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Bacteria are present in natural environments, both on biotic and abiotic surfaces, in the state of biofilm, where even more than one microbial species can be present at the same time. Because not all the available location are not colonized by such microbial consortia, it has been suggested that microbes possess suitable mechanisms to perceive specific features of the surrounding environment and their changes. Furthermore specific communication system could be involved in the spatial and functional organization of biofilm network, in order to coordinate all the organisms within the biofilm matrix and their metabolic activities. These mechanisms are based on small signal molcules, which can easily diffuse in the environment as well as inside the bacterial cell and can modify gene regulation and thus physiological response of a microorganism or a microbial community. The concentration of these signals is depending on the bacterial density and thus bacterial species can monitor population density and modify their metabolic pathways accordingly. Because of the concentration-dependent response, this communication system has been called quorum sensing. This mechanism has been related with several biological microbial activities such as toxin production, bioluminescence and biofilm formation.
The heterogeneity of microbial species has led also to a diversified production of signal molecules: while Gram negative are able to produce N-acyl homoserine lactones (AHLs), modified oligopetides are frequently associate to Graqm positive bacteria. Despite this difference, both these two microbial groups share AI-2 quorum sensing pathway based on the activity of the enzyme LuxS. LuxS acts, within bacterial cell, as component of activated methyl cycle (AMC), which, starting from S-adenosyl-L-methionine (SAM), produces activated methyl substituents, necessary for protein methylation, RNA, DNA and other metabolites. This reaction is catalyzed by dedicated transmethylases with consequent formation of S-adenosyl-L-methionine (SAH). Because of its highly toxic characteristics for the cell, species of both kingdoms Archaea and Eukarya employ a one-step pathway of detoxification through conversion of SAH into adenosine and homocysteine: the involved enzyme is SahH. In order to achieve detoxification of SAH, there is an alternative possible process, demonstrated in all Firmicutes and some species of proteobacteria: in this case SAH is converted in adenosine and S-ribosyl homocysteine (SRH) by Pfs and LuxS transforms SRH into homocysteine (HCY) and 4,5-dihydroxy-2,3-pentanedione (DPD). This latter one is the effective precursor of AI-2, which can be formed through different internal ciclizations due to unstable nature of DPD.
Begin of research on luxS could be found in Greenberg et al. (1979), where culture surnatant of diverse non bioluminescent microbes caused bioluminescence of V. harvey. After identification of AI-1 and its related system, researchers hypothesized the contemporary presence of an additional autoinducer-based process which allows interspecies communication with V. harvey. Demonstration of this was given by Bassler et al. (1997), where AI-2 was presented as "interspecies communication signal". After few years, structure of AI-2 and its corresponding receptor LuxP was determined. Once AI-2, diverse studies were conducted on presence and effects off luxS in several bacterial species, whose modified phenotypes were attributed to luxS-based QS signalling. Until now more than 70 diverse bacterial species are known to produce AI-2 and luxS has been identified in many bacterial genomes sequenced, leading to the general conclusion that AI-2 are accepted by scientific community as "universal cell-to-cell signal in prokaryotic microorganism" (Turovskiy, et al. 2007).
Researches concerning AI-2 and QS have been in the most recent period associate to another well exploited topic, biofilm formation on surfaces. Different authors have exploited these two themes in their published reports, some of which are briefly summarized in the following lines. Belval et al. (2006) examined the influence of AI-2 on attachment to surfaces by L. monocytogenes EGD-e.Â Compared to wild-type, the Î”luxS strain produced denser biofilms on stainless steel. External additions of AI-2 did not alter number of adhered cells, although SRH could affect significantly biofilm cellular density of both wild-type and mutant strains. The authors conclude that altered phenotype is more probably to attribute to an accumulation of SRH instead of QS gene disruption. Similar conclusion were demonstrated with regard to Lactobacillus reuterii, by Tannock et al. (2005), where a luxS mutant of L. reuteri 100-23 was constructed and evaluated for adhesion capacity on plastic surface. Additionally luxS disrurption seem to have wider range of effect as ATP content of mutant was 35% lower than parental strain.
Doherty et al. (2006) showed that luxS-null mutant of S. aureus did not differs significantly from parental strain, when it was grown in rich growth medium. Also virulence and hemolysis were not affected from gene knock-out as well as biofilm formation, although growth in sulfur-limited condition was heavily compromised. Authors hypothesized that AMC cycle was substituted by intake of methionine from environment with consequent modification of metabolism. From test of competition involving wild-type and luxS null strain, authors concluded that cell grown in coculture have same pool of autoindicers and modified phenoptype could not be attributed to QS. LuxS has been demonstrated by Schauder et al. (2005) to have similar observed effect also in N. meningitidis. Beside effect of biofilm, luxS seem to have more wide range influence on general metabolism. Sperandio et al. (2006) from their study of trascriptomics on luxS-null mutant have demonstrated an altered phenotype in biosynthesis, metabolism and transport of aminoacids as well as in carbon compounds metabolism, but comprehension of actual range of luxS influence is still far from complete exploitation.
