Mechanism of Antibacterial Activity of Bacillus amyloliquefaciens C-1 Lipopeptide toward Anaerobic Clostridium difficile

Probiotics may offer an attractive alternative for standard antibiotic therapy to treat Clostridium difficile infections (CDI). In this study, the antibacterial mechanism in vitro of newly isolated B. amyloliquefaciens C-1 against C. difficile was investigated. The lipopeptides surfactin, iturin, and fengycin produced by C-1 strongly inhibited C. difficile growth and viability. Systematic research of the bacteriostatic mechanism showed that the C-1 lipopeptides damage the integrity of the C. difficile cell wall and cell membrane. In addition, the lipopeptide binds to C. difficile genomic DNA, leading to cell death. Genome resequencing revealed many important antimicrobial compound-encoding clusters, including six nonribosomal peptides (surfactins (srfABCD), iturins (ituABCD), fengycins (fenABCDE), bacillibactin (bmyABC), teichuronic, and bacilysin) and three polyketides (bacillaene (baeEDLMNJRS), difficidin (difABCDEFGHIJ), and macrolactin (mlnABCDEFGHI)). In addition, there were other beneficial genes, such as phospholipase and seven siderophore biosynthesis gene clusters, which may contribute synergistically to the antibacterial activity of B. amyloliquefaciens C-1. We suggest that proper application of antimicrobial peptides may be effective in C. difficile control.


Introduction
Clostridium difficile is an anaerobic, gram-positive, sporeforming bacterium. Clinical signs of C. difficile infection (CDI) range from mild diarrhea to fulminant colitis [1]. The incidence and severity of CDI have increased significantly, especially by the recently emerged and highly virulent epidemic strain BI/NAP1/027 [2]. With increasing antibiotic resistance of C. difficile, there is an urgent need to develop new agents and efficient methods for the treatment and control of CDI [1,3]. Distinct from the traditional antibiotics, many novel antimicrobial agents, such as ramoplanin, surotomycin, and cadazolid, are currently being investigated in clinical trials for the treatment of CDI. Surotomycin is an orally dosed lipopeptide antibiotic that acts by disrupting the cell membrane [4].
Bacteriocins are ribosomally synthesized antimicrobial peptides with high activity against other bacteria. Bacteriocins are secreted by some probiotic microorganisms, such as Lactobacillus species, Saccharomyces boulardii, and bifidobacteria [5]. Thus, further evaluation should be given to the bacterial resources, antimicrobial mechanisms, and biosafety of probiotics in considering bacteriocins as an alternative or adjunctive therapeutic method for CDI.
Except the ribosomally synthesized antimicrobial peptides, Bacillus species could synthetize a mixture of lipopeptides by nonribosomal peptide synthetases, which mainly include surfactin, iturin, lichenysin, and fengycin families, with broad-spectrum biological activities [6][7][8]. With the described functional secondary metabolites, many Bacillus spp. strains have been developed as biofertilizers and biopesticides, and they are currently regarded as promising environmentally friendly means for plant protection and plant growth promotion, and as secondary metabolite factories [9]. However, for B. amyloliquefaciens, there are only a few reports that describe an antimicrobial activity against toxin-producing C. difficile [10].
We isolated B. amyloliquefaciens C-1 from ready-to-eat fruit samples. The bacterial strain is stored in the China Center for Type Culture Collection as a patent strain with the number of CCTCC M2010177. The supernatant of C-1 showed high antioxidant activity and inhibitory activity against foodborne pathogens (Escherichia coli O157:H7, B. cereus, S. aureus, etc.) and human pathogens (C. difficile, Klebsiella pneumoniae, Enterococcus faecium, etc.) [11,12]; however, no effect against fungi was found.
Comparative genomic analysis showed evolutionary traits for B. amyloliquefaciens strain adaptation to host habitats [13]. The C-1 strain exhibited a biosurfactant activity phenotype against pathogens. The molecular bases/mechanisms of this pathogen-specific activity were unknown. In this study, we investigated the anti-C. difficile mechanisms of the secreted B. amyloliquefaciens C-1 extracellular lipopeptides. We systematically investigated the effects of C-1 lipopeptide on C. difficile cell growth, morphological structure, cell wall and membrane integrity, and genome. Then, we resequenced the entire C-1 genome and identified relevant gene clusters, locations, and potential regulatory sequences, including genes for bacteriocins, ribosomally synthesized antibacterial peptides, phospholipase, siderophores, and genes that provide resistance to toxic compounds.
