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Floris, R. Bioactive compounds from the Gut of Grey Mullets. Encyclopedia. Available online: https://encyclopedia.pub/entry/17570 (accessed on 17 June 2024).
Floris R. Bioactive compounds from the Gut of Grey Mullets. Encyclopedia. Available at: https://encyclopedia.pub/entry/17570. Accessed June 17, 2024.
Floris, Rosanna. "Bioactive compounds from the Gut of Grey Mullets" Encyclopedia, https://encyclopedia.pub/entry/17570 (accessed June 17, 2024).
Floris, R. (2021, December 27). Bioactive compounds from the Gut of Grey Mullets. In Encyclopedia. https://encyclopedia.pub/entry/17570
Floris, Rosanna. "Bioactive compounds from the Gut of Grey Mullets." Encyclopedia. Web. 27 December, 2021.
Bioactive compounds from the Gut of Grey Mullets
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Fish gut represents a peculiar ecological niche where bacteria can transit and reside to play vital roles by producing bio-compounds with nutritional, immunomodulatory and other functions. This complex microbial ecosystem reflects several factors (environment, feeding regimen, fish species etc.). The objective of the present study was the identification of intestinal microbial strains able to produce molecules called biosurfactants (BSs) which were tested for surface and antibacterial activity in order to select a group of probiotic bacteria for aquaculture use. This works indicated that fish gut is a source of bioactive compounds which deserves to be explored for applicative purposes.

grey mullets natural antibiotics gut microbiota probiotics

1. Enumeration of Bacteria and Colony Isolation

Forty-two bacterial colonies were isolated from the gut of different mullet species captured from a Mediterranean lagoon in autumn and in winter in Sardinia (Table 1).

