% (E. coli), 0.707% (Salmonella Typhimurium), and 0.051% (C. jejuni).

Influence of oligosaccharides on bacterial adhesion

A group of oligosaccharides was tested for their ability to inhibit the attachment of the 4 aforementioned pathogens to HT-29 cells. The concentration used was 2.5 mg/mL, which was the concentration used in the previous studies (Rhoades et al., 2006; Ganan et al., 2010). Results are shown in Table 2.

Table 2.

Influence of Carbohydrates on the Adhesion of Bacteria to HT-29 Cells

	Mean % ARC (±SD) ^b
Saccharides ^a	Vibrio cholerae	Escherichia coli	Salmonella Typhimurium	Campylobacter jejuni
d-Mannose	39.8±8.3	80.5±2.9	76.9±2.4	69.3±4.7
d-Galactose	102±16	83.5±1.2	83.9±1.4	78.2±4.0
d-Fructose	37.7±3.0	95.8±10.4	62.2±5.7	101±3.3
d-Fucose	69.6±2.8	90.4±9.8	72.9±6.6	72.1±3.2
d-Glucose	62.9±9.0	90.4±9.8	102±7.6	79.3±7.9
l-Fucose	107±8.5	108±5.4	103±3.5	98.3±6.5
POS	16.1±3.1	98.9±3.2	80.6±3.3	95.9±3.8
AOS	104±6.7	85.5±4.1	103±7.1	109±5.7
COS	18.9±5.1	41.1±10.3	86.6±2.4	92.6±2.9
FOS	97.6±2.8	103±9.8	28.6±4.7	93.2±5.7
GOS	99.8±17.3	93.7±13.9	45.5±2.8	101±7.7
KOS	109±16.5	107±14.8	96.9±1.9	90.5±5.7
TOS	126±11.5	98.5±21.3	37.9±3.8	103±0.7

The carbohydrate concentration used is 2.5 mg/mL.

Results are expressed as the percentage of bacteria adhered relative to control (ARC). Values shown are the means of three independent experiments.

d-Configuration monosaccharides, including d-mannose, d-fucose, d-galactose, and d-glucose, can decrease the number of attached C. jejuni to approximately 70% and 80%. Despite the fact that adhesion ability varies among different strains, previous studies have demonstrated that several clinically isolated infective strains can be inhibited in vitro by α-1, 2-fucosylated carbohydrate moieties containing sugars (Ruiz-Palacios et al., 2003). The structure of FOS used in the present study is complicated and mainly consists of α-1,3-fucosylated moieties, which resulted in the failed inhibition of oligosaccharide to C. jejuni adhesion.

The FOS, TOS, and GOS oligosaccharides can decrease the adherence of Salmonella Typhimurium to <50%. In the absence of oligosaccharide treatment, the bacteria showed great adhesive ability to the cells. For example, the adhesion of Salmonella Typhimurium without oligosaccharide treatment (Fig. 1a) was stronger than that in the presence of FOS (Fig. 1b). Several other carbohydrates, including d-mannose, d-galactose, d-fructose, and d-fucose, also exhibited antiadhesive activity to Salmonella Typhimurium.

FIG. 1.

Adherence of Salmonella Typhimurium strain to HT-29 cell lines in the absence (a) and in the presence of fucoidan oligosaccharide (b). (Magnification×1000; scale bar=20 μm.)

COS significantly decreased the ARC values of E. coli and V. cholerae to 41.1% and 18.9%, respectively (Table 2). COS is mainly composed of glucosamine and N-acetylglucosamine, which can function as receptors for many pathogens (Klemm and Schembri, 2000). In a previous study, COS at 2.5 mg/mL could not decrease the adhesion of verotoxigenic E. coli to human cells, but significantly inhibited the adhesion of enteropathogenic E. coli to <30% (Rhoades et al., 2006). Moreover, POS is an oligomer obtained from pectin-rich byproducts and contains a backbone of α-(1, 4)-d-galacturonic acid residue (Martínez Sabajanes et al., 2012). The greatest adherence inhibition on V. cholerae was exhibited by POS, which reduced the ARC to about 16.1%. d-Configuration monosaccharides, including d-mannose, d-fucose, d-fructose, and d-glucose, could also inhibit V. cholerae binding, which is consistent with previous observations (Holmgren et al., 1983).

The antiadhesive ability of several monosaccharides and oligosaccharides was confirmed by the aforementioned experiments (Table 2). The most effective saccharides were selected for dose-dependent experiments. The concentrations of the tested oligosaccharides were 2.5, 2.0, 1.5, 1.0, and 0.5 mg/mL. Results are shown in Figure 2. The antiadhesive effect of many oligosaccharides was dependent on their concentrations. The maximum inhibition of adhesion was obtained at 2.5 mg/mL, which also presented the least ARC. When the concentration was decreased, a concomitant decrease in the antiadhesive effect was observed. A significant antiadhesive effect on V. cholerae was observed in POS, which decreased the ARC to 28.4% even at the lowest concentration level of 0.5 mg/mL. The antiadhesive effect was the highest at a concentration of 2.5 mg/mL of POS, with an ARC of 16.1%. Although all oligosaccharides tested as shown in Figure 2 were capable of inhibiting the adhesion of pathogens to some degree, some oligosaccharides exhibited a different pattern of dose-dependent effect. For example, d-fructose and COS against V. cholerae exhibited the highest antiadhesive activity at 1.5 mg/mL, and decreased the ARC to about 20 and 7.2%, respectively.

FIG. 2.

Effect of oligosaccharide concentration on the adhesion of HT-29 cells by intestinal pathogens (Salmonella Typhimurium, Vibrio cholerae, Escherichia coli, and Campylobacter jejuni). The results represent the mean values of the adhesion number relative to control (ARC). Error bars indicate standard errors of the mean of triplicate assays. *Represents significant differences compared to control with p<0.05. GOS, guar gum oligosaccharide; TOS, tara gum oligosaccharide; FOS, fucoidan oligosaccharide; POS, pectin oligosaccharide; COS, chitooligosaccharide.

Discussion

Compared with traditional chemotherapy approaches, oligosaccharides are more gentle, safe, and ecologically friendly (Sharon, 2006; Mulvey et al., 2001). Many researchers have focused on natural oligosaccharides as a source of potential antiadhesion agents during the past few decades. For example, human milk oligosaccharides could inhibit the attachment of several pathogens to the intestinal tracts of infants (Coppa et al., 1990; Kunz et al., 2000). Cranberry juice, an inhibitive agent of uropathogenic E. coli, could significantly decrease the rate of urinary tract infections in women consuming the juice (Sharon, 2006; Kontiokari et al., 2001). Feeding a diet supplemented with β-1,4-mannobiose during the first 2 weeks after hatching could reduce the susceptibility to Salmonella Enteritidis infection in broilers (Agunos et al., 2007). Thus, the addition of oligosaccharides and analogs in diets has an excellent market prospect. In their project named “Foods for Special Medical Uses,” the Japanese government has already recommended the addition of specific oligosaccharides to food (Tomomatsu, 1994). In the field of animal husbandry, which is currently limited by restriction on antibiotics use (Regulation (EC) No. 1831/2003), functional oligosaccharides used as feed additives to commercial animal diets can greatly reduce the bacterial contaminations, particularly C. jejuni infection (Lengsfeld et al., 2007).

In this work, oligosaccharides were prepared through enzymatic hydrolysis from natural polysaccharides. These food-grade polysaccharides are safe, biodegradable, and are considered good materials for developing commercial products in the broad field of food and pharmacy (Yang and Zhang, 2009). As new enzyme-based technology for degrading polysaccharides has emerged in the past few years, low-cost mass production of oligosaccharides has become economically feasible. Microbial polysaccharide depolymerases, including bacteria-borne and bacteriophage-borne enzymes, represent a natural, nontoxic and highly specific tool for manufacturing novel oligosaccharides with particular biological functions (Rastall, 2010; Chai et al., 2014). Our work will be theoretically beneficial for the study and preparation of antiadhesive oligosaccharides, which could be used as dietary supplements in the food and health-product industries.

For certain oligosaccharides, linear relationships between the antiadhesive effects and their oligosaccharide concentrations were observed. Results shown in Figure 2 suggested that the antiadhesive capability of COS to E. coli increased with the increasing concentration in a suitable concentration range. Previous studies have also reported the dose-dependent effect of oligosaccharides against the adhesion of specific foodborne pathogens (Rhoades et al., 2005; Shoaf et al., 2006). Thus, a sufficient dose is necessary for certain oligosaccharides to block the adhesins located on the bacterial surfaces. Nevertheless, some oligosaccharides exhibited a different pattern of dose-dependent effect. Excessive concentrations of oligosaccharides might attenuate the optimal inhibitory effect. This result indicates that suitable concentration is one of the important factors for future development of antiadhesive oligosaccharides. In addition, factors other than adhesin–receptor interactions, such as hydrophobic and other nonspecific interactions, can also influence the dose-dependent effect in practical applications (Ofek et al., 2003).

Although antiadhesive oligosaccharides have been proven to be effective in in vitro and in vivo animal studies (Mysore et al., 1999; Ofek et al., 2003), more works are still needed in human clinical application to demonstrate their real values. This study showed that no single saccharide treatment can totally eliminate the bacterial adhesion. This consequence may be ascribed to the different types of adhesins expressed by the pathogens that enabled adherence to distinct oligosaccharide receptors located on the host cells (Shoaf et al., 2006). This finding emphasizes the possibility of creating a mixture of several inhibitive sugars that can impede bacterial adhesion in further investigations (Thomas and Brooks, 2004). Possible measures for further development of antiadhesive oligosaccharides include adjusting the oligosaccharide concentration, or a cocktail of oligosaccharides therapy helping to combat certain pathogens that express multiple adhesins (Sharon, 2006).

Conclusions

Results of this work suggest that suitable oligosaccharides derived from natural sources through enzymatic hydrolysis had the potential to reduce the adhesive abilities of intestinal pathogens. Differences in oligosaccharide types and concentrations could affect their antiadhesive abilities. Particularly, pectin oligosaccharides and chitooligosaccharides had a significant effect on reducing the adhesion of V. cholerae to epithelial cells.

Footnotes

Acknowledgments

This work was supported by the Shandong Science and Technology Development Project (2014GHY115037), National Natural Science Foundation of China (30771646 and 41076087), and the Fundamental Research Funds for the Central Universities (201362041).

Disclosure Statement

No competing financial interests exist.

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