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. 2023 Jan 20;18(1):e0280676. doi: 10.1371/journal.pone.0280676

Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolates

Noriaki Ide 1,#, Miki Kawada-Matsuo 2,3,#, Mi Nguyen-Tra Le 2,3, Junzo Hisatsune 3,4, Hiromi Nishi 1, Toshinori Hara 4,5,6, Norikazu Kitamura 4, Seiya Kashiyama 4,5,6, Michiya Yokozaki 4,6, Hiroyuki Kawaguchi 1, Hiroki Ohge 4,7, Motoyuki Sugai 3,4, Hitoshi Komatsuzawa 2,3,*
Editor: Guadalupe Virginia Nevárez-Moorillón8
PMCID: PMC9858009  PMID: 36662820

Abstract

Clinical isolates of Clostridioides difficile sometimes exhibit multidrug resistance and cause diarrhea after antibiotic administration. Metronidazole and vancomycin are often used as therapeutic agents, but resistance to these antibiotics has been found clinically. Therefore, the development of alternative antimicrobial agents is needed. Nisin A, produced by Lactococcus lactis, has been demonstrated to be effective against C. difficile infection. In this study, we evaluated the susceptibility of 11 C. difficile clinical isolates to nisin A and found that they could be divided into 2 groups: high and low susceptibility. Since CprABC and DltDABC, which are responsible for nisin A efflux and cell surface charge, respectively, have been reported to be related to nisin A susceptibility, we investigated the expression of cprA and dltA among the 11 strains. cprA expression in all strains was induced by nisin A, but dltA expression was not. The expression levels of both genes did not correlate with nisin A susceptibility in these clinical isolates. To evaluate cell surface charge, we performed a cytochrome C binding assay and found no relationship between charge and nisin A susceptibility. Then, we determined the whole genome sequence of each clinical isolate and carried out phylogenetic analysis. The 11 isolates separated into two major clusters, which were consistent with the differences in nisin A susceptibility. Furthermore, we found common differences in several amino acids in the sequences of CprA, CprB, and CprC between the two clusters. Therefore, we speculated that the different amino acid sequences of CprABC might be related to nisin A susceptibility. In addition, C. difficile strains could be divided in the same two groups based on susceptibility to epidermin and mutacin III, which are structurally similar to nisin A. These results suggest that genotypic variations in C. difficile strains confer different susceptibilities to bacteriocins.

Introduction

Clostridioides difficile is an obligatory anaerobic, spore-forming, gram-positive bacterium. This bacterium produces toxins, toxin A (TcdA), toxin B (TcdB), or binary toxin CDT [1, 2]. In particular, toxin A and toxin B have been demonstrated to be significantly involved in the onset of diarrhea; thus, this bacterium causes colitis related to antibiotic administration [3]. Since some clinical C. difficile strains exhibit multidrug resistance, and the administration of several antibiotics, such as β-lactams and quinolones, sometimes disrupts the composition of normal gut microbiota is then followed by the proliferation of C. difficile [4, 5]. Therefore, this disease is mostly found in health care-associated infections. Metronidazole and vancomycin have generally been used to treat C. difficile infection, but recently, resistance to these antibiotics has been found clinically [4]. Similar to C. difficile, many drug-resistant gram-positive and gram-negative bacteria have significantly challenged the available therapeutic drugs [6]. Therefore, actions against antimicrobial resistance (AMR) have been performed around the world. In the 2019 report of antibiotic resistance threats in the US by the Centers for Disease Control and Prevention (CDC), C. difficile was ranked as an urgent threat, together with carbapenem-resistant Acinetobacter/Enterobacteriaceae [7]. Therefore, the development of new antibacterial agents against C. difficile infection is needed.

Bacteriocins are considered to be new candidate antibiotic agents [8, 9]. Bacteriocins are peptides or proteins that are ribosomally synthesized in some bacterial species, including gram-positive and gram-negative bacteria [10, 11]. Bacteriocins are mainly classified into 2 groups, classes I and II [12, 13]. Class I bacteriocins are post-translationally modified peptides that include the unusual amino acids lanthionine and β-methyllanthionine. Class I bacteriocins include lantibiotics, sactibiotics, linaridins, thiopeptides, glycocins, circular peptides, and bottromycins from gram-positive bacteria and nucleotide peptides and siderophore peptides from gram-negative bacteria [13]. Class II bacteriocins consist of unmodified peptides [14]. Among the many bacteriocins, nisin A produced by Lactococcus lactis has been well characterized and is widely used as a food additive worldwide [15, 16]. Nisin A binds to lipid II on the cell membrane and causes pore formation, resulting in bacterial death [12, 17]. Nisin A also inhibits cell wall peptidoglycan biosynthesis because lipid II is associated with its biosynthesis. Therefore, many investigations regarding the effects of nisin A on pathogenic bacteria, including Staphylococcus aureus, C. difficile and Clostridium perfringens, have been performed [18–22]. However, gram-positive bacteria, including S. aureus, S. mutans and C. difficile, have a two-component system (TCS)-mediated factor for resistance to nisin A [23–27]. In Clostridioides difficile, CprRK is a TCS that regulates the expression of CprABC, an ABC transporter responsible for nisin A resistance [26, 27]. CprK senses nisin A, which is followed by activation of CprR. In addition, it was reported that the Dlt system, which is associated with cell surface charge, was involved in resistance to cationic antimicrobial agents, including nisin A [28].

In this study, we investigated the susceptibility of 11 clinically isolated C. difficile strains to nisin A and found that they could be mainly divided into 2 groups: high and low susceptibility. To elucidate their differences, we performed whole-genome analysis and compared the high- and low-susceptibility strains.

