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Fifteen bifidobacterial strains were obtained from faeces of Rousettus aegyptiacus; after grouping them by RAPD PCR only eight were selected and characterized. Analysis of 16S rRNA and of five housekeeping (hsp60, rpoB, clpC, dnaJ, dna G) genes revealed that these eight strains were classified into five clusters: Cluster I (RST 8 and RST 16 T ), Cluster II (RST 9 T and RST 27), Cluster III (RST 7 and RST 11), Cluster IV (RST 19), Cluster V (RST 17) were closest to Bifidobacterium avesanii DSM 100685 T (96.3%), Bifidobacterium callitrichos DSM 23973 T (99.2% and 99.7%), Bifidobacterium tissieri DSM 100201 T (99.7 and 99.2%), Bifidobacterium reuteri DSM 23975 T (98.9%) and Bifidobacterium myosotis DSM 100196 T (99.3%), respectively. Strains in Cluster I and strain RST 9 in Cluster II could not be placed within any recognized species while the other ones were identified as known species. The average nucleotide identity values between two novel strains, RST 16 T and RST 9 T and their closest relatives were lower than 79% and 89%, respectively. In silico DNA-DNA hybridization values for those closest relatives were 32.5 and 42.1%, respectively. Phenotypic and genotypic tests demonstrated that strains in Cluster I and RST 9 T in Cluster II represent two novel species for which the names Bifidobacterium vespertilionis sp. nov. (RST 16 T =BCRC 81138 T =NBRC 113380 T =DSM 106025 T ; RST 8=BCRC 81135=NBRC 113377) and Bifidobacterium rousetti sp. nov. (RST 9 T =BCRC 81136 T =NBRC 113378 T =DSM 106027 T ) are proposed., (Copyright © 2019 Elsevier GmbH. All rights reserved.)