Biofilms represent a quite extensively exploited topic: Djordevic et al. (2002) and Harvey et al. (2007) employed a microtiter plate assay to assess quantitatively the adhesive properties of L. monocytogenes strains in their investigations, as well as Kushwaha and Muriana (2009) did. Another quite often used device was stainless steel coupon (Kalmokoff, et al. 2001; Mai, et al. 2007; Fuster-Valls, et al. 2008), where testing strains were grown at different temperatures and with diverse growth media to evaluate the relevance of such parameters. Other scientists (Sternberg, et al. 1999; Heydorn, et al. 2000; Stoodley, et al. 2001; Perni, et al. 2006,) use a dynamic approach: tested surfaces were put into flow chambers or cells, where nutrients solution was flown at defined fastness and, through suitable microscopy strumentation, kinetics and developing architecture of biofilm were observed.
Unfortunately both these approaches have intrinsic disadvantages, which make them not perfectly suitable to simulate the process of bacterial setting on surfaces. Through static devices bacterial adhesivity capacity on different material can be investigated, but, instead of biofilm, cells deposition is supposed more probably to take place. Furthermore, shear forces and other factors related to flow rate and mobility in fluids environments are absent in similar conditions, leading to modifications of architecture and complexity of biofilm architecture: cells are enveloped in a multilayered structure of organic matter, which can increase just its thickness within certain limits. When subjected in turbulent regime, voids within the matrix and tower-like complexes are observed and detachment of cells-organic matter occurs.
This aspect is strongly related to antimicrobial resistance: in a multilayered complex biocide cannot penetrate the whole thickness acting only in top levels, while in dynamic regimen cells can significantly modify their metabolic pathways expressing the so-called "`biofilm phenotype", physiological state in which cells are less susceptible to antimicrobials and more suitable to grow in absence or low levels of nutrients. Equipment like flow chambers can overcome this limit by application of flow rate of solution through a peristaltic pump, but the final result is a qualitative or semi-quantitative measurement because of monitoring all the stages of the process by epifluorescence microscopy or other optical techniques.
Aim of the present work was investigating on influence of luxS on biofilm formation. To carry out this purpose, luxS in-frame deletion was performed on L. innocua UC 8410 through Campbell-like single crossover event. Both wild type and luxS-null mutant were evaluated for surface adhesivity both in static and dynamic conditions as well as for biocide resistance. Also modifications of other traits of interest (e.g. hydrophobicity) due to applied genetic deletion were evaluated.
Materials and methods
Bacterial strains and plasmids
Three isolates of Listeria innocua (CLIP 11262, UC 8409 and UC 8410) were cultured in BHI broth at 37Â°C. E. coli TB1 was grown in Luria-Bertani (LB) at 37Â°C in shaking conditions. L. innocua 7117 (Listeria innocua Î”luxS UC 8410) was grown at 37Â°C in BHI supplemented with 32 Î¼g/ml erythromycin. V. harvey BB117 (ATCCÂ® BAA-116) was grown in aerobic conditions in Marine Broth (MB, Difco) at 30Â°C. Cloning procedure has involved p-GEM T-easy Vector (Promega) and pRV300. Electrocompetent containing the cloning pGEM T-easy- and pRV300-based vectors were cultured in LB broth supplemented with 100 Î¼g/ml of suitable antibiotic (ampicillin and erythromycin, respectively).
Stainless steel coupons were used to test Listeria adhesion in static conditions of incubation. 0,1 mm thickness wire of stainless steel (SS), polyethylene terephtalate (PET), and Copper (C), which were acquired from local supermarkets, were used as surface test materials in dynamic regime tests, covering a large range of possible materials present in food transforming locations.
Benzalkonium chloride (BC) (50% in water, Fluka) was prepared at concentrations of 200 Î¼g/ml and 5% (equal to 5,8 mg/ml), respectively, as routinely used for sanitation in food processing environments.
Single colonies from ALOA (Agar Listeria & Ottaviani Agosti, Biolife Italiana Spa) were used as inoculums for 10 ml BHI broth. 1 Âµl of an overnight subculture of Listeria innocua was treated with 19 Âµl of microLysis (Labogen), as indicated by the manufacturer. 1 Î¼l of the resulting solution was used as DNA template in 50 Âµl PCR reaction using GoTaq Green Master Mix (Promega), 0.4 ÂµM luxS primers (LuxS1-LuxS2). luxS primers sequence and PCR thermal conditions are reported in T. In parallel, PCR of iap was performed on same samples following protocol reported in Jordan, et al. (2008) as control. Experimental conditions are reported in Tab. 1.