2.2. Isolation and Identification of B. amyloliquefaciens C-1 Extracellular Lipopeptide. Lipopeptide isolation was performed by acid precipitation according to Zhang et al. [14]. Briefly, the crude cell-free culture was adjusted to pH 2.0 with 6 M HCl and placed overnight at 4°C. After centrifugation, the precipitate was extracted twice with methanol. The solution was dried in a vacuum freeze dryer, and the dry residue was dissolved in 50 mM Tris-HCl (pH 7.5) and passed through a 0.22 μm filter. The extracted lipopeptide sample was analyzed with a UV-VIS spectrophotometer (UV5, Mettler Toledo).

2.3.
Thin-Layer Chromatography. The purified lipopeptide was examined by thin-layer chromatography (TLC) on a silica gel G plate [15]. TLC assay was performed on a silica gel G plate (10 × 20 cm, Silica gel 60, Germany). A chloroformmethanol mixture (10 : 1, v/v) was used as the mobile phase. A sample was spotted onto the TLC plate and hydrolyzed with 6 M HCl for 2 h in a sealed container. Once dried, the plate was developed in the mobile phase. After development, the plate was sprayed with 0.5% ninhydrin and placed in an oven at 110°C for 10 min to detect the peptides as red spots.
2.4. Semipreparative HPLC Analysis. The putative lipopeptides were identified by HPLC analysis. Briefly, crude extract spots were removed from the TLC plates and dissolved in 10% methanol; the supernatants were analyzed by semipreparative high-pressure liquid chromatography using an Agilent LC 1200 system. The chromatographic separation was performed with a C-18 Column (4:6 × 250 mm). The column outlet was coupled to an Agilent MSD Ion Trap XCT mass spectrometer equipped with an ESI ion source. The lipopeptide fragments were selectively desorbed with methanol gradients from 35% to 65% within 140 min. All elution programs used a flow rate of 0.5 ml/min at 214 nm and detection occurred using the negative ion mode at m/z ranging from 400 to 2000. The isolated fragments were collected for the following experiments.
2.6. The Inhibitory Activity of Lipopeptide against C. difficile. Antimicrobial activities of the lipopeptides were detected by disc diffusion assay. 0.5 McF (10 6 CFU/ml) of C. difficile cells was inoculated onto the surface of blood agar plates. An Oxford cup (6 mm diameter), containing 100 μl lipopeptide with concentrations of 5, 10, and 15 μg/ml dissolved in 10% methanol, was placed on test C. difficile-seeded plates. A cup containing 100 μl 10% methanol was used as negative control. Each C. difficile strain was plated in triplicate. The plates were incubated anaerobically overnight at 35°C, and antimicrobial activity was evaluated by measuring inhibition zones against the tested C. difficile cells. The minimal inhibitory concentrations (MIC) against C. difficile strains were 2 BioMed Research International determined by the broth microdilution method in 96-well microplates with a final concentration of 10 5 CFU/ml, and the final concentration of the added lipopeptide ranged from 10 μg/ml to 0.0095 μg/ml; bacterial growth without lipopeptide was set as control [16]. The MIC was defined as the lowest lipopeptide concentration at which growth was completely inhibited after overnight anaerobic incubation of the plates at 35°C. To clearly detect an effect on the plasma membrane, 0.5 MIC of the purified lipopeptide was incubated with C. difficile cells (10 7 cells/ml) in RCM liquid medium. The mixture was incubated anaerobically at 35°C for 1 h. Then, 10 μl of 100 μg/ml PI solution was added to the cell suspension, and the mixture was incubated for 30 min in the dark. Finally, the treated C. difficile cells were observed with a Nikon TI-S fluorescence microscope with the filters set at an excitation wavelength of 488 nm and an emission wavelength of 538 nm. Cells treated with the same amount of sterile water were used as a negative control [19]. All experiments were repeated three times.

Determination of Extracellular Alkaline Phosphatase
Activity of C. difficile Cells Treated with Lipopeptide. An overnight culture of C. difficile was subcultured into fresh RCM liquid medium. Lipopeptide was added separately into the C. difficile culture at final concentrations of 0.25 and 0.5 MIC. Bacterial supernatant (0.5 ml) was collected every 12 h for the measurement of extracellular alkaline phosphatase (AKPase) activity using an AKP assay kit (Nanjing Jiancheng Technology Co., Ltd., Nanjing, China) as described in [20]. The AKPase unit was defined as 1 mg of phenol produced by 100 ml of bacterial culture supernatant reacted with the substrate at 37°C for 15 minutes. Cells treated with the same amount of sterile water were used as negative control.