Materials and methods

Bacterial strains and culture medium

The C. difficile strains used in this study are listed in Table 1. These strains except JCM1296T and JCM5243 were isolated from patients at Hiroshima University Hospital. C. difficile JCM1296T (NCTC 11209) and JCM5243 were obtained from the Japan Collection of Microorganisms (JCM). The C. difficile strains were cultured in brain heart infusion (BHI) broth (Becton, Dickinson and Company, New Jersey, USA) at 37°C under anaerobic conditions using the GasPack system (Mitsubishi Gas Chemical Company Inc., Tokyo, Japan). All fresh media were kept under anaerobic conditions for at least 5 hours. Lactococcus lactis (ATCC11454 [25], QU5 [29]), Staphylococcus epidermidis (KSE56, KSE650 [30]), Streptococcus mutans (KSM2, KSM13, KSM157, KSM170 [31]) and Enterococcus mundtii QU2 [32], were grown aerobically in trypticase soy broth (TSB) at 37 °C with (Streptococcus, L. lactis and Enterococcus) or without (S. epidermidis) 5% CO2.

Table 1. Strains used in this study.

Strains Character Reference
Clostridioides difficile JCM5243 Wild type, laboratory strain, ST13 Riken BRC 2
Clostridioides difficile JCM1296T Wild type, laboratory strain, ST3 Riken BRC
Clostridioides difficile H39 Clinical isolate, ST109 This study
Clostridioides difficile H139 Clinical isolate, ST48 This study
Clostridioides difficile H153 Clinical isolate, ST8 This study
Clostridioides difficile H156 Clinical isolate, ST81 This study
Clostridioides difficile H176 Clinical isolate, ST203 This study
Clostridioides difficile H197 Clinical isolate, ST183 This study
Clostridioides difficile H299 Clinical isolate, ST81 This study
Clostridioides difficile H329 Clinical isolate, ST39 This study
Clostridioides difficile H333 Clinical isolate, ST109 This study
Lactococcus lactis ATCC11454 Nisin A-producing strain 26
Lactococcus lactis QU5 Lacticin Q-producing strain 30
Staphylococcus epidermidis KSE56 Epidermin-producing strain 31
Staphylococcus epidermidis KSE650 Nukacin KSE650-producing strain 31
Streptococcus mutans KSM157 Mutacin I-producing strain 32
Streptococcus mutans KSM2 Mutacin III-producing strain 32
Streptococcus mutans KSM170 Mutacin IIIb-producing strain 32
Streptococcus mutans KSM13 Mutacin IV-producing strain 32
Enterococcus mundtii QU2 Mundticin KS-producing strain 33
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1. Sequence type

2. Japan Collection of Microorganisms.

Ethics

All the clinical C. difficile isolates were anonymized prior to obtaining by us, and there was no information about patients. The Ethical Committee for Epidemiology of Hiroshima University reviewed our application and approved it (E2019-1941).

Susceptibility test

Two methods were used for the evaluation of antibacterial activity. To measure the MIC value of nisin A (Sigma–Aldrich, St. Louis, USA), the microdilution method was used as described elsewhere [23]. Bacitracin (Fujifilm Wako chemicals, Osaka, Japan), vancomycin (Sigma–Aldrich), Metronidazole (Fujifilm Wako chemicals), ampicillin (Nacalai Tesque, Kyoto, Japan), chloramphenicol (Wako chemicals), gentamicin (Nacalai Tesque), ofloxacin (Sigma–Aldrich) and imipenem (Fujifilm Wako chemicals) were also used for MIC evaluations.

To assess the antibacterial activity of the bacteriocins, a direct assay was performed by a method described elsewhere [23]. An overnight culture (3 μl) of the bacteriocin-producing strain, as indicator bacteria, was spotted on a TSA plate and cultured at 37°C for 24 h. Then, 3.5 ml of prewarmed BHI soft agar (0.75%) containing C. difficile cells (108 cells/ml) was poured over the TSA plate. The plates were incubated anaerobically at 37°C for 24 h. Then, the diameters of the growth inhibitory zones were measured in three directions. Three independent experiments were performed, and the average diameters were calculated.

Cytochrome c binding assay

A cytochrome c binding assay was performed to evaluate the cell surface charge as described elsewhere [33]. Bacterial cells in the exponential phase were collected and suspended in 10 mM sodium phosphate buffer (pH 6.8) by adjusting to a concentration of 109 cells/ml. Cytochrome c (Sigma Aldrich) was added to the bacterial suspensions at a final concentration of 250 μg/ml. After 10 min of incubation at room temperature, each bacterial suspension was centrifuged at 15 000 rpm for 5 min. The absorbance of the supernatant at 530 nm was measured. By comparing the absorbance values with and without bacterial cells, the absorption ratio was calculated to reflect the bacterial surface charge.

Gene expression analysis by quantitative PCR

Quantitative PCR (qPCR) was performed to investigate the expression of genes involved in bacteriocin susceptibility. Overnight cultures (500 μl each) of the C. difficile strains were inoculated into 5 ml of BHI broth and then grown at 37 °C under anaerobic conditions. After 5 h (0.2 by optical density at 660 nm), nisin A (final concentration: 16 μg/ml) was added to each bacterial culture. After 30 min, the bacterial cells were collected. RNA extraction, cDNA synthesis and qPCR were performed as described previously [23]. The primers used in this study are listed in S1 Table. The expression of each gene was quantified against 16S rRNA expression.

