Degradation of N-Acyl-l-Homoserine Lactones by Bacillus cereus in Culture Media and Pork Extract

Abstract

Degradation of the quorum-sensing signal molecule N-acyl-l-homoserine lactone (AHL) in cocultures was verified with Bacillus cereus and Yersinia enterocolitica in culture medium and in pork extract. Results showed evidence of microbial interaction when the AHL-degrading bacterium and AHL-producing bacterium were cocultured in a food-simulating condition.

The term “quorum sensing” has been proposed to describe the ability of bacteria to monitor their own population density and modulate gene expression accordingly (17). This communication system uses chemical signal molecules called autoinducers, which are produced and released by the bacterial cell. Intracellular response occurs when the concentration of autoinducers rises above the minimum concentration threshold. Some gram-negative food-associated bacteria can produce N-acyl-l-homoserine lactones (AHLs) as signal molecules. Examples include Aeromonas hydrophila, Pseudomonas aeruginosa, and Yersinia enterocolitica (11, 12, 20, 24, 26). In these bacteria, a relationship between AHL production and expression of some mechanisms such as biofilm formation, motility, and exoprotease production has been described previously (2, 15, 17, 25).

The signal molecule AHL is the key factor in this bacterial communication process. Degradation of the molecule prevents its sufficient accumulation in the immediate vicinity of the bacterial cell and will consequently lead to a disruption of the communication system. In several fields, blocking of quorum sensing by degradation of AHL molecules has been proposed as a promising alternative to diminish bacterial virulence (4, 5, 6, 9, 18). In food systems quorum sensing may have a role in food spoilage, the growth and/or toxin production of pathogens present in food, biofilm formation, bacteriocin production, virulence responses, etc. A possible role of some bacteria in biological degradation of AHLs has been suggested (5, 6, 7, 10, 13, 14, 19, 22). Most of the reports on quorum-sensing degradation have been focused on plant pathogens (5, 6, 18). Although it is well known that the lactonase gene is widely spread among certain strains of food-borne Bacillus spp., no actual data are available on the interaction of mixed populations of AHL-producing and AHL-degrading bacteria in food-simulating conditions.

The aim of the present study was to screen for the AHL-degrading capacity of Bacillus cereus and some other current and former Bacillus spp. and to establish whether degradation capacity is noted in the coculture environment. The AHL-degrading capacity of Bacillus sp. strains was evaluated with both synthetic AHL molecules and AHL produced by Y. enterocolitica. The AHL degradation ability was further assessed in one B. cereus strain inoculated in coculture with a Y. enterocolitica strain in culture medium and pork extract.

Screening of synthetic-AHL degradation at 30°C.

Twenty B. cereus and 29 other current and former Bacillus isolates were screened for their AHL degradation capacities. N-Hexanoyl-l-homoserine lactone (C₆-HSL) (Biochemika Sigma-Aldrich) and 3-oxo-hexanoyl-l-homoserine lactone (3-oxo-C₆-HSL) (Sigma-Aldrich) were used as target molecules in the degradation assay. Bacillus strains were inoculated with a loopful of culture in 50 ml LB medium and incubated at 30°C for 14 h. After incubation, a volume of 900 μl of each culture was mixed with 100 μl of each of the synthetic standards to obtain the final concentrations of C₆-HSL and 3-oxo-C₆-HSL of 50 μmol liter⁻¹ and 46 μmol liter⁻¹, respectively. The mixtures were incubated for 24 h at 30°C, and the sterile supernatant was prepared by centrifuging cultures at 6,000 × g for 5 min in a microcentrifuge (Biofuge; Pico, Osterode, Germany) and by filtration (0.45-μm high-pressure liquid chromatography filters; Alltech, IL). The sterile supernatants were screened for the presence of AHL using an indirect fluorescence-based method (18). Three replicate fluorescence measurements per supernatant were performed. Escherichia coli JB523, containing the plasmid pJBA130, responsible for the production of a green fluorescent protein, was used as an AHL biosensor (1).

Controls prepared with each synthetic standard in LB medium were run in parallel. The recombinant Pseudomonas fluorescens P3/pME6863 strain and the derivate P. fluorescens P3/pME6000 were used as degrader and nondegrader reference strains, respectively (18). The screening results showed evidence of AHL-degrading factors occurring in B. cereus and Paenibacillus polymyxa (formerly Bacillus polymyxa). At 30°C, 15 out of 20 (75%) tested B. cereus isolates were able to degrade AHLs (Table 1). These results are in agreement with other reports presenting AHL degradation by several Bacillus species (5, 10). The two P. polymyxa strains degraded both AHL synthetic standards. Antibacterial properties of P. polymyxa have been reported elsewhere (21, 23); however, to the best of our knowledge no reports on the AHL degradation ability of this microorganism have been presented so far.

