Improved culture of fastidious Gemmata spp. bacteria using marine sponge skeletons

Gemmata are Planctomycetes bacteria recalcitrant to traditional cultivation in the clinical microbiology laboratory and they have been seldom documented in patients. Based on previously known relationships of Planctomycetes with marine sponges, we designed a new culture medium A incorporating marine sponge skeleton of Spongia sp. to the standard culture medium; and culture medium B incorporating Spongia sp. skeleton heat aqueous filtrate into medium A; and inoculating the three culture media (standard, A and B) with Gemmata obscuriglobus DSM 5831^T and Gemmata massiliana DSM 26013^T in the presence of negative controls. Cultures were observed by naked eyes for 7 days and bacterial growth was quantified by microscopic observations and culture-based enumerations. Macroscopic observations at day-3 revealed a pink bacterial pellet in medium B tubes while standard medium tubes remained limpid until day-8. Growing Gemmata spp. bacteria in medium A yielded air bubbles released by bacterial respiration, whereas control tubes remained bubble-free. The number of colonies in standard medium (1.363 ± 115 for G. obscuriglobus, 1.288 ± 83 for G. massiliana) was significantly lower than those counted from medium B (2.552 ± 128 for G. obscuriglobus, 1.870 ± 112 for G. massiliana) and from medium A (2.851 ± 137 for G. obscuriglobus, 2.035 ± 163 for G. massiliana) (p < 0.10^-4) at day-2 incubation. At day-3 incubation, the number of colonies counted from supplemented media A and B increased up to one log than those counted from the control medium (p < 0.10^-4). Along the following day-4-7 incubation, the number of colonies counted from media A and B remained significantly higher compared to standard medium (p < 0.10^-4). These data indicate that incorporation of spongin-based marine sponge skeleton and heat aqueous filtrate of sponge skeleton significantly improved growth of Gemmata spp. bacteria. These observations pave the way towards improved isolation and culture of Gemmata spp. from environmental and clinical specimens.