Effect of nisin A on germination

Prior to the germination assay, spores were purified by a method described elsewhere [34]. To evaluate the effect of nisin A on germination, we selected 2 strains with different DNA type, JCM5243 (Group 1) and H39 (Group 2). C. difficile JCM5243 and H39 were grown in BHI broth with 0.5% yeast extract and 0.1% L-cysteine at 37 °C under anaerobic conditions. The overnight culture was then plated on BHI agar with 0.1% L-cysteine and anaerobically incubated at 37 °C. After 7 days of incubation, the plate was placed at 4 °C for 24 h. Bacterial cells were scraped from the agar plate and washed with phosphate-buffered saline (PBS) 2 times. Then, the cells were suspended in PBS containing 125 mM Tris, 200 mM EDTA and 0.3 mg/ml proteinase K and incubated aerobically with gentle agitation at 37 °C for 2 h. Then, the spores were washed with sterilized water 10 times. The spore solution was stored for up to 1 year at 4 °C prior to use. We confirmed the number of spores in each experiment before use.

Before the germination assay, spore stocks were incubated at 65°C for 30 min. Spores (105 spores) were added to BHI broth with or without 0.1% taurocholate (an inducer for germination) in the presence or absence of nisin A (32 or 512 μg/ml). The spore solution was incubated anaerobically at 37°C for 30 min. Then, appropriate dilutions were plated on BHI agar with 0.1% taurocholate and incubated anaerobically at 37°C for 2 days. Colony-forming units (CFU) were counted. Three independent experiments were performed.

Genome sequence analysis of the C. difficile isolates

To perform whole-genome sequencing of the C. difficile isolates, DNA was extracted from each isolate. Genomic DNA was purified from the lysate using AMPure XP beads (Beckman Coulter, USA). DNA libraries were prepared as described previously [35]. Whole-genome sequencing (WGS) of C. difficile isolates was performed using the Illumina MiSeq sequencing platform, followed by annotation with Rapid Annotation using Subsystem Technology (RAST) version 2.0 [36]. A phylogenetic tree was constructed using the CSI Phylogeny 1.4 pipeline available from the Center for Genomic Epidemiology (Lungby, Denmark) for SNP calling and then annotated using the iTOL web-based tool [37]. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

We also compared the 11 clinical genomes with 50 C. difficile genomes that are available in the NCBI database. Fifty genomes were randomly selected from the database. Then, we performed phylogenetic tree analysis and compared the amino acid sequences of CprA, B and C among the 61 genomes, including 11 genomes sequenced in this study.

Accession number

The genome data of the C. difficile isolates used in this study have been deposited into the DDBJ Sequence Read Archive (DRA) accession number DRA014785 under the BioProject accession no. PRJDB14281.

Results

Susceptibility of C. difficile to nisin A

The susceptibility to nisin A varied among the 11 clinical C. difficile isolates (Table 2). The MIC values ranged from 2 to 64 μg/ml. Since the final nisin A concentration used in this study was 2.5%, the calculated MIC range was 0.05 to 1.6 μg/ml. The MIC value for each isolate correlated with the results of the direct assays, as the isolates with higher MIC values showed lower inhibitory zones (Table 2, S1 Fig).

Table 2. Susceptibility to nisin A by MIC and direct assay method.

DNA Group MIC (μg/ml) Halo size1 (mm)
JCM1296T 1 16 16.6 ± 0.00
JCM5243 1 32 15.5 ± 1.19
H139 1 16 16.4 ± 0.99
H153 1 8 16.9 ± 0.14
H176 1 64 15.3 ± 0.31
H197 1 32 15.8 ± 0.85
H39 2 4 17.8 ± 0.65
H156 2 2 19.7 ± 2.83
H299 2 8 17.7 ± 0.50
H329 2 4 19.9 ± 1.98
H333 2 2 20.0 ± 1.59
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1. diameter of inhibition zone.

Based on phylogenetic tree analysis, the 11 isolates were divided into 2 clusters designated Groups 1 and 2 (Fig 1). Group 1 included 6 isolates, showing nisin A MICs of 8 to 64 μg/ml, while Group 2 included 5 isolates, showing nisin A MICs of 2 to 8 μg/ml (Fig 2A). In the direct assay, the diameter length of Group 1 isolates showed smaller than that of Group 2 isolates (Fig 2B). In both assays, the susceptibility of the strains to nisin A in Group 1 was lower than that of Group 2.

Fig 1. Phylogenetic analysis of 11 C. difficile strains.

Fig 1

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A phylogenetic tree was constructed with 11 genome sequences by the method described in Materials and Methods.

Fig 2. Comparison of susceptibility to nisin A in Groups 1 and 2.

Fig 2

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Susceptibility to nisin A between Group1 and Group 2 was compared by (A) MIC method and (B) the direct assay.

Susceptibility of C. difficile strains to several bacteriocins and antibiotics

Since C. difficile displayed susceptibility to nisin A, we evaluated its susceptibility to several lantibiotics and non-lantibiotics (Table 3, Fig 3). The two groups divided by nisin A susceptibility also showed a significant difference in epidermin susceptibility (p = 0.0081, Fig 3). Susceptibility against mutacin I (p = 0.027), mutacin III (p = 0.024) and mutacin IIIb (p = 0.025) also tended to be higher in Group 2 than in Group 1. In contrast, no significant difference in susceptibility to mutacin IV (p = 0.35) was found between the two groups. Mundticin KS and lacticin Q showed no antibacterial activity of 9 strains and 7 strains among 12 strains, and we found no significant differences between the two groups (mundticin KS; p = 0.31, lacticin Q; p = 0.21). In addition, nukacin ISK-1 showed no antibacterial activity against all C. difficile strains. Therefore, lantibiotics with structurally similar to nisin A showed a similar pattern of the susceptibility to nisin A among C. difficile isolates.