The amplified fragment was purified by microClean (Labogen) and quantified by Marker II (Roche). The obtained fragment was inserted in p-GEM T-easy vector (Promega) according to manufacturer protocols, obtaining the plasmid pPC 7048. The so constructed cloning vector was electroporated in electrocompetent E. coli TB1 at 200 Î©, 25 Î¼F and 12.5 kV/cm (time constant = 4.0÷4.4) in 0.2 cm electroporation cuvette (Biorad). The final mixture was plated by spreading onto LB agar plates supplemented with 100 Âµg/ml ampicillin. The selected colonies were cultured in 10 ml LB broth incubated at 37Â°C in shaking overnight and the plasmid was extracted by Wizards SV MInipreps (Promega). Gene insertion in pGEM T-easy Vector was verified by PCR. 10 Âµl plasmidic DNA were digested with SalI at 37Â°C for 3 hours and loaded into 1x agarose gel in 1x TAE.
The 240 bp band was cut and purified by GenEluteâ„¢ Gel Extraction Kit (Sigma-Aldrich). The so treated fragment was ligated in pRV300, previously digested with SalI and dephosphorylated by calf alkaline phosphatase (Promega). 1 Âµl of treated plasmid was mixed with 3 Î¼l of 240 bp fragment and ligated by using T4 ligase (Promega). The final mixtures were incubated at 4Â°C over-night and electroporated in E. coli TB1, as previously described. The so treated E. coli was plated by spreading onto LB agar plates containing 100 Âµg/ml erythromycin and incubated over-night at 37Â°C. One colony was picked up to inoculate 10 ml LB containing 100 Âµg/ml erythromycin. The so constructed shuttle vector was extracted as above mentioned and verified by a PCR screening with the primers T7 promoter-LuxS2 and LuxS1-LuxS2.
Preparation of electrocompetent cells
Electrocompetent L. innocua cells were obtained using procedure of Monk et al. (2008) with slight modifications. A shaken overnight BHI culture was diluted 1:100 in 500 ml of BHI containing 500 ÂµM sucrose (BHIS), resulting in an initial optical density at 600 (OD600) of 0.01 ÷ 0.02, and then grown to an OD600 of 0.4 ÷ 0.5. Cells were cooled on ice for 10 min and centrifuged (5,000 x g for 10 min at 4Â°C). Cell pellets were resuspended in 500 ml of ice-cold sucrose-glycerol wash buffer (SGWB) (10% glycerol, 500 ÂµM sucrose; pH adjusted to 7 with NaOH; filter- sterilized) by swirling on ice. Cells were centrifuged two more times; they were resuspended in 175 ml of SGWB after the first centrifugation and in 50 ml of SGWB after the second centrifugation. Cells were centrifuged (3,000 x g for 10 min at 4Â°C) and resuspended in 20 ml of SGWB. Cells were finally centrifuged, the final volume was adjusted to 2.5 ml by pipetting, and 50 Âµl aliquots were frozen at -80Â°C.
Electroporation into L. innocua
A 50 Âµl aliquot of electrocompetent cells was mixed with 2 of plasmid DNA and incubated on ice for 5. The mixture was transferred to a chilled 1 electroporation cuvette (Bio-Rad) and pulsed at 10 kV/cm, 400 Î© and 25 ÂµF. Time constants between 7 and 8 were observed with the protocol described above. To regenerate the cells, 1 ml of room temperature autoclaved BHIS was pipetted immediately into the cuvette and incubated statically at 37Â°C for 1,5h. Regenerated cells were serially diluted and plated on BHI agar containing erythromycin.
Adhesion in static condition
Stainless steel coupons were used as test surface: each coupon has ten squares (1 cm x 1.cm x 0.1 cm), organized in two rows, for an overall superficial area of 1 cm2. L. innocua strains were subcultured in BHI broth for 18 h at 200 rpm at 37Â°C. Each strain was harvested at 4000 xg for 5 min and washed twice with sterile peptone water. Cell numbers were determined by serial dilutions and plating on TSA (Tryptone Soy Agar, LABM, United Kingdom) plates, after incubation for at 37Â°C 24 h. In parallel to plate counting, optical density at 600 nm (OD600) was measured on the same dilutions, in order to have a rapid evaluation of bacterial density. Aliquots were taken from aqueous cell suspensions and inoculated into 25 ml BHI Broth. The volume of aliquots to be added into 25 ml BHI broth, was calculated in order to provide a density of 105-106 CFU/ml. Inoculated medium was added into a Petri plate containing a previously washed and autoclaved steel coupon. After 24 h incubation at 37Â°C, the coupon was transferred into a new Petri plate, into which 25 ml of 10% diluted BHI (dBHI). The so treated coupon was incubated up to 7 days at 37Â°C. At defined periods coupons were scraped with wet cotton swabs. Swabs were serially diluted into sterile peptone water and plated onto TSA plates, which were incubated as above. Results were expressed as average of three replicates.