TABLE 1.

AHL degradation assay for Bacillus cereus and other current or former Bacillus spp. at 30°C^a

Isolate	C6-HSL	3-Oxo-C6-HSL
B. cereus isolates
527	+++	+
720	+++	+
396	+++	+
729	+++	+
254	+++	−
710	+++	−
711	+++	−
712	+++	−
718	+++	−
727	+++	−
005	++	+
004	++	+
724	+	+
431	++	−
435	−	+
258	−	−
426	−	−
428	−	−
434	−	−
552	−	−
Other current or former Bacillus sp. isolates
Bacillus alvei 110	−	−
Bacillus brevis
107	−	−
111	−	−
Bacillus circulans
196	−	−
681	−	−
682	−	−
683	−	−
684	−	−
685	−	−
Bacillus firmus 118	+	−
Bacillus lentus 631	−	−
Bacillus licheniformis
628	−	−
629	−	−
Bacillus megaterium
104	−	−
630	−	−
P. polymyxa
625	+++	+++
115	+++	+++
072	−	−
Bacillus pumilus
108	−	−
626	−	−
627	−	−
Bacillus sphaericus 109	−	−
Geobacillus stearothermophilus
007	−	−
113	−	−
008	−	−
Bacillus subtilis
102	−	−
623	−	−
624	−	−
Bacillus thuringiensis 092	−	−

^aAs determined with fluorescence assay (18). Symbols: −, no degrading activity observed; +, weak degrading activity; ++, medium degrading activity; +++, high degrading activity.

Screening of synthetic-AHL degradation at 7°C.

The ability of some psychrotrophic B. cereus isolates to degrade synthetic-AHL molecules at 7°C was investigated. A selection of psychrotrophic strains, previously confirmed as AHL degraders at 30°C, showed AHL-degrading capacity at 7°C, too.

Degradation of naturally produced AHL.

P. polymyxa 625 and B. cereus 720, previously determined as C₆-HSL- and 3-oxo-C₆-HSL-degrading strains, were selected as AHL degraders. Y. enterocolitica 057 was selected as an AHL producer (16). The sterile supernatant of a 24-h culture of Y. enterocolitica was mixed with overnight cultures of two AHL-degrading strains separately, and AHL degradation was evaluated as described above. Results obtained indicated a reduction of relative fluorescence unit (RFU) values and thus degradation of AHLs present in the sterile supernatants of cocultures of Y. enterocolitica 057 with B. cereus 720 or P. polymyxa 625, in comparison to the sterile supernatant of monoculture of Y. enterocolitica 057.

AHL degradation with sterile Bacillus sp. supernatants.

In order to determine if the degrading factor is excreted out of the cell or is cell bound, the filter-sterilized supernatants of the overnight AHL-degrading cultures of B. cereus 720 and P. polymyxa 625 were tested in the degradation assay, as described above. Current findings suggest that the degrading factor is not excreted to the cell exterior, which agrees with the report by Molina et al. (18) hypothesizing that AHL molecules diffuse into the AHL-degrader bacterial cells, where molecule inactivation takes place.

AHL degradation in cocultures.

Different bacterial relationships and behaviors can occur when mixed bacterial populations are present, which is often the case in food. Therefore, the performance of these AHL-degrading mechanisms was evaluated in food-simulating conditions using an AHL producer-AHL degrader model. The assay comprised cocultures of B. cereus 720 and Y. enterocolitica 057 on solid and in liquid culture media. Pork extract (solid and liquid) was used as a model food matrix. For coculture on solid medium Y. enterocolitica 057 and Bacillus cereus 720 were streaked perpendicularly to each other onto the surface of LB agar previously inoculated with E. coli JB523. LB agar inoculated with E. coli JB523 was prepared as described for top-layer agar used in thin-layer chromatography identification of AHLs (16). The pork extract, prepared as described elsewhere (3), was mixed with agar (Agar Bacteriological no. 1; Oxoid Ltd., Basingstoke, Hampshire, England) (1.6%) and sterilized. Pork extract agar was inoculated with the E. coli JB523 culture, as described above for LB agar. The non-AHL degrader B. cereus 258 was used as a negative control. After 24 h of incubation at 30°C, a fluorescent signal was determined using a Transilluminator (Clare Chemical Research DR; 45 M) under the conditions of 230 V, 50 Hz, and 9 W. The absence of fluorescence near the point of contact of B. cereus 720 and Y. enterocolitica 057 compared to the obvious fluorescence with nondegrading B. cereus 258 indicates that B. cereus 720 can degrade AHL molecules produced by Y. enterocolitica if present nearby (Fig. 1). Appropriate controls showed that pure cultures of B. cereus 720 and B. cereus 258 did not produce detectable AHL by the fluorescence assay. Similar results for AHL degradation were observed in pork extract agar. The possible explanation may lie in the diffusion of Y. enterocolitica short-chain AHLs through the medium into B. cereus cells, where they are then degraded.

