Multilocus sequence typing of Clostridium innocuum isolates

We selected housekeeping genes used for typing Clostridium and Clostridioides species from the PubMLST database (https://pubmlst.org/organisms). The homologous of ten house-keeping genes (atpA, ddl, dxr, groL, gyrA, gyrB, mdh, pgk, recA, and rpoB) were identified in C. innocuum, amplified by polymerase chain reaction, and subjected to amplicon sequencing. Simpson’s index of diversity [22, 23], used to evaluate the discriminatory power of the 10 selected genes, was calculated for 52 isolates of C. innocuum from Chang Gung Memorial Hospital at Linkou (CGMH, Taiwan) and 60 sequence-available C. innocuum isolates from the National Center for Biotechnolgy Information (NCBI) Genome Database (https://www.ncbi.nlm.nih.gov/datasets/taxonomy/1522/), as shown in Table 2. Multilocus sequence typing (MLST) analyses typically use seven loci to type bacterial isolates (https://pubmlst.org). Simpson’s index of diversity of groL, pgk, and recA from 112 isolates was less than 0.7 (CGMH + NCBI, Table 2); therefore, these genes were excluded from MLST analysis. Finally, seven genes, including atpA, ddl, dxr, gyrA, gyrB, mdh, and rpoB, were utilized to type C. innocuum isolates.

Table 2 Simpson’s index of the selected genes

Among 52 isolates from CGMH (Taiwan) and 60 isolates from the NCBI Genome Database, 36 and 25 different sequence types (STs) were identified, respectively (Fig. 1A). Among 58 STs, ST1 (5.4%, 6/112), ST6 (11.6%, 13/112) and ST42 (5.4%, 6/112) exhibited a prevalence greater than 5%. Fifty-three STs were exclusive to isolates from specific geographic regions (Fig. 1B). ST1, ST6, and ST51 were found in C. innocuum isolates from both CGMH and the NCBI Genome Database, representing isolates from Taiwan and the USA (Fig. 1A and B). Furthermore, ST37 was identified in C. innocuum isolates from Australia and China, and ST42 was identified in isolates from the USA and Canada (Fig. 1B). Noticeable, there is only one isolate from Australia, Switzerland and the UK; therefore, these results cannot represent the prevalence of STs in these geographic regions.

Fig. 1

Multilocus sequence typing for 52 C. innocuum isolates in Taiwan (CGMH) and 60 sequence-available C. innocuum isolates from the NCBI Genome Database. The housekeeping genes atpA, ddl, dxr, gyrA, gyrB, mdh, and rpoB were utilized for typing C. innocuum isolates. (A) The sequence types (STs) of C. innocuum from CGMH and NCBI Genome Database. (B) The STs of C. innocuum with corresponding geographic distribution information

Phylogenetic analysis and biofilm production activity of C. innocuum isolates

The intestinal microbiota can form a biofilm adhering to the intestinal mucus surface under healthy conditions [11]. To evaluate the biofilm production activity of C. innocuum, we cultured 52 isolates from CGMH for 24 h and analyzed their biofilm production using the crystal violet staining assay. All analyzed C. innocuum isolates were found to be biofilm producers (OD562 > background; acetic acid washes from empty wells). The OD562 values ranged from 0.069 to 3.746, with a median value of 0.184 (Fig. 2A). We categorized C. innocuum isolates according to biofilm formation levels: isolates with OD562 < 0.184 (median) exhibited low-level biofilm formation (25/52, 48%), isolates with 0.184 < OD562 < 0.276 (1.5× median) exhibited medium-level biofilm formation (9/52, 17%), isolates with 0.276 < OD562 < 0.552 (3× median) exhibited high-level biofilm formation (7/52, 14%), and isolates with OD562 > 0.552 exhibited very high-level biofilm formation (11/52, 21%) (Fig. 2B). 52% of C. innocuum isolates (27/52) showed medium to very high levels of biofilm production activity. C. innocuum isolates with ST2, 4, 15, 18, 21, 26, 28, and 58 exhibited a very high-level biofilm mass (Fig. 2A).

