Co-culture models illustrate the digestion of Gemmata spp. by phagocytes

Odilon D Kaboré,Sylvain Godreuil,Michel Drancourt,Ahmed Loukil

doi:10.1038/s41598-018-31667-0

Abstract

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.

Highlights

Free-living amoebae (FLA) are unicellular eukaryotes commonly found in natural aquatic and soil environments[1,2,3]
Extra-phagocyte bacteria were killed by antibiotic treatment with 82 ± 5% and 92 ± 2% of G. obscuriglobus death in Page’s amoebal saline (PAS) and RPMI medium, respectively; and 86 ± 7% and 89 ± 4% of G. massiliana death in PAS and RPMI medium, respectively
The sterility of the third washing and of G. obscuriglobus and G. masiliana-inoculated amoebae as well as that of the THP-1 cells not submitted to thermal shock confirm that further culture observations from the thermal shock lysates derive from intracellular bacteria

Summary

Introduction

Free-living amoebae (FLA) are unicellular eukaryotes commonly found in natural aquatic and soil environments[1,2,3]. Planctomycetes are members of terrestrial and aquatic microbial communities[15,16,17,18,19,20] These organisms have been regularly isolated from various habitats including soil, freshwater lakes, seawater, brackish water lagoon, wastewater habitats and river biofilms[15,16,17,18,19,20,21,22,23]. Some studies reported the relationship between planctomycetes and aquatic microbial communities such as sponges[23,30] and macroalgae[31,32,33], but the interactions between amoebae and Gemmata bacteria have not been explored. After preliminary experiments ensuring the viability of both Gemmata species in the medium used to culture amoebae and in the medium used to culture macrophages, both Gemmata species were exposed to amoebae and macrophages and the intracellular uptake and survival of bacteria were observed by optic and confocal microscopy, and colony-forming unit measurement

Methods

Results

Conclusion