Biofilm formation in presence of a turbulent flow
The apparatus was composed by 5 glass tubes (10 cm long, internal diameter 0. 4 mm), into which the wire test material was introduced, linked with teflon junctions to a solution dispenser (30 cm long, i.d. 20 mm). The apparatus was chemically disinfected with an succession of disinfection solutions (NaOH 0.5 mol and 50% EtOH) and washed with sterile physiological solution. An over-night culture of L. innocua strains in skimmed milk was introduced into the system at 1 ml/min flow rate and, when the pipeline was full, the flow was stopped and the whole system was incubated at 37Â°C for 1h. A washing step with physiological solution was performed to remove only slightly adhered cells. After this step, the nutrient solution was flown inside the appatatus at a flow rate for 1 ml/min. At defined time points (0, 3, 6, 18, and 24 h), the flow was stopped and aliquots of both tested materials and eluate were taken and subjected to serial dilutions and plating on BHI agar, to count both sessile and plancktonic cells, respectively. All the experiments were performed in triplicate.
Minimal biocidal concentration (MBC)
Minimal biocidal concentration was determined through the two available broth microdilution protocols, NCCLS guidelines and EURECAST protocol. L. innocua UC 8410 and UC 7117 were subcultured in BHI broth for 18 hours at 37Â°C in aerobic shaking. Both the strains were washed with sterile saline solution twice and diluted until OD625=0.15-0.20. The so-treated subcultures were exposed to different increasing concentrations of benzalkonium chloride in 96-wells polystyrene plates and incubated for 24 hours at 37Â°C. The plates were read through a microplate reader at three different concentrations. Experiments were performed onto six replicates.
Bactericidal efficiency of disinfectants
Both L. innocua UC 8410 and L. innocua UC 7117 were grown in biofilm state using apparatus described in previous paragraph. Bactericidal effectiveness of above mentioned biocides on planktonic and sessile cells was evaluated on aliquots of nutrient solution (milk) and tested surfaces (1 cm wire), respectively during 24 h monitoring period by using UNI EN 1040:2006 protocol.
Monitoring of Listeria growth kinetic through semi-automated apparatus
Bacterial cultures were washed and adjusted with 0.9% NaCl in order to get OD625 of 0.080÷0.100. 100 Î¼l of cell suspension were added into a 100 wells honeycomb plates (Honeycomb, ThermoLabsystems, Finland), where 300 Î¼l of test medium and 4 Î¼l of testing antimicrobial were added. Plates were incubated for 24-48 h at hours with low shaking and reading of optical density were performed every 15 min by using a wide band filter (420-580 nm). At least two independent experiments were performed with approximately 3-5 replicates for each of them, while data elaboration was conducted on average of each replicate. Test medium was BHI broth. Results were fitted into a modified equation of Gompertz, as reported in Begot, et al. (1996).
Microbial adhesion to solvents (MATS)
For the assay four solvents were used: hexadecane (apolar) and chloroform This hydrofobicity assay was performed as described by Briandet et al. (1999): 18h liquid subculture was harvested at 2500 xg for 5 min and washed twice with 0.85 % NaCl in water. Cells suspension was divided in 2.4 aliquots, to which 0.4 ml of solvent was added. The mixture was vortexed for 1 min and then incubated at room temperature for 15 min, to allow complete separation of the two phases composing the mixture. 1 ml was taken from the mixture and optical density at 400 nm was measured. Results were collected from three replicates obtaining by using two independent subcultures and were expressed as percentage of affinity with solvent by using the equation:
% affinity with the solvent = 100 x [1-(A/A0)]
AI2 bioluminescence assay
Bioluminescence assay for quantitative detection of AI-2 was performed as described from Vilchez et al. (2007). Briefly, an aliquot form -70Â°C stock was grown onto an AB medium plate over-night. Plate was washed with fresh AB medium and then cultured in AB-Fe medium (AB medium supplemented with iron solution) for 1.5 in shaking conditions. Once checked optical density, culture was diluted in order to get reference value (3000-5000 CPS). All measurements were performed in triplicate using Victor 1420 Multilabel Plate Reader (Perkin Elmer).
In parallel method described by Bassler et al. (1993) was carried out with slight modification. A over-night subculture of V. harvey BB117 in 3 ml of Marine Broth (MB, Difco) was diluted 1:5000 in fresh MB and used as working solution. In parallel testing solutions were prepared as following. 1%-inoculated liquid subculture was harvested at 2500 xg for 10 min at room temperature. Cell surnatant of L. innocua was recovered, adjusted to pH 7 and sterilized through filtration with 0.45 Î¼m membrane filter. The so-treated surnatant was divided into aliquots and kept at -20Â°C. 900 Î¼l of working solution were mixed with 100 Î¼l of sterile pH-adjusted cell surnatant and incubated for 6 h. Measurement was performed through a systemSUREâ„¢ portable luminometer (Celsisâ€¢Lumac, Cambridge). As negative control, pH-adjusted and filter-sterilized MB was used. All obtained data were obtained from three replicates.