2. Screening of Bacteria for BS Production

The application of different screening methods allowed the selection of a “group” of intestinal strains as surfactant producers. The drop-collapse method (Bodour et al., 2003) was used as a first screening test for identifying the “bioactive” microbes. Table 1 shows the results of all the used screening tests. 
Table 1. BS-producing bacteria from grey mullets’ guts: bacterial affiliations (similarity 99–100%), performed tests: (mean ± SD) and TLC results. Highest E24 values are highlighted in bold.
Strain Fish
Species
Bacterial
Affiliation
GeneBank
Accession Number
Drop Collapse E-24
(%)
Surface
TensionmN·m−1
BS
Type
1 CR Pseudomonas aeruginosa MW369461 +++ 70.5 ± 9.1 36.5 ± 0.1 Rhamnolipid
3 CR Pseudomonas aeruginosa OK342256 +++ 68.0 ± 12.7 37.1 ± 0.1 Rhamnolipid
5 CR Pseudomonas aeruginosa OK342257 +++ 77.0 ± 0.0 36.9 ± 0.4 Rhamnolipid
6 CR Pseudomonas aeruginosa MW369462 +++ 56.4 ± 0.0 37.1 ± 0.1 Less polar compound
8 CR Pseudomonas aeruginosa OK342258 ++ 0.0 ± 0.0 37.1 ± 0.1 nd
9 CR Pseudomonas aeruginosa OK342259 +++ 57.7 ± 1.8 37.2 ± 0.3 nd
10 CR Pseudomonas alcaligenes MW369463 + 50.0 ± 1.8 37.2 ± 0.3 nd
11 CR Aeromonas caviae MW369464 - 0.0 ± 0.0 43.0 ± 0.1 nd
12 CR - - +++ 15.4 ± 21.8 36.9 ± 0.1 nd
13 CR Pseudomonas aeruginosa MW369465 +++ 51.3 ± 3.6 36.9 ± 0.1 Rhamnolipid
15 CR Pseudomonas aeruginosa MW369466 +++ 59.0 ± 3.6 36.6 ± 0.6 Rhamnolipid
16 CR - - weak 0.0 ± 0.0 35.35 ± 0.6 nd
17 CR Pseudomonas mendocina MW369467 - 20.5 ± 0 nd nd
18 CR Pseudomonas putida OK342260 weak 33.3 ± 3.6 36.1 ± 0.1 Less polar compound
19 MC Pseudomonas sp. OK342261 + 28.2 ± 3.6 35.2 ± 0.0 Less polar compound
20 MC Pseudomonas alcaliphila MW369468 weak 0.0 ± 0.0 35.0 ± 0.4 nd
21 MC - - weak 25.6 ± 14.5 nd nd
22 MC Pseudomonas sp. OK342262 + 25.6 ± 0.0 35.1 ± 0.2 Less polar compound
23 MC Pseudomonas sp. OK342263 weak 0.0 ± 0.0 36.5 ± 0.1 nd
24 MC Pseudomonas khazarica MW369469 weak 0.0 ± 0.0 nd nd
25 MC Pseudomonas sp. OK342264 + 0.0 ± 0.0 35.3 ± 0.1 Less polar compounds
26 MC Pseudomonas aeruginosa MW369470 ++ 33.3 ± 0.0 37.6 ± 0.3 Less polar compound
28 CS Enterobacter ludwigii MW369471 + 0.0 ± 0.0 37.9 ± 0.1 nd
30 CS Aeromonas media MW369472 weak 0.0 ± 0.0 39.4 ± 0.9 nd
35 CS Aeromonas taiwanensis MW369473 weak 0.0 ± 0.0 43.2 ± 0.1 nd
37 CL Aeromonas media MW369474 - 0.0 ± 0.0 35.9 ± 0.1 nd
40 CL Aeromonas media MW369476 - 0.0 ± 0.0 46.1 ± 0.3 nd
41 CL Pseudomonas anguilliseptica MW369477 + 32.1 ± 5.4 35.2 ± 0.6 nd
45 CL Pseudomonas stutzeri OK342265 + 0.0 ± 0.0 36.3 ± 0.1 nd
47 CL Pseudomonas protegens MW369478 weak 0.0 ± 0.0 40.5 ± 0.4 nd
51 CL Pseudomonas protegens OK342266 + 0.0 ± 0.0 35.5 ± 0.1 nd
55 CL Pseudomonas protegens MW369480 - 0.0 ± 0.0 37.7 ± 0.1 nd
56 CL Pseudomonas sp. OK342267 + 0.0 ± 0.0 39.9 ± 0.1 nd
 Figure 1 shows the emulsification indexes E-24(%) and the surface tension activity of the detected “bioactive” intestinal bacteria.
Figure 1. Emulsification index E-24(%) and the surface tension activity (mN·m−1) of intestinal bacterial cultures from mullet species.
The most interesting strains are represented by eight isolates (strain 1, 3, 5, 6, 9, 10, 13, 15), showing an emulsification index E-24(%) from 50.0 to 77.0% and a surface tension from 36.5 to 37.2 (mN·m−1).

3. Bacterial Identification

Thirty strains, which showed bioactivity, were identified by 16S rRNA gene partial sequencing. Table 1 shows fish origin, phylogenetic affiliation and accession number of the studied intestinal strains. Figure 2 shows the phylogenetic tree reconstructed using the 16S rRNA gene sequences. Different groups and subgroups were obtained with respect to the corresponding reference species type strains and among themselves. The most heterogeneous group was represented by Pseudomonas spp., while Aeromonas spp. and Enterobacter sp. formed well distinguished clusters. The outgroup strain NR074804 Cellvibrio japonicus strain Ueda 107 was separated from all the others.
Figure 2. Phylogenetic tree based on 16S rRNA gene sequences comparison between the intestinal strains and reference collection strains. NR074804 Cellvibrio japonicus strain Ueda 107 was used as the outgroup strain. Each node indicates the percentage of the obtained bootstrap values higher than 50% of 1000 replicates. The scale bar indicates sequence divergence.

