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Co-Culture with Listeria monocytogenes within a Dual-
Species Biofilm Community Strongly Increases
Resistance of Pseudomonas putida
to Benzalkonium
Chloride

Efstathios Giaouris, Nikos Chorianopoulos
1 Department of Food Science and Nutrition, University of the Aegean, Myrina, Lemnos island, Greece, 2 Veterinary Research Institute of Athens, Greek
Agricultural Organization "Demeter", Aghia Paraskeui, Greece, 3 Department of Food Science and Human Nutrition, Laboratory of Microbiology and
Biotechnology of Foods, Agricultural University of Athens (AUA), Athens, Greece
Biofilm formation is a phenomenon occurring almost wherever microorganisms and surfaces exist in close proximity.
This study aimed to evaluate the possible influence of bacterial interactions on the ability of Listeria monocytogenes and Pseudomonas putida to develop a dual-species biofilm community on stainless steel (SS), as well as on the subsequent resistance of their sessile cells to benzalkonium chloride (BC) used in inadequate (sub-lethal) concentration (50 ppm). The possible progressive adaptability of mixed-culture biofilms to BC was also investigated.
To accomplish these, 3 strains per species were left to develop mixed-culture biofilms on SS coupons, incubated in daily renewable growth medium for a total period of 10 days, under either mono- or dual-species conditions. Each day, biofilm cells were exposed to disinfection treatment. Results revealed that the simultaneous presence of L. monocytogenes strongly increased the resistance of P. putida biofilm cells to BC, while culture conditions (mono-/ dual-species) did not seem to significantly influence the resistance of L. monocytogenes biofilm cells. BC mainly killed L. monocytogenes cells when this was applied against the dual-species sessile community during the whole incubation period, despite the fact that from the 2nd day this community was mainly composed (>90%) of P. putida cells. No obvious adaptation to BC was observed in either L. monocytogenes or P. putida biofilm cells. Pulsed field gel electrophoresis (PFGE) analysis showed that the different strains behaved differently with regard to biofilm formation and antimicrobial resistance. Such knowledge on the physiological behavior of mixed-culture biofilms could provide the information necessary to control their formation.
Citation: Giaouris E, Chorianopoulos N, Doulgeraki A, Nychas G-J (2013) Co-Culture with Listeria monocytogenes within a Dual-Species Biofilm
Community Strongly Increases Resistance of Pseudomonas putida to Benzalkonium Chloride . PLoS ONE 8(10): e77276. doi:10.1371/journal.pone.
0077276
Editor: Mark Alexander Webber, University of Birmingham, United Kingdom
Received May 9, 2013; Accepted September 1, 2013; Published October 10, 2013
Copyright: 2013 GIAOURIS et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work received funding through the following sources: Action THALIS "Biological Investigation Of the Forces that Influence the Life of
pathogens having as Mission to Survive in various lifestyles; BIOFILMS", which falls under the Operational Programme (OP) "Education and Lifelong
Learning (EdLL)", cofinanced by the European Social Fund (ESF) and National Resources. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: stagiaouris@aegean.gr biofilm structure can lead to the cross-contamination of thefood products, causing food spoilage and / or foodborne Biofilms are commonly defined as communities of diseases The risk becomes even more serious, since it microorganisms attached to a surface or interface and has been observed that the antimicrobial resistance of biofilm microorganisms are embedded ]. In the food industry, In food processing environments, a variety of different biofilm formation by spoilage and pathogenic bacteria has been bacteria are known to attach to surfaces, survive, grow and of considerable interest and has provoked the interest of many form sessile biofilm communities Spatial and metabolic research groups [ interactions between species are believed to contribute to the onto food-contact surfaces and the subsequent biofilm organization of multispecies biofilms, and the production of a formation is undesirable, since the detachment of cells from the dynamic local environment []. Mixed-species biofilms are PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm usually more stable than mono-species biofilms, while cell-to- In recent years, the study of mixed species sessile cell interactions and communication have been demonstrated communities composed of various foodborne bacteria has to play a key role in initial cell attachment, biofilm growth and increased the understanding of interactions and dynamics of structure, cell dispersion, as well as in the resistance of biofilm surface attached bacteria and biofilms under conditions relevant to food processing Evidently, Listeria monocytogenes is a ubiquitous Gram-positive multi-species biofilms are dynamic communities with extensive facultative intracellular bacterial pathogen capable of surviving interactions taking place between the different species and and growing under a wide range of adverse environmental strains which probably have a significant effect on the conditions, such as high acidity, high salinity and at structure, composition (extracellular matrix constituents), refrigeration temperatures [It is the causal agent of population dynamics (i.e. which species and / or strain is listeriosis, a severe foodborne disease which provoked 134 present / dominates) and physiology (i.e. function, metabolism, human deaths in European Union in 2011, with a reported high resistance, virulence) of mature communities. Particularly case fatality ratio of 12.7% [Notably, many L. challenging is the attempt to understand the complexity of all monocytogenes strains are capable of adhering to various both these interactions encountered within such communities, and biotic (e.g. animal tissues) and abiotic (e.g. stainless steel, how this may influence the final community outcome and plastic) surfaces and create biofilms (for a review see Attachment to surfaces is believed to be important for survival To this direction, in this study, some selected L. monocytogenes and P. putida strains (three strains per and persistence of this pathogen in food processing species) were left to form biofilms on stainless steel (SS) environments, with some strains being able to remain on surfaces, under either mono- or dual-species conditions, at 18 equipment surfaces even for several years [ °C, for a total period of 10 days, with daily medium renewal.
