Romyasamit Chonticha, Thatrimontrichai Anucha, Aroonkesorn Aratee, Chanket Wannarat, Ingviya Natnicha, Saengsuwan Phanvasri, Singkhamanan Kamonnut
Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand.
Department of Pediatrics, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand.
Front Pediatr. 2020 Sep 25;8:572633. doi: 10.3389/fped.2020.572633. eCollection 2020.
infection is implicated as a major cause of antibiotic-associated diarrhea in hospitals worldwide. Probiotics, especially lactic acid bacteria, are the most frequently used alternative treatment. This study aims to identify potential probiotic enterococci strains that act against strains and exert a protective effect on colon adenocarcinoma cells (HT-29 cells). To this end, nine strains isolated from the feces of breast-fed infants were investigated. They were identified as by 16s rRNA sequencing and MALDI-TOF. The probiotic properties including their viabilities in simulated gastrointestinal condition, cell adhesion ability, and their safety were evaluated. All strains exhibited more tolerance toward both pepsin and bile salts and adhered more tightly to HT-29 cells compared with the reference probiotic strain ATCC 14917. Polymerase chain reaction (PCR) results exhibited that six of nine strains carried at least one virulence determinant gene; however, none exhibited virulence phenotypes or carried transferable antibiotic resistance genes. These strains did not infect when compared to pathogenic strain ( < 0.05). Moreover, their antibacterial activities against were examined using agar well-diffusion, spore production, and germination tests. The six safe strains inhibited spore germination (100 - 98.20% ± 2.17%) and sporulation, particularly in ATCC 630 treated with PK 1302. Furthermore, immunofluorescence assay showed that the cytopathic effects of of HT-29 cells were reduced by the treatment with the cell-free supernatant of strains. These strains prevented rounding of HT-29 cells and preserved the -actin microstructure and tight junctions between adjacent cells, which indicated their ability to reduce the clostridial cytopathic effects. Thus, the study identified six isolates that have anti- activity. These could be promising probiotics with potential applications in the prevention of colonization and treatment of infection.
感染被认为是全球医院中抗生素相关性腹泻的主要原因。益生菌，尤其是乳酸菌，是最常用的替代治疗方法。本研究旨在鉴定对艰难梭菌菌株有作用并对结肠腺癌细胞（HT-29细胞）发挥保护作用的潜在益生菌肠球菌菌株。为此，对从母乳喂养婴儿粪便中分离出的9株艰难梭菌菌株进行了研究。通过16s rRNA测序和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）将它们鉴定为艰难梭菌。评估了它们在模拟胃肠道条件下的活力、细胞粘附能力及其安全性等益生菌特性。与参考益生菌菌株ATCC 14917相比，所有菌株对胃蛋白酶和胆盐均表现出更高的耐受性，并且与HT-29细胞的粘附更紧密。聚合酶链反应（PCR）结果显示，9株菌株中有6株携带至少一个毒力决定因素基因；然而，没有一株表现出毒力表型或携带可转移的抗生素耐药基因。与致病性艰难梭菌菌株相比，这些菌株不会感染（P<0.05）。此外，使用琼脂孔扩散法、孢子产生和萌发试验检测了它们对艰难梭菌的抗菌活性。这6株安全菌株抑制孢子萌发（100 - 98.20%±2.17%）和孢子形成，特别是在用PK 1302处理的艰难梭菌ATCC 630中。此外，免疫荧光分析表明，用艰难梭菌菌株的无细胞上清液处理可降低HT-29细胞中艰难梭菌的细胞病变效应。这些菌株可防止HT-29细胞变圆，并保留肌动蛋白微结构和相邻细胞之间的紧密连接，这表明它们有能力降低梭菌的细胞病变效应。因此，该研究鉴定出6株具有抗艰难梭菌活性的分离株。这些可能是有前景的益生菌，在预防艰难梭菌定植和治疗艰难梭菌感染方面具有潜在应用价值。
