Binary Biofilms Research Articles

Evaluation of approaches to increase the effectiveness of various disinfectants against biofilm communities of different ages

Disinfectants are used as the main agents against microorganisms circulating on the surfaces of food enterprises. However, the adaptive ability of microorganisms to form biofilms complicates the process of surface cleaning and reduces the effectiveness of disinfectants. Modern disinfectants act against freely circulating microflora, but it is known that they are not always effective against biofilms. The purpose of this study was to investigate effective disinfectant compositions with bactericidal effect on binary bacterial biofilms of different ages. The article describes the effects of disinfectants based on chlorine, peracetic acid and quaternary ammonium compounds with enzymes in concentrations recommended by the manufacturer and increased several times on Salmonella 38, Brochothrix thermosphacta 2726 and Staphylococcus equorum 2736 planktonic cultures and binary biofilms. Binary biofilms of different ages (2 and 9 days old) were exposed to disinfectants with various active ingredients in combination with adjuvants, i. e. hydrogen peroxide 6% and various concentrations of isopropyl alcohol (30%). All products in concentrations recommended by the manufacturer did not have a disinfectant effect against the studied biofilm cultures. As a result of the work, it was found that the most effective disinfectants against multispecies biofilms were quaternary ammonium compounds in combination with enzymes and chlorine in combination with isopropyl alcohol (30%). The results obtained allow to expand knowledge about effective methods for controlling biofilms.

A-Type Natriuretic Peptide Alters the Impact of Azithromycin on Planktonic Culture and on (Monospecies and Binary) Biofilms of Skin Bacteria Kytococcus schroeteri and Staphylococcus aureus.

It has been established that the human atrial natriuretic peptide is able to alter the effect of azithromycin on Kytococcus schroeteri H01 and Staphylococcus aureus 209P monospecies and binary biofilms. The effect of the hormone depends on the surface type and cultivation system, and it may have both enhancing and counteracting effects. The antagonistic effect of the hormone was observed mostly on hydrophobic surfaces, whereas the additive effect was observed on hydrophilic surfaces like glass. Also, the effect of the hormone depends on the antibiotic concentration and bacterial species. The combination of azithromycin and ANP led to an amplification of cell aggregation in biofilms, to the potential increase in matrix synthesis, and to a decrease in S. aureus in the binary community. Also, ANP, azithromycin, and their combinations caused the differential expression of genes of resistance to different antibiotics, like macrolides (mostly increasing expression in kytococci), fluoroquinolones, aminoglycosides, and others, in both bacteria.

Evaluating the effect of various types of disinfectants on bacterial biofilms

Biofilm formation on equipment surfaces is a potential food safety hazard, providing increased resistance and persistence of pathogens and spoilage microorganisms in food production environments. The issue of preventing the biofilm formation is extremely important, since a wide range of disinfectants does not always provide the proper effect. The article discusses the antimicrobial effectiveness of disinfectants with various active ingredients (based on active chlorine, peracetic acid and quaternary ammonium compounds (QAC) with enzymatic substances) on binary biofilms. The objects of the study were the strains of pathogenic and opportunistic microorganisms isolated from abiotic surfaces of food production environments and food products. Different effects of disinfectants on biofilms formed by bacteria have been established. Disinfectant based on peracetic acid and chlorine had the greatest effect on binary biofilms of Brochothrix thermosphacta/Salmonella spp. and Staphylococcus equorum/Salmonella spp. The greatest antimicrobial effect on biofilm of Listeria monocytogenes 12/Pseudomonas azotoformans 6 was shown by a chlorine-based disinfectant. Disinfectants based on chlorine and QAC with enzymatic substances were most effective against the binary biofilm of L. monocytogenes 12/Salmonella spp. 14. However, none of the disinfectants had absolute antimicrobial effectiveness against the studied binary biofilms. Biofilm-forming microorganisms have shown resistance to the recommended concentrations of disinfectants. Therefore, currently, it is extremely important to revise approaches to hygiene at enterprises by finding working concentrations of new antimicrobial agents and new procedure that are effective for destroying biofilms.

Phylogenetic Constitution and Survival of Microbial Biofilms Formed on the Surface of Polyethylene Composites Protected with Polyguanidine Biocides

A series of biocide-containing polyethylene composites were obtained using novel guanidine-containing copolymers immobilized on an inert mineral carrier. Multispecies microbial communities were isolated from the surface of polyethylene samples either incubated or found in the environment, and their taxonomic composition was determined. Biofilms reconstructed using microorganisms obtained from different ecotopes were shown to intensively foul polyethylene surfaces. The presence of polyguanidine biocide suppressed the growth and survival of both binary and multispecies biofilms, with a cumulative effect during long-term incubation. When microorganisms were co-cultivated in binary biofilms, the phenomenon of a decrease in biocide effectiveness was demonstrated. This protective effect is potentially based on cooperative interactions inside the binary biofilm community. Scanning electron microscopy showed a pronounced difference in the architecture of reconstructed biofilms incubated in the presence of biocide in comparison to control samples, where biocide suppressed the formation of dense and well-organized three-dimensional structures. Biofilm disruption by immobilized biocides occurred primarily during the later stages of biofilm formation, probably caused by polycation interaction with their negatively charged extracellular components.

Characterization of Binary Biofilms of Listeria monocytogenes and Lactobacillus and Their Response to Chlorine Treatment.

In nature, Listeria may interact competitively and cooperatively with other organisms, resulting in unique spatial organization and functions for cells within the community. This study was undertaken to characterize the biofilm architecture of binary biofilms of Listeria monocytogenes and Lactobacillus species and to assess their effect on the survival of Listeria during exposure to hypochlorite. Three L. monocytogenes strains, ATCC 19115 (Lm5), ATCC 19117 (Lm7), and Coleslaw (LmC), were selected and combined individually with three Lactobacillus strains: L. fermentum (Lf), L. bavaricus (Lb), and L. plantarum (Lp). In binary Lm-Lp biofilms, the Lm cell counts were similar to single-species biofilms (8.5 log CFU/well), and the Lp cell numbers declined by 1.0 log CFU/well. In the presence of Lb, the Lm cell counts were reduced by 1.5 log CFU/well (p < 0.05), whereas the Lf cell counts increased at least by 3.5 log CFU/well. Confocal laser scanning microscopy (CLSM) determined that interspecies interactions significantly