Pseudomonas spp. are Gram-negative obligatory aerobic Both types of biofilms (mono-/dual-species) were daily bacteria, capable of degrading a variety of low molecular subjected to chemical disinfection by applying inadequate weight organic components and are very common in fresh (sublethal) concentration of BC (50 ppm). The microbial foods, mainly because of their widespread existence in water, composition of the mono-species sessile communities the first soil and vegetation. Many Pseudomonas species are day of incubation, as well as of the dual-species community the last day of incubation, with regard to strain occurrence, just microorganisms of raw foods stored under refrigeration before and following disinfection, was also evaluated by using Pseudomonads are among the better-studied microorganisms a pulsed field gel electrophoresis (PFGE) approach. Results with respect to phenotypic changes taking place throughout the obtained highlight the impact of bacterial interactions taking process of biofilm formation and the genetic determinants place inside such mixed-culture sessile communities on both involved These are commonly found in the food- their population dynamics and chemical disinfection resistance processing environment ] and have been previously and could be helpful in our efforts to control mixed-culture documented as good producers of extracellular polymeric biofilm formation by unwanted bacteria in food processing substances, including polysaccharides, nucleic acids, and proteins making them ideal organisms with which toinvestigate the growth of L. monocytogenes within multispecies Materials and Methods
biofilms. Notably, P. putida bacteria are capable of adhering tovarious food contact surfaces and form strong biofilms Bacterial strains and growth conditions
This species is also known to produce Three L. monocytogenes (FMCC_B-125, FMCC_B-129, biosurfactants, amphipathic molecules which have been shown FMCC_B-169) and three P. putida (CK119, CK120, CK148) able to influence biofilm development and moreover to break strains, isolated from different origins, were used in this study.
Regarding the L. monocytogenes strains, FMCC_B-125 was Benzalkonium chloride (BC) is a biocide belonging to the the clinical reference strain ScottA (serotype 4b, lineage I, group of quaternary ammonium compounds (QACs) that are epidemic strain, human isolate, [originally supplied by the commonly used in both food and medical environments [ Agrotechnological Research Institute ATO-DLO (Wageningen, QACs possess antimicrobial effect against a broad range of the Netherlands), FMCC_B-129 was isolated from a ready-to- microorganisms, since they act on general membrane eat minced meat based frozen meal [and FMCC_B-169 permeability, causing cytolytic leakage of cytoplasmic material was isolated from the environment of an Italian food processing at low concentrations. At high concentrations, they target the plant (strain 2UD of DSA collection, []). The selection of the carboxylic groups and cause general coagulation in the three L. monocytogenes strains was based on previous bacterial cytoplasm ]. However, the frequent use comparative results of biofilm formation on polystyrene combined with the misuse of disinfectants, such as BC, in food microplates by 11 L. monocytogenes strains in total under environments can lead to the development of cellular different growth conditions []. Regarding the P. putida adaptation mechanisms and the emergence of disinfectant resistant cells As thus, resistance to QACs has been Before utilization, all the microorganisms were stored frozen reported in many Gram positive as well as Gram negative (at -80°C) in bead vials (Protect; Technical Service Consultants bacteria associated with food [ Ltd, Heywood, Lancashire, UK) and each one was then PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm resuscitated by adding one bead to 100 ml of Brain Heart shaking" which was previously followed (following the initial 3 h Infusion (BHI) broth (LAB M; International Diagnostics Group attachment step and daily in the course of biofilm formation).
Plc, Bury, Lancashire, UK) in a conical flask and incubating at After this rinsing, coupon was individually introduced in new 30°C for 24 h under agitation (precultures). Working cultures glass test tube containing 5 ml of 50 ppm of benzalkonium were prepared by adding a 100-μl aliquot of each preculture to chloride (BC) solution. BC was purchased from Sigma-Aldrich 100 ml of BHI broth and incubating at 30°C for 16 h (under (Life Science Chemilab S.A., Athens, Greece) and its desired agitation), at which time late exponential phase was attained solution was prepared in distilled water. Before use for for each strain (data not shown). Cells from final working disinfection treatments, BC was checked for sterility by agar cultures were harvested by centrifugation (5000 x g, 10 min, at plating. Disinfection was carried out at 18°C for 6 min to imitate 4 °C), washed twice with ¼ Ringer solution (Ringer's tablets; disinfection conditions encountered in real food processing Merck, Darmstadt, Germany) and finally resuspended in ¼ Ringer solution (ca. 109 CFU ml-1), in order to be used asinocula for the biofilm development assays.
