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

Degradation of the quorum-sensing signal molecule N-acyl-L-homoserinelactone (AHL) in cocultures was verified with Bacillus cereusand Yersinia enterocolitica in culture medium and in pork extract.Results showed evidence of microbial interaction when the AHL-degradingbacterium and AHL-producing bacterium were cocultured in a food-simulatingcondition.

INTRODUCTION

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Introduction

The term "quorum sensing" has been proposed to describe theability of bacteria to monitor their own population densityand modulate gene expression accordingly (17). This communicationsystem uses chemical signal molecules called autoinducers, whichare produced and released by the bacterial cell. Intracellularresponse occurs when the concentration of autoinducers risesabove the minimum concentration threshold. Some gram-negativefood-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 productionand 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 bacterialcommunication process. Degradation of the molecule preventsits sufficient accumulation in the immediate vicinity of thebacterial cell and will consequently lead to a disruption ofthe communication system. In several fields, blocking of quorumsensing by degradation of AHL molecules has been proposed asa promising alternative to diminish bacterial virulence (4,5, 6, 9, 18). In food systems quorum sensing may have a rolein food spoilage, the growth and/or toxin production of pathogenspresent in food, biofilm formation, bacteriocin production,virulence responses, etc. A possible role of some bacteria inbiological degradation of AHLs has been suggested (5, 6, 7,10, 13, 14, 19, 22). Most of the reports on quorum-sensing degradationhave been focused on plant pathogens (5, 6, 18). Although itis well known that the lactonase gene is widely spread amongcertain strains of food-borne Bacillus spp., no actual dataare available on the interaction of mixed populations of AHL-producingand AHL-degrading bacteria in food-simulating conditions.

The aim of the present study was to screen for the AHL-degradingcapacity of Bacillus cereus and some other current and formerBacillus spp. and to establish whether degradation capacityis noted in the coculture environment. The AHL-degrading capacityof Bacillus sp. strains was evaluated with both synthetic AHLmolecules and AHL produced by Y. enterocolitica. The AHL degradationability was further assessed in one B. cereus strain inoculatedin coculture with a Y. enterocolitica strain in culture mediumand pork extract.

Screening of synthetic-AHL degradation at 30°C.

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Screening of synthetic-ahl...

Twenty B. cereus and 29 other current and former Bacillus isolateswere screened for their AHL degradation capacities. N-Hexanoyl-L-homoserinelactone (C₆-HSL) (Biochemika Sigma-Aldrich) and 3-oxo-hexanoyl-L-homoserinelactone (3-oxo-C₆-HSL) (Sigma-Aldrich) were used as target moleculesin the degradation assay. Bacillus strains were inoculated witha loopful of culture in 50 ml LB medium and incubated at 30°Cfor 14 h. After incubation, a volume of 900 µl of eachculture was mixed with 100 µl of each of the syntheticstandards to obtain the final concentrations of C₆-HSL and 3-oxo-C₆-HSLof 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 culturesat 6,000 x g for 5 min in a microcentrifuge (Biofuge; Pico,Osterode, Germany) and by filtration (0.45-µm high-pressureliquid chromatography filters; Alltech, IL). The sterile supernatantswere screened for the presence of AHL using an indirect fluorescence-basedmethod (18). Three replicate fluorescence measurements per supernatantwere performed. Escherichia coli JB523, containing the plasmidpJBA130, responsible for the production of a green fluorescentprotein, was used as an AHL biosensor (1).

Controls prepared with each synthetic standard in LB mediumwere run in parallel. The recombinant Pseudomonas fluorescensP3/pME6863 strain and the derivate P. fluorescens P3/pME6000were used as degrader and nondegrader reference strains, respectively(18). The screening results showed evidence of AHL-degradingfactors occurring in B. cereus and Paenibacillus polymyxa (formerlyBacillus polymyxa). At 30°C, 15 out of 20 (75%) tested B.cereus isolates were able to degrade AHLs (Table 1). These resultsare in agreement with other reports presenting AHL degradationby several Bacillus species (5, 10). The two P. polymyxa strainsdegraded both AHL synthetic standards. Antibacterial propertiesof P. polymyxa have been reported elsewhere (21, 23); however,to the best of our knowledge no reports on the AHL degradationability of this microorganism have been presented so far.

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TABLE 1. AHL degradation assay for Bacillus cereus and other current or former Bacillus spp. at 30°C^a

Screening of synthetic-AHL degradation at 7°C.

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

Degradation of naturally produced AHL.

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Degradation of naturally...

P. polymyxa 625 and B. cereus 720, previously determined asC₆-HSL- and 3-oxo-C₆-HSL-degrading strains, were selected asAHL degraders. Y. enterocolitica 057 was selected as an AHLproducer (16). The sterile supernatant of a 24-h culture ofY. enterocolitica was mixed with overnight cultures of two AHL-degradingstrains separately, and AHL degradation was evaluated as describedabove. Results obtained indicated a reduction of relative fluorescenceunit (RFU) values and thus degradation of AHLs present in thesterile supernatants of cocultures of Y. enterocolitica 057with B. cereus 720 or P. polymyxa 625, in comparison to thesterile supernatant of monoculture of Y. enterocolitica 057.

AHL degradation with sterile Bacillus sp. supernatants.

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Ahl degradation with sterile...

In order to determine if the degrading factor is excreted outof the cell or is cell bound, the filter-sterilized supernatantsof the overnight AHL-degrading cultures of B. cereus 720 andP. polymyxa 625 were tested in the degradation assay, as describedabove. Current findings suggest that the degrading factor isnot excreted to the cell exterior, which agrees with the reportby Molina et al. (18) hypothesizing that AHL molecules diffuseinto the AHL-degrader bacterial cells, where molecule inactivationtakes place.