Fig. 2

The phylogenetic distance and biofilm production activity of C. innocuum isolates. (A) The sequence type (ST) and biofilm production activity of 52 C. innocuum isolates from the Chang Gung Memorial Hospital at Linkou (CGMH, Taiwan). (B) The percentage distribution of C. innocuum isolates exhibited low (OD562 < 0.184), medium (0.184 < OD562 < 0.276), high (0.276 < OD562 < 0.552), and very high (OD562 > 0.552) levels of biofilm. 0.184 is the median value of the biofilm mass of 52 analyzed C. innocuum isolates. The maximum likelihood phylogenetic tree constructed from the sequence of concentrated housekeeping genes of C. innocuum isolates (C) from the CGMH (n = 51; excluding the outgroup isolate CIN117) or (D) in both CGMH (n = 52) and NCBI Genome Database (n = 60). The scale bar represents nucleotide substitutions per site. The phylogenetical clade and biofilm production activity of C. innocuum isolates from CGMH are labeled in (C). Two biological replicates were performed for biofilm production activity

To assess the relationship between the biofilm production phenotype and the genetic phylogenetics of C. innocuum, the concentrated sequences of the 7 selected loci were used for the phylogenetic analysis based on maximum likelihood estimation. The results showed that the 52 C. innocuum isolates from CGMH could be separated into phylogenetical clade I (13/52, 25%) and clade II (39/52, 75%) (Fig. 2C), with CIN117 as an outgroup isolate (Supplementary Fig. S1). Noticeably, among 11 isolates exhibiting very high-level biofilm formation, 8 isolates were classified as clade I (Fig. 2C), suggesting that the strong biofilm production phenotype was associated with a specific clade of C. innocuum. The phylogenetic distance of 52 isolates from CGMH and 60 isolates from the NCBI database was shown in Fig. 2D. Most analyzed isolates showed close phylogenetic distance except 4 outgroup isolates (CIN117, OF1-2LB, DFI.7.33, and DFI.1.206, Fig. 2D).

Vancomycin treatments did not activate the production of biofilm in C. innocuum

C. innocuum is intrinsically resistant to vancomycin [4, 5]. As vancomycin is one of the primary drugs used to treat Clostridioides difficile infection, it may serve as an external signal or environmental stress for C. innocuum. To evaluate whether vancomycin influences the biofilm production activity of C. innocuum, we selected isolates from clade I and clade IIa (Fig. 2C) with varying levels of biofilm production activities (Fig. 3A) for analysis. The growth of the selected isolates was inhibited by 8 µg/mL vancomycin (except for CIN152) while remaining unaffected by 2 µg/mL and 4 µg/mL vancomycin treatments (Fig. 3B). The crystal violet staining assay showed that the biofilm production of the selected isolates remained similar under 0 µg/mL and 4 µg/mL vancomycin culture conditions (Fig. 3C). This suggests that vancomycin does not appear to be a factor that affect the biofilm production activity of C. innocuum isolates.

Fig. 3

The sublethal concentration of vancomycin treatments did not induce biofilm production in C. innocuum isolates. (A) Biofilm production activity of the selected clade I and IIa C. innocuum isolates. (B) The growth activity of the selected C. innocuum isolates under vancomycin concentrations ranged from 0 to 8 µg/mL. (C) Biofilm production activity of the selected C. innocuum isolates under 0 and 4 µg/mL vancomycin treatments. Two biological replicates were performed for growth activity and biofilm production activity

Biofilm-embedded C. innocuum survived the high concentration of vancomycin treatments

The biofilm provides protection for bacteria against antibiotic elimination. Therefore, this study evaluated whether vancomycin treatments could effectively eliminate C. innocuum inside the biofilm. The selected isolates of clade I and IIa were cultured in the 6-well polypropylene plate to form biofilms under vancomycin-free conditions. After 24 h of incubation, planktonic cells were removed and the biofilm mass in the plate was exposed to vancomycin at concentrations ranging from 4 to 16 µg/mL for an additional 24 h. Crystal violet staining showed that only CIN152 and CIN164 exhibited a statistically significant decrease (ANOVA, p < 0.01) in biofilm mass under 16 µg/mL vancomycin treatments (Fig. 4A).

Fig. 4

Biofilm protection by C. innocuum isolates against high concentrations of vancomycin treatments. (A) Biofilm mass of the C. innocuum isolates after exposure to vancomycin treatments at 0, 4, 8, and 16 µg/mL. (B) The vancomycin biofilm inhibitory concentration (BIC) of C. innocuum isolates, categorized based on their levels of biofilm formation, ranging from low to very high. Three biological replicates were performed for biofilm production and vancomycin BIC. *, P < 0.05

To further verify whether the biofilm could protect C. innocuum isolates from high concentrations of vancomycin treatments, and whether strong biofilm producers would be more resistant to vancomycin than weak biofilm producers, the vancomycin biofilm inhibitory concentration (BIC) [21] was compared for the selected C. innocuum isolates with different levels of biofilm production. Among the 9 isolates that exhibited low to medium levels of biofilm formation, 8 isolates had vancomycin BIC 8 µg/mL, and one isolate (CIN146) showed vancomycin BIC > 256 µg/mL (Fig. 4B). Among 8 isolates exhibiting high to very-high levels of biofilm formation, 7 isolates had vancomycin BIC > 256 µg/mL (Fig. 4B). These results suggest that strong biofilm producers of C. innocuum can survive very high concentrations of vancomycin treatments.