Table 3. Susceptibility to various bacteriocin-producing strains by direct assay.

Halo size (mm)
Strains DNA Group Epidermin1 Mutacin I2 Mutacin III3 Mutacin IIIb4 Mutacin IV5 Mundticin KS6 Lacticin Q7
JCM 1296T 1 16.5 ± 0.08 9.4 ± 0.04 10.7 ± 0.45 14.7 ± 0.83 6.2 ± 0.05 0 0
JCM 5243 1 15.7 ± 0.57 9.5 ± 0.08 11.5 ± 0.16 15.1 ± 0.22 6.3 ± 0.37 0 0
H139 1 16.7 ± 0.61 9.5 ± 0.00 13.1 ± 0.22 17.4 ± 0.37 9.5 ± 0.79 0 15.6±0.1
H153 1 18.9 ± 0.37 8.8 ± 0.08 11.8 ± 0.14 15.9 ± 0.28 6.4 ± 0.50 0 13.1±0.3
H176 1 16.5 ± 0.29 10.6 ± 0.16 12.4 ± 0.28 16.4 ± 0.42 6.4 ± 0.08 0 0
H197 1 18.2 ± 0.51 10.0 ± 0.37 11.8 ± 0.54 15.8 ± 0.22 6.4 ± 0.51 10.6±0 0
H39 2 17.6 ± 0.38 9.7 ± 0.43 11.7 ± 0.57 16.3 ± 0.37 6.4 ± 0.08 0 0
H156 2 19.0 ± 0.00 11.1 ± 0.22 12.9 ± 0.08 16.4 ± 0.59 7.4 ± 0.16 0 7.8±0
H299 2 24.1 ± 0.43 12.4 ± 1.02 15.5 ± 0.51 19.6 ± 1.77 7.1 ± 0.16 0 19.6±0.06
H329 2 20.2 ± 0.42 10.1 ± 0.22 13.0 ± 0.99 17.2 ± 0.26 7.1 ± 0.08 10.5±0 0
H333 2 20.0 ± 0.91 10.1 ± 1.14 13.2 ± 0.78 16.9 ± 0.65 7.1 ± 0.16 12.7±0 13.8±0
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1, Staphylococcus epidermidis KSE56

2, Streptococcus mutans KSM157

3, Streptococcus mutans KSM2

4, Streptococcus mutans KSM170

5, Streptococcus mutans KSM13

6, Enterococcus mundtii QU2

7, Lactococcus lactis QU5

Fig 3. Comparison of susceptibility to epidermin, and mutacin I/II/III in Groups 1 and 2.

Fig 3

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The distribution of the inhibitory zones (diameter: mm) are shown. Student’s t test was used to determine significant differences between the Group 1 and Group 2 C. difficile strains.

Then, we further investigated the susceptibility of C. difficile strains to several antimicrobial agents (Table 4, Fig 4). However, susceptibility of Groups 1 and 2 to the other antibacterial agents showed no significant differences.

Table 4. MIC of various antibiotics.

Strains DNA Group MIC (μg/ml)
AMP1 CP2 GM3 IP4 MNZ5 OFLX6 BC7 VC8
JCM1296T 1 2 8 256 4 0.5 8 512 1
JCM5243 1 1 2 128 4 16 8 512 2
H139 1 0.25 1 128 2 0.5 8 256 1
H153 1 2 1 128 4 0.25 32 256 1
H176 1 0.5 1 128 2 0.25 8 256 0.5
H197 1 0.5 1 1024 4 0.125 16 64 2
H39 2 0. 5 2 512 4 16 8 256 0.5
H156 2 1 2 64 4 16 32 512 2
H299 2 4 1 64 2 0.25 32 256 0.5
H329 2 0.5 1 64 1 0.125 32 256 0.25
H333 2 1 1 64 4 0.125 8 32 0.5
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1, Ampicillin

2, Chloramphenicol

3, Gentamicin

4, Imipenem

5, Metronidazole

6, Ofloxacin

7, Bacitracin

8, Vancomycin

Fig 4. Comparison of susceptibility of Groups 1 and Group 2 to antibacterial agents.

Fig 4

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The distribution of MICs is shown. Student’s t test was used to determine significant differences between the Group 1 and Group 2 C. difficile strains.

Expression of cprA and dltD in C. difficile strains

Since cprA-C and dltA-D were previously reported to be associated with nisin A resistance [26–28], we investigated the expression of cprA and dltD in the strains with or without nisin A addition (Fig 5). All isolates showed that cprA expression was significantly induced by the addition of sub-MICs of nisin A, although the expression levels varied among the strains. The expression level in each strain was not separated by the groups classified defined by nisin A susceptibility. In the absence of nisin A, the cprA expression level was quite low in all strains. dltD expression was not induced by the addition of nisin A, and was very low in both the presence and absence of nisin A. The expression level of dltD among the 11 isolates showed no correlation with grouping.

Fig 5. cprA and dltD expression among the 11 C. difficile strains.

Fig 5

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cprA and dltD expression in 11 C. difficile strains with or without nisin A treatment was investigated. Total RNA was extracted from cells in the exponential phase. After cDNA synthesis, quantitative PCR was performed by using specific primers for cprA and dltD. 16S rRNA was used as an internal control.

Cytochrome c binding assay

We further investigated the cell surface charge because cell surface charge was associated with the susceptibility to cationic peptides including nisin A [28]. To evaluate the cell surface charge, a cytochrome binding assay was performed (Fig 6). Binding affinity was similar among all strains. There was no relationship between the binding affinity and nisin A susceptibility.

Fig 6. Cytochrome c binding assay with C. difficile strains.