Recovery and quantification of viable biofilm cells
Abiotic substratum and biofilm formation procedure
On 1st, 2nd, 4th, 6th, 8th and 10th day of incubation, the viable biofilm bacteria on SS coupons, just before and following the 6- Stainless steel (SS) coupons (3 x 1 x 0.1 cm, type AISI-304, min exposure to BC disinfection, were quantified by using the Halyvourgiki Inc., Athens, Greece) were the abiotic substrates "bead vortexing method" described by Kostaki et al. [], in used for biofilm formation, since this material is frequently usedfor the manufacture of food processing equipment. Prior to use, which strong vortexing of each coupon with glass beads is coupons were cleaned according to the procedure described used to detach the biofilm cells. It should be noted that by Kostaki et al. ]. Following cleaning, coupons were following BC disinfection, SS coupons were incubated for 10 individually placed in empty glass test tubes (length, 10 cm; min in Dey-Engley neutralizing broth in order to stop BC action diameter, 1.5 cm) and autoclaved at 121°C for 15 min.
and also help viable stressed / injured cells to recover.
To produce biofilms on SS coupons, three strains per Detached cells were subsequently enumerated by agar plating, species were selected and left to produce biofilms, under either after ten-fold serial dilutions. In the case of mono-species mono- or dual-species conditions, according to the protocol biofilm development, Tryptone Soy Agar (TSA; LAB M) was described by Kostaki et al. []. Briefly, two subsequent steps used for the enumeration of bacteria. In the case of dual- were followed: sterile coupons were initially incubated in saline species biofilm development, the cells of each one species bacterial suspensions (bacterial attachment step), and were enumerated using the following selective media: afterwards coupons carrying strongly attached bacteria were PALCAM Listeria Selective agar (PALCAM; LAB M) for L. incubated in daily renewable growth medium (biofilm formation monocytogenes and Pseudomonas Agar (CFC Agar; LAB M) for P. putida. Previously, it had been confirmed that P. putida For the attachment step, 5 ml of bacterial suspension in ¼ cells were not able to grow on PALCAM plates, while L. Ringer solution, containing ca. 108 CFU ml-1 for each strain, monocytogenes cells were not able to grow on CFC plates.
was poured into each one glass test tube containing a sterilized Additionally, TSA was also used in the case of dual-species SS coupon, followed by incubation at 18°C for 3 h, under static biofilm monitoring to have a comparison with the results conditions. Care was taken in order the bacterial suspension to obtained by the selective media (results not shown). In all contain approximately the same number of cells for each strain cases, plates were incubated at 30°C for 48 - 72 h.
(ca. 108 CFU ml-1).
Following attachment step, each coupon was carefully Isolation of strains from mixed-culture biofilm
removed from glass test tube using sterile forceps and was thereafter rinsed by immersing it, for 5 min, in 5 ml of ¼ Ringersolution, with shaking, in order to remove the loosely attached In order to monitor the individual contribution of each L. cells. After rinsing, coupon was individually introduced in new monocytogenes and P. putida strain in both the development sterile glass test tube containing 5 ml of Tryptone Soy Broth and resistance of mixed-culture (mono-/dual-species) biofilm (TSB; LAB M) and subsequently incubated at 18°C for a total communities, 12 colonies were randomly selected from the period of 10 days (240 h), under static conditions, to allow highest dilutions of agar plates (used to enumerate the viable biofilm development on the coupon. Growth medium was bacteria recovered from SS coupons by the bead vortexing renewed every 24 h. During each medium renewal, loosely method just before and following disinfection) the first and the attached cells were removed by rinsing (as described above).
last day of incubation. In particular, out of a total number of 96colonies picked, half of them (48) were recovered from the Exposure of biofilm cells to disinfection
mono-species biofilms the 1st day of incubation (containing Every 24 hours, each SS coupon – carrying biofilm cells onto either L. monocytogenes or P. putida strains), while the other it – was carefully removed from glass test tube using sterile half (48) were recovered from the dual-species biofilm the 10th forceps and was thereafter rinsed by pipetting two times 10 ml day of incubation (containing both L. monocytogenes and P. of ¼ Ringer solution (each time), in order to remove the loosely putida strains). All isolated colonies were stored at -80°C in attached cells. "Strong twice pipetting" was chosen here as a TSB containing 20% (v/v) glycerol (Serva GmbH, Heidelberg, more harsh method to do this, compared to "immersing with Germany), until PFGE analysis.
PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm Pulsed-field gel electrophoresis (PFGE) analysis
Effect of culture conditions (mono-/dual-species) on BC
PFGE of L. monocytogenes strains was performed according resistance of L. monocytogenes and P. putida biofilm
to Kostaki et al. ], while the PFGE protocol described by Doulgeraki and Nychas [] was followed for the P. putida The effect of the 6-min disinfection treatment with 50 ppm of BC solution on L. monocytogenes and P. putida biofilm cells,cultured under either mono- or dual-species conditions, was expressed as biofilm population log-reduction (difference in log Each experiment included three replicate coupons and was repeated twice using independent bacterial cultures.
Regarding L. monocytogenes it was observed Microbiological counts were transformed to logarithms before that, in general, culture conditions (mono-/dual-species) do not means and standard deviations were computed. All data were seem to significantly influence the resistance of its biofilm cells analyzed by analysis of variance (ANOVA) by the general against BC (except the 8th day of incubation). Thus, for linear model procedure of SPSS data analysis software for instance, the last (10th) day of incubation, log reductions of 1.89 Windows (release 10.0.1; SPSS Inc., Chicago, IL). Least and 1.82 log CFU/cm2 were monitored for mono- and dual- square means were separated by Fisher's least significant species conditions, respectively. Under current experimental difference (LSD) test. All differences are reported at asignificance level of an alpha of 0.05.
setup, this bacterium presented the higher susceptibility the 8thday under mono-species conditions (log reduction 2.64 log CFU/cm2), while the most resistance was recorded on the 4thday (log reductions of 0.94 and 0.88 for mono- and dual- Effect of culture conditions (mono-/dual-species) on
species conditions, respectively).
biofilm formation by L. monocytogenes and P. putida
Regarding P. putida (culture conditions (mono-/ dual-species) significantly influenced the resistance of its The results regarding the populations (log CFU/cm2) of L. biofilm cells against BC. In particular, the simultaneous monocytogenes and P. putida biofilm cells on SS coupons, at presence of L. monocytogenes cells led to a significant the different incubation (sampling) days, just before the increase of the resistance of P. putida biofilm cells to the disinfection treatment, either under mono- or dual-species chemical disinfection during whole incubation period. Thus, conditions, are presented in In general, it is observed while under mono-species conditions log reduction of P. putida that the simultaneous presence of both bacterial species in the cells ranged from 1.74 log CFU/cm2 (the 10th day) to 3.33 log biofilm community seems to lead to a reduction of their sessile CFU/cm2 (the 8th day), under dual-species conditions the populations, compared to when each species was left to form highest log reduction recorded was just 0.79 log CFU/cm2 (the biofilm separately from the other.
Regarding L. monocytogenes culture conditions (mono-/dual-species) significantly (p < 0.05) influenced its final Microbial (species) composition of the dual-species
(after 10 days) biofilm population level. Thus, under mono- species conditions, L. monocytogenes reached a sessile The results on the microbial (species) composition of the population of 6.05 log CFU/cm2, while under dual-speciesconditions, this population was approximately 30 times less dual-species biofilm communities formed on SS coupons, at (4.57 log CFU/cm2). Besides the 10th day of incubation, the the different incubation (sampling) days, for L. monocytogenes simultaneous presence of P. putida cells also significantly and P. putida cells, just before and following the 6-min reduced the population of L. monocytogenes biofilm cells the 2nd, 4th and 8th day of incubation (fold difference ranged from According to these results, while L. monocytogenes 4.5 times less the 4th day to more than 234 times less the 8th dominated in the dual-species sessile community the 1st day of day). Rather strangely, the 1st and 6th day of incubation, L. incubation (91.6% out of the total biofilm cells belonged to it), monocytogenes biofilm counts did not significantly differ all other next days the dual-species community was mainly between mono- and dual-species conditions.
composed of P. putida cells (>90%) (In the same Regarding P. putida (culture conditions (mono-/ way, for each sampling day, following disinfection, the dual- dual-species) do not seem to significantly influence its final species community was mainly composed of P. putida cells.