Each test was performed in three biological replicates.
2.11. Lipopeptide Binding to C. difficile Genomic DNA. Gel retardation experiment assays were performed to identify the DNA binding activity of the lipopeptide as described in [21]. Briefly, 50 ng of C. difficile genomic DNA was mixed with 1 μl of 1, 2, and 5 μg/ml lipopeptide in 20 μl of binding buffer (10 mM Tris-HCl, 1 mM EDTA buffer, pH 8.0). One μl of sterilized water mixed with C. difficile genomic DNA was used as negative control. Mixtures were incubated at 35°C for 1 h. All samples were subjected to 1.0% agarose gel electrophoresis and stained with ethidium bromide.
The genomic DNA of strain C-1 was isolated and purified by a kit (Applied Biosystems® 4413021) and sequenced on the Pacific Bioscience (PacBio) RS II system at Genefund, Shanghai, China. The genome was assembled with SMRT analysis v.2.3 and RS_HGAP_Assembly.3, and the genome assembly was improved by using the software Pilon. Identification of protein-coding open reading frames (ORFs) and annotation of ORFs were performed by using the NCBI Prokaryotic Genome Annotation Pipeline. Genes were functionally annotated by BLAST search in COG (Gene Ontology Consortium), Nr (NCBI RefSeq), and Pfam Databases [22,23]. KEGG (Kyoto Encyclopedia of Genes and Genomes) database was used in the analysis of metabolic pathways of lipopeptide-producing Bacillus species. All amino acid sequences derived from the Bacillus genomes were submitted to the KEGG database, and the metabolic functions of these sequences were annotated by KASS. The KO (KEGG Orthology) term and corresponding KEGG pathway for each ORF were automatically generated. Secondary metabolite clusters present in the genome of the B. amyloliquefaciens collection have been evaluated using antiSMASH 5.0 [24]. The 16S rRNA gene sequences of Bacillus species were extracted from genome sequences and aligned using the CLUSTALX [25]. Phylogenetic trees were constructed using the neighbor-joining method implemented in the software package MEGA version 7.0.26 [26]. Evolutionary distances were calculated using Kimura's two-parameter model.
The C-1 genome sequence data were deposited into the Sequence Read Archive (SRA) of NCBI and can be accessed via accession number SRP127533.
2.13. Statistical Analysis. All experimental data are expressed as the average with standard deviation of at least three independent replicates. Statistical analyses were performed using the t-test and analysis of variance (ANOVA), JMP pro (SAS Institute Inc., NC, US), STAMP 10 , and SPSS V20.0 (IBM Inc., IL, US). Significant differences were considered at P < 0:05.

Results and Discussion
3.1. Production, Purification, and Identification of C-1 Lipopeptide. Bacillus spp. produces abundant secondary metabolites, such as proteinase, amylase, bacteriocin, and exopolysaccharide. In this study, we focused on the lipopeptide-producing B. amyloliquefaciens strain C-1. The growth profile of C-1 is shown in Figure 1. The maximum growth and cell dry weight were reached at 48 h; the maximum lipopeptide production (3:49 ± 0:26 mg/ml) was reached at 72 h. Lipopeptide production was conducted in a fermentation medium. The active compound from the culture supernatant was scanned from 190 to 900 nm, and the maximum absorption occurred at 213 nm, which is the typical absorption wavelength of peptides. Three spots were observed by thin-layer chromatography, and three component peaks were detected by RP-HPLC. Mass spectroscopy showed that the molecular masses of the three components at m/z were 1067 Da, 1477 Da, and 1506 Da, which corresponded to surfactin, fengycin, and iurin, respectively. PCR products of 675 bp, 1400 bp, and 482 bp corresponded to Srf, FenB, and ItuD genes, and the PCR fragments were sequenced and showed 99% identity with surfactin, fengycin, and iturin biosynthesis gene clusters, individually (S1).

3.2.
Antimicrobial Activity of C-1 Lipopeptide against C. difficile. Bacillus spp. lipopeptides have an inhibitory activity against plant pathogenic fungi and pathogenic bacteria and have been developed as biocontrol agents [27]. Although, in our previous report, C-1 did not show any inhibitory activity against fungi, it did have antibacterial activity toward several human pathogens, and this antibacterial activity in the C-1 supernatant was verified to be the contribution of lipopeptide, not exopolysaccharide [11,12].
