Introduction

In 2016, the genus Propionibacterium was restructured on the basis of genomic evidence, and separated into Propionibacterium sensu stricto and three novel genera: Acidipropionibacterium, Cutibacterium, and Pseudopropionibacterium (Scholz and Kilian 2016). The cutaneous species earlier classified as Propionibacterium acnes, P. avidum, P. granulosum, and P. namnetense (Aubin et al. 2016) were assigned to the new genus Cutibacterium. While C. acnes is well-known for its potential to cause acne vulgaris, post-surgical infections and other human infections, less is known for the other species. In 2011, a novel Propionibacterium species was reported in a patient with humeral infection after revision of a total shoulder arthroplasty. This new species was tentatively named “P. humerusii” based on genomic data (Butler-Wu et al. 2011). Very recently, a Japanese group described an isolate from a patient with inflamed meibomian glands for which they formally proposed the name Cutibacterium modestum sp. nov. (Dekio et al. 2020).

Here, we describe a novel clinical isolate belonging to Cutibacterium modestum from a patient with an infected hip implant. During our analyses of this strain and retrospective data analysis of similar 16S rRNA gene and whole genome sequences, we found evidence that “P. humerusii” and C. modestum represent the same species and that this species is often misidentified as C. acnes.

Material and methods

Culture, identification methods, and antimicrobial susceptibility testing (AST)

Aerobic and anaerobic culture was performed according routine microbiological procedures. For tentative identification, we compared the obtained spectra from the MALDI-TOF MS (microflex LT, Bruker Daltonics) to the current MALDI-TOF database version (MBT 8468 MSP Library, BDAL V9.0.0.0_7854-8468). Partial 16S rRNA gene sequencing was executed as described previously (Hinic et al. 2014) and AST was performed using the gradient diffusion technology (MIC Test Strip; Liofilchem) against 20 antimicrobial agents under anaerobic conditions.

Genome sequencing, assembly and phylogenetic analysis

DNA extracted from isolate 602588-20-USB was sequenced on the Illumina NextSeq platform (PE150) following library creation with Nexteraflex (Illumina). Digital DNA-DNA hybridisation (dDDH) used GGDC2.1 (http://ggdc.dsmz.de/ggdc.php#) and the DDH cut off of < 70%) (Auch et al. 2010). Reads (mean 92 × coverage) were assembled using Unicycler (Wick et al. 2017) to produce an assembly of 2.6 Mb in 22 contigs. The neighbour joining tree using whole genome SNPs was created in CLC genomic workbench v20.0.2 using parameters described in the figure legend (Fig. 1).

Fig. 1
figure 1

Whole genome SNP tree comparing isolates belonging to “P. humerusii” and C. modestum Neighbour joining SNP tree created using CLC genomic workbench v20.0.2. All relevant genomes were downloaded from NCBI, accession numbers are given in the figure. The newly described isolate genome is in bold. Tree rooted using the genome of the type strain of C. acnes: ATCC6919 (GCA_003030305, not shown). Map**, variant calling and single nucleotide polymorphism (SNP) tree creation used parameters that differed from the default as: variant calling with single ploidy, 10 × minimum coverage, 10 minimum count and 70% minimum frequency, and SNP tree creation with 10 × minimum coverage, 10% minimum coverage, 0 prune distance and including multi-nucleotide variants (MNVs). Where required, assemblies were shredded into reads using SAMtools wgsim prior to phylogeny construction. The genome of T33958 was used as the reference (italics) as the M12 assembly is in nine contigs. All data mapped to over 97% of the reference assembly

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Retrospective sequence analysis

Very similar 16S rRNA gene sequences and genome sequences associated with C. modestum were retrieved using the BLAST algorithm and were compared with the sequences from C. modestum strain M12T and our isolate 602588-20-USB.

Results

A 39 year old male patient presented with an implant-related hip infection following internal fixation of a femoral neck fracture. After debridement surgery and antibiotic treatment of Staphylococcus capitis according to susceptibility testing, no signs of persistent infection were identified at the 6 months postoperative control. C. modestum was not covered by antimicrobial therapy. From three cannulated screws sent for culture, growth of Staphylococcus capitis (> 1000 CFU/ml) and Cutibacterium species (80 CFU/ml) was observed. After six days of anaerobic incubation on BD Brucella blood agar (Becton Dickinson), we detected characteristic white colonies and corresponding typical Gram-positive rods that was compatible with presumptive identification of C. acnes. Catalase and indole reactions were positive and confirmed this suggestion. MALDI-TOF was not able to provide valid identification. The first three MALDI-TOF hits were Propionibacterium sp. score 1.64, C. acnes score 1.61 and C. acnes 1.51. Subsequent partial 16S rRNA gene analysis (693 bp) showed 100% identity to 9 sequences of Propionibacterium acnes / Propionibacterium spp./ “P. humerusii” isolates, followed by identities to type reference sequences of C. acnes ATCC6919 (97.7%), C. namnetense NTS31307302 (97.3%), C. avidum ATCC25577 (95.1%) and C. granulosum DSM20700 (93.4%). AST showed that isolate 602588-20-USB had similar patterns to other Cutibacterium spp., thus susceptible to common antimicrobial substances, whereas it was resistant to metronidazole and gentamicin (see supplementary materials table S1).