(after 10 days) biofilm population level. Thus, this bacterium Thus, the percentage of viable P. putida cells out of the total reached final sessile populations of 6.50 and 5.93 log CFU/cm2 number of biofilm cells ranged from 82.3% the 1st day to 99.9% for mono- and dual-species conditions, respectively. However, the 6th day Interestingly, under current applied the simultaneous presence of L. monocytogenes cellssignificantly (p < 0.05) reduced the population of P. putida experimental setup, BC mainly killed L. monocytogenes biofilm biofilm cells the 1st, 4th and 8th day of incubation. For instance, cells, when this was applied against the dual-species biofilm the 8th day, P. putida reached under mono-species conditions a communities, during the whole incubation period ( sessile population of 7.46 log CFU/cm2, while under dual- Thus, the percentage of killed L. monocytogenes cells out of species conditions, this population was more than 20 times the total number of killed cells ranged from 82.8% the 2nd day less (6.09 log CFU/cm2).
to 99.5% the 10th PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm Figure 1. Populations (log CFU/cm2) of biofilm cells on SS coupons, just before disinfection. (A) L. monocytogenes; (B) P.
putida
. Biofilms were left to be formed on coupons incubated at 18 °C for a total period of 10 days in daily renewable TSB, under
either mono-species (□, three strains of each one species together), or dual-species conditions (■, six strains of the two different
species together) and subjected daily to disinfection (6-min exposure to 50 ppm of BC solution). The bars represent the mean
values ± standard deviations (n=6, two independent experiments, each performed three times). For each graph separately, mean
values sharing at least one common lower case letter shown above the bars are not significantly different at a P value of <0.05.
doi: 10.1371/journal.pone.0077276.g001
Distribution of L. monocytogenes and P. putida strains
plant (strain 2UD of DSA collection, [was found to in the mixed-culture biofilm communities
dominate in all biofilm communities tested. Thus, under mono- The individual contribution of each L. monocytogenes strain species conditions, this strain completely dominated from the (FMCC_B-125, FMCC_B-129, FMCC_B-169) and each P. 1st day of incubation. Under dual-species conditions (the 10th putida strain (CK119, CK120, CK148) in the composition of the day), this strain consisted the 91.7% of the total fraction of L. mono-species biofilm communities (the 1st day of incubation), monocytogenes biofilm cells before the disinfection treatment.
as well as in the dual-species biofilm community (the 10th day This percentage was reduced to 75% following disinfection. In of incubation), just before and after the 6-min exposure to 50 the latter case, the other 25% belonged to FMCC_B-129 strain, ppm of BC solution is illustrated in originally isolated from a ready-to-eat frozen meal [It's According to these results, it is obvious that the different worth to be noted that the clinical FMCC_B-125 (ScottA) strain, strains employed here (3 strains / species) did not contribute at originally isolated from human ], seemed to be unable to the same levels to either the formation of these mixed-culture develop biofilm on SS coupons, under current experimental sessile communities or their antimicrobial recalcitrance.
conditions and sampling days.
Regarding L. monocytogenes Regarding P. putida (CK119 and CK148 strains originally isolated from the environment of a food processing equally contributed to the formation of the mono-species biofilm PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm Figure 2. Log reductions (log CFU/cm2) of biofilm cells on SS coupons, following disinfection. (A) L. monocytogenes; (B) P.
putida
. Biofilms were initially left to be formed on coupons incubated at 18 °C for a total period of 10 days in daily renewable TSB,
under either mono-species (□, three strains of each one species together), or dual-species conditions (■, six strains of the two
different species together) and subjected daily to disinfection (6-min exposure to 50 ppm of BC solution). The bars represent the
mean values ± standard deviations (n=6, two independent experiments, each performed three times). For each graph separately,
mean values sharing at least one common lower case letter shown above the bars are not significantly different at a P value of
<0.05.
doi: 10.1371/journal.pone.0077276.g002
community the 1st day of incubation, while the CK120 strain seemed to be unable to compete the other two strains in thecourse of biofilm formation. Following disinfection, the mono- In the majority of natural and man-made environments, species biofilm community composed exclusively of the CK148 microorganisms usually associate with surfaces in complex strain, which proved to be more resistant to the BC multi-species communities The structural andfunctional dynamics of multi-species biofilms are largely due to antimicrobial action. On the contrary, under dual-species the interactions between the different species conditions, CK148 strain was totally absent the 10th day of However, this complexity is not taken into consideration when incubation. In the latter case and with regard to P. putida cells, growing microorganisms in monocultures under laboratory dual-species community was composed by 91.7% of CK120 conditions. In this study, the simultaneous biofilm formation by strain, while the other 8.3% belonged to CK119 strain.
six selected L. monocytogenes and P. putida strains was Following disinfection, dual-species community was equally investigated (3 strains per species). These were left to develop composed by the CK119 and CK120 strains.
mixed-culture biofilms on SS coupons incubated in dailyrenewable growth medium, while biofilm communities were PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm Figure 3. Percentages of viable L. monocytogenes (filled bars) and P. putida (open bars) cells in the dual-species
biofilms.
