Aix Marseille Univ., IRD, MEPHI, IHU Méditerranée Infection, Marseille, France.
Université de Montpellier UMR 1058 UMR MIVEGEC, UMR IRD 224-CNRS Inserm, 1058, Montpellier, France.
Sci Rep. 2018 Sep 6;8(1):13311. doi: 10.1038/s41598-018-31667-0.
Gemmata spp. bacteria thrive in the same aquatic environments as free-living amoebae. DNA-based detection of Gemmata spp. sequences in the microbiota of the human digestive tract and blood further questioned the susceptibility of Gemmata spp. to phagocytes. Here, Gemmata obscuriglobus and Gemmata massiliana were co-cultured with the amoebae Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba griffini and THP-1 macrophage-like phagocytes. All experiments were performed in five independant replicates. The ratio amoeba/bacteria was 1:20 and the ratio THP-1/bacteria was 1:10. After a 2-hour co-culture, extracellular bacteria were killed by kanamycin or amikacin and eliminated. The intracellular location of Gemmata bacteria was specified by confocal microscopy. Microscopic enumerations and culture-based enumerations of colony-forming units were performed at T = 0, 1, 2, 3, 4, 8, 16, 24, 48 and 72 hours post-infection. Then, Gemmata bacteria were engulfed into the phagocytes' cytoplasmic vacuoles, more than (98 ± 2)% of Gemmata bacteria, compared to controls, were destroyed by phagocytic cells after a 48-h co-culture according to microscopy and culture results, and no positive culture was observed at T = 72-hours. Under our co-culture conditions, Gemmata bacteria were therefore susceptible to the environmental and host phagocytes here investigated. These data suggest that these Acanthamoeba species and THP-1 cells cannot be used to isolate G. massiliana and G. obscuriglobus under the co-culture conditions applied in this study. Although the THP-1 response can point towards potential responses that might occur in vivo, these responses should first bevalidated by in vivo studies to draw definite conclusions.
Gemmata 细菌在与自由生活的变形虫相同的水生环境中繁衍生息。在人类消化道和血液的微生物群中，基于 DNA 的 Gemmata 序列检测进一步质疑了 Gemmata 对吞噬细胞的易感性。在这里，将 Gemmata obscuriglobus 和 Gemmata massiliana 与变形虫 Acanthamoeba polyphaga、Acanthamoeba castellanii、Acanthamoeba griffini 和 THP-1 巨噬细胞样吞噬细胞共培养。所有实验均在五个独立重复中进行。变形虫/细菌的比例为 1:20，THP-1/细菌的比例为 1:10。共培养 2 小时后，用卡那霉素或阿米卡星杀死并消除细胞外细菌。通过共聚焦显微镜确定 Gemmata 细菌的细胞内位置。在感染后 T=0、1、2、3、4、8、16、24、48 和 72 小时时进行显微镜计数和基于培养的菌落形成单位计数。然后，Gemmata 细菌被吞噬细胞的细胞质空泡吞噬，与对照相比，在 48 小时共培养后，超过（98±2）%的 Gemmata 细菌被吞噬细胞破坏，在 T=72 小时时未观察到阳性培养。在我们的共培养条件下，Gemmata 细菌因此易受环境和宿主吞噬细胞的影响。这些数据表明，在本研究中应用的共培养条件下，这些棘阿米巴物种和 THP-1 细胞不能用于分离 G. massiliana 和 G. obscuriglobus。尽管 THP-1 的反应可以指出体内可能发生的潜在反应，但这些反应首先应通过体内研究进行验证，以得出明确的结论。
