Differential biofilm formation and chemical disinfection resistance of sessile cells of listeria monocytogenes strains under monospecies and dual-species (with Salmonella enterica) conditions

This study aimed to investigate the possible influence of bacterial intra- and interspecies interactions on the ability of Listeria monocytogenes and Salmonella enterica to develop mixed-culture biofilms on an abiotic substratum, as well as on the subsequent resistance of sessile cells to chemical disinfection. Initially, three strains from each species were selected and left to attach and form biofilms on stainless steel (SS) coupons incubated at 15°C for 144 h, in periodically renewable tryptone soy broth (TSB), under either monoculture or mixed-culture (mono-/dual-species) conditions. Following biofilm formation, mixed-culture sessile communities were subjected to 6-min disinfection treatments with (i) benzalkonium chloride (50 ppm), (ii) sodium hypochlorite (10 ppm), (iii) peracetic acid (10 ppm), and (iv) a mixture of hydrogen peroxide (5 ppm) and peracetic acid (5 ppm). Results revealed that both species reached similar biofilm counts (ca. 10(5) CFU cm(-2)) and that, in general, interspecies interactions did not have any significant effect either on the biofilm-forming ability (as this was assessed by agar plating enumeration of the mechanically detached biofilm bacteria) or on the antimicrobial resistance of each individual species. Interestingly, pulsed-field gel electrophoresis (PFGE) analysis clearly showed that the three L. monocytogenes strains did not contribute at the same level either to the formation of mixed-culture sessile communities (mono-/dual species) or to their antimicrobial recalcitrance. Additionally, the simultaneous existence inside the biofilm structure of S. enterica cells seemed to influence the occurrence and resistance pattern of L. monocytogenes strains. In sum, this study highlights the impact of microbial interactions taking place inside a mixed-culture sessile community on both its population dynamics and disinfection resistance.     

Spatial competition with Lactococcus lactis in mixed-species continuous-flow biofilms inhibits Listeria monocytogenes growth

Surfaces in industrial settings provide a home for resident biofilms that are likely to interact with the attachment, growth and survival of pathogens such as Listeria monocytogenes. Experimental results have indicated that L. monocytogenes cells were inhibited by the presence of a model resident flora (Lactococcus lactis) in dual-species continuous flow-biofilms, and are spatially restricted to the lower biofilm layers. Using a new, simplified individual-based model (IBM) that simulates bacterial cell growth in a three-dimensional space, the spatial arrangements of the two species were reconstructed and their cell counts successfully predicted. This model showed that the difference in generation times between L. monocytogenes and L. lactis cells during the initial stages of dual-species biofilm formation was probably responsible for the species spatialization observed and the subsequent inhibition of growth of the pathogen.     

Rapid methods to assess sanitizing efficacy of benzalkonium chloride to Listeria monocytogenes biofilms

Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P = 0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.     

Extracellular DNA in Single- and Multiple-Species Unsaturated Biofilms

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.     

Listeria monocytogenes Can Form Biofilms in Tap Water and Enter Into the Viable but Non-Cultivable State

Listeria monocytogenes is a foodborne pathogen that can be transmitted through contaminated raw food or by ready-to-eat products that have been in contact with contaminated surfaces. Tap water (TW) is used to wash produce, as a processed food constituent and to wash processing surfaces and floors. The main aim of this work was to investigate the formation and survival of L. monocytogenes biofilms on stainless steel (SS) coupons in TW at 4, 22, 30 and 37 °C. For that, coupons with biofilm were visualised in situ while other coupons were scraped to quantify total cells by SYTO 9, cultivable numbers by plating onto brain heart infusion agar and viable numbers by the direct viable count method. Results showed that L. monocytogenes can form biofilms on SS surfaces in TW at any temperature, including at 4 °C. The number of total cells was similar for all the conditions tested while cultivable numbers varied between the level of detection (<8.3 CFU cm^?2) and 3.5?×?10⁵ CFU cm^?2, meaning between 7.0?×?10⁴ and 1.1?×?10⁷ cells cm^?2 have entered the viable but non-cultivable (VBNC) state. This work clearly demonstrates that L. monocytogenes can form biofilms in TW and that sessile cells can remain viable and cultivable in some conditions for at least the 48 h investigated. On the other hand, VBNC adaptation suggests that the pathogen can remain undetectable using traditional culture recovery techniques, which may give a false indication of processing surface hygiene status, leading to potential cross-contamination of food products.     