(A) Just before disinfection; (B) Following disinfection. Dual-species biofilms were initially left to be formed on SS
coupons incubated at 18 °C for a total period of 10 days in daily renewable TSB and subjected daily to disinfection (6-min exposure
to 50 ppm of BC solution).
doi: 10.1371/journal.pone.0077276.g003
also, in daily basis, subjected to inadequate (sub-lethal) something rather expected given the well-known ability of disinfection treatment with 50 ppm of BC solution. "Glass bead Pseudomonads, including P. putida, to produce a variety of vortexing" was used to dislodge biofilm cells for counting onto extracellular polymeric substances (EPS, e.g. cellulose, selective media, since it has been previously proven effective alginate, Pel and PsI exopolysaccharides), which help them to to give "rough" estimations of biofilm counts ]. Although form strong biofilm communities either on abiotic or even biotic this method may not be effective to completely remove all the surfaces [The increased tolerance to biofilm cells from the surface, it still gives quite more accurate BC of the Gram-negative P. putida compared to the Gram- results compared to other methods employed for this purpose positive L. monocytogenes may also account for this (e.g. swabbing, sonication). Undoubtedly, the possibility to use observation. Additionally, P. putida, together with other species in parallel a sensitive microscopic technique for obtaining high- of the same genus, is also known to produce biosurfactants resolution optical images of the formed biofilm (e.g. by using well-known anti-biofilm compounds which have been different fluorescent probes for the two different species and shown to inhibit both attachment and biofilm formation by other observing them under confocal laser scanning microscopy, species or even disperse already established biofilms CLSM) would be a valuable help in our effort to unravel the microbial (species) composition of the dual-species biofilm On the contrary, the foodborne pathogen L. monocytogenes does not always have a high potential for forming strong mono- Obtained results ) revealed that under such species biofilms in vitro on food contact materials at relevant conditions, both bacteria were able to develop a dual-species food industry conditions ], but surfaces already biofilm, in which however P. putida was found to dominate from colonized by other bacteria may significantly increase its the 2nd day of incubation (Thus more than 90% out of adherence and biofilm formation ]. In this context, the total number of biofilm cells belonged to this species. The Hassan et al. [noticed, when studied the behaviour of L. observed dominance of P. putida over L. monocytogenes was monocytogenes on condensate-covered stainless steel with a PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm Figure 4. Percentages of killed L. monocytogenes (filled bars) and P. putida (open bars) cells in the dual-species
biofilms.
Dual-species biofilms were initially left to be formed on SS coupons incubated at 18 °C for a total period of 10 days in
daily renewable TSB and subjected daily to disinfection (6-min exposure to 50 ppm of BC solution).
doi: 10.1371/journal.pone.0077276.g004
P. putida biofilm over a total period of 35 days, that L. observed that the simultaneous presence of both bacterial monocytogenes attached in significantly greater numbers (> 3- species in the dual-species biofilm community seems to lead to log difference) to surfaces with pre-existing P. putida biofilms a reduction of their sessile populations, compared to mono- than to Pseudomonas-free surfaces. In another study and in species conditions (It's worth to be noted that each accordance with current results, Chorianopoulos et al. one of the six strains employed here was also screened for found, when co-cultured on SS surfaces 5 strains belonging to inhibitory activity against the other strains using the well Salmonella enterica, monocytogenes, diffusion assay. However, such activity was not revealed (data Staphylococcus simulans and Lactobacillus fermentum, that not shown). Likewise, Norwood and Gilmour [] found, when mixed-culture biofilm was mainly composed of P. putida cells determined the differential adherence capabilities at three (97.8%), while S. enterica and L. monocytogenes represented different temperatures of two L. monocytogenes strains to SS together only the 2.2% of it. Similarly, Lourenço et al. [ by submerging stainless steel coupons in both 48-h Listeria observed, when evaluated biofilm formation by 4 selected L. monocultures and mixed cultures additionally containing monocytogenes strains (at 12 and 37 °C) either on pure Staphylococcus xylosus and P. fragi, that the monoculture cultures or on co-cultures with P. aeruginosa (PAO1), that in biofilms consistently contained greater L. monocytogenes co-culture biofilms, P. aeruginosa was the dominant species, at numbers than the multispecies biofilms.
both temperatures, representing 99% of the total biofilm In recent years, several studies have also been conducted on the resistance of mixed species biofilms to disinfectants In a number of previously published studies, attachment and ]. However, most of these biofilm formation by L. monocytogenes and P. putida have relevant studies performed did not include results of single been shown to be influenced by either the natural in situ species biofilm resistance, making it impossible to judge presence of other species or just their metabolic by-products whether there is any effect of interspecies interactions on the resistance of each individual species in the mixed community.