Cultivation of bacterial biofilms of Listeria innocua in microtiter plates
A 18 hours liquid subculture in BHI broth was centrifuged at 2700 xg for 10 minutes, washed twice with PBS pH 7.4 and resuspended in 5 ml PBS. After dilution up to approximately 5 x 106 CFU/ml, 200 Î¼l of each bacterial suspension were added in 96-well sterile polystyrene microtiter plate. The so-treated plate was incubated for 48 h at 37Â°C. Bacterial growth was measured at 620 nm after 24 and 48 h using Thermo Scientific Plate Reader. Growth media used were 1% (w/v) glucose-supplemented Tryptone Soy Broth (TSB; LABM, United Kingdom), TSB, 10% diluted TSB (dTSB), BHI and 10% diluted BHI (dBHI).
Determination of extracellular polysaccharides (EPS) through red ruthenium staining
This assay was performed as described by Boruchi et al. (2003) and Zameer et al. (2009). Bacterial biofilms grwon as above described were washed with PBS and stained with ruthenium red. After the removal of exhausted liquid medium, 200 Î¼l of an aqueous suspension of 0.1% ruthenium red was added to each well and incubated for 45 min at room temperature. The liquid was transferred into a new microtiter plate and the optical density at 450 nm was measured in order to quantify the amount of dye bound to the EPS within the biofilms themselves. Results were expressed as average of six replicates.
Statistical analysis and treatment of data
Data were subjected, through SPSS 14.0, to One-way ANOVA, t test and post-hoc analysis, in order to detect significant parameters and to give a quantitative definition of influence of selected parameters (i.e. strain, temperature, nutrients, surface material).
luxS gene inactivation in L. innocua
As first step, the presence of luxS was verified into the studied strains. Primers used (LuxS1-LuxS2) were constructed basing on the sequences of luxS of L. innocua CLIP 11262present in Genebank. Both UC 8409 and UC 8410 harbor this gene, as confirmed by the sequence analysis of the amplified fragment, as reported in Fig. . In order to evaluate its influence on biofilm formation, luxS gene disruption was performed by a Campbell-like gene inactivation. To achieve this goal, the plasmids pRV300 was used as shuttle vector in L. innocua.
A 240 bp fragment of luxS gene (from bp base 135 to bp base 455) was amplified in L. innocua UC 8410, cloned in the multi-cloning site site of pGEMÂ®-T Easy vector and then introduced in the vector pRV300, a plasmid harbouring an origin of replication suitable for E. coli but not for Firmuctes as L. innocua (Leloup et al. 1997) as well as the gene for erythromycin resistance. The knock-out vector harboring the 240 bp luxS fragment was replicated in E. coli TB1. PCR screening was conducted on plasmids isolated from E. coli TB1 to verify that they were properly constructed: primers used were able to amplify the sequence between the plasmid promoter (T7 for) and investigated gene. The verified vector, named pPC 7051, was then electroporated into electrocompetent L. innocua cells.
Once regenerated with sucrose supplemented medium, cells were plated on agar plates supplemented with 5 Î¼g/ml of erythromycin agar plates and were then screened by PCR assay to verify plasmid integration in bacterial chromosome and thus genetic disruption of target gene: the couple of primers T7-LuxS2 was able to amplify the sequence between the T7 promoter and the inserted fragment harboured by the knock out vector (380 bp), while a 320 bp was the amplicone of chromosomal luxS gene obtained by using the primers LuxS1-LuxS2 (Fig. 1). luxS-deleted L. innocua strains were coded as UC 7117.
Effect of luxS gene disruption through MATS and bioluminescence
To verify the physiological effect of luxs inactivation, the bioassay for AI-2 detection through the bioluminescence of V. harvey was performed on the cell supernatant of both L. innocua UC 8410 and UC 7117. For this purpose a culture of V. harvey in stationary phase was exposed to filter-sterilized cell surpernatant, whose pH was adjusted to pH 7. Results are reported in Fig. 3. Overnight subculture of L. innocua UC 8410 was demonstrated to produce a signal of 0.10 RLU, while this feature was completely abolished in the luxS-null mutant UC 7117.
Moreover, the autoinducer production was monitored during 24 h of growth through bioluminescence of V. harvey BB170 with AB medium as previously reported (Vilchez, et al. 2007). Results showed clearly that UC 7117 had a sharply decrease ability in producing AI-2 compared to wild type version (Fig. 2 and Fig. 3). The effect of genetic mutation on cell hydrophocbicity was investigated through microbial adhesion to solvents (MATS) assay. This method was performed with four different solvents, as reported by Briandet et al. (1999) and Hamadi and Latrache (2008). The solvents used were:
Hexadecane, an apolar solvent, and chloroform, acid monopolar solvent with weak basic properties;
Diethyl ether, strong basic solvent, and hexane.