Establishment of New Genetic Traits in a Microbial Biofilm Community

Conjugational transfer of the TOL plasmid (pWWO) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. The community consisted of three species, Pseudomonas putida RI, Acinetobacter sp. strain C6, and an unidentified isolate, D8. Only P. putida RI could act as a recipient for the TOL plasmid. Cells carrying a chromosomally integrated lacI^q gene and a lacp-gfp-tagged version of the TOL plasmid were introduced as donor strains in the biofilm community after its formation. The occurrence of plasmid-carrying cells was analyzed by viable-count-based enumeration of donors and transconjugants. Upon transfer of the plasmids to the recipient cells, expression of green fluorescence was activated as a result of zygotic induction of the gfp gene. This allowed a direct in situ identification of cells receiving the gfp-tagged version of the TOL plasmid. Our data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmid establishment in the biofilm community. Employment of scanning confocal laser microscopy on fixed biofilms, combined with simultaneous identification of P. putida cells and transconjugants by 16S rRNA hybridization and expression of green fluorescence, showed that transconjugants were always associated with noninfected P. putida RI recipient microcolonies. Pure colonies of transconjugants were never observed, indicating that proliferation of transconjugant cells preferentially took place on preexisting P. putida RI microcolonies in the biofilm.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Variation in Biofilm Formation among Strains of Listeria monocytogenes

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.     

Biofilm-growing intestinal anaerobic bacteria

Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum.     

Competition of Listeria monocytogenes Serotype 1/2a and 4b Strains in Mixed-Culture Biofilms