Fig 6

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A cytochrome c binding assay with C. difficile strains was performed. Student’s t test was used to determine significant differences between the Group 1 and Group 2 C. difficile strains.

Comparison of CprA-C amino acid sequences

Since we found no correlation between nisin A susceptibility and the expression of cprA and dltD, we compared the amino acid sequences of CprA-C, which are intrinsic factors for nisin A resistance, among the strains (Fig 7). We found some consensus differences in several amino acids of CprA-C between Groups 1 and 2. The amino acid residues of CprA at positions 30 (serine), 70 (threonine), 116 (threonine) and 193 (glutamic acid) in Group 1 were different from those in CprA of Group 2 (30 [proline], 70 [serine], 116 [isoleucine] and 193 [lysine]). CprB and CprC in Group 1 also showed differences in 3 and 4 amino acids compared to those in Group 2, respectively.

Fig 7. Comparison of CprABC amino acid sequences among 11 C. difficile strains.

Fig 7

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(A) Phylogenetic analysis of CprA, B and C among 11 C. difficile strains including Group 1 (circle) and Group 2 (square). (B) Conserved amino acid differences between Groups 1 and Group 2.

We further expanded our analysis by using 50 C. difficile genomes randomly selected from the NCBI database (Fig 8, S2 and S3 Figs). Although the clusters groups by phylogenetic tree analysis were further divided into several subgroups, the common differences in CprA-C amino acid patterns between Group 1 and Group 2 were also observed in the other genomes that originated somewhere else.

Fig 8. Phylogenetic analysis of CprA among 61 C. difficile strains.

Fig 8

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A phylogenetic tree was constructed with 11 the genome sequences determined in this study and 50 sequences obtained from the NCBI database. The blue-dashed square and green-dashed square show strains in Group 1 and Group 2, respectively.

Effect of nisin A on C. difficile spores and germinated cells

To know the susceptibility to nisin A on spores and germinated cells in Group 1 and 2, we investigated the susceptibility in JCM5243 (Group 1) and H39 (Group 2). In the presence of 0.1% taurocholate, the addition of nisin A (32 or 512 μg/ml) for 30 min drastically reduced the number of cells of both C. difficile strains JCM5243 and H39 (Fig 9). In particular, nisin A at 512 μg/ml completely killed the bacterial cells of both strains. In the absence of 0.1% taurocholate, the number of spores in JCM5243 (MIC: 32 μg/ml) in the presence of 32 and 512 μg/ml showed 10.8.0% and 12.5% reduction, respectively, while the number of spores in H39 (MIC: 4 μg/ml) in the presence of 32 and 512 μg/ml showed 24.0% and 87.2% reduction, respectively.

Fig 9. Effect of nisin A on spores and germinated cells.

Fig 9

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The effect of nisin A on the spores and germinated cells of strains JCM5243 and H39. Spores were treated with nisin A (32 or 512 μg/ml) for 30 min in the presence and absence of 0.1% taurocholate.

Discussion

In this study, we found that nisin A susceptibility was different among C. difficile strains, which could be divided into 2 groups: high and low nisin A susceptibility. We also found that these groups correlated with the clusters based on whole-genome sequence phylogenetic analysis. Furthermore, it is noteworthy that the amino acid sequences of CprA-C were also divided into 2 groups corresponding to nisin A susceptibility and phylogenetic tree analysis. The cprA-C genes encode an ABC transporter, which consists of 2 permeases and 1 ATPase [26, 27]. CprABC was demonstrated to be responsible for resistance to several lantibiotics, including nisin A. cprK, encoding histidine kinase, was located downstream of cprA-C, while cprR, encoding a response regulator, was separate from the cprA-K regions. CprKR regulates the expression of cprA-C in response to nisin A. We also found that the amino acid sequences of CprK and CprR were divided into 2 groups according to nisin A susceptibility (S4 Fig). However, cprA expression in all tested strains was increased by nisin A addition (Fig 5). The expression levels varied among the strains, but were not correlated with nisin A susceptibility. Therefore, we concluded that the different amino acid sequences of CprRK between the 2 groups were not related to nisin A susceptibility.

By comparing the amino acid sequence of CprA-C, we found that several amino acids (4, 3, and 4 amino acid differences in CprA, B and C, respectively) were commonly different in the 2 groups. We further investigated these differences in CprA-C by randomly selecting 50 C. difficile genome datasets from the NCBI database. The number of clusters was increased after phylogenetic tree analysis, but these common amino acid differences were still found in each cluster (Fig 8, S2 and S3 Figs). Previously, we found amino acid sequence variations in MutFEG, an ABC transporter responsible for nisin A and mutacin I/III/IIIb resistance in Streptococcus mutans [38]. MutFEG was classified into 3 groups (MutFEGs α, β, and γ) based on amino acid sequence. Several amino acid substitutions of MutFEG altered susceptibility to nisin A, mutacin I and mutacin III/IIIb. Based on these results, we suggest that several amino acid differences in CprA-C are associated with susceptibility to nisin A, although we did not directly demonstrate whether these differences affect nisin A susceptibility by replacing cprA-C in each type of strain.

Since we found that the C. difficile strains separated into 2 groups based on nisin A susceptibility, we further investigated their susceptibility to other bacteriocins, including bacteriocins with structures similar to nisin A. The first and second ring structures of epidermin and mutacin I/III/IIIb showed a structure similar to that of nisin A [39] (S5 Fig). In particular, the secondary ring structures of epidermin and mutacin III/IIIb were identical to those of nisin A. In Table 3 and Fig 3, we found tendencies of susceptibility to epidermin and mutacin I/III/IIIb similar to that of nisin A, while we did not find the tendencies of susceptibility to class II bacteriocins. Suárez JM demonstrated that the expression of cprA was induced by several lantibiotics closely related to nisin A, including gallidermin and mutacin III [26]. From these results, it is speculated that the amino acid differences in CprA-C strongly contributed to susceptibility to nisin A and bacteriocins closely related to nisin A, although their contributions to the level of susceptibility were different.

