Chester Clinic

 

Gaia.aua.gr

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: [email protected] 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.
Different strains were found to dominate, according to 1. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott 9. Bagge-Ravn D, Ng Y, Hjelm M, Christiansen JN, Johansen C et al.
HM (1995) Microbial biofilms. Annu Rev Microbiol 49: 711-745. doi: (2003) The microbial ecology of processing equipment in different fish industries-analysis of the microflora during processing and following 2. Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: cleaning and disinfection. Int J Food Microbiol 87: 239-250. doi: from the natural environment to infectious diseases. Nat Rev Microbiol 10. Carpentier B, Chassaing D (2004) Interactions in biofilms between 3. Shi X, Zhu X (2009) Biofilm formation and food safety in food Listeria monocytogenes and resident microorganisms from food industries. Trends Food Sci Technol 20: 407-413. industry premises. Int J Food Microbiol 97: 111-122. doi: 4. Brooks JD, Flint SH (2008) Biofilms in the food industry: problems and 11. Cruz CD, Fletcher GC (2011) Prevalence and biofilm-forming ability of potential solutions. Int J Food Sci Technol 43: 2163-2176. doi: Listeria monocytogenes in New Zealand mussel (Perna canaliculus) processing plants. Food Microbiol 28: 1387-1393. doi: 5. Midelet G, Carpentier B (2002) Transfer of microorganisms, including Listeria monocytogenes, from various materials to beef. Appl Environ 12. Didienne R, Defargues C, Callon C, Meylheuc T, Hulin S et al. (2012) Microbiol 68: 4015-4024. Characteristics of microbial biofilm on wooden vats ('gerles') in PDO 6. Papadopoulou OS, Chorianopoulos NG, Gkana EN, Grounta AV, Salers cheese. Int J Food Microbiol 156: 91-101. doi: Koutsoumanis KP et al. (2012) Transfer of foodborne pathogenic bacteria to non-inoculated beef fillets through meat mincing machine.
13. Latorre AA, Van Kessel JS, Karns JS, Zurakowski MJ, Pradhan AK et Meat Sci 90: 865-869. doi:. PubMed: al. (2010) Biofilm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with Listeria monocytogenes. J 7. Bridier A, Briandet R, Thomas V, Dubois-Brissonnet F (2011) Dairy Sci 93: 2792-2802. doi:. PubMed: Resistance of bacterial biofilms to disinfectants: a review. Biofouling 27: 1017-1032. doi:.
14. Licitra G, Ogier JC, Parayre S, Pediliggieri C, Carnemolla TM et al.
8. Austin JW, Bergeron G (1995) Development of bacterial biofilms in (2007) Variability of bacterial biofilms of the "tina" wood vats used in the dairy processing lines. J Dairy Res 62: 509-519. doi: ragusano cheese-making process. Appl Environ Microbiol 73: 6980-6987. doi:.
PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm 15. Sharma M, Anand SK (2002) Characterization of constitutive microflora 34. Uhlich GA, Rogers DP, Mosier DA (2010) Escherichia coli serotype of biofilms in dairy processing lines. Food Microbiol 19: 627-636. doi: O157:H7 retention on solid surfaces and peroxide resistance is enhanced by dual-strain biofilm formation. Foodborne Pathog Dis 7: 16. Timke M, Wang-Lieu NQ, Altendorf K, Lipski A (2005) Community structure and diversity of biofilms from a beer bottling plant as revealed 35. van der Veen S, Abee T (2011) Mixed species biofilms of Listeria using 16S rRNA gene clone libraries. Appl Environ Microbiol 71: monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid. Int J Food 17. Moons P, Michiels CW, Aertsen A (2009) Bacterial interactions in 36. Zhao T, Doyle MP, Zhao P (2004) Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms. Appl Environ 18. Nadell CD, Xavier JB, Foster KR (2009) The sociobiology of biofilms.
