General microbiological characterization of C. pyruviciproducens strain WYJY-01

C. pyruviciproducens strain WYJY-01 isolated and cultured from abscess drainage fluid was cultured on Columbia blood plates at 37 °C for 48 h, and its colony morphology was round and smooth, and the colony center was convex and transparent (Fig. 1A). The strain WYJY-01 was Gram-positive under the light microscope after Gram staining, and its shape was rod-shaped (Fig. 1B). However, the strain WYJY-01 was identified as Kocuria rosea by VITEK 2 Compact full-automatic microbial analyzer. Because Kocuria rosea is the Gram-positive cocci, it was evident that the results were not consistent with each other [14].

Fig. 1

General identification of the clinical isolate of C. pyruviciproducens strain WYJY-01. A Colony characteristics of the strain WYJY-01 cultured on Columbia blood agar plates. B Morphological characteristics of the strain WYJY-01 under the light microscope after Gram staining. C Lipid requirement test and growth curve determination of the strain WYJY-01

The comparison results based on the GenBank database showed that the 16S rRNA gene sequence of the strain WYJY-01 was the closest to Corynebacterium sp. 718260/2011 (GenBank JX501770.1), with a sequence similarity of 100%. The rpoB gene sequence of the strain WYJY-01 was the closest to that of Corynebacterium sp. NML00-0179 (GenBank GU304660.1), with a sequence similarity of 99.75%. These two strains were clinical isolates of C. pyruviciproducens. Subsequently, a phylogenetic analysis of the 16S rRNA gene with a tree constructed based on the Neighbor-Joining method showed that the strain WYJY-01 was a member of the genus Corynebacterium (Fig. 2).

Fig. 2

The phylogenetic tree of C. pyruviciproducens strain WYJY-01 obtained by the Neighbor-Joining method based on the 1393 bp 16S rRNA gene sequences. This tree shows the phylogenetic relationships between the strain WYJY-01 and closely related species

The difference in biochemical characteristics between the strain WYJY-01 and the strain ATCC BAA-1742 T was (Additional file 1: Table S1): CAMP reaction and reduction of nitrates of strain WYJY-01 are positive; fermentation of D-ribose, D-xylose, D-glucose, maltose and sucrose of strain WYJY-01 were negative.

According to the Clinical and Laboratory Standards Institute (CLSI) guidelines [15], the drug sensitivity of the strain WYJY-01 was measured by the Etest method (Table 1). The strain WYJY-01 was resistant to ceftriaxone, gentamicin, erythromycin, ciprofloxacin, and clindamycin. However, the strain ATCC BAA-1742 T was sensitive to gentamicin and ciprofloxacin.

Table 1 Antimicrobial susceptibility testing results

C. pyruviciproducens strain WYJY-01 was cultured at 37 °C in nutrient broth (NB) medium and NB medium with 1% Tween 80 added. The OD600 of bacterial liquid in different media was measured at intervals to obtain the growth curve of the strain WYJY-01 (Fig. 1C). According to the growth curve, the strain WYJY-01 belongs to the lipophilic microorganism. The strain WYJY-01 was incubated at 37 °C for about 40 h in NB medium supplemented with 1% Tween 80 at a plateau stage.

Pathogenic phenotype of C. pyruviciproducens strain WYJY-01

The macroscopic changes in the organs of BALB/c mice in each group were observed after being dissected. Compared with BALB/c mice injected intraperitoneally with normal saline (Fig. 3A), BALB/c mice injected intraperitoneally with the strain WYJY-01 showed scattered small abscesses on the surface of the liver (Fig. 3B). Compared with BALB/c mice injected with normal saline subcutaneously (Fig. 3C), BALB/c mice injected with the strain WYJY-01 subcutaneously had abscess formation at the injection site (Fig. 3D).