The majority of Listeria monocytogenes isolates recovered from foods and the environment are strains of serogroup 1/2, especially serotypes 1/2a and 1/2b. However, serotype 4b strains cause the majority of human listeriosis outbreaks. Our investigation of L. monocytogenes biofilms used a simulated food-processing system that consisted of repeated cycles of growth, sanitation treatment, and starvation to determine the competitive fitness of strains of serotypes 1/2a and 4b in pure and mixed-culture biofilms. Selective enumeration of strains of a certain serotype in mixed-culture biofilms on stainless steel coupons was accomplished by using serotype-specific quantitative PCR and propidium monoazide treatment to prevent amplification of extracellular DNA or DNA from dead cells. The results showed that the serotype 1/2a strains tested were generally more efficient at forming biofilms and predominated in the mixed-culture biofilms. The growth and survival of strains of one serotype were not inhibited by strains of the other serotype in mixed-culture biofilms. However, we found that a cocktail of serotype 4b strains survived and grew significantly better in mixed-culture biofilms containing a specific strain of serotype 1/2a (strain SK1387), with final cell densities averaging 0.5 log₁₀ CFU/cm² higher than without the serotype 1/2a strain. The methodology used in this study contributed to our understanding of how environmental stresses and microbial competition influence the survival and growth of L. monocytogenes in pure and mixed-culture biofilms.A prominent food-borne pathogen, Listeria monocytogenes can cause severe infections in humans, primarily in high-risk populations, though the disease (listeriosis) is relatively rare (11, 30, 43). Outbreaks of listeriosis have resulted from the contamination of a variety of foods by L. monocytogenes, especially meat and dairy products (27). L. monocytogenes is ubiquitous in the environment, able to grow at refrigeration temperature, and tolerant of the low pHs (3 to 4) typical of acidified foods (28, 32, 44). The capacity to produce biofilms confers protection against stresses common in the food-processing environment (13, 33).Biofilms are characterized by dense clusters of bacterial cells embedded in extracellular polymeric substances which are secreted by cells to aid in adhesion to surfaces and to other cells (4, 5). Strains of L. monocytogenes have been known to persist for years in food-processing environments, presumably in biofilms. Of the 13 known serotypes of L. monocytogenes, three (1/2a, 1/2b, and 4b) account for >95% of the isolates from human illness (21). Serotype 1/2a accounts for >50% of the L. monocytogenes isolates recovered from foods and the environment, while most major outbreaks of human listeriosis have been caused by serotype 4b strains (1, 3, 14, 15, 17, 22, 29, 31, 41, 47, 49,). No correlation between L. monocytogenes strain fitness and serotype has been identified (16, 19). Some studies have reported that strains repeatedly isolated from food and environmental samples (defined as persistent strains) had a higher adherence capacity than strains that were sporadically isolated (2, 36), while this phenomenon was not observed by others (7). Serotype 4b strains exhibited a higher capacity for biofilm formation than did serotype 1/2a strains (36), whereas this was not observed by Di Bonaventura and colleagues (6). It has been suggested that serotype 1/2a strains could be more robust than serotype 4b strains in biofilm formation under a variety of environmental conditions. Furthermore, strains of these serotypes differ in terms of the medium that promotes biofilm formation. Biofilm formation by serotype 4b strains was higher in full-strength tryptic soy broth than in diluted medium, whereas the opposite was observed with serotype 1/2a strains, which produced more biofilm in diluted medium (12).There is limited information on microbial competition between strains of different serotypes in biofilms or on how the environmental stresses present in food-processing environments may affect the biofilm formation and survival of L. monocytogenes of different serotypes. In food-processing plants, the environmental stresses encountered by bacteria are more complex and variable than most laboratory systems used for microbial ecology and biofilm studies. A simulated food-processing (SFP) system has been developed to address this issue (38). The SFP system incorporates several stresses that may affect bacteria in biofilms in the food-processing environment, including exposure to sanitizing agents, dehydration, and starvation. When biofilms were subjected to the SFP regimen over a period of several weeks, the cell numbers of L. monocytogenes strains in the biofilms initially were reduced and then increased as the culture adapted (38). The development of resistance to sanitizing agents was specific to the biofilm-associated cells and was not apparent in the detached cells (38). This suggested that extracellular polymeric substances present in the biofilm matrix were responsible for the resistance to sanitizing agents. It was subsequently found that real-time PCR, in combination with propidium monoazide (PMA) treatment of samples prior to DNA isolation, was an effective method for enumerating viable cells in biofilms (37).The objective of this study was to determine if strains of serotype 1/2a or 4b have a selective advantage under stress conditions. We investigated and compared the initial attachment and biofilm formation capabilities of L. monocytogenes strains of these two serotypes and analyzed the survival and growth of bacteria of each serotype in mixed-serotype biofilms in the SFP system by using PMA with quantitative PCR.     

Effect of growth temperature,surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.     

Commensal Interactions in a Dual-Species Biofilm Exposed to Mixed Organic Compounds

There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the p-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at high p-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and Escherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.     

Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes–Escherichia coli dual-species biofilms

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes–Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r² = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.     

Population dynamics of a dual Pseudomonas putida–Pseudomonas fluorescens biofilm in a capillary bioreactor

Most biofilm studies employ single species, yet in nature biofilms exist as mixed cultures, with inevitable effects on growth and development of each species present. To investigate how related species of bacteria interact in biofilms, two Pseudomonas spp., Pseudomonas fluorescens and Pseudomonas putida, were cultured in capillary bioreactors and their growth measured by confocal microscopy and cell counting. When inoculated in pure culture, both bacteria formed healthy biofilms within 72?h with uniform coverage of the surface. However, when the bioreactors were inoculated with both bacteria simultaneously, P. putida was completely dominant after 48?h. Even when the inoculation by P. putida was delayed for 24?h, P. fluorescens was eliminated from the capillary within 48?h. It is proposed that production of the lipopeptide putisolvin by P. putida is the likely reason for the reduction of P. fluorescens. Putisolvin biosynthesis in the dual-species biofilm was confirmed by mass spectrometry.     