4.  BSs yield and Thin Layer Chromatography (TLC) 

Figure 3 shows the BSs yield extracts of representative intestinal bacteria. The BS producers which gave the highest yield extracts (values from 6–6.42 g L−1) were strains 6, 13, 15.
Figure 3. Yield of BS extracts (g L1) from intestinal bacterial cell-free supernatants. Error bars indicate standard error (SE).
The chromatographic analyses (TLC) of the BSs extracted from intestinal bacterial supernatants showed two types of glycolipid compounds (Figure 4). The TLC silica gel glass plates stained by anisaldehyde (carbohydrates) (Figure 4a,c) and cerium sulphate (lipids) (Figure 4b,d) indicate a group of specific TLC fractions (retardation factor Rf = 0.42), which presumably represents the di-rhamnolipid structures, while a group of other fractions (Rf = 0.75) detects the mono-rhamnolipid molecules (Figure 4a,b).  Another type of molecule was also detected by the TLC analyses but not separated using the first solvent system indicated above (strains 19 and 26, Figure 4a,b). These less polar compounds from strains 6, 18, 19, 22, 25 and 26 were separated using a less polar solvent system, as described above, and gave different profiles of the TLC fractions (from the bottom, Rf = 0.20, 0.30, 0.60) (Figure 4c,d).
Figure 4. Examples of TLC plates of intestinal BS extracts stained for detecting sugars (a) and (c) and lipids (b) and (d). BH: Bushnell–Haas broth; S: sophorolipids; T: trealose lipids; R: rhamnolipids; PL: phospholipids; (a) and (b) = solvent system: chloroform: acetic acid:methanol:water (65:15:1:1); (c) and (d) = solvent system: chloroform:exane:ether:acetic acid (70:30:2).

