Rätsep M, Kõljalg S, Sepp E, Smidt I, Truusalu K, Songisepp E, Stsepetova J, Naaber P, Mikelsaar R H, Mikelsaar M
Institute of Biomedicine and Translational Medicine, University of Tartu, EE 50411, Estonia; Bio-Competence Centre of Healthy Dairy Products LLC, EE 51014, Estonia.
Institute of Biomedicine and Translational Medicine, University of Tartu, EE 50411, Estonia.
Anaerobe. 2017 Oct;47:94-103. doi: 10.1016/j.anaerobe.2017.03.019. Epub 2017 Apr 29.
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 10 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.
艰难梭菌感染（CDI）是医院和疗养院中最常见的医疗保健相关感染之一。预防CDI采用了不同的方法。住院患者肠道中缺乏乳酸杆菌和双歧杆菌与艰难梭菌定植有关。我们的目的是测试：a）不同来源的艰难梭菌菌株和肠道益生菌植物乳杆菌Inducia（DSM 21379）对包括甲硝唑、万古霉素在内的各种抗菌制剂的敏感性；b）艰难梭菌菌株对益生菌植物乳杆菌Inducia、益生元木糖醇（Xyl）及其作为合生元（Syn）产品的组合的拮抗作用的敏感性；c）在艰难梭菌感染动物模型中，用Inducia、Xyl和Syn处理在体外和体内对艰难梭菌孢子萌发的抑制作用。来自挪威和爱沙尼亚的艰难梭菌VPI菌株10463（ATCC 43255）、流行菌株（M 13042）和临床分离株（n = 12）对万古霉素、甲硝唑和环丙沙星敏感，相反，植物乳杆菌Inducia对这些药物耐药。Inducia的完整细胞、天然和中和的无细胞上清液在共培养后，在体外抑制了受试艰难梭菌参考菌株VPI以及爱沙尼亚和挪威艰难梭菌临床分离株的生长。在氨苄青霉素（0.75μg/ml）和Xyl（5%）存在的情况下，这种对艰难梭菌的作用在液体培养基中持续存在。此外，将Inducia在含5% Xyl的培养基中孵育48小时后，完全阻止了艰难梭菌VPI菌株孢子的萌发。在感染模型中，给48只仓鼠施用氨苄青霉素（30mg/kg）和10 - 30个艰难梭菌VPI菌株的孢子。在攻毒前后五天，它们还接受了合生元预处理（通过灌胃每天单剂量给予1ml含10CFU/ml植物乳杆菌Inducia和1ml含20%木糖醇）。与未接受治疗的仓鼠13%的存活率相比，接受治疗的仓鼠存活率提高到了78%（p = 0.003）。当给予木糖醇时，仓鼠的存活率达到56%，而未治疗组为13%（p = 0.06）。在合生元组（6/9，p = 0.003）和木糖醇组（3/9，p = 0.042）中，未被艰难梭菌定植的动物数量均显著增加。总之，木糖醇与植物乳杆菌Inducia的组合可抑制艰难梭菌孢子的萌发并阻止其生长为产生毒素的营养细胞，减少肠道中该病原体的定植。可能的治疗方法包括在抗菌治疗期间使用合生元以预防CDI及其降低CDI复发的可能性。