Potential of antimicrobial treatment of linear low-density polyethylene with poly((tert-butyl-amino)-methyl-styrene) to reduce biofilm formation in the food industry

Antimicrobial surfaces are one approach to prevent biofilms in the food industry. The aim of this study was to investigate the effect of poly((tert-butyl-amino)-methyl-styrene) (poly(TBAMS)) incorporated into linear low-density polyethylene (LLDPE) on the formation of mono- and mixed-species biofilms. The biofilm on untreated and treated LLDPE was determined after 48 and 168 h. The comparison of the results indicated that the ability of Listeria monocytogenes to form biofilms was completely suppressed by poly(TBAMS) (Δ_168 h 3.2 log₁₀ cfu cm^?2) and colonization of Staphylococcus aureus and Escherichia coli was significantly delayed, but no effect on Pseudomonas fluorescens was observed. The results of dual-species biofilms showed complex interactions between the microorganisms, but comparable effects on the individual bacteria by poly(TBAMS) were identified. Antimicrobial treatment with poly(TBAMS) shows great potential to prevent biofilms on polymeric surfaces. However, a further development of the material is necessary to reduce the colonization of strong biofilm formers.     

Degradation of chlorotoluenes and chlorobenzenes by the dual-species biofilm of Comamonas testosteroni strain KT5 and Bacillus subtilis strain DKT

The cooperation of Bacillus subtilis strain DKT and Comamonas testosteroni KT5 was investigated for biofilm development and toluenes and chlorobenzenes degradation. Bacillus subtilis strain DKT and C. testosteroni KT5 were co-cultured in liquid media with toluenes and chlorobenzenes to determine the degradation of these substrates and formation of dual-species biofilm used for the degradation process. Bacillus subtilis strain DKT utilized benzene, mono- and dichlorinated benzenes as carbon and energy sources. The catabolism of chlorobenzenes was via hydroxylation, in which chlorine atoms were replaced by hydroxyl groups to form catechol, followed by ring fission via the ortho-cleavage pathway. The investigation of the dual-species biofilm composed of B. subtilis DKT and C. testosteroni KT5 (a toluene and chlorotoluene-degrading isolate with low biofilm formation) showed that B. subtilis DKT synergistically promoted C. testosteroni KT5 to develop biofilm. The bacterial growth in dual-species biofilm overcame the inhibitory effects caused by monochlorobenzene and 2-chlorotoluene. Moreover, the dual-species biofilm showed effective degradability toward the mixture of these substrates. This study provides knowledge about the commensal relationships in a dual-culture biofilm for designing multispecies biofilms applied for the biodegradation of toxic organic substrates that cannot be metabolized by single-organism biofilms.

     

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.     

In situ monitoring of biofilm formations ofEscherichia coli andPseudomonas putida by use of lux and GFP reporters

A plasmid vector containing two reporter genes,mer-lux andlac-GFP, was transformed to bothEscherichia coli andPseudomonas putida. Their cellular activities and biofilm characteristics were investigated in flow-cell units by measuring bioluminescent lights and fluorescent levels of GFP. Bioluminescence was effective to monitor temporal cell activities, whereas fluorescent level of GFP was useful to indicate the overall cell activities during biofilm development. The light production rates ofE. coli andP. putida cultures were dependent upon concentrations of HgCl₂. Mercury molecules entrapped inP. putida biofilms were hardly washed out in comparison with those inE. coli biofilms, indicating thatP. putida biofilms may have higher affinity to mercury molecules thanE. coli biofilms. It was observed thatP. putida expressed GFP cDNA in biofilms but not in liquid cultures. This may indicate that the genetic mechanisms ofP. putida were favorably altered in biofilm conditions to make a foreign gene expression possible.     

Effect of Batch and Fed-Batch Growth Modes on Biofilm Formation by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> at Different Temperatures

The influence of Listeria monocytogenes (L. monocytogenes) biofilm formation feeding conditions (batch and fed-batch) at different temperatures on biofilm biomass and activity was determined. Biofilm biomass and cellular metabolic activity were assessed by Crystal Violet (CV) staining and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) colorimetric method, respectively. Live/Dead staining was also performed in order to get microscopic visualization of the different biofilms. Results revealed that at refrigeration temperature (4°C) a higher amount of biofilm was produced when batch conditions were applied, while at higher temperatures the fed-batch feeding condition was the most effective on biofilm formation. Moreover, independently of the temperature used, biofilms formed under fed-batch conditions were metabolically more active than those formed in batch mode. In conclusion, this work shows that different growth modes significantly influence L. monocytogenes biofilm formation on abiotic surfaces as well as the metabolic activity of cells within biofilms.