In addition to CprA-C, the Dlt system in gram-positive bacteria including C. difficile has been reported to be involved in resistance to cationic antimicrobial peptides [28, 40–42]. Dlt is involved in alanine incorporation into teichoic or lipoteichoic acid. This incorporation adds a positive charge to these molecules, causing a shift to a weak negative charge on the bacterial cell surface. In this study, we investigated the expression of dltD in all strains in the presence and absence of nisin A, and we found that dltD expression had no correlation with nisin A susceptibility. In addition, the surface charge evaluated by the cytochrome C binding assay showed no correlation between cell surface charge and nisin A susceptibility. Therefore, we think that DltABCD is not associated with the different susceptibilities to nisin A found in this study.

In this study, we also found no correlation between antibiotic susceptibility and the 2 groups. However, we found that the vanXYG genes, which were reported to be involved in vancomycin susceptibility [43], were also only found in type 1 strains, but we did not find a difference in vancomycin susceptibility between the 2 groups. Previous reports also demonstrated that the presence of vanG did not contribute to vancomycin resistance [44]. It is interesting to note that the presence of antibiotic resistance genes is linked to C. difficile types defined by nisin A susceptibility, although the biological interaction between them remains unknown. Bacteriocin resistance is considered to be important for the survival of bacterial microbiota in certain environments, including the human body. Therefore, type 1 strains might have a strong ability to cause host infection compared to type 2 strains. Furthermore, we investigated the genes for CDT, toxin A (TcdA) and toxin B (TcdB) which are major virulence factors in C. difficile [1, 2]. Interestingly, cdtAB genes were only found in Group I except H176 strain, while tcdAB genes were found in 4 (JCM1296T, JCM5243, H153 and H197) of 6 strains in Group1 and 2 (H156 and H299) of 5 strains in Group 2. Therefore, the classification of susceptibility by nisin A is also shown to be related to the presence of the gene for CDT.

To know the different susceptibility to nisin A between Group 1 and Group 2 is found in spores or germinated cells, we also investigated the effect of nisin A on spores and germinated cells (Fig 9). Above the MIC of nisin A, germinated cells induced by 0.1% taurocholate were completely killed after 30 min. In contrast, in the absence of 0.1% taurocholate, strain JCM5243, which has low susceptibility to nisin A, did not show a reduction in viable spores after treatment with nisin A (32 or 512 μg/ml) for 30 min, but the H39 strain showed a reduction in viable spores after the addition of nisin A, although the spores were not completely killed. The surface of C. difficile spores consists of several layers, including the exosporium, coat, and cortex, which exhibit resistance to many environmental stimuli, such as heat, antibiotics and disinfectants [34]. Therefore, it seems that bacteriocins are not effective against C. difficile spores. However, treatment of spores with a high concentration of nisin A in the absence of 0.1% taurocholate reduced the cell (spore) numbers. This result could be explained by the fact that some portions of nisin A attach to the spore surface and show antibacterial activity against the highly susceptible H39 strain after spore germination induced by 0.1% taurocholate.

In conclusion, we first demonstrated the variation in nisin A susceptibility and some lantibiotics that are structurally similar to nisin A. This variation was considered to be due to the differences in CprA-C amino acid sequences that are responsible for nisin A resistance. Moreover, the different amino acid sequences of CprA-C are a result of genomic variations in C. difficile. Our results provide a new characteristic feature of C. difficile strains and give some considerations for the clinical application of nisin A against C. difficile infection.

Supporting information

S1 Fig. Susceptibility to nisin A and epidermin against C. difficile strains.

Direct assay was performed to evaluate the susceptibility to nisin A and epidermin.

(TIF)

Click here for additional data file. (2.4MB, tif)
S2 Fig. Phylogenetic analysis of CprB among 61 C. difficile strains.

A phylogenetic tree was constructed with 11 the genome sequences determined in this study and 50 sequences obtained from the NCBI database. The blue-dashed square and green-dashed square show strains in Group 1 and Group 2, respectively.

(TIF)

Click here for additional data file. (1.4MB, tif)
S3 Fig. Phylogenetic analysis of CprC among 61 C. difficile strains.

A phylogenetic tree was constructed with 11 the genome sequences determined in this study and 50 sequences obtained from the NCBI database. The blue-dashed square and green-dashed square show strains in Group 1 and Group 2, respectively.

(TIF)

Click here for additional data file. (1.1MB, tif)
S4 Fig. Comparison of CprKR amino acid sequences among 11 C. difficile strains.

Phylogenetic analysis of CprK and CprR among 11 C. difficile strains.

(TIF)

Click here for additional data file. (314.6KB, tif)
S5 Fig. Structure of nisin A and its structurally related bacteriocins.

(TIF)

Click here for additional data file. (886.4KB, tif)
S1 Table. Primers used in this study.

(TIF)

Click here for additional data file. (135.6KB, tif)

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Guadalupe Virginia Nevárez-Moorillón

28 Dec 2022

PONE-D-22-27105Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolatesPLOS ONE

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Reviewer #1: The work is well thought out, and carried out the related tests.

The perspectives that the author proposes are appropriate to the results found.

