Four Novel Leaderless Bacteriocins, Bacin A1, A2, A3, and A4 Exhibit Potent Antimicrobial and Antibiofilm Activities against Methicillin-Resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a major bacterial pathogen that causes hospital- and community-acquired infections. Owing to its multidrug resistance, it is imperative to develop new antimicrobial agents to treat MRSA infections. In this study, using genome mining analysis and a culture-based screening method to detect bacteriocin activity, we screened a strain, Bacillus sp. TL12, which harbored a putative leaderless bacteriocin gene cluster (bac gene cluster) and exhibited potent anti-MRSA activity. The antimicrobial agents, products of the bac gene cluster, were purified and identified as four novel leaderless bacteriocins: bacin A1, A2, A3, and A4. Bacin A2 was evaluated as a representative antimicrobial agent and showed remarkable antimicrobial activity against S. aureus, MRSA, and the foodborne pathogens Listeria monocytogenes and Bacillus cereus. Mechanistic experiments revealed that bacin A2 damaged cell membranes and exhibited bactericidal activity against MRSA. Bacin A2 effectively inhibited the formation of S. aureus and MRSA biofilms (>0.5× MIC) and killed the cells in their established biofilms (>4× MIC). The hemolytic and NIH/3T3 cytotoxicity assay results for bacin A2 confirmed its biosafety. Thus, bacins have potential as alternative antimicrobial agents for treating MRSA infections. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that is difficult to treat because of its resistance to several widely used antibiotics. The present study aimed to identify novel anti-MRSA bacteriocins in a prominent producer of bacteriocins, Bacillus cereus group. Four novel leaderless bacteriocins, bacin A1, A2, A3, and A4, which show potent bactericidal effect against S. aureus and MRSA, were identified in Bacillus sp. TL12. Moreover, bacins inhibited biofilm formation and killed cells in the established biofilms of S. aureus and MRSA. These findings suggest that bacins are promising alternatives to treat MRSA infections.

Keywords: bacin; leaderless bacteriocin; methicillin-resistant Staphylococcus aureus (MRSA).

FIG 1

Screening strategy for novel bacteriocin-producing strains of the Bacillus cereus group with antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA).

FIG 2

Screening of the bacteriocin-producing strain Bacillus sp. TL12. (A) Colony morphology of Bacillus sp. TL12. (B) Scanning electron microscope observation of Bacillus sp. TL12. (C) The putative leaderless bacteriocin gene cluster (bac gene cluster) in the genome of Bacillus sp. TL12 and multiple-sequence alignments of their precursor peptides (BacA1, BacA2, BacA3, and BacA4) with LnqZ (the precursor peptide of a leaderless bacteriocin, Lacticin Z). Black, precursor peptides; red, transport or immune related proteins; green, membrane proteins. (D) Antimicrobial activity of the supernatant of Bacillus sp. TL12 against MRSA ATCC 43300. The outer well was added to the untreated supernatant, and the inner well was added to the corresponding supernatant treated with mixed enzymes. (E) Growth kinetics of Bacillus sp. TL12 in Luria-Bertani broth (LB), nutrient broth (NB), and trypticase soy broth (TSB) media. All experiments were performed in triplicate, and the data are shown as the means ± SD.

FIG 3

Purification and identification of four novel leaderless bacteriocins produced by Bacillus sp. TL12. (A) High-performance liquid chromatography (HPLC) analysis of a CE of Bacillus sp. T1L12 culture supernatant. Fractions A and B have antimicrobial activity against MRSA ATCC 43300. No synergistic antimicrobial effect was observed between fractions A and B. (B) Mass spectrometry (MS) analysis of antimicrobials in fraction A. (C) MS analysis of antimicrobials in fraction B. (D) Analysis of the molecular mass of four putative leaderless bacteriocins of Bacillus sp. TL12. fM, formylmethionine.

FIG 4

MS/MS spectra and proposed structure of bacin A2. The b and y ions in the MS/MS spectra of bacin A2 were manually assigned upon fragmentation.

FIG 5

Bacin A2 targets the cell membrane of MRSA cells and exerts bactericidal activity. (A) Growth curve of MRSA ATCC 43300 treated with various concentrations of bacin A2. Bacin A2 was dissolved and diluted with NB medium. NB medium was used as the negative control. The data are expressed as the means ± SD of three independent replicates. (B) Bacin A2 treatment resulted in the lysis of MRSA cells. The figure is representative of three independent experiments. (C) Potassium release from MRSA cells after treated with 2× and 4× MIC of bacin A2. Bacin A2 was dissolved and diluted with normal saline. Normal saline was used as the negative control. The data are expressed as the means ± SD of three independent replicates. (D) Morphology observation of MRSA ATCC 43300 cells after treated with 2× and 4× MIC of bacin A2 with SEM. Bacin A2 was dissolved and diluted with NB medium. NB medium was used as the negative control. The red circles indicate the significant membrane damage of MRSA cells.

FIG 6

Growth and biofilm formation of S. aureus ATCC 6538 (A, B) and MRSA ATCC 43300 (C, D) in the presence of bacin A2 (1/4×, 1/2×, 1×, 2× MIC). Bacin A2 was dissolved and diluted with TSB medium. Nisin A (5 μM) and TSB medium were used as positive and negative controls, respectively. Asterisk ratings indicate statistically significant differences: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤ 0.0001.

FIG 7

The effect of bacin A2 on cell viability in 1-day-old biofilms of S. aureus ATCC 6538 and MRSA 43300. The viability of bacterial cells in 1-day-old biofilms was detected by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) assay (A, B) and viable count (C, D). Bacin A2 was dissolved and diluted with TSB medium. Nisin A (50 μM) and TSB medium were used as positive and negative controls, respectively. ****, P ≤ 0.0001.

FIG 8

Hemolytic activity and cytotoxicity of bacin A2. Hemolytic activity of bacin A2 in sheep blood cells (A). Bacin A2 was dissolved and diluted with phosphate-buffered saline (PBS). PBS was taken as the negative control, and 1% Triton X-100 was taken as the positive control. Cytotoxicity of bacin A2 in NIH/3T3 cells (B). Bacin A2 was dissolved and diluted with PBS. PBS was taken as the negative control, and paclitaxel (10 μg/mL) was taken as the positive control. ****, P ≤ 0.0001.