A total of 61 isolates, 41 (67%) Gram positive and 20 (33%) Gram negative strains, were not identifiable using routine methods and were included in the NOVA study within the study period. Thirty-five (57%) organisms were identified to be novel bacterial species and 26 (43%) isolates represented difficult to identify organisms.

Predominant genus was Corynebacterium with 11 isolates (2 C. pseudogenitalium, 1 C. hindlerae, 1 “C. phoceense”, 1 “C. provencense”, and 6 Corynebacterium sp. nov. (Fig. 2), followed by Schaalia sp. with 5 strains.

TYGS GBDP tree showing relationships between Corynebacterium genomes and references. Figure was generated by TYGS (https://doi.org/10.1038/s41467-019-10210-3), with 11 genomes sequenced for this publication shown in blue colour. Species clusters are defined by dDDH with a 70% cutoff shown in the first metadatacolumn. Isolates USB_NOVA_40 and USB_NOVA_41 represent C. pseudogenitalium, isolates USB_NOVA_47, USB_NOVA_57, and USB_NOVA_60 cluster with species C. hindlerae, “C. provencense”, and “C. phoceense”, respectively; the other 6 genomes do not form clusters with any sequenced type strains

The anatomical localization of these 61 clinical samples are indicated in Tables 1 and 2. Predominant specimen was blood culture (n = 9). Detailed microbiological results from the 61 cases including type of specimen, microscopy, cultured isolates, MALDI-TOF MS, and partial 16 S rRNA gene sequencing are listed in Table S1.

Overall, medical history and information on clinical relevance were available from 47/61 cases. In 15/47 of cases, the respective bacterial isolate was considered clinically relevant, and in 21 cases as not clinically relevant. In the remaining 11 cases, clinical relevance was unclear. In 3/15 cases classified as clinically relevant, culture growth was monomicrobial. In 2 of these 3 cases, patients had received antibiotics for > 3 days at the time of sample collection.

The age range of the 47 patients was from 7 to 94, median age 61 years. Thirty (64%) were males and 17 (36%) females.

Twenty-one isolates grew under aerobic and 14 under anaerobic conditions. Twenty-four (69%) strains were Gram positive and 11 (31%) Gram negative. Six isolates belong to novel species within the genus Corynebacterium (Fig. 2), followed by Schaalia (n = 5). There were two strains representative for each of the following genera: Anaerooccus, Clostridium, Desulfovibrio, and Peptoniphilus. For each of the following genera one new species was identified: Citrobacter, Dermabacter, Helcococcus, Lancefieldella, Neisseria, Ochrobactrum (Brucella), Paenibacillus, Pantoea, Porphyromonas, Pseudoclavibacter, Pseudomonas, Psychrobacter, Pusillimonas, Rothia, Sneathia, and Tessaracoccus (Table 1).

The following isolates represent the same novel species based on an ANI index ≥ 96.0: Corynebacterium sp. nov.: isolate USB_NOVA_02 and USB_NOVA_03, ANI 99.96 (Fig. 2); Desulfovibrio sp. nov. USB_NOVA_16 and USB_NOVA_17, ANI 98.5; Peptoniphilus sp. nov. USB_NOVA_18 und USB_NOVA_19, ANI 97.7.

Clinical data were available from 26 cases. Seven/26, 6/26, and 13/26 were classified clinically relevant, unclear, and clinically not relevant, respectively (Table 1).

Twenty-six isolates belong to previously described species which could not be identified by standard identification methods, but only by WGS. These strains represent 19 species already validly published and three species not yet validly published. (Table 2). Seventeen (65.4%) strains were Gram-stain-positive and 9 (34.6%) Gram-stain-negative. Four isolates were identified as Gulosibacter hominis, and one isolate as Pseudoclavibacter triregionum. Both aerobic Gram-stain-positive bacilli have been described taxonomically from our group in collaboration with the BCCM/LMG Bacteria Collection, Ghent, Belgium in 2021 and 2022, respectively [13, 14]. Two isolates represent Corynebacterium pseudogenitalium that has been published validly very recently [16]. In addition, one isolate for each of the following species were identified: Anaerococcus degeneri, Corynebacterium hindlerae, Corynebacterium phoceense, Corynebacterium provencense, Cutibacterium modestum, Devosia equisanguinis, Enterococcus dongliensis, Fenollaria massiliensis, Kingella negevensis, Kingella pumchi, Mogibacter kristiansenii, Pantoea agglomerans, Parvimonas parva, Prevotella brunnea, Pseudomonas yangonensis, Pseudoramibacter alactolyticus, Saezia sanguinis, Slackia exigua, and Vandamella animalimorsus. Of these, “Corynebacterium phoceense”, “Corynebacterium provencense”, and “Kingella pumchi” represent not yet validly published bacterial species. Fifteen (63.6%) from a total of 22 species have been described recently (≥ year 2019) and 14 (53.8%) out of 26 isolates have been isolated before the valid description of the species.

Clinical data were available from 21 cases. Eight/21, 5/21, and 8/21 were classified clinically relevant, unclear, and clinically not relevant, respectively (Table 2).