FIG. 1.

Degradation assay on LB agar. (Left) Y. enterocolitica 057 and B. cereus 258 (AHL-nondegrading strain); (right) Yersinia enterocolitica 057 and B. cereus 720 (AHL-degrading strain). The area around B. cereus 258 (fluorescence) and that around B. cereus 720 (lack of fluorescence) indicate the inability of the former and the ability of the latter to degrade AHL molecules.

For coculture in liquid medium, Y. enterocolitica 057 and test strain B. cereus 720 were cultured in 7 ml LB medium and incubated overnight at 30°C. The AHL nondegrader B. cereus 258 was used as a negative control for AHL degradation. Afterward, a mix of B. cereus and Y. enterocolitica (10:1) was prepared and 30 μl of this mix was used to inoculate 30 ml of LB broth and/or pork extract (initial inoculum level of approximately 3 log CFU ml⁻¹ and approximately 4 log CFU ml⁻¹ for Y. enterocolitica and B. cereus, respectively). The cocultures were incubated for 24 h at 30°C and then 10-fold serially diluted in physiological peptone solution. The spread plate enumeration (detection limit, 100 CFU ml⁻¹) was performed on the surface of egg yolk polymyxin agar (LAB M; Lancashire, United Kingdom) and plate count agar (Oxoid, Hampshire, England) containing crystal violet (1.5%, wt/vol) to enumerate B. cereus and Y. enterocolitica, respectively. Centrifugation, filter sterilization, and screening for the presence of AHLs in the supernatant with fluorescence assay were performed as described for synthetic-AHL degradation. Results obtained indicated no inhibition of the bacterial growth. Both microorganisms reached similar stationary-phase levels when grown in coculture in comparison to respective monoculture. A reduction in the fluorescence signal (RFU) values was noted for the coculture of B. cereus 720 and Y. enterocolitica 057, indicating AHL degradation (Fig. 2). In contrast, in the coculture of B. cereus 258 and Y. enterocolitica 057 the obtained fluorescence signal (RFU value) was similar to the one produced by Y. enterocolitica in monoculture, indicating lack of AHL degradation (Fig. 2). However, no difference in the counts of B. cereus 720 and B. cereus 258 in the respective cocultures was observed. A reduction of RFU values with cocultures of B. cereus 720 and Y. enterocolitica 057 was also observed in pork extract (Fig. 3). However, in pork extract, a limited AHL degradation was observed also with B. cereus 258. As AHL molecules are chemically unstable under alkaline conditions (8), the pH value of pork extract (pH 5.7) could not have caused the observed AHL degradation. Further investigation is suggested in order to elucidate the observed results.

FIG. 2.

AHL detection by fluorescence assay in supernatant derived from coculture of Y. enterocolitica and B. cereus in LB medium. (a) Symbols: •, Y. enterocolitica 057 (positive control); □, Y. enterocolitica 057 and B. cereus 720 (AHL degrader); ▪, LB medium (blank). (b) •, Y. enterocolitica 057 (positive control); □, Y. enterocolitica 057 and B. cereus 258 (AHL nondegrader); ▪, LB medium (blank).

FIG. 3.

AHL detection by fluorescence assay in supernatant derived from coculture of B. cereus and Y. enterocolitica in pork extract. Symbols: □, Y. enterocolitica 057 and B. cereus 720 (AHL degrader); ▴, Y. enterocolitica 057 and B. cereus 258 (AHL nondegrader); ▪, Y. enterocolitica 057 (positive control); •, pork extract (blank).

The present study provides evidence of the AHL-degrading capacities of B. cereus and P. polymyxa, also under food-simulating conditions. These bacteria may use this AHL-degrading mechanism as a competitive advantage over bacterial competitors, and it may help them to dominate their ecological niche (6, 19).