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. 2021 Apr 26;6(18):12261-12273.
doi: 10.1021/acsomega.1c01137. eCollection 2021 May 11.

Gut Bacteria of Rattus rattus (Rat) Produce Broad-Spectrum Antibacterial Lipopeptides

Noor Akbar  1   2 , Ruqaiyyah Siddiqui  2 , Mazhar Iqbal  3 , Kuppusamy Sagathevan  1 , Kwang Sik Kim  4 , Fazal Habib  3 , Naveed Ahmed Khan  5
Affiliations

Gut Bacteria of Rattus rattus (Rat) Produce Broad-Spectrum Antibacterial Lipopeptides

Noor Akbar et al. ACS Omega. .

Abstract

Among several animals, Rattus rattus (rat) lives in polluted environments and feeds on organic waste/small invertebrates, suggesting the presence of inherent mechanisms to thwart infections. In this study, we isolated gut bacteria of rats for their antibacterial activities. Using antibacterial assays, the findings showed that the conditioned media from selected bacteria exhibited bactericidal activities against Gram-negative (Escherichia coli K1, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, and Salmonella enterica) and Gram-positive (Bacillus cereus, methicillin-resistant Staphylococcus aureus, and Streptococcus pyogenes) pathogenic bacteria. The conditioned media retained their antibacterial properties upon heat treatment at boiling temperature for 10 min. Using MTT assays, the conditioned media showed minimal cytotoxic effects against human keratinocyte cells. Active conditioned media were subjected to tandem mass spectrometry, and the results showed that conditioned media from Bacillus subtilis produced a large repertoire of surfactin and iturin A (lipopeptides) molecules. To our knowledge, this is the first report of isolation of lipopeptides from bacteria isolated from the rat gut. In short, these findings are important and provide a platform to develop effective antibacterial drugs.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Phylogenetic tree representing 15 Bacillus strains with an E. coli strain U 5/41 (purple bold) as the outgroup and the B. subtilis (CM2) strain used in this study (red bold) based on the phylogenetic analysis of 16S rRNA genes. The phylogenetic tree was reconstructed using the maximum likelihood (ML) method (MEGA 7.) based on a GTR+G (8) model with concatenated 16S rRNA sequences. Percentage bootstrap values that were higher than 50% of 1000 replicates are indicated at branching nodes.
Figure 2
Figure 2
Conditioned media of rat gut bacteria possessed significant antibacterial activities against Gram-negative bacteria. CM were incubated with 1 million bacteria for 2 h at 37 °C. Next, cultures were serially diluted and plated onto nutrient agar plates, and plates were incubated overnight at 37 °C. Viable bacterial cfu were calculated, and results were recorded. (a) CM tested against S. enterica, (b) against P. aeruginosa, (c) against K. pneumoniae, (d) against E. coli K1, and (e) against S. marcescens and (f) representative effects of CM against S. enterica and P. aeruginosa. Data are expressed as the mean ± standard error of several independent experiments performed in duplicate. P values were determined using Student’s t-test. An asterisk (*) denotes P ≤ 0.05.
Figure 3
Figure 3
Conditioned media exhibited important bactericidal effects against Gram-positive bacteria. Approximately 1 × 106 bacterial cells were exposed to CM from rat gut bacteria and incubated at 37 °C for 2 h. After this incubation, cultures were serially diluted, plated on nutrient agar, and incubated at 37 °C for 24 h. Bacterial colonies were enumerated on the following day. Data are expressed as the mean ± standard error of several independent experiments performed in duplicate. P values were determined using two sample t-tests. An asterisk (*) denotes P ≤ 0.05 (a) when CM were tested against B. cereus, (b) against S. pyogenes, and (c) against MRSA and (d) demonstrative effects of CM against MRSA and S. pyogenes.
Figure 4
Figure 4
Heat-treated CM-mediated antibacterial properties against MRSA and S. pyogenes. Heat-treated CM were incubated with MRSA and S. pyogenes for 2 h at 37 °C. Cultures were serially diluted and subsequently plated on nutrient agar plates, and plates were incubated at 37 °C overnight. The next day, bacterial cfu were enumerated, and results were recorded. Data are expressed as the mean ± standard error of three independent experiments performed in duplicate.
Figure 5
Figure 5
Conditioned media showed limited cytotoxicity properties against HaCaT cell lines. Briefly, HaCaT cells were challenged with CM from the rat gut bacteria for 24 h at 37 °C in the presence of 95% humidity and 5% CO2. The next day, LDH released from HaCaT cells was measured as described in the materials and methods. (a) CM tested were nontoxic against HaCaT cells except CM1 (55%) and (b) representation of CM cytotoxic effects incubated with a human cell monolayer.
Figure 6
Figure 6
Conditioned media of rat gut bacteria produced negligible cytotoxicity against human cell lines. CM from all three Bacillus species were tested against human cells at their graduated volume/concentration overnight at 37 °C with 5% CO2 and 95% humidity. Only CM1 (B. cereus) showed moderate to high cytotoxic effects, and other CM showed limited effects. Data are presented as the mean ± SE of three independent experiments performed in duplicate.
Figure 7
Figure 7
LCMS spectrum of B. subtilis isolated from the rat gut revealed the presence of several homologues of lipopeptides labeled as (a) surfactin molecules and (b) spectrum illustrating iturin A molecular ion species at positive ion mode.
Figure 8
Figure 8
LCMS spectrum representing surfactin molecular ion species. The fragmentation pattern and product ion spectra of sodiated molecules of surfactin at m/z 1030.67 [M+Na]+at positive ion mode.
Figure 9
Figure 9
Profiling of fragmentation data obtained from tandem mass spectrometry of a surfactin homologue at m/z 1058.75 [M+Na]+ at positive ion mode.
Figure 10
Figure 10
Fragmentation pattern and product ion spectra of sodiated molecules of the iturin A homologue having m/z at 1043.83 [M+H]+.
Figure 11
Figure 11
ESI–MS/MS spectrum of the iturin A precursor ion [M+Na]+ at m/z 1065.42 and their daughter ion peaks upon CID.
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