I have brief doubts, which I will write here considering the line number:

Line 18: replace gut microflora with gut microbiota

Line 121: include a “.” after “Table 1”

Line 125: Missing to include the country of Becton Dickinson and Company

Table 1. Why are the recent isolates written as Clostridium, considering that they are now called Clostridioides?

Line 15. Metronidazole should be lowercase in this case.

Line 176: mentions that qPCR, as previously indicated, however, does not include any reference.

Line 182: what is the reason for only using the two indicated strains (JCM5243 and H39)?

Line 190: what was the maximum time before the use of the spores?

Line 225: remove spaces between Table 2 and throughout the text, and check the space between words.

Line 293: unify “Fig.” throughout the text; sometimes, it is not written the same.

Table 4: Is there any parameter according to the CLSI to determine the multi-resistance or susceptibility of C. difficile?

Line 313: from here on, there are values between “( )” bibliographical references that clarify and homogenize the text.

Line 450: replace flora with microbiota

Line 515. Homogenize references; here, the year appears in parentheses, and in the others it does not.

Reviewer #2: In recent years, the problem of drug-resistant bacteria has been the most important issue in the therapeutic treatment of bacterial infections. In the case of Clostridium spp. infection, the occurrence of drug-resistant bacteria has also become serious problem, and the response to this problem needs to be addressed.　In this paper, using 11 clinical isolates of Clostridium difficile, the authors examined the susceptibility to several bacteriocins, including nisin A, and antibiotics. The results of the nisin A susceptibility test showed that the isolates were classified into two major groups, and the classification was similar for the other bacteriocin susceptibility except Mutacin IV. Additionally, no correlation was found between the expression levels of cpr or dlt genes, a group of molecules involved in nisin A resistance, and its susceptibility. Thus, the authors performed a comparative analysis of the whole genome sequences among the isolates to determine the cause of the differences in nisin A susceptibility. As results, the exact same two group formations seen in the susceptibility were confirmed by the genetic information analysis. Furthermore, the authors found that the amino acid sequences of Cpr molecules are unique to each group of bacteria, and speculated that the differences in the amino acid sequences affects the susceptibility to nisinA.

The reviewer considers the current manuscript to be well organized and sufficient for a publication of PLOS ONE if the author will provide suitable responses to the following minor comments;

1. The authors test susceptibility to several bacteriocins in the manuscript. In most bacteriocin susceptibilities, there is a significant difference between the two groups formed by nisinA susceptibility, but not in one. It is strongly expected that this may be caused by structural features of the bacteriocins. However, the current manuscript lacks information on the structure of bacteriocins and their structure-activity relationships. The reviewer recommend that the authors provide a figure showing bacteriocin structure in this regard.

2. Related to comment #1.

What would happen to the susceptibility of isolates if other bacteriocins with less structural homology to nisinA, as like mutacin IV, were used in the test? If the authors have any information on this point, they should provide it or discuss it.

3. The pathogenicity of C. difficile has been well studied, and the typical toxins Tcds and CDT are known to be closely related to its virulence. In this study, the authors have divided the isolates into two groups according to nisinA susceptibility, are there any commonalities in the virulence factors in each group? The authors could add an explanation on this point based on whole genome information, which would further strengthen the value of this paper.

4. The authors need to provide more detailed information regarding the source of the bacteria used in this paper.

5. The authors use taurocholate in the experiments. The purpose of its use should be stated in the Methods section.

6. (Line121) Add period after “Table 1”.

7. (Line176) The authors should add a reference paper regarding qPCR experiment.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2023 Jan 20;18(1):e0280676. doi: 10.1371/journal.pone.0280676.r002

Author response to Decision Letter 0

4 Jan 2023

Reviewer #1: The work is well thought out, and carried out the related tests.

The perspectives that the author proposes are appropriate to the results found.

I have brief doubts, which I will write here considering the line number:

Line 18: replace gut microflora with gut microbiota

Our response: According to the suggestion, we revised it.

Line 121: include a “.” after “Table 1”

Our response: According to the suggestion, we revised it.

Line 125: Missing to include the country of Becton Dickinson and Company

Our response: According to the suggestion, we revised it.

Table 1. Why are the recent isolates written as Clostridium, considering that they are now called Clostridioides?

Our response: According to the suggestion, we revised it.

Line 15. Metronidazole should be lowercase in this case.

Our response: According to the suggestion, we deleted this sentence.

Line 176: mentions that qPCR, as previously indicated, however, does not include any reference.

Our response: According to the suggestion, we put the reference about qPCR method.

Line 182: what is the reason for only using the two indicated strains (JCM5243 and H39)?

Our response: According to the suggestion, we revised it to explain the reason.

Line 190: what was the maximum time before the use of the spores?

Our response: According to the suggestion, we revised it.

Line 225: remove spaces between Table 2 and throughout the text, and check the space between words.

Our response: According to the suggestion, we revised it.

Line 293: unify “Fig.” throughout the text; sometimes, it is not written the same.

Our response: According to the suggestion, we revised it throughout the text.

Table 4: Is there any parameter according to the CLSI to determine the multi-resistance or susceptibility of C. difficile?

Our Response: There are MIC breakpoints of several antibiotics according to CLSI. However, among antibiotics used in this study, vancomycin and metronidazole are included in the MIC breakpoints of C. difficile by CLSI, but other antibiotics are not included. However, we wanted to compare the MIC value between 2 groups and found no correlation between 2 groups.

Line 313: from here on, there are values between “( )” bibliographical references that clarify and homogenize the text.

Our response: According to the suggestion, we revised it.

Line 450: replace flora with microbiota

Our response: According to the suggestion, we revised it.

Line 515. Homogenize references; here, the year appears in parentheses, and in the others it does not.