presence of Staphylococcus xylosus and Pseudomonas fragi In a recent study, Saá Ibusquiza et al. ] studied the affected the numbers of L. monocytogenes biofilm cells on resistance to BC and the microscopic structure between mixed- stainless steel ], while Carpentier and Chassaing [] species biofilms formed by four different strains of L. reported that among 29 tested dairy environmental strains, monocytogenes and one strain of P. putida under different 53% and 13%, reduced or enhanced L. monocytogenes biofilm scenarios. They found that the presence of P. putida in L. formation, respectively. In the present study, it was in general monocytogenes biofilms quickened biofilm formation and PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm Figure 5. Percentages of viable cells for each strain in the mixed-culture biofilm communities. (A) L. monocytogenes; (B) P.
putida
. Mixed-culture (mono-/dual-species) biofilms were left to be formed on SS coupons incubated at 18 °C for a total period of 10
days in daily renewable TSB and subjected daily to disinfection (6-min exposure to 50 ppm of BC solution). Graphs present the
distribution of viable strains in the mono-species biofilm communities (the 1st day of incubation), as well as in the dual-species
biofilm community (the 10th day of incubation), just before and after disinfection.
doi: 10.1371/journal.pone.0077276.g005
significantly increased their resistance to BC with respect to the resistance of mixed-species biofilms of L. monocytogenes and resistance of mono-species L. monocytogenes biofilms after 4 P. putida to BC seems to be related to their microscopic days of incubation at 25 °C. These authors suggested that the structure and to the association between the involved species.
PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm On the contrary, in the present study, culture conditions It is generally acknowledged that microbial resistance to (mono-/dual-species) did not seem to significantly influence the disinfectants may develop following exposure to sublethal resistance of L. monocytogenes biofilm cells to BC, while these concentrations of them [Thus, many studies have had a profound effect on the resistance of P. putida cells demonstrated that bacteria, including L. monocytogenes and (In particular, it was observed that the simultaneous pseudomonads, are capable of adapting to disinfectants used presence of L. monocytogenes strongly increased resistance of in industrial settings after prolonged exposure to sublethal P. putida biofilm cells to BC. It should be noted that the effect concentrations ,BC-resistance among L. of the 6-min disinfection treatment on biofilm cells was monocytogenes strains isolated from food sources can vary expressed as biofilm population log-reduction (difference in log from 10% to over 40% ], while more than 30% of CFU/cm2 values just before and after the treatment) in order to the pseudomonads isolated from poultry carcasses were found take also into account the initial biofilm counts (before able to grow in the presence of BC at the concentrations used disinfection). In a recent relevant study of dual-species biofilm in poultry plants ]. However, in the present study no formation between three L. monocytogenes and three S. obvious adaptation to BC was observed in either L. enterica strains, interspecies interactions did not significantly monocytogenes or P. putida biofilm cells, since log reductions, influence neither the biofilm forming ability, nor the consequence of the 6-min exposure to 50 ppm of BC solution, antimicrobial resistance of each individual species [ did not significantly differ between first and last day of Contradictory literature results on biofilm research data are incubation, for both growth conditions (mono-/dual-species) probably not solely explained by the inherent differences ). In a biofilm study examining the morphological and between the different strains, but also by the fact that the biochemical changes in P. fluorescens biofilms grown in the interactions encountered in mixed-culture biofilm communities presence of subinhibitory concentrations of 4 antimicrobial depend on a number of factors, such as nutritional conditions, agents including BC, it was observed that P. fluorescens bacterial co-aggregation, metabolic requirements, exposure to exhibited adaptation to BC at 10 mg / ml ]. In another study antimicrobial agents and other environmental factors (e.g.
examining biofilm formation by 95 L. monocytogenes strains and also aiming to determine the extent to which biofilmproduction protects this pathogen against QAC challenge (50 in these factors can drastically impact the structure, dynamics or 150 ppm for 60 sec), it was concluded that it is the maturity and thus the behaviour of the biofilm community [ of the biofilm, rather than the strain itself, which is actually Obviously, the above examples emphasize the principle that studies on biofilm formation by foodborne bacteria should be In current study, a promising PFGE approach was also used performed under relevant mixed-culture conditions employing a to monitor the individual contribution of each L. monocytogenes variety of different strains and species. Additionally, special and P. putida strain in the formation and maintenance of mono- care should be taken when educing conclusions based on the and dual-species biofilm communities, whether or not these results of a single study, since biofilms seem to be very diverse were exposed to BC disinfection. In order to achieve this, two and unique, not just to the microorganism, but to the particular time periods in the course of biofilm development were environment in which they are being formed.