In plate tests, the C-1 lipopeptide displayed antagonistic activities against three C. difficile strains. Inhibition zone diameters ranged between 7.05 mm and 22.00 mm, and the largest inhibition zone was from 15 μg/ml lipopeptide against strain C. difficile ATCC 9689 ( Table 1). The MICs against C. difficile strains ATCC 9689, ATCC 700057, and ATCC BAA-1870 were 0.75, 2.5, and 2.5 μg/ml, separately. Within a certain concentration range (0.0095 μg/ml-10 μg/ml), the inhibitory effect was positively correlated with the concentration of the C-1 lipopeptide. To analyze the inhibitory effect, we determined the growth curves of the three C. difficile strains. At 24 h of continuous measurement, the maximum OD 600 of C. difficile ATCC 9689, ATCC 700057, and ATCC BAA-1870 treated with 1/4 MIC lipopeptide reached 51.57%, 51.54%, and 56.12% of the control. For the 1/2 MIC treatment, growth was reduced to 43.15%, 46.39%, and 46.12% of the control. For the 3/4 MIC treatment, growth was further reduced to 38.95%, 40.21%, and 41.84% of the control. The C. difficile ATCC9689 was significantly the most sensitive to the treatment (P < 0:01), and the inhibitory effect was stronger with an increased concentration of the C-1 lipopeptide ( Figure 2).
Because C. difficile ATCC9689 was more sensitive to the C-1 lipopeptide, and it was also a tcdA and tcdB positive strain, we assessed the following antibacterial mechanism toward this strain.

Effect of C-1 Lipopeptide on C. difficile Morphology, Cell
Wall Permeability. Scanning electron microscopy showed that the surface of C. difficile ATCC 9689 was damaged after treatment with the C-1 lipopeptide. Exudates surrounded the bacteria, and the cell wall and cell membrane were interrupted and indistinct, whereas untreated cells were smooth and uninterrupted (Figures 3(a) and 3(b)). In addition, with increasing concentrations of lipopeptide, the bacteria were surrounded by exudate that may have been cytoplasm extruded from the cells. Propidium iodide (PI) penetrates only a damaged cell membrane, after which it binds to double-stranded DNA and fluoresces red with illumination at 488 nm. We stained the C-1 lipopeptide-treated and C-1 lipopeptide-untreated C. difficile cells with PI and observed the cells with a fluorescence microscope. C. difficile ATCC 9689 cells treated with lipopeptide were stained with PI as shown by red fluorescence, which indicated a damaged cell membrane (Figures 3(d) and 3(f)), whereas the untreated cells did not show any fluorescence (Figures 3(c) and 3(e)).
The destroyed cell membrane increased the permeability of C. difficile ATCC 9689 after C-1 lipopeptide treatment. To verify the effect of lipopeptide on cell wall permeability, we measured alkaline phosphatase (AKPase) activity. A damaged cell wall and cell membrane increased cell permeability and caused an increase in extracellular AKPase. After lipopeptide treatment, extracellular AKPase activity increased continuously and was significantly higher than that of the untreated cells (Fig S2). With 36 h of incubation, the extracellular AKPase content of C. difficile ATCC 9689 treated with 1/4 MIC and 1/2 MIC of lipopeptide increased 4.7-fold and 7.7-fold.
The antibacterial activity of Bacillus spp. lipopeptide was observed with other pathogens, such as S. aureus [28], Vibrio anguillarum [18], and E. clocae [8]. However, our report is the first to document the effects of lipopeptide on toxinproducing C. difficile. The inhibitory mechanism of the C-1 lipopeptide against C. difficile could be explained by destroying the bacterial cell by forming ion-conducting channels in the cell membrane as described by Etchegaray et al. [29]. This mode of action drastically reduces the chance of the    Figure 4(a)). Genome annotation at the RAST server showed that the C-1 genome encodes 4013 proteins, and the corresponding functional categorization by COG annotation is in Figure 4(b). The sequence data of the B. amyloliquefaciens C-1 genome were deposited into NCBI and can be accessed via accession number SRP127533. Significant difference of C-1 lipopeptide treatments vs. negative control (P < 0:01). b Significant difference of lipopeptide treatments between C. difficile strains ATCC 9689 and ATCC 70057, ATCC 9689, and ATCC BAA-1870 (P < 0:01).   Blast searches of the 16S rRNA gene sequence of C-1 showed that it was most similar to other B. amyloliquefaciens isolates. B. amyloliquefaciens isolates appear to group into two clades indicated by phylogenetic tree analysis (Fig S3). Although C-1 is in the same clade with known strains such as DSM7, ATCC19217, and ATCC 14580, it appears to be phylogenetically distant from most other isolates. In the carbohydrate transport and metabolism cate-gory, 437 genes (10.9% of total genes) were predicted in the C-1 genome. This finding suggests that C-1 possesses a broad battery of genes coding for enzymes required to release a variety of environmental carbon sources.
