- The assay performed was performed using Chromobacterium violaceum CV026 strain, capable of producing violacein in response to the presence of short chain N-aclyhomoserine lactones.
- Ecobiol®, prevented the production of violacein demonstrating the quorum quenching activity on C. violaceum CV026, using either C6 HSL (homoserine lactone) as a substrate.
"Quorum sensing" (QS) is a bacterial communication system based on production and secretion of small signal molecules called autoinducers, accumulated in the extracellular environment when a high bacterial cell density is reached (Fuqua et al., 1994). Once an intracellular concentration threshold of autoinducers is reached, the signal molecule triggers the synchronized expression of multiple genes in the population, thus regulating important biological functions such as the transfer of plasmids, motility, aggregation, luminescence, antibiotic biosynthesis, and virulence (Swift et al., 2001; Waters and Bassler, 2005; Williams et al, 2007). The best characterized autoinducer are N-acylhomoserin lactones (AHLs), a family of molecules formed by a ring of homoserine lactone (HSL), N-acylated with a fatty acyl group in the alpha position. The mechanisms leading to the inactivation of the QS communication system have been generally referred to as "Quorum Quenching" (QQ) (Dong et al., 2001, 2007), although some authors prefer to restrict the term to enzymatic degradation of the AHL signals (Kjelleberg et al., 2008). One of the possible mode of action of probiotics is through QQ to block the communication (i.e., quorum sensing) between pathogenic bacteria, thus preventing outgrowth, biofilm formation and expression of virulence.
Therefore, an in vitro study was conducted to assess the QQ activity of Bacillus amyloliquefaciens CECT 5940, strain with an inherent capacity to produce a wide range of secondary metabolites which has demonstrate to interact with different bacterial populations.
Materials and Methods
Probiotic: Bacillus amyloliquefaciens CECT 5940 (Ecobiol®)
Biosensor: Chromobacterium violaceum CECT 5999 (CV026) (McClean et al., 1997)
The wild strain of the species Chromobacterium violaceum produces a purple pigment (violacein) and its production is regulated via QS. The strain used in the study was a mutant of this species, which has lost its ability to constitutively produce the purple pigment and is therefore known as a white mutant (CV026). In this mutant, however, the occurrence of violacein can be induced by applying AHLs to the culture medium.
Synthetic Homoserine lactones (commercial compounds analogous to AHLs):
- C6-HSL: N-hexanoyl-L-homoserine lactone. (Sigma-Aldrich 09926)
- C8-HSL: N-octanoyl-L-homoserine lactone (Sigma-Aldrich 10940)
These compounds (inducers) were prepared as stock solutions in sterile distilled water at a concentration of 1mg / ml for C6 and at 0.7mg / ml for C8.
Quorum Quenching screening (Florez et al., 2014):
Growth of Bacillus amyloliquefaciens CECT 5940 with analogous: Bacillus amyloliquefaciens CECT 5940 was grown overnight at 30°C in LB (Luria Bertani) medium in 250 mL flasks with 50 mL of medium. The above pre-inoculum was used to inoculate a new flask with 25 mL of LB medium, to maintain an approximate OD600 of 1.1. The AHL (autoinducers) analogs, C6-HSL or C8-HSL were added to the culture medium at 4µg / ml final concentration and were incubated at 30 °C with agitation. Samples were taken at different time intervals: 0 (just after adding the inducer), 1, 2, 4, 6 and 24 hours. Each of the samples taken was centrifuged at 3000 rpm for 10 minutes to remove cell debris. The supernatant was recovered after centrifugation.
LB agar plates were covered with 4 mL of LB 0.8% containing 1 ml of the overnight culture of the indicator C. violaceum, in LB with kanamycin 25 µg/ml. Coated LB plates were allowed to solidify. Wells plates of 6 mm in diameter were prepared with sterile tips. The different samples to be tested were added to each well plate at 50 µl per well. The plates were then incubated for 24 h at 30°C to enable the growth of the indicator microorganism.
Controls and Interpretation of Results:
Where purple halos exist, QS was successful and no QQ was observed. Where there was QQ activity from samples added to different plate wells, halos will be opaque and colorless although the inducer is present.
In the detection assay the following controls were used. The color expected in the trial as well as the nomenclature used in the plates (in brackets) is detailed below:
• Colorless opaque halo (no inducer present)
- Sterile distilled water (A). Colorless opaque halo as the mutant is white and there is no inducer molecule.
- Sterile LB culture medium (M). Colorless opaque halo as there is no induction
- LB medium + probiotic (no inducer). Probiotic cultures were incubated for the same number of hours as the test probiotic with the C6 or C8 inducer molecule samples and used as a control to check if the probiotic produces some compound stimulating the production of violacein that could give a false negative result. These controls are expected to have a colorless opaque halo and are labelled with an “E” followed by the incubation time in hours (Exhours).
• Purple halo (inducer present)
- Inducer molecules (C6 HSL or C8 HSL). Purple halo was expected as these compounds are violacein inducers.
- LB medium + C6 or C8 as appropriate. These cultures were incubated for the same number of hours as the test probiotic with C6 or C8, in order to check for inducer degradation over time in the absence of the microorganism. These controls are expected to have purple halos and were labelled with an “M” followed by the incubation time in hours (Mxhours)
Test samples (inducer molecule + probiotic) were labelled with the incubation hours only: 0, 1, 2, 4, 6, 24. As the inducer is present violet color was expected, and lack of this pigment indicated that QS was blocked with a QQ mechanism.
Results and Discussion
The assay performed allows the easy visualization (via inhibition of the purple violacein production) of the capacity in any compound to block induction of QS,
Supernatants from different incubation times of the probiotic (B. amyloliquefaciens CECT 5940) grown in the presence of inducers C6 were analyzed.
The wells with water (A), culture medium (M) or culture medium with Ecobiol® alone (E0-24 hours) did not produce pigment (Figure 1.) This was to be expected as these wells did not contain inducer C6, and Ecobiol® alone does not produce any compound stimulating production of violacein in the biosensor C. violaceum. When media with the C6 inducer was added to wells and incubated for 24 hours (0-24 M) a pigmented halo was consistently observed (Figure 1). This suggests that the C6 inducer does not degrade over time as the violacein halo was observed even after 24 hours.
In wells containing media with Ecobiol® and the inducer C6 (0-24), pigmented halo was observed within the first 6 hours, showing that the C6 is present and stimulating violacein production. However, after 24 hours the pigment was no longer observed, indicating that Ecobiol®, prevented the production of violacein and therefore shows QQ activity.
Figure 1. Induction of violacein synthesis in C. violaceum CV0526 strain by HSLs synthetic (C6HSL) and inhibition. A: distilled water, M: Medium LB, M0-24: Medium LB + C6-HSL at different incubation times, E0-24: Medium LB + Ecobiol without C6 at different incubation times. 0-24: Medium LB + Ecobiol + C6-HSL at different incubation times.
The assay was repeated using C8 HSL as inducer obtaining the same results.
The results of this study demonstrate the QQ activity of B. amyloliquefaciens CECT 5940 on C. violaceumCV026, using either C6-HSL or C8-HSL as a substrate.