5. Antibacterial Activities

Cell free supernatans (CFSs) of the bioactive strains exhibited inhibitory activity against the target strains S. aureus H1610 and P. mirabilis H1643, while no inhibition was evidenced against the target strains P. aeruginosa H1328, K. pneumoniae H1637 and A. hydrophila H1563 (Table 2).
Table 2. Antibacterial activity of supernatants and crude extracts (mm) in agar diffusion assay against bacterial pathogens. Values are expressed as the mean ± standard deviation of three replicates. Highest values are highlighted in bold.
  Cell-Free Supernatants (CFSs) and Crude Extracts (CEs) (mm)
Test S. aureus H1610 P. mirabilis H1643 K. pneumoniae H1637 A. hydrophila H1563
  CFSs CEs CFSs CEs CFSs CEs CFSs CEs
Pseudomonas aeruginosa 1 13.5 ± 0.7 5.5 ± 0.7 15 ± 0.0 - - 17.5 ± 0.7 - -
Pseudomonas aeruginosa 3 10.5 ± 2.1 5.5 ± 0.7 18.5 ± 0.7 - - 8.0 ± 0.0 - -
Pseudomonas aeruginosa 5 - 5.5 ± 0.7 - 15.5 ± 0.7 - 6.0 ± 0.0 - -
Pseudomonas aeruginosa 6 + 8.5 ± 0.7 - 12.5 ± 0.7 - 11.5 ± 0.7 - -
Pseudomonas aeruginosa 8 - 6.5 ± 0.7 + - - 7.0 ± 0.0 - 6.5 ± 0.7
Pseudomonas aeruginosa 9 - 14.5 ± 0.7 - - - 12.0 ± 0.0 - -
Pseudomonas alcaligenes 10 12 ± 0.0 7.5 ± 0.7 - 16.0 ± 0.0 - + - -
Aeromonas caviae 11 - 8.5 ± 0.7 - - - + - -
Unidentified 12 9 ± 1.4 7.5 ± 0.7 17.0 ± 1.4 - - 7.5 ± 0.7 - -
Pseudomonas aeruginosa 13 12.5 ± 0.7 12.5 ± 0.7 7.0 ± 0.0 - - 12.5 ± 0.7 - 7.0 ± 0.0
Pseudomonas aeruginosa 15 14 ± 0.0 12.5 ± 0.7 - 14.0 ± 0.0 - 12.5 ± 0.7 - -
Unidentified 16 - - 10.0 ± 0.0 - - - - -
Pseudomonas putida 18 - - + - - - - -
Pseudomonas sp. 19 - 7.5 ± 0.7 - - - 8.0 ± 0.0 - +
Pseudomonas alcaliphila 20 - - - - - - - -
Pseudomonas sp. 22 - 7.5 ± 0.7 13.0 ± 0.0 - - 5.5 ± 0.7 - -
Pseudomonas sp. 23 - + + - - - - -
Pseudomonas sp. 25 9 ± 0.0 + - 12.0 ± 0.0 - 6.5 ± 0.7 - -
Pseudomonas aeruginosa 26 - - 10 ± 0.0 - - - - -
Enterococcus ludwigii 28 - 5.5 ± 0.7 9.0 ± 0.0 - + + - -
Aeromonas media 30 - 5.5 ± 0.7 - - - 6.0 ± 0.0 - -
Aeromonas taiwanensis 35 - 5.5 ± 0.7 - - - 6.0 ± 0.0 - -
Pseudomonas protegens 37 - - 10.0 ± 0.0 - - - - -
Aeromonas media 40 - 7.5 ± 0.7 - 15.5 ± 0.7 - + - +
Pseudomonas anguilliseptica 41 - + - - - - - 8.0 ± 0.0
Pseudomonas stutzeri 45 - - + - - - - -
Pseudomonas protegens 47 - 9.5 ± 0.7 8.0 ± 0.0 - - - - 6.5 ± 0.7
Pseudomonas protegens 51 - 7.0 ± 0.7 - - - 7.5 ± 0.7 - -
Pseudomonas protegens 55 10 ± 0.0 9.5 ± 0.7 - - - - - -
Pseudomonas sp. 56 13.5 ± 0.7 5.5 ± 0.7 8.0 ± 0.0 - - + - -
Negative control 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
Chloramphenicol 21 ± 0.0 - + 30.0 ± 0.0
Gentamycin CN30 - 14 8.0 ± 0.0 18.0 ± 0.0
Amoxycillin - - - -
Of the tested CFSs, 26.6% exhibited antibacterial activity, with halos ≥10 mm compared to the target strain S. aureus H1610 (Figure 5)
 Microorganisms 09 02555 g005a 550[1]Microorganisms 09 02555 g005b 550
Figure 5. Inhibitory activity exhibited against the target S. aureus H1610 (a), P. mirabilis H1643 (b), K. pneumoniae H1637 (c) and A. hydrophila H1563 (d) by concentrated supernatants (CFSs green) and crude extracts (CEs blue) obtained by bacterial isolates.
On the other hand, the crude extracts (CEs), obtained from the bacterial supernatants, evidenced antibacterial activity against more target strains, namely S. aureusH1610, P. mirabilisH1643, K. pneumoniaeH1637 and A. hydrophila H1563, while no activity was recorded against P. aeruginosa H1628. Of the CEs, 76.7% and 66.7% were active against S. aureus H1610 and K. pneumoniae H1637, respectively, while 20% of the CEs showed inhibitory activity against P. mirabilis H1643 and A. hydrophilaH1563 (Figure 5). 

6. Conclusions

The present entry let to select bacterial strains from the gut of grey mullets with interesting biotechnologically traits and has confirmed that intestinal microbiota is a promising source of new and biologically active pharmaceutical agents to control fish health and to preserve the environment. Additionally, the study of BS-producing bacteria associated with fish intestine is of relevance for our understanding of their ecological role in the symbiotic and antagonist interaction with the host and between themselves and for understanding whether the production of bioactive compounds might represent a biological strategy for protecting fish against gut and liver inflammations, an immune response and for survival with respect to the surrounding environment

References

  1. Bodour, A.A.; Drees, K.P.; Maier R.M.; Distribution of biosurfactant-producing bacteria in undisturbed contaminated arid southwestern soils. . Appl. Environ. Microbiol. 2003, 69, 3280-3287, 10.1128/AEM.69.6.3280-3287.2003.
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