AHL degradation in cocultures.

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Ahl degradation in cocultures.

Different bacterial relationships and behaviors can occur whenmixed bacterial populations are present, which is often thecase in food. Therefore, the performance of these AHL-degradingmechanisms was evaluated in food-simulating conditions usingan AHL producer-AHL degrader model. The assay comprised coculturesof B. cereus 720 and Y. enterocolitica 057 on solid and in liquidculture media. Pork extract (solid and liquid) was used as amodel food matrix. For coculture on solid medium Y. enterocolitica057 and Bacillus cereus 720 were streaked perpendicularly toeach other onto the surface of LB agar previously inoculatedwith E. coli JB523. LB agar inoculated with E. coli JB523 wasprepared as described for top-layer agar used in thin-layerchromatography identification of AHLs (16). The pork extract,prepared as described elsewhere (3), was mixed with agar (AgarBacteriological no. 1; Oxoid Ltd., Basingstoke, Hampshire, England)(1.6%) and sterilized. Pork extract agar was inoculated withthe E. coli JB523 culture, as described above for LB agar. Thenon-AHL degrader B. cereus 258 was used as a negative control.After 24 h of incubation at 30°C, a fluorescent signal wasdetermined using a Transilluminator (Clare Chemical ResearchDR; 45 M) under the conditions of 230 V, 50 Hz, and 9 W. Theabsence of fluorescence near the point of contact of B. cereus720 and Y. enterocolitica 057 compared to the obvious fluorescencewith nondegrading B. cereus 258 indicates that B. cereus 720can degrade AHL molecules produced by Y. enterocolitica if presentnearby (Fig. 1). Appropriate controls showed that pure culturesof B. cereus 720 and B. cereus 258 did not produce detectableAHL by the fluorescence assay. Similar results for AHL degradationwere observed in pork extract agar. The possible explanationmay lie in the diffusion of Y. enterocolitica short-chain AHLsthrough the medium into B. cereus cells, where they are thendegraded.

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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 teststrain B. cereus 720 were cultured in 7 ml LB medium and incubatedovernight at 30°C. The AHL nondegrader B. cereus 258 wasused as a negative control for AHL degradation. Afterward, amix of B. cereus and Y. enterocolitica (10:1) was prepared and30 µl of this mix was used to inoculate 30 ml of LB brothand/or pork extract (initial inoculum level of approximately3 log CFU ml^–1 and approximately 4 log CFU ml^–1for Y. enterocolitica and B. cereus, respectively). The cocultureswere incubated for 24 h at 30°C and then 10-fold seriallydiluted in physiological peptone solution. The spread plateenumeration (detection limit, 100 CFU ml^–1) was performedon 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. cereusand Y. enterocolitica, respectively. Centrifugation, filtersterilization, and screening for the presence of AHLs in thesupernatant with fluorescence assay were performed as describedfor synthetic-AHL degradation. Results obtained indicated noinhibition of the bacterial growth. Both microorganisms reachedsimilar stationary-phase levels when grown in coculture in comparisonto respective monoculture. A reduction in the fluorescence signal(RFU) values was noted for the coculture of B. cereus 720 andY. enterocolitica 057, indicating AHL degradation (Fig. 2).In contrast, in the coculture of B. cereus 258 and Y. enterocolitica057 the obtained fluorescence signal (RFU value) was similarto the one produced by Y. enterocolitica in monoculture, indicatinglack of AHL degradation (Fig. 2). However, no difference inthe counts of B. cereus 720 and B. cereus 258 in the respectivecocultures was observed. A reduction of RFU values with coculturesof B. cereus 720 and Y. enterocolitica 057 was also observedin pork extract (Fig. 3). However, in pork extract, a limitedAHL degradation was observed also with B. cereus 258. As AHLmolecules are chemically unstable under alkaline conditions(8), the pH value of pork extract (pH 5.7) could not have causedthe observed AHL degradation. Further investigation is suggestedin order to elucidate the observed results.

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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); ${square}$ , Y. enterocolitica 057 and B. cereus 720 (AHL degrader); ${blacksquare}$ , LB medium (blank). (b) •, Y. enterocolitica 057 (positive control); ${square}$ , Y. enterocolitica 057 and B. cereus 258 (AHL nondegrader); ${blacksquare}$ , LB medium (blank).

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FIG. 3. AHL detection by fluorescence assay in supernatant derived from coculture of B. cereus and Y. enterocolitica in pork extract. Symbols: ${square}$ , Y. enterocolitica 057 and B. cereus 720 (AHL degrader); ${blacktriangleup}$ , Y. enterocolitica 057 and B. cereus 258 (AHL nondegrader); ${blacksquare}$ , Y. enterocolitica 057 (positive control); •, pork extract (blank).

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

ACKNOWLEDGMENTS

We thank Tom Defoirdt of the Department of Biochemical and MicrobialTechnology, Faculty of Bioscience Engineering, Ghent University,for providing the E. coli JB523 strain.

We thank the Consejo de Desarrollo Científico y Humanísticoof Universidad Central de Venezuela for the Ph.D. scholarshipof M. S. Medina-Martínez.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Food Microbiology and Food Preservation, FBW-UGent, Coupure Links 653, 9000 Ghent, Belgium. Phone: 32 9 264 6178. Fax: 32 9 225 5510. E-mail: Mieke.Uyttendaele{at}UGent.be.

Published ahead of print on 9 February 2007.

REFERENCES

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