selected; one at the first day of incubation and the other at the Under current applied experimental conditions, it was last (10th) day. Results revealed that different strains behave observed that BC mainly killed L. monocytogenes biofilm cells, differently with regard to their ability to develop mixed-culture when this was applied against the dual-species biofilm biofilms and their antimicrobial recalcitrance (). Under communities, during the whole incubation period ( mono-species conditions, from the 1st day of incubation, only This was rather expected given that it is well known that Gram one strain for each species dominates (this is FMCC_B-169 for negative bacteria are less susceptible to QACs than Gram L. monocytogenes and CK148 for P. putida). As expected, this positive bacteria, and additionally Pseudomonas spp. have situation was found to remain the same until the end of generally high intrinsic resistance compared with other Gram incubation (10th day) (results not shown). The higher initial negative bacteria [However, it is still quite interesting attachment ability, the higher specific growth rate and / or the that the percentage of killed L. monocytogenes cells out of the better entrapment ability in the developing biofilm structure total number of killed cells exceeded 80% (), in dual- (and as thus released dispersal), as well as the increased species communities containing more that 90% of P. putida resistance to BC of some strains, compared to the other strains cells Interactions leading to specific spatial also being present, may explain why some strains were found distribution of cells having different resistance to disinfectant in to dominate in each mixed-culture biofilm community. However, mixed-species biofilms may also explain observed resistance it should be noted that the BC susceptibility of each individual of P. putida biofilm cells [,By applying different strain (i.e. under mono-culture conditions) was on purpose disinfectants, Fatemi and Frank ] investigated the ability of chosen not to be examined, since under mixed-culture peracetic acid and peroctanoic acid sanitizers to inactivate conditions, bacterial interactions may influence both biofilm mixed-culture biofilms of a Pseudomonas sp. and L. formation ability and BC susceptibility of each individual strain.
monocytogenes on SS and they found that Pseudomonas and The last becomes more evident if we take into account the L. monocytogenes were inactivated to similar levels by the possible complex 3D biofilm structure which may develop when sanitizer treatments, even though Pseudomonas predominated the different strains are left to develop biofilm together.
in the initial biofilm population.
Although under dual-species conditions the strain distribution PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm could have been monitored each sampling day, this was surrounding environmental conditions. Obviously, the results of examined only at last (10th) incubation day (mainly due to another relevant study on biofilm formation of these two practical reasons). Despite this, it is still obvious that bacterial species (under both mono- and dual-species conditions) interactions among the different strains and species seem to without any disinfection treatment could help us to more clearly have a significant influence on the growth potential, survival unravel the effect of culture conditions (mono-/dual-species) on and more generally in the "individual behaviour" of each strain.
the biofilm forming ability of each individual species and strain, In the same way, in a recently published study ], both intra- and also to unravel the possible effect of biofilm maturation on and inter-species bacteria interactions encountered inside biofilm composition. However, in this study, the conditions mono- and dual-species biofilms of L. monocytogenes and S. encountered in real food processing areas, that are daily enterica were found to have a profound effect on both the disinfection of biofilm cells, were deliberately chosen.
population dynamics, as well as on the resistance pattern of Evidently, bacterial pathogens, such as L. monocytogenes each L. monocytogenes strain being present. Competitive and spoilage bacteria such as P. putida, can be entrapped in interactions among L. monocytogenes strains in mixed-culture multi-species sessile communities formed on inadequately biofilms have also been previously observed between serotype cleaned and disinfected food processing surfaces. However, in real food environments, the possible presence of many othermicrobial species clearly adds additional complexity to the behaviour of multi-species biofilms, since all incorporatedmicroorganisms are able to compete, cooperate, and In summary, present results highlight the impact of bacterial communicate with each other. Undoubtedly, further research is interactions taking place inside a mixed-culture sessile required to improve our understanding on the physiology of community on both its population dynamics and chemical multi-species biofilms formed by food related bacteria. This disinfection resistance. Interestingly, under dual-species could probably facilitate the development of methods for conditions, the simultaneous presence of L. monocytogenes controlling them in food areas and therefore reduce the strongly increased resistance of P. putida biofilm cells to BC.
contamination of the food products.
Following disinfection of dual-species sessile community withBC, the vast majority of cells killed belonged to L. monocytogenes, while the remaining viable community wasmainly composed of P. putida cells (>90%). In general, under We are grateful to Theodora Kouklada and Olga Hondrodimou dual-species conditions, the simultaneous presence of both for their valuable technical assistance.
bacterial species led to a reduction of their sessile populations, compared to mono-species conditions. Additionally, besidesthe differences observed in the dual-species biofilms with Conceived and designed the experiments: EG NC GJN.
regard to species occurrence, differences in strain dominance Performed the experiments: EG NC AD. Analyzed the data: EG were also observed in mixed-culture biofilm communities.
GJN. Wrote the manuscript: EG.
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  • Effect of culture conditions (mono-/dual-species) on BC resistance of L. monocytogenes and P. putida biofilm cells
  • Microbial (species) composition of the dual-species biofilm communities
  • Distribution of L. monocytogenes and P. putida strains in the mixed-culture biofilm communities
  • Source: http://gaia.aua.gr/xmlui/bitstream/handle/123456789/5981/Nichas1.pdf?sequence=1

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