UC 8410, in tests with hexadecane, were compared to other available strains of same species and with its luxS null derivative version, to assess if cell surface property could explain different adhesion capacity (Fig. 7). Relevant discrepancies were observed among tested isolates (even if belonging to same environmental niche). Most of tested strains showed low affinity to hexadecane, demonstrating that cells can be correctly assumed as strongly hydrophilic. UC 8409 revealed higher affinity to employed solvent (17%, respectively), which was considered as not significant in statistical elaboration. Although luxS-null strains expressed quantitatively different hydrophobicity from their wild-type analogue, L. innocua UC 7117 has revealed marked hydrophilic properties, while its parental strain showed slight hydrophobicity. This conclusion was confirmed by further tests using all the four solvents (Fig. 8).
Growth kinetic was significantly affected by luxS
luxS in L. innocua was investigated if it couldetermine further effects beside the attachment ability on abiotic surfaces. For this purpose growth kinetic of L. innocua UC 8410 was periodically monitored through the turbidometer BioscreenC in parallel with its luxS-null version, UC 7117. Apparently the growth kinetic curve of both the strains differs slightly between each other: while UC 8410 grew sharply up to 1.856 Â± 0.143 and then decreased equally rapidly, UC 7117 raised more gradually until 1.67 Â± 0.106, which was kept almost constantly for 48 h (Fig. 6). UC 8410 possessed a 30% higher generation time, while the growth rate of luxS-null strain was 52% more reduced than its parental strain. Another source of variation among these two strains is related to the lag time, which was increased by almost 50% in UC 7117. By observing these two strains in both Petry plate and broth test tube, a sort of slime was produced in UC 7117, whilst the parental strain did not produce anything similar. Microscope observation of this slime allowed to state that this gelatinous product was a sort of network-like organic matter in which large amounts of cells were found (data not shown).
Role of luxS in biofilm formation on abiotic surfaces
The adhesion of L. innocua UC 8410 and its luxS-null mutant L. innocua UC 7117 was evaluated within a 24 h both in static conditions. In this case the experimental procedure was performed using BHI broth, instead of dBHI. During the 24 h monitoring period, L. innocua UC 7117 demonstrated to adhere on SS coupon in lesser amounts when compared to UC 8410, although this difference ranged between 1-2 logarithms during the whole period of analysis (Fig. 12). Experiments were extended until 168 h to assess luxS influence in prolonged nutrient-limited conditions (Fig. 14). L. innocua UC 8410 was reduced by one logarithm and three logarithms at 72 h and 168 h, respectively, during starvation, while population density of UC 7117 decreased by two logarithms and four logarithms at 72 and 168 h, respectively, in same experimental conditions. These data demonstrated that L. innocua was susceptible to prolonged starvation in strictly limiting conditions. Moreover, luxS disruption made L. innocua slightly more susceptible to nutrient depletion for long period, although higher cellular densities could probably mask an increased sensitivity due to genetic modifications.
The dynamic apparatus described was used to monitor adhesion of UC 8410 and UC 7117 in presence of 1 ml/min of milk. In presence of a turbulent flow of milk, luxS did not alter significantly density of planktonic cells (which were costant at 108 CFU/cm2), although more than 108 CFU/cm2 were found at 3 h (Fig. 13). Differently from what observed in static adhesion on the same surface, obtained results showed clearly that gene disruption alter strongly the strain originary adhesive capacity: despite the high amounts of cells in the flowing medium, luxS-null mutant strain lost completely ability to adhere on all tested surfaces, including SS, which showed to be the most propitious support for establishment of sessile communities in testing conditions.
In presence of a turbulent flow of milk, luxS did not alter significantly density of planktonic cells (which were constant at 108 CFU/cm2), although more than 108 CFU/cm2 were found at 3 h. Differently from what observed in static adhesion on the same surface, obtained results showed clearly that gene disruption alter strongly the parental strain adhesive capacity: despite the high amounts of cells in the flowing medium, luxS-null mutant strain lost completely ability to adhere on SS, which showed to be the most propitious support for establishment of sessile communities in testing conditions.
The adhesion capacity of L. innocua UC 7117 on different surfaces in presence of a flow of milk was investigated and compared with the one of its parental strain. Beside SS, also other materials like PET or C were investigated. While the amount of planktonic cells was not markedly modified, L. innocua UC 7117 was not detected even at the lowest dilution on all the tested materials, demonstrating that the genetic disruption abolished completely the adhesion capacity of the used L. innocua strain (Fig. 15).