Fig. 3

The macroscopic changes in the livers and skins of BALB/c mice infected with C. pyruviciproducens strain WYJY-01 in different ways

The liver, spleen, and skin of normal and diseased BALB/c mice were taken and paraffin sectioned. After hematoxylin–eosin (HE) staining, the pathological changes in the tissue structure were observed under the light microscope (Fig. 4). The liver portal area of BALB/c mice injected intraperitoneally with the strain WYJY-01 showed cysts of different sizes and irregular shapes, suggesting cyst formation in the portal area (Fig. 4D) and partial central vein dilatation (Fig. 4G); Lymphocyte and neutrophil infiltration can be seen in the liver (Fig. 4H). The spleen of BALB/c mice injected intraperitoneally with the strain WYJY-01 showed reactive proliferation of splenic corpuscles (Fig. 4E). The subcutaneous tissue of BALB/c mice injected subcutaneously with the strain WYJY-01 showed inflammatory infiltration (Fig. 4F), inflammatory necrosis, cellulose-like exudation, and granulation tissue hyperplasia (Fig. 4I).

Fig. 4

Pathological changes of BALB/c mice infected with C. pyruviciproducens strain WYJY-01 in different ways. A HE staining of normal liver tissue. B HE staining of normal spleen tissue. C HE staining of normal skin tissue. D Cysts of different sizes appear in the portal area of ​​the liver. E Reactive hyperplasia of splenic bodies. F Inflammatory infiltration of the subcutaneous tissue. G Central vein dilation in part of the liver. H Infiltration of lymphocytes and neutrophils in liver tissue. I Inflammatory necrosis, fibrinoid exudate, and granulation tissue hyperplasia in the subcutaneous tissue

After the subcutaneous abscess drainage fluid of BALB/c mice that were subcutaneously injected with the strain WYJY-01 was smeared directly, heated, and fixed. Gram-positive and rod-shaped bacteria were seen after Gram staining. The abscess drainage fluid was cultured on Columbia blood plate at 37 °C for 48 h. The colonies isolated and cultured are round, smooth, central bulge, and transparent (Additional file 2: Fig. S1A). Some of the isolated colonies in the blood plate were Gram-stained. Gram-positive and rod-shaped bacteria (Additional file 2: Fig. S1B) were observed under the light microscope, and their morphology was the same as that of the strain WYJY-01. Moreover, the 16S rRNA gene sequence of the isolated colonies from the subcutaneous abscess drainage fluid is consistent with the 16S rRNA gene sequence of the strain WYJY-01. This proves that the colonies recovered as C. pyruviciproducens strain WYJY-01.

Genomic characteristics of C. pyruviciproducens strain WYJY-01

The genome of the strain WYJY-01 was extracted and electrophoresed through agarose gel (Additional file 3: Fig. S2A). Based on the Oxford Nanopore technology platform, the raw sequencing results of the strain WYJY-01 genome showed that the Reads N50 length was 18,025 bp (Additional file 3: Fig. S2B), and the MeanQual was 10.7. Therefore, the sequencing result of the strain WYJY-01 genome was high quality.

The assembled contig sequence was aligned with the Nucleotide database to determine the chromosome type. The assembly generated a single circular chromosome with total size of 2,881,050 bp, and the GC content was 60.76%. The GC content of the strain ATCC BAA-1742 T genome was 61.1% (BioProject ID PRJNA78965). The GC content of the strain UMB0763 genome was 60.8% (BioProject ID PRJNA316969). It can be seen that the difference in GC content among the three C. pyruviciproducens species was less than 1%. The results based on the GC content also supplemented the identification of the species of the strain WYJY-01.

The genome of the strain WYJY-01 was compared with the most similar sequence in the Nr database by BLAST. Thus, the species distribution of the matched sequence was calculated (Additional file 4: Fig. S3A). The statistical chart showed that 85.71% of the sequences in the genome of the strain WYJY-01 were most similar to C. pyruviciproducens, and 4.34% of the sequences were similar to C.glucuronolyticum. Based on the genome components predicted by the database and the gene COG function classification predicted by the eggNOG database (Additional file 4: Fig. S3B), the location of annotated loci between the genome components of the strain WYJY-01 was presented by Circos v0.66 (Fig. 5).