Microbiol 70: 3996-4003. 37. Zhao T, Podtburg TC, Zhao P, Chen D, Baker DA et al. (2013) 19. Remis JP, Costerton JW, Auer M (2010) Biofilms: structures that may Reduction by competitive bacteria of Listeria monocytogenes in biofilms facilitate cell-cell interactions. ISME J 4: 1085-1087. and Listeria bacteria in floor drains in a ready-to-eat poultry processing 20. Tolker-Nielsen T, Molin S (2000) Spatial organization of microbial 38. Zhao T, Podtburg TC, Zhao P, Schmidt BE, Baker DA et al. (2006) biofilm communities. Microb Ecol 40: 75-84. PubMed: Control of Listeria spp. by competitive-exclusion bacteria in floor drains 21. Behnke S, Parker AE, Woodall D, Camper AK (2011) Comparing the of a poultry processing plant. Appl Environ Microbiol 72: 3314-3320.
chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single- and dual-species cultures. Appl 39. Gandhi M, Chikindas ML (2007) Listeria: A foodborne pathogen that Environ Microbiol 77: 7176-7184. doi: knows how to survive. Int J Food Microbiol 113: 1-15. doi: 22. Burmølle M, Webb JS, Rao D, Hansen LH, Sørensen SJ et al. (2006) 40. Kathariou S (2002) Listeria monocytogenes virulence and Enhanced biofilm formation and increased resistance to antimicrobial pathogenicity, a food safety perspective. J Food Protect 65: 1811-1829.
agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms. Appl Environ Microbiol 72: 3916-3923. doi: 41. EFSA ECDC (2013) The European Union summary report on trends and sources of zoonoses, zoonotic agents and foodborne outbreaks in 23. Habimana O, Heir E, Langsrud S, Asli AW, Møretrø T (2010) Enhanced 2011. EFSA J 11(4): 3129.
surface colonization by Escherichia coli O157:H7 in biofilms formed by 42. Renier S, Hébraud M, Desvaux M (2011) Molecular biology of surface an Acinetobacter calcoaceticus isolate from meat-processing colonization by Listeria monocytogenes: an additional facet of an environments. Appl Environ Microbiol 76: 4557-4559. doi: opportunistic Gram-positive foodborne pathogen. Environ Microbiol 13: 24. Klayman BJ, Volden PA, Stewart PS, Camper AK (2009) Escherichia 43. Carpentier B, Cerf O (2011) Review – Persistence of Listeria coli O157:H7 requires colonizing partner to adhere and persist in a monocytogenes in food industry equipment and premises. Int J Food capillary flow cell. Environ Sci Technol 43: 2105-2111. doi: Microbiol 145: 1-8. PubMed: 25. Kostaki M, Chorianopoulos N, Braxou E, Nychas G-J, Giaouris E 44. Holah JT, Bird J, Hall KE (2004) The microbial ecology of high-risk, (2012) Differential biofilm formation and chemical disinfection chilled food factories; evidence for persistent Listeria spp. and resistance of sessile cells of Listeria monocytogenes strains under Escherichia coli strains. J Appl Microbiol 97: 68-77. monospecies and dual-species (with Salmonella enterica) conditions.
Appl Environ Microbiol 78: 2586-2595. doi: 45. Lundén JM, Miettinen MK, Autio TJ, Korkeala HJ (2000) Persistent Listeria monocytogenes strains show enhanced adherence to food 26. Leriche V, Briandet R, Carpentier B (2003) Ecology of mixed biofilms contact surface after short contact times. J Food Protect 63: subjected daily to a chlorinated alkaline solution: spatial distribution of 1204-1207. PubMed: .
bacterial species suggests a protective effect of one species to another.