Our response: According to the suggestion, we revised it.

Reviewer #2: In recent years, the problem of drug-resistant bacteria has been the most important issue in the therapeutic treatment of bacterial infections. In the case of Clostridium spp. infection, the occurrence of drug-resistant bacteria has also become serious problem, and the response to this problem needs to be addressed.　In this paper, using 11 clinical isolates of Clostridium difficile, the authors examined the susceptibility to several bacteriocins, including nisin A, and antibiotics. The results of the nisin A susceptibility test showed that the isolates were classified into two major groups, and the classification was similar for the other bacteriocin susceptibility except Mutacin IV. Additionally, no correlation was found between the expression levels of cpr or dlt genes, a group of molecules involved in nisin A resistance, and its susceptibility. Thus, the authors performed a comparative analysis of the whole genome sequences among the isolates to determine the cause of the differences in nisin A susceptibility. As results, the exact same two group formations seen in the susceptibility were confirmed by the genetic information analysis. Furthermore, the authors found that the amino acid sequences of Cpr molecules are unique to each group of bacteria, and speculated that the differences in the amino acid sequences affects the susceptibility to nisinA.

The reviewer considers the current manuscript to be well organized and sufficient for a publication of PLOS ONE if the author will provide suitable responses to the following minor comments;

1.The authors test susceptibility to several bacteriocins in the manuscript. In most bacteriocin susceptibilities, there is a significant difference between the two groups formed by nisinA susceptibility, but not in one. It is strongly expected that this may be caused by structural features of the bacteriocins. However, the current manuscript lacks information on the structure of bacteriocins and their structure-activity relationships. The reviewer recommend that the authors provide a figure showing bacteriocin structure in this regard.

Our responses: According to the suggestion, we showed the structure of bacteriocins including nisin A and its structural related bacteriocins which were discussed in Discussion section as the supplemental figure (Supplemental Fig. 5).

2. Related to comment #1.

What would happen to the susceptibility of isolates if other bacteriocins with less structural homology to nisinA, as like mutacin IV, were used in the test? If the authors have any information on this point, they should provide it or discuss it.

Our responses: According to the suggestion, we investigated the susceptibility of other bacteriocins with no structural relation to nisin A. As the result, we found no tendency as nisin A susceptibility. Therefore, the different amino acid sequences of CprA-C only affected the susceptibility of nisin A and its-related bacteriocins. We revised Table 3 and Fig. 3 by adding two bacteriocins, and also discussed it in Discussion section.

3. The pathogenicity of C. difficile has been well studied, and the typical toxins Tcds and CDT are known to be closely related to its virulence. In this study, the authors have divided the isolates into two groups according to nisinA susceptibility, are there any commonalities in the virulence factors in each group? The authors could add an explanation on this point based on whole genome information, which would further strengthen the value of this paper.

Our responses: We investigated the genes coding for Cdt and Txoin AB (TcdAB), and found a tendency of cdt genes in Group I and II, but did not find the tendency of tcdAB. We added these results in Discussion section.

4. The authors need to provide more detailed information regarding the source of the bacteria used in this paper.

Our responses: According to the suggestion, we added the detailed information of the strains in Methods section. Also, the strain name of JCM1296 was corrected to JCM1296T.

5. The authors use taurocholate in the experiments. The purpose of its use should be stated in the Methods section.

Our responses: According to the suggestion, we describe the purpose of taurocholate in Material and Methods section.

6. (Line121) Add period after “Table 1”.

Our responses: According to the suggestion, we revised it.

7. (Line176) The authors should add a reference paper regarding qPCR experiment.

Our responses: According to the suggestion, we put the reference.

Attachment

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Decision Letter 1

Guadalupe Virginia Nevárez-Moorillón

6 Jan 2023

Different CprABC amino acid sequences affect nisin A susceptibility in  Clostridioides difficile isolates

PONE-D-22-27105R1

Dear Dr. Komatsuzawa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewers' comments:

Acceptance letter

Guadalupe Virginia Nevárez-Moorillón

12 Jan 2023

PONE-D-22-27105R1

Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolates

Dear Dr. Komatsuzawa:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Guadalupe Virginia Nevárez-Moorillón

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Susceptibility to nisin A and epidermin against C. difficile strains.

    Direct assay was performed to evaluate the susceptibility to nisin A and epidermin.

    (TIF)

    Click here for additional data file. (2.4MB, tif)
    S2 Fig. Phylogenetic analysis of CprB among 61 C. difficile strains.

    A phylogenetic tree was constructed with 11 the genome sequences determined in this study and 50 sequences obtained from the NCBI database. The blue-dashed square and green-dashed square show strains in Group 1 and Group 2, respectively.

    (TIF)

    Click here for additional data file. (1.4MB, tif)
    S3 Fig. Phylogenetic analysis of CprC among 61 C. difficile strains.

    A phylogenetic tree was constructed with 11 the genome sequences determined in this study and 50 sequences obtained from the NCBI database. The blue-dashed square and green-dashed square show strains in Group 1 and Group 2, respectively.

    (TIF)

    Click here for additional data file. (1.1MB, tif)
    S4 Fig. Comparison of CprKR amino acid sequences among 11 C. difficile strains.

    Phylogenetic analysis of CprK and CprR among 11 C. difficile strains.

    (TIF)

    Click here for additional data file. (314.6KB, tif)
    S5 Fig. Structure of nisin A and its structurally related bacteriocins.

    (TIF)

    Click here for additional data file. (886.4KB, tif)
    S1 Table. Primers used in this study.

    (TIF)

    Click here for additional data file. (135.6KB, tif)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (18.8KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting information files.

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