Fig. 5

Circular genome map of C. pyruviciproducens strain WYJY-01. The outermost circle is the indication of genome size, each scale is 5 kb; the second and third circles are genes on the positive and negative strands of the genome, respectively, and different colors represent different COG functional classifications; the fourth circle is repetition Sequence; the fifth circle is tRNA and rRNA, blue is tRNA, and purple is rRNA; the sixth circle is GC content, the light yellow part indicates that the GC content in this region is higher than the average GC content of the genome, and the higher the peak, the higher the average GC content. The greater the difference, the blue part indicates that the GC content of the region is lower than the average GC content of the genome; the innermost circle is the GC-skew, dark gray represents the region with G content greater than C, and red represents the region with C content greater than G

The ANI value based on the strain WYJY-01 and ATCC BAA-1742 T was 97.44%. And the ANI value based on the strain WYJY-01 and UMB0763 was 98.56%. The dDDH value based on the strain WYJY-01 and ATCC BAA-1742 T was 76.70%. And the dDDH value based on the strain WYJY-01 and UMB0763 was 87.00%. Venn diagram of gene families of the strain WYJY-01, ATCC BAA-1742 T, and UMB0763 genome showed (Additional file 5: Fig. S4) that there were 19 unique gene families in the genome of the strain WYJY-01, and 128 gene families different from strain ATCC BAA-1742 T genome. Furthermore, the number of unique genes of the strain WYJY-01 was 314, and the number of differential genes different from strain ATCC BAA-1742 T was 441.

Virulence factors and antimicrobial resistance genes

By searching the Comprehensive Antibiotic Resistance Database (CARD) using BLSAT, the predicted resistance genes in the genome of the strain WYJY-01 were ermX, aph(3')-Ia,aph(3″)-Ib,aph(6)-Id, cmx. ErmX is an rRNA methyltransferase that protected the ribosome from inactivation due to antibiotic binding. Therefore, ErmX made bacteria resistant to macrolide antibiotics, lincosamide antibiotics, and streptogramin antibiotics by changing the target of antibiotics. APH(3')-Ia, APH(3″)-Ib, APH(6)-Id were aminoglycoside phosphotransferases, which can enable bacteria to acquire resistance against aminoglycoside antibiotics by inactivating antibiotics. Cmx was a chloramphenicol exporter, which can make bacteria resistant to phenicol antibiotics and chloramphenicol by exuding antibiotics.

By searching the Virulence Factor Database (VFDB) using BLAST, it was predicted that the genes in the genome of the strain WYJY-01 might be involved in encoding the virulence factors, including capsule, intercellular adhesion proteins, hemolysin, type III secretion system (TTSS), type IV secretion system, type VI secretion system, type VII secretion system, rhamnolipid, mucoid exopolysaccharide, SpaA-type pilus, polar flagella, biofilm, adhesin, metal ions transporter (mainly iron ions), P60 extracellular protein, etc.

Horizontal gene transfer is the exchange of genetic information between different species of bacteria through the mobile components. Mobile components can carry virulence factor genes and resistance genes, including genomic islands, prophages, insertion sequences, integrons, transposons, plasmids, etc. [16, 17].

The prophage region of the genome of the strain WYJY-01 predicted by PhiSpy v2.3 was 69,911 bp in length (starting at 814,919 bp and ending at 884,829 bp). Combined with the VFDB prediction results, the virulence factor genes in the prophage region of the genome include farB, clpP, hspX, and flpF (Fig. 6A). Among them, FarB was a fatty acid efflux system protein, and it mediated the resistance to antimicrobial long-chain fatty acids. HspX stabilized cell structure during long-term survival and permitted the bacilli to survive within the low-oxygen environment of the granuloma.