As a Bacillus. spp., B. amyloliquefaciens possesses an enormous potential to synthesize bioactive secondary metabolites, especially nonribosomal-synthesized peptides and polyketides. For the nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKS), we used anti-SMASH to identify related giant gene clusters (Table 3,  Table S1). C-1 was found to harbor genes encoding six nonribosomal peptides (surfactins (srfABCD), iturins (ituABCD), fengycins (fenABCDE), bacillibactin (bmyABC), teichuronic, and bacilysin) and three polyketides (bacillaene (baeEDLMNJRS), difficidin (difABCDEFGHIJ), and macrolactin (mlnABCDEFGHI)). Compared with other sequenced B. amyloliquefaciens strains, fengycin, difficidin, bacillibactin, bacilysin, macrolactin, and bacillanen showed  Figure 4: The genome map (a) of B. amyloliquefaciens C-1 and overview of the subsystem category coverage of the C-1 genome based on RAST serve (b). The red circle is the CDS of the forward strand, and the blue circle is the CDS of the reverse strand. The outer circle represents the categorization of predicted protein coding sequences in the C-1 genome in COG annotation, and the inner circle represents the genes encoding function RNA. 8 BioMed Research International 100% identity, whereas surfactins had 82% identity (Table S1). Surfactin can inhibit a wide range of microorganisms due to its ability to insert into the cell wall and create ion pores. Bacillomycin D, iturin, and fengycin have antifungal properties primarily based on their ability to disrupt the cell wall [30,31]. Macrolactins, important 24-membered macrolactones produced by Bacillus spp., exhibit antimicrobial activities, where macrolactin A and E and succinyl macrolactin are the representative compounds. And it is assembled by a modular PKS system like macrolides, which could inhibit the H + -transporting ATPase of the bacterial cells [32]. Polyketide compounds inhibit a wide range of microorganisms by preventing protein synthesis [31]. The identity of surfactin among different strains varied from 82% to 96%, including subunit genes of SrfAA, SrfAB, SrfAC, and SrfAD ( Fig S4). C-1 had all the subunit genes, SrfAA, SrfAB, SrfAC, and SrfAD, and the mutation of subunit genes in other strains may indicate loss of the ability to synthesize secondary metabolites [33]. PCR experiments detected C-1 surfactin, iturin, and fengycin genes, an intact Bac operon that included Bacilysin biosynthesis proteins BacA, BacB, BacC, BacD, and BacE (ORF3909-3913), and the oligopeptide permease operon (ORF1362-1366). Bac proteins, nonribosomally synthesized dipeptides active against a range of bacteria and some fungi, are involved in the biosynthesis of bacilysin. The proteolysis of this dipeptide releases the nonproteinogenic amino acid L-anticapsin, which functions as a competitive inhibitor of glucosamine synthase and can cause lysis of fungal cells [34]. Because there was 100% sequence identity of bacilysin, whereas there was no antifungal activity of the C-1 lipopeptide and exopolysaccharide, the regulation and expression of the encoded Bac operon, and modification of produced bacilysin, deserve more analysis.
Other antimicrobial gene clusters were predicted by anti-SMASH, such as the lantibiotic amylolysin [35], the bacteriocin amylocyclicin [36], and the aminoglycoside antibiotic butirosin [37]. These antibacterials have not been detected by chemical analysis from C-1 supernatants, possibly because of the fermentation medium or condition that had been used [38]. Potential gene clusters may explain the broad activity of C-1 against pathogens.
Moreover, we also detected genes for amphiphilic membrane-active biosurfactants and peptide antibiotics that have powerful antibacterial and mosquito larvicidal activity. The giant gene clusters add to the capacity of the C-1 bacterium to contribute to the antimicrobial activity against C. difficile. And we also checked the biosafety of the C-1 strain by using Galleria mellonella and intestinal epithelial