Influence of growth media and luxS in EPS production
Diverse growth media were used to test if nutrients could affect EPS production within bacterial biofilms of L. innocua UC 8410. Results are shown in Fig. 4. Glucose-supplemented TSB was not demonstrated to support EPS synthesis as well as TSB, although the addition of glucose increased slightly the amount of exopolysaccharide matrix produced (0.287 Â± 0.049 and 0.265 Â± 0.015, respectively).The highest amount of EPS was detected when BHI was used (1.029 Â± 0.1), while lower synthesis of EPS was demonstrated with dTSb and more dBHI in comparison with not diluted media (0.152 Â± 0.013 and 0.116 Â± 0.005, respectively). The luxS-null strain possessed a limited capacity to produce EPS in all tested media, although BHI resulted in a 3 fold higher production of EPS (0.356 Â± 0.070) than other liquid media.
Influence of luxS on biocide resistance
Planktonic subcultures of the two above mentioned L. innocua strains, UC 8410 and UC 7117, were tested, through microdilution protocols in according to both NCCLS standard and EURECAST guidelines, for their susceptibility to benzalkonium chloride. Results are summarized in Fig. 9, Fig. 10 and Fig. 11. UC 8410 showed slightly lesser tolerance to the biocidal agent than its null mutant version (2-fold dilution), although not all tested medium did not has same growth performances. In fact, Mueller-Hinton broth led to lower optical density than European test medium (OD620 value of 0.14 Â± 0.1 and 0.25 Â± 0.02, respectively).
In parallel to superficial adhesion on used wires, both L. innocua UC 8410 and UC 7117 were evaluated for their susceptibility against BC. From the above mentioned device for biofilm formation under dynamic conditions, aliquots of wire and milk, within a 24 h monitoring period, were exposed for 30 min at 200 Âµg/ml BC, as reported in the standard UNI EN 1040:2006, to evaluate bactericidal effectiveness on sessile and planktonic cells. This investigation was performed on different materials, such as SS, PET and C, to investigate possible effect of influence of tested surfaces (Fig. 16).
In terms of adhered cells, UC 7117 showed a higher susceptibility to BC than UC 8410, probably because of its reduced capacity to adhere on SS. BC was strongly effective against planktonic cells at 0 and 24 h, while, from 3 until 18 h, none quantitative difference was observed between two examined strains. In presence of PET no colony was detectable at all stages of sampling for both the investigated groups of cells, whereas BC acted efficiently only to sessile cells. In presence of copper, the antimicrobial effect of BC produced a reduction even stronger than the one observed for L. innocua UC 8410 in same experimental conditions. Statistical analysis revealed that wild-type and mutant significantly differs from each other, although test materials represent a further level of variability. Same conclusion was obtained from statistical analysis run on bacterial recoveries after disinfectant exposure.
Listeria monocytogenes was often tested in former scientific papers for its aility to adhere on stainless steel. Moltz and Martin (2005) monitored adhesive capacity of L. monocytogenes strains on both microtiter plate and stainless steel chips, in order to exploit if material could be considered as a parameter significatively influencing this phenomenon. Tresse et al (2007) used a stainless steel microtiter plate to examine same phenomenon but on larger collection of L. monocytogenes strains, investigating also if genetic traits or source of origin could affect adhesivity observed in such surface. Silva et al. (2008) evaluated different materials for L. monocytogenes attachment and biofilm formation, observing that plastic surfaces supported markedly growth on surfaces in agreement with Oulahl et al. (2008). Mai and Conner (2007) demonstrated that rich growth medium allowed to obtain higher level of bacterial densities compared to minimal mediums, although also incubation temperature was described as a significantly discriminating parameter. Fuster-Valls et al. (2008) clearly stated that environmental condition could be also considered in experimental design as influencing factor.
All the above mentioned reports used static apparatus, while few papers reported adhesion under dynamic regimen (Perni et al. 2006, Perni et al. 2007). In this investigation a laboratory-sized system was used to monitor bacterial attachment on different materials in presence of a flowing solution, and obtained results were compared to those collected from stainless steel coupon. Both the system did not show any significative difference between each other, confirming often use of static device for evaluation of bacterial attachment o surfaces. But, as underlined by Perni et al. (2007), information collected by these two different approaches is completely different from a theoretical point of view. Actually, static system could give information about adhesive strength of a species and/or a strain, but, instead of biofilm development, cells deposition it is more likely to occur in such devices.
The recent research have been focusing on communication systems present within spatially organized and coordinated communities, with particular mention to quorum sensing processes. Among the diverse quorum sensing signal processes demonstrated in bacteria, LuxS-based signal processing have been found in several both Gram-positive and Gram-negative species and AI-2 (signal molecule indirectly derived by LuxS activity) was recognized as "interspeciesÂ communication signal". Despite extensively exploitation in several articles (e.g. Xavier and Bassler, 2003; Vendeville, et al. 2005; De Keersmaecker, et al. 2006), Turovskiy et al. 2007) considered AI-2-based communication a weak hypothesis due to several conflicting theoretical aspects concerning this topic, although further clarifying investigations focused on QS are were not excluded from the authors themselves. Analogue conclusion were reported by Garmin et al. (2009), who recognized agr as the "only effective communication system" present in the genus Listeria, although no experimental approach was proposed to support such affirmation. AI-2 were commented by these latter authors as not being able to fit with the definition of a signaling molecule, attributing importance to agr-based communication.