Fig. 6

Horizontal gene transfer of virulence factor genes and resistance genes in the genome of C. pyruviciproducens strain WYJY-01. A Virulence factor genes in prophage regions in the genome of the strain WYJY-01. B-E Resistance genes and virulence factor genes in genomic islands in the genome of the strain WYJY-01. Diff, expressed as the differential gene between the strain WYJY-01 and ATCC BAA-1742 T

There were 13 genomic islands predicted by IslandPath-DIMOB v0.1 in the genome of the strain WYJY-01. Genomic island 4 (Fig. 6B) started at 613,513 bp and ended at 647,861 bp. The virulence factor genes in this genomic island include irp2, fbpC, irtB, irtA, pchC, rhlB, cap8J, and cylA. Meanwhile, these genes were the different genes that were different from the genome of the strain ATCC BAA-1742 T. Genomic island 6 (Fig. 6C) started at 885,274 bp and ended at 928,269 bp. The genomic island contained virulence factor genes, including irtA, iroC, phoR, phoP, and aha1389. It was worth noting that the five resistance genes in the genome of the strain WYJY-01 searched by the CARD were all located on Genomic island 6. Genomic island 8 (Fig. 6D) started at 1,494,249 bp and ended at 1,505,409 bp, and Genomic island 10 (Fig. 6E) started at 1,916,529 bp and ended at 1,928,888 bp. The virulence factor genes contained in these two genomic islands included bopD, fbpC, wzt, cpsE, and ugd, and these genes were also differential genes from the genome of the strain ATCC BAA-1742 T.

The results of gene function annotated by the KEGG database (Fig. 7) showed the functions of the differential genes between the strain WYJY-01 and the strain ATCC BAA-1742 T were mainly clustered in environmental information processing (Fig. 7B). The differential genes clustered into ABC transporter and predicted by VFDB included fbpC on Genomic island 4, fbpC on Genomic island 8, and wzt on Genomic island 10, shuU, and fepC. One of the differential genes clustered into the two-component system was the phoR of Genomic island 6. The results of gene function annotated by the GO database showed (Additional file 6: Fig. S5) that the proportion of differential genes in the two categories of the extracellular region and electron carrier activity in each functional category was significantly higher than that of the genome of the strain WYJY-01.

Fig. 7

The KEGG database annotations of the genome of C. pyruviciproducens strain WYJY-01 and its differential genes with that of ATCC BAA-1742 T. A KEGG cluster analysis of the genome of the strain WYJY-01. B KEGG cluster analysis of differential genes between the genomes of the strain WYJY-01 and ATCC BAA-1742 T

Secondary metabolite gene clusters

The secondary metabolite gene clusters of the strain WYJY-01 predicted by antiSMASH v5.0.0 consists of two regions. The non-ribosomal polypeptides (NRPS) gene cluster (Fig. 8A) in the genome started at 585,737 bp and ended at 641,084 bp, and the secondary metabolites were roughly predicted as (cys) + (cys—cys), peptide bonds linked three Cysteines. The polyketides (T1PKS) gene cluster (Fig. 8B) in the genome started at 1,167,876 bp and ended at 1,212,552 bp, and the secondary metabolite was roughly predicted to be pyruvic acid (CH3COCOOH). It was worth noting that the core structure region of the NRPS gene cluster had overlapping genes with Genomic island 4 (Fig. 6B), and most of the genes in the core structure region were the differential genes from the genomic of the strain ATCC BAA-1742 T.

Fig. 8

Secondary metabolite gene clusters in the genome of C. pyruviciproducens strain WYJY-01. A Prediction of non-ribosomal polypeptide (NRPS) gene clusters in the genome of the strain WYJY-01. B Prediction of the polyketide (T1PKS) gene cluster in the genome of the strain WYJY-01. Diff, expressed as the differential gene between the strain WYJY-01 and ATCC BAA-1742 T