Retrospective analysis of 16S rRNA gene sequences in public databases resulted in 11 entries with highly similar (99.2–100%) 16S rRNA gene sequences since 2001 (excluding C. modestum M12T). The corresponding isolates originated from different countries and sources showing that this organism has a clinical relevance (Table 1), (Dekio et al. 2020; Kunishima et al. 2001; Lin et al. 2010). Five of these 11 isolates were identified as C. acnes despite considerable differences (97.5–98.2%) towards the 16S rRNA gene sequence of the C. acnes type strain (ATCC 6919T). We also detected within the 16S rRNA gene sequence of C. modestum M12T (LC466959) three mismatches compared to the whole-genome-sequence of M12T (BJEN01000000): position 3 a G instead of A, position 11 a T instead of C, and position 1484 an A instead of G (Dekio et al. 2020) (see also Table 1).

Table 1 List of probable C. modestum isolates (n = 13)
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Genome sequences of six additional “P. humerusii” strains are present in public databases (Table 2), (Butler-Wu et al. 2011; Dekio et al. 2020). Table 2 also includes genome sequence data of our isolate 602588-20-USB, C. modestum strain M12T, and C. acnes ATCC 6919T for comparison. 16S rRNA gene sequences extracted from the “P. humerusii”, 602588-20-USB and C. modestum M12T genomes were 100% identical and dDDH and ANI values indicated that these organisms represented a single species, i.e. C. modestum. In Fig. 1, a whole genome SNP phylogenetic tree of the isolates listed in Table 2 is presented and reveales a high genomic homogeneity among all C. modestum isolates.

Table 2 List of available C. modestum genomes
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In addition, we retrieved more than 100 sequence database entries of uncultured bacterium clone sequences from the human skin with 99.9–100% identity of a length of 1330 bp or longer compared to extracted C. modestum 16S rRNA sequence M12T. These sequences all belong to human skin microbiome data from three different studies (Grice et al. 2009; Kong et al. 2012; Oh et al. 2013).

Genome data availability

Genome data is deposited in ENA project PRJEB41775 and the 16SrRNA gene sequence under accession no. HG992826.

Discussion

We report a novel clinical isolate belonging to the recently described C. modestum (Dekio et al. 2020). Correct species identification was enabled only by partial 16S rRNA gene sequencing and whole genome analysis. Unfortunately, the 16S rRNA gene analysis was hampered by three incorrect nucleotides within the 16S rRNA reference sequence of C. modestum M12T (accession no. LC466959), and incorrect C. acnes species designations in multiple 16S rRNA sequence entries.

The catalase test is an important biochemical characteristic for preliminary identification of Cutibacterium sp. Despite performing API Coryne analysis that comprises the catalase reaction, this result was not reported in the taxonomic proposal of C. modestum as a novel species (Dekio et al. 2020). The indole test is another basic biochemical reaction for biochemical identification of Cutibacterium sp. which was reported negative in the Dekio et al. study (Dekio et al. 2020). In contrast, our strain showed a clear positive indole and positive catalase reaction which confirms data reported for the tentatively characterised “P. humerusii” strain (Butler-Wu et al. 2011).

Routine MALDI-TOF MS failed to identify C. modestum because this species is not yet recorded in the commercial database. Dekio et al. (2020) reported four predominant MALDI-TOF MS peaks at 3493, 3712, 6986 and 7424 Da in C. modestum M12T. The same m/z peaks were also present in the spectrum of our isolate confirming their diagnostic value for future identification of C. modestum with MALDI-TOF MS (see supplementary materials figure S2).

The retrospective analysis of 16S rRNA sequence entries showed that C. modestum represents a bacterial organism of considerable clinical significance which often has been misidentified as C. acnes. Unfortunately, these incorrect data may lead to further misidentifications in current diagnostic applications based on 16S rRNA gene BLAST analysis.

Comparative analysis of the available genomes of this bacterium clearly indicates that C. modestum and “P. humerusii” represent the same species and consequently the “P. humerusii” database entries should be renamed (Table 2). Surprisingly, the corresponding phylogenetic tree only shows minimal genomic differences with exception of strain P5998 among the C. modestum and “P. humerusii” strains despite the origin of the isolates from different continents (Fig. 1).

We demonstrate for the first time that novel C. modestum might represent an organism of the normal skin microbiota. The high similarity to multiple uncultured clone sequences could indicate that this organism is difficult to detect using conventional cultural methods.

To conclude, the isolation and identification of Cutibacterium spp. remains challenging. The correct description of very recently named C. modestum and the availability of a correct 16S rRNA sequence of the type strain may help to clarify the taxonomical uncertainty concerning “P. humerusii”. C. modestum is identical to the previously named “P. humerusii” and represents a further clinically important species within the genus Cutibacterium.