Apart from study of Belval et al. (2006), Sela et al. (2006) performed gentic deletion of luxS in L. monocytogenes EGD by mutagenesis through plasmid pKSV7::luxSLm and evaluated biofilm formation on glass and in 24-well polystyrene plate in parallel to bioluminescence assay for detection of AI-2: mutant strain showed massively increased amounts of cells on glass slide, although more compromised growth was observed. In this research cloning procedure of luxS was performed by using pRV300 (Berthier et al. 1996, Leloup et al. 1997) for the construction of the knock-out vector.
pRV300 was originally designed for Lactobacillus spp. to integrate, within a certain locus of bacterial chromosome, through Campbell-like integration mechanism and to create a separation of selected gene from its natural regulation machinery, but here it was successfully demonstrated a possible application also for Gram-positive bacteria such Listeria. After verifying the correct construction of the knock-out vector harbouring a 240 bp internal fragment of luxS, the plasmid pPC 7051 was successfully electroporated into UC 8410. All the successfully constructed clones of the so treated L. innocua strain were designed UC 7117. MATS assay as well as bioluminesce assay for AI-2 detection, which were performed both on L. innocua UC 8410 and L. innocua UC 7117, successfully demonstrated the genetic disruption of luxS.
Bacterial attachment of wild-type L. innocua was examined in parallel to its luxS-null version both in static and dynamic approaches: obtained results on statically incubated stainless steel allowed to confirm results of both Belval et al. (2006) and Sela et al. (2006), while, in presence of shear forces, the luxS-null mutant strain was not able to adhere even to the most propitious surface, SS. A possible explanation could be related to the reduced synthesis of EPS, which varied with the used medium. BHI allowed obtaining higher levels of optical density at 37Â°C and much more at 30Â°C than TSB in same experimental conditions, while a limited extent of polysaccharides were observed in both diluted media.
These data suggested that monitoring the adhesion under strict nutrient depletion could give a realistic evaluation of adhesive properties of the examined strains. Further research should be required to investigate EPS production in both food simulating models as well as in apparatus similar to food processing environments, in order to give a clear picture of microbial contamination within such conditions.
Polysaccharide production is affected not only by the growth medium, but also by the incubation temperature: higher levels were determined at 30Â°C, whereas limited production was observed at both 37 and 20Â°C. UC 7117 was demonstrated to be unable to produce significantly high amounts of EPS at all the investigated temperatures. Furthermore, when mutant was evaluated for its adhesion capacity on static devices, a sort of slime and limited growth during prolonged incubation under nutrient depletion were observed, suggesting that luxS could affect not only growth kinetic but also susceptibility under adverse conditions.
Another trait modified by genetic disruption was the sensitivity to biocides: BC was tested, through the European Standard 1040:2006, for its bactericidal effectiveness on UC 7117 in comparison with L. innocua UC 8410. The obtained results showed that the used biocide had an increased efficacy in reducing bacterial population up to 108CFU/ml for the planktonic cells, demonstrating that gene knock-out performed on L. innocua markedly modified the sensitivity to antimicrobials. Apparently these results seem in conflict with those observed in broth microdilution test, where the luxS-null strain seem to be 2-fold more tolerant than the parental effect, but this effect could be more probably attributed to the general higher cellular density of such strains which could lead to an underestimation of the real inhibitory effect.
AI-2 have been recognized as "universal cell-to-cell communication signal" for the presence of the correlated gene luxS in over 50 bacterial species and has been associated to biofilm forming ability on abiotic surfaces, although final results varied depending on the considered microbial genus and/or species.
Actually there were a limited number of studies conducted above the influence of this gene on Listeria monocytogenes biofilm forming capacity on materials such as stainless steel, and the actual opinion of scientific community had attributed to luxS a negative influence of bacterial attachment on surfaces. However, a new and innovative point of view was reached through extensive tests.
luxS was demonstrated to possess a wider gamma of effects, which influence (i) the mechanism/s controlling the bacterial density in a defined environment, (ii) bacterial cell surface properties, (iii) overall metabolic efficiency, and (iv) the ability to produce EPS, which are the fundamental structural components of bacterial biofilms organic matrix.
Further research should be performed to elucidate the specific molecular pathways affected by luxS, in order to suggest new and promising possible solutions to prevent biofilm formation within industrial locations, and to assess its real range of effect in Listeria as well as in other microbial species, especially in foodborne pathogens.