46. Møretrø T, Langsrud S (2004) Listeria monocytogenes: biofilm Environ Microbiol 5: 64-71. doi: formation and persistence in food-processing environments. Biofilms 1: 27. Lindsay D, Brözel VS, Mostert JF, von Holy A (2002) Differential 47. De Jonghe V, Coorevits A, Van Hoorde K, Messens W, Van efficacy of a chlorine dioxide-containing sanitizer against single species Landschoot A et al. (2011) Influence of storage conditions on the and binary biofilms of a dairy-associated Bacillus cereus and a growth of Pseudomonas species in refrigerated raw milk. Appl Environ Pseudomonas fluorescens isolate. J Appl Microbiol 92: 352-361. doi: 28. Moons P, Van Houdt R, Aertsen A, Vanoirbeek K, Engelborghs Y et al.
48. Doulgeraki AI, Nychas G-J (2013) Monitoring the succession of the (2006) Role of quorum sensing and antimicrobial component biota grown on a selective medium for pseudomonads during storage production by Serratia plymuthica in formation of biofilms, including of minced beef with molecular-based methods. Food Microbiol 34: mixed biofilms with Escherichia coli. Appl Environ Microbiol 72: 7294-7300. doi:.
49. Tryfinopoulou P, Tsakalidou E, Nychas G-J (2002) Characterization of 29. Rieu A, Lemaître JP, Guzzo J, Piveteau P (2008) Interactions in dual Pseudomonas spp. associated with spoilage of gilt-head sea bream species biofilms between Listeria monocytogenes EGD-e and several stored under various conditions. Appl Environ Microbiol 68: 65-72. doi: strains of Staphylococcus aureus. Int J Food Microbiol 126: 76-82. doi: 50. Harmsen M, Yang L, Pamp SJ, Tolker-Nielsen T (2010) An update on 30. Simões LC, Lemos M, Araújo P, Pereira AM, Simões M (2011) The Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal.
effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens. Biofouling 27: 337-346.
51. Klausen M, Gjermansen M, Kreft JU, Tolker-Nielsen T (2006) Dynamics of development and dispersal in sessile microbial 31. Simões M, Simões LC, Vieira MJ (2007) Biofilm interactions between communities: examples from Pseudomonas aeruginosa and distinct bacterial genera isolated from drinking water. Appl Environ Pseudomonas putida model biofilms. FEMS Microbiol Lett 261: 1-11.
Microbiol 73: 6192-6200. PubMed: 52. O'Toole GA, Kolter R (1998) Initiation of biofilm formation in 32. Simões M, Simões LC, Vieira MJ (2008) Physiology and behavior of Pseudomonas fluorescens WCS365 proceeds via multiple, convergent Pseudomonas fluorescens single and dual strain biofilms under diverse signalling pathways: a genetic analysis. Mol Microbiol 28: 449-461. doi: hydrodynamics stresses. Int J Food Microbiol 128: 309-316. doi: 53. Sauer K, Camper AK (2001) Characterization of phenotypic changes in 33. Simões M, Simões LC, Vieira MJ (2009) Species association increases Pseudomonas putida in response to surface-associated growth. J biofilm resistance to chemical and mechanical treatments. Water Res Bacteriol 183: 6579-6589. 43: 229-237. doi: PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm 54. Hood SK, Zottola EA (1997) Isolation and identification of adherent 73. Russell AD (2004) Bacterial adaptation and resistance to antiseptics, gram-negative micro-organisms from four meat-processing facilities. J disinfectants and preservatives is not a new phenomenon. J Hosp Food Protect 60: 1135–1138.
55. Jeong DK, Frank JF (1994) Growth of Listeria monocytogenes at 21°C 74. Saá Ibusquiza PS, Herrera JJ, Cabo ML (2011) Resistance to in biofilms with micro-organisms isolated from meat and dairy benzalkonium chloride, peracetic acid and nisin during formation of processing environments. Lebensm Wiss Technol 27: 415–424. doi: mature biofilms by Listeria monocytogenes. Food Microbiol 28: 56. Ghafoor A, Hay ID, Rehm BH (2011) Role of exopolysaccharides in 75. Langsrud S, Sundheim G, Borgmann-Strahsen R (2003) Intrinsic and Pseudomonas aeruginosa biofilm formation and architecture. Appl acquired resistance to quaternary ammonium compounds in food- Environ Microbiol 77: 5238-5246. doi: related Pseudomonas spp. J Appl Microbiol 95: 874-882.
76. Sidhu MS, Sørum H, Holck A (2002) Resistance to quaternary 57. Mann EE, Wozniak DJ (2012) Pseudomonas biofilm matrix composition ammonium compounds in food-related bacteria. Microb Drug Resist 8: and niche biology. FEMS Microbiol Rev 36: 893-916. doi: 77. Marouani-Gadri N, Augier G, Carpentier B (2009) Characterization of 58. Ude S, Arnold DL, Moon CD, Timms-Wilson T, Spiers AJ (2006) Biofilm bacterial strains isolated from a beef-processing plant following formation and cellulose expression among diverse environmental cleaning and disinfection - Influence of isolated strains on biofilm Pseudomonas isolates. Environ Microbiol 8: 1997-2011. doi: formation by Sakaï and EDL 933 E. coli O157:H7. Int J Food Microbiol 59. Antoniou K, Frank JF (2005) Removal of Pseudomonas putida biofilm 78. Saá Ibusquiza PS, Herrera JJR, Vázquez-Sánchez D, Cabo ML (2012) and associated extracellular polymeric substances from stainless steel Adherence kinetics, resistance to benzalkonium chloride and by alkali cleaning. J Food Protect 68: 277-281. PubMed: .
microscopic analysis of mixed biofilms formed by Listeria 60. Auerbach ID, Sorensen C, Hansma HG, Holden PA (2000) Physical monocytogenes and Pseudomonas putida. Food Control 25: 202-210.
morphology and surface properties of unsaturated Pseudomonas putida biofilms. J Bacteriol 182: 3809-3815. doi: 79. Skandamis PN, Nychas GJ (2012) Quorum sensing in the context of food microbiology. Appl Environ Microbiol 78: 5473-5482. doi: 61. Chorianopoulos NG, Giaouris ED, Skandamis PN, Haroutounian SA, Nychas G-JE (2008) Disinfectant test against monoculture and mixed- 80. Parsek MR, Greenberg EP (2005) Sociomicrobiology: the connections culture biofilms composed of technological, spoilage and pathogenic between quorum sensing and biofilms. Trends Microbiol 13: 27-33. doi: bacteria: bactericidal effect of essential oil and hydrosol of Satureja thymbra and comparison with standard acid-base sanitizers. J Appl 81. Schlech WF 3rd, Lavigne PM, Bortolussi RA, Allen AC, Haldane EV et Microbiol 104: 1586-1596. doi: al. (1983) Epidemic listeriosis: evidence for transmission by food. N Engl J Med 308: 203-206. doi: 62. Chumkhunthod P, Schraft H, Griffiths MW (1998) Rapid monitoring method to assess efficacy of sanitizers against Pseudomonas putida 82. Cocolin L, Stella S, Nappi R, Bozzetta E, Cantoni C et al. (2005) Analysis of PCR-based methods for characterization of Listeria 63. Kruijt M, Tran H, Raaijmakers JM (2009) Functional, genetic and monocytogenes strains isolated from different sources. Int J Food chemical characterization of biosurfactants produced by plant growth- promoting Pseudomonas putida 267. J Appl Microbiol 107: 546-556.
83. Davey ME, O'Toole GA (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64: 847-867. doi: 64. Kuiper I, Lagendijk EL, Pickford R, Derrick JP, Lamers GE et al. (2004) Characterization Pseudomonas 84. Elias S, Banin E (2012) Multi-species biofilms: living with friendly biosurfactants, putisolvin I and II, which inhibit biofilm formation and neighbors. FEMS Microbiol Rev 36: 990-1004. PubMed: break down existing biofilms. Mol Microbiol 51: 97-113. PubMed: 85. Rickard AH, Gilbert P, High NJ, Kolenbrander PE, Handley PS (2003) Bacterial coaggregation: an integral process in the development of 65. Buffet-Bataillon S, Tattevin P, Bonnaure-Mallet M, Jolivet-Gougeon A multi-species biofilms. Trends Microbiol 11: 94-100. doi: (2012) Emergence of resistance to antibacterial agents: the role of quaternary ammonium compounds - a critical review. Int J Antimicrob 86. Wimpenny J, Manz W, Szewzyk U (2000) Heterogeneity in biofilms.
Agents 39: 381-389. PubMed: 66. Hegstad K, Langsrud S, Lunestad BT, Scheie AA, Sunde M et al.
87. Yang L, Liu Y, Wu H, Hóiby N, Molin S et al. (2011) Current (2010) Does the wide use of quaternary ammonium compounds understanding of multi-species biofilms. Int J Oral Sci 3: 74-81. doi: enhance the selection and spread of antimicrobial resistance and thus threaten our health? Microb Drug Resist 16: 91-104. doi: 88. Goller CC, Romeo T (2008) Environmental influences on biofilm development. Curr Top Microbiol Immunol 322: 37-66. doi: 67. McDonnell G, Russell AD (1999) Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 12: 147-179. PubMed: 89. James GA, Beaudette L, Costerton JW (1995) Interspecies bacterial interactions in biofilms. J Ind Microbiol 15: 257-262. doi: 68. Tischer M, Pradel G, Ohlsen K, Holzgrabe U (2012) Quaternary ammonium salts and their antimicrobial potential: targets or nonspecific 90. Rendueles O, Ghigo JM (2012) Multi-species biofilms: how to avoid interactions? Chemmedchem 7: 22-31. unfriendly neighbors. FEMS Microbiol Rev 36: 972-989. PubMed: 69. Dynes JJ, Lawrence JR, Korber DR, Swerhone GD, Leppard GG et al.
91. Nielsen L, Li X, Halverson LJ (2011) Cell-cell and cell-surface (2009) Morphological and biochemical changes in Pseudomonas interactions mediated by cellulose and a novel exopolysaccharide fluorescens biofilms induced by sub-inhibitory exposure to antimicrobial contribute to Pseudomonas putida biofilm formation and fitness under agents. Can J Microbiol 55: 163-178. doi:. PubMed: water-limiting conditions. Environ Microbiol 13: 1342-1356. doi: 70. Mangalappalli-Illathu AK, Vidović S, Korber DR (2008) Differential 92. Yang L, Hu Y, Liu Y, Zhang J, Ulstrup J et al. (2011) Distinct roles of adaptive response and survival of Salmonella enterica serovar extracellular polymeric substances in Pseudomonas aeruginosa biofilm enteritidis planktonic and biofilm cells exposed to benzalkonium development. Environ Microbiol 13: 1705-1717. chloride. Antimicrob Agents Chemother 52: 3669-3680. doi: 93. Yousef-Coronado F, Travieso ML, Espinosa-Urgel M (2008) Different, 71. Pagedar A, Singh J, Batish VK (2012) Adaptation to benzalkonium overlapping mechanisms for colonization of abiotic and plant surfaces chloride and ciprofloxacin affects biofilm formation potential, efflux by Pseudomonas putida. FEMS Microbiol Lett 288: 118-124. doi: pump and haemolysin activity of Escherichia coli of dairy origin. J Dairy 94. Pamp SJ, Tolker-Nielsen T (2007) Multiple roles of biosurfactants in 72. Russell AD (2001) Mechanisms of bacterial insusceptibility to biocides.
structural biofilm development by Pseudomonas aeruginosa. J Am J Infect Control 29: 259-261. Bacteriol 189: 2531-2539. PubMed: PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276 L. monocytogenes, P. putida Mixed-Culture Biofilm 95. Irie Y, O'Toole GA, Yuk MH (2005) Pseudomonas aeruginosa Enterococcus faecium on Listeria monocytogenes biofilm formation. J rhamnolipids disperse Bordetella bronchiseptica biofilms. FEMS Food Protect 71: 634-638. PubMed: .
Microbiol Lett 250: 237-243. 112. Norwood DE, Gilmour A (2000) The growth and resistance to sodium hypochlorite of Listeria monocytogenes in a steady-state multispecies 96. Janek T, Łukaszewicz M, Krasowska A (2012) Antiadhesive activity of the biosurfactant pseudofactin II secreted by the Arctic bacterium Pseudomonas fluorescens BD5. BMC Microbiol 12: 24. doi: 113. Norwood DE, Gilmour A (2001) The differential adherence capabilities of two Listeria monocytogenes strains in monoculture and multispecies 97. Meylheuc T, Renault M, Bellon-Fontaine MN (2006) Adsorption of a biofilms as a function of temperature. Lett Appl Microbiol 33: 320-324.
biosurfactant on surfaces to enhance the disinfection of surfaces contaminated with Listeria monocytogenes. Int J Food Microbiol 109: 114. Bourion F, Cerf O, Meylheuc T (1996) Disinfection efficacy against 71-78. doi:.
pure-culture and mixed-population biofilms of Listeria innocua and 98. Harvey J, Keenan KP, Gilmour A (2007) Assessing biofilm formation by Pseudomonas aeruginosa on stainless steel, Teflon ® and rubber. Sci Listeria monocytogenes strains. Food Microbiol 24: 380-392. doi: Aliments 16: 151-166.
115. Bremer PJ, Monk I, Butler R (2002) Inactivation of Listeria 99. Kalmokoff ML, Austin JW, Wan XD, Sanders G, Banerjee S et al.
monocytogenes/Flavobacterium spp. biofilms using chlorine: impact of (2001) Adsorption, attachment and biofilm formation among isolates of substrate, pH, time and concentration. Lett Appl Microbiol 35: 321-325.
Listeria monocytogenes using model conditions. J Appl Microbiol 91: 116. Fatemi P, Frank JF (1999) Inactivation of Listeria monocytogenes/ 100. Nilsson RE, Ross T, Bowman JP (2011) Variability in biofilm production Pseudomonas biofilms by peracid sanitizers. J Food Protect 62: by Listeria monocytogenes correlated to strain origin and growth conditions. Int J Food Microbiol 150: 14-24. doi: 117. Lebert I, Leroy S, Talon R (2007) Effect of industrial and natural biocides on spoilage, pathogenic and technological strains grown in 101. Takahashi H, Miya S, Igarashi K, Suda T, Kuramoto S et al. (2009) biofilm. Food Microbiol 24: 281-287. doi: Biofilm formation ability of Listeria monocytogenes isolates from raw ready-to-eat seafood. J Food Protect 72: 1476-1480. PubMed: 118. Zhang W, Sileika T, Packman AI (2013) Effects of fluid flow conditions on interactions between species in biofilms. FEMS Microbiol Ecol 84: 102. Tresse O, Shannon K, Pinon A, Malle P, Vialette M et al. (2007) Variable adhesion of Listeria monocytogenes isolates from food- 119. Russell AD, Chopra I (1996) Understanding antibacterial action and processing facilities and clinical cases to inert surfaces. J Food Protect resistance. Ellis Horwood Ltd.
120. Meyer B (2006) Does microbial resistance to biocides create a hazard 103. Habimana O, Meyrand M, Meylheuc T, Kulakauskas S, Briandet R to food hygiene? Int J Food Microbiol 112: 275-279. doi: (2009) Genetic features of resident biofilms determine attachment of Listeria monocytogenes. Appl Environ Microbiol 75: 7814-7821. doi: 121. Joynson JA, Forbes B, Lambert RJ (2002) Adaptive resistance to benzalkonium chloride, amikacin and tobramycin: the effect on 104. Hassan AN, Birt DM, Frank JF (2004) Behavior of Listeria susceptibility to other antimicrobials. J Appl Microbiol 93: 96-107. doi: monocytogenes in a Pseudomonas putida biofilm on a condensate- forming surface. J Food Protect 67: 322-327.
122. To MS, Favrin S, Romanova N, Griffiths MW (2002) Postadaptational 105. Sasahara KC, Zottola EA (1993) Biofilm formation by Listeria resistance to benzalkonium chloride and subsequent physicochemical monocytogenes utilizes a primary colonizing microorganism in flowing modifications of Listeria monocytogenes. Appl Environ Microbiol 68: systems. J Food Protect 56: 1022-1028.
106. Lourenço A, Machado H, Brito L (2011) Biofilms of Listeria monocytogenes produced at 12 °C either in pure culture or in co-culture 123. Aase B, Sundheim G, Langsrud S, Rørvik LM (2000) Occurrence of with Pseudomonas aeruginosa showed reduced susceptibility to and a possible mechanism for resistance to a quaternary ammonium compound in Listeria monocytogenes. Int J Food Microbiol 62: 57-63.
107. Bremer PJ, Monk I, Osborne CM (2001) Survival of Listeria 124. Mereghetti L, Quentin R, Marquet-Van Der Mee N, Audurier A (2000) monocytogenes attached to stainless steel surfaces in the presence or Low sensitivity of Listeria monocytogenes to quaternary ammonium absence of Flavobacterium spp. J Food Prot 64: 1369-1376.
compounds. Appl Environ Microbiol 66: 5083-5086. 108. Habimana O, Guillier L, Kulakauskas S, Briandet R (2011) Spatial competition with Lactococcus lactis in mixed-species continuous-flow 125. Soumet C, Ragimbeau C, Maris P (2005) Screening of benzalkonium biofilms inhibits Listeria monocytogenes growth. Biofouling 27: chloride resistance in Listeria monocytogenes strains isolated during 1065-1072. doi:.
cold smoked fish production. Lett Appl Microbiol 41: 291-296. doi: 109. Leriche V, Carpentier B (2000) Limitation of adhesion and growth of Listeria monocytogenes on stainless steel surfaces by Staphylococcus 126. Langsrud S, Sundheim G (1997) Factors contributing to the survival of sciuri biofilms. J Appl Microbiol 88: 594-605. doi: poultry associated Pseudomonas spp. exposed to a quaternary ammonium compound. J Appl Microbiol 82: 705-712. 110. Leriche V, Chassaing D, Carpentier B (1999) Behaviour of L. monocytogenes in an artificially made biofilm of a nisin-producing strain 127. Pan Y, Breidt F Jr, Kathariou S (2009) Competition of Listeria of Lactococcus lactis. Int J Food Microbiol 51: 169-182. doi: monocytogenes serotype 1/2a and 4b strains in mixed-culture biofilms.
Appl Environ Microbiol 75: 5846-5852. doi: 111. Minei CC, Gomes BC, Ratti RP, D'Angelis CE, De Martinis EC (2008) Influence of peroxyacetic acid and nisin and coculture with PLOS ONE www.plosone.org October 2013 Volume 8 Issue 10 e77276

Bacterial strains and growth conditions

Abiotic substratum and biofilm formation procedure

Exposure of biofilm cells to disinfection

Recovery and quantification of viable biofilm cells

Isolation of strains from mixed-culture biofilm communities

Pulsed-field gel electrophoresis (PFGE) analysis

Effect of culture conditions (mono-/dual-species) on biofilm formation by L. monocytogenes and P. putida strains

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