A combination of the probiotic and prebiotic product can prevent the germination of Clostridium difficile spores and infection

Highlights

• For prevention of C. difficile infection (CDI) a synbiotic product comprising Lactobacillus plantarum Inducia and prebiotic xylitol was applied.

• The synbiotic composition suppressed growth of different C. difficile strains of Estonian and Norwegian origin in vitro.

• The synbiotic composition fully stopped the germination of C. difficile VPI 10463 reference strain in vitro.

• The prefeeding of synbiotic prevented the death of experimental hamsters and colonization of the gut with C. difficile.

• The possibility to apply the synbiotic during antimicrobial treatment for prevention of CDI and its recurrencies is suggested.

Abstract

Clostridium difficile infection (CDI) is one of the most prevalent healthcare associated infections in hospitals and nursing homes. Different approaches are used for prevention of CDI. Absence of intestinal lactobacilli and bifidobacteria has been associated with C. difficile colonization in hospitalized patients.

Our aim was to test a) the susceptibility of C. difficile strains of different origin and the intestinal probiotic Lactobacillus plantarum Inducia (DSM 21379) to various antimicrobial preparations incl. metronidazole, vancomycin; b) the susceptibility of C. difficile strains to antagonistic effects of the probiotic L. plantarum Inducia, prebiotic xylitol (Xyl) and their combination as a synbiotic (Syn) product; c) the suppression of germination of C. difficile spores in vitro and in vivo in animal model of C. difficile infection with Inducia, Xyl and Syn treatment. The VPI strain 10463 (ATCC 43255), epidemic strain (M 13042) and clinical isolates (n = 12) of C. difficile from Norway and Estonia were susceptible and contrarily L. plantarum Inducia resistant to vancomycin, metronidazole and ciprofloxacin. The intact cells of Inducia, natural and neutralized cell free supernatant inhibited in vitro the growth of tested C. difficile reference strain VPI and Estonian and Norwegian clinical isolates of C. difficile after co-cultivation. This effect against C. difficile sustained in liquid media under ampicillin (0.75 μg/ml) and Xyl (5%) application. Further, incubation of Inducia in the media with 5% Xyl fully stopped germination of spores of C. difficile VPI strain after 48 h. In infection model the 48 hamsters were administered ampicillin (30 mg/kg) and 10–30 spores of C. difficile VPI strain. They also received five days before and after the challenge a pretreatment with a synbiotic (single daily dose of L. plantarum Inducia 1 ml of 10¹⁰ CFU/ml and 20% xylitol in 1 ml by orogastric gavage). The survival rate of hamsters was increased to 78% compared to 13% (p = 0.003) survival rate of hamsters who received no treatment. When administered Xyl the survival rate of hamsters reached 56% vs.13% (p = 0.06). In both Syn (6/9, p = 0.003) and Xyl (3/9, p = 0.042) groups the number of animals not colonized with C. difficile significantly increased.

In conclusion, the combination of xylitol with L. plantarum Inducia suppresses the germination of spores and outgrowth into vegetative toxin producing cells of C. difficile and reduces the colonization of gut with the pathogen. Putative therapeutical approach includes usage of the synbiotic during antimicrobial therapy for prevention of CDI and its potential to reduce recurrences of CDI.

Introduction

Clostridium difficile is a gram-positive rod, spore forming anaerobic bacillus and is part of the normal intestinal microbiota. First discovered in 1935 [1] the C. difficile was identified as a cause of pseudomembranous colitis in humans at 1978 [2]. The less severe disorder caused by this bacterium is C. difficile associated diarrhea (CDAD). C. difficile is the cause of approximately 25–30% of all cases of AAD, exposing it as one of the most prevalent healthcare associated infections in hospitals and nursing homes [3], [4], [5], [6], [7]. It is defined as diarrhea occurring two hours to two months after use of antibiotics [4]. C. difficile infection (CDI) symptoms vary amongst patients, ranging from mild to severe cases. The latter may result in toxic megacolon or death. Symptomatic recurrence of CDI occurs in approximately 20% of patients and they can be characterized with increasing age, initial disease severity and hospital exposure [8], [9].

C. difficile infections can currently be controlled by using antibiotic therapy either vancomycin (complicated disease) or metronidazole (mild disease) [10]. Concerning healthcare associated CDI, especially due to the relapses and re-infections, it is necessary to check the susceptibility of C. difficile strains of different origin to special antimicrobials.

The high spread of the intestinal pathogen in hospitals and nursing homes is associated with massive amounts of endospores of high environmental resistance [11], [12]. In order to cause disease, these spores must infect a person, germinate and return to growth of vegetative cells in intestinal tract. Further, the disease pathogenesis involves the actions of secreted toxins, which are produced by vegetative cells, not by spores [13]. Different metabolites of intestinal microbiota may intervene into CDI pathogenesis to stop germination of C. difficile spores. The alternative therapies have suggested the application of intestinal beneficial bacteria in form of probiotics or fecal transplants [14] especially in recurrent infections for correction of the intestinal imbalance e.g. dysbiosis [15], [16], [17].

Disruption of intestinal microecological balance due to antimicrobial treatment is a key factor in the pathogenesis of C. difficile colonization and disease [4], [7], [18]. Absence of intestinal lactobacilli and bifidobacteria has been associated with C. difficile colonization in hospitalized patients [19], [20]. Previous studies for prevention of CDI with probiotics suggest combinations of lactobacilli such as L. acidophilus and L. casei [21], or L. rhamnosus [22], [23]. The ingested probiotics mostly act by stabilizing the gut microbiota and maintaining colonization resistance by prevention of settlement of C. difficile or binding its toxins [24], [25]. The synbiotic (combination of probiotic and prebiotic compounds) may reduce the favorability of the environment for C. difficile.

Prebiotic xylitol is a 5-C sugar alcohol, e.g. pentitol, and is detected in plants, fungi and algae. It is an important intermediate product in carbohydrate metabolism found also in human blood. Xylitol stimulates the growth and activities of some species of microbiota in the large intestines [26]. In the studies on CACO-2 cell lines xylitol prevented the adhesion of vegetative cells of C. difficile reference strain VPI 10463, seemingly blocking the receptors on cells.

However, effective application of antagonistic beneficial bacteria (Lactobacillus spp. probiotic products) against C. difficile substantially depend of strain specificity of applied lactobacillus strain, but also of the used reference strain of C difficile [27]. The strain Lactobacillus plantarum DSM 21379 acronym Inducia^® has several beneficial properties: produces H₂O₂, has strong antioxidative potential and moderate bile salt hydrolase activity. It reduces the level of low-density lipoprotein (LDL)-cholesterol in blood and the oxidative stress level of human body [28], [29].

Our aim was to test a) the susceptibility of C. difficile strains of different origin and the intestinal probiotic L. plantarum Inducia to various antimicrobial preparations incl. metronidazole, vancomycin; b) the susceptibility of C. difficile strains to antagonistic effects of the probiotic L. plantarum strain Inducia, prebiotic xylitol (Xyl), and their combination as a synbiotic product (Syn); c) the suppression of germination of C. difficile spores in vitro and in animal model of C. difficile infection with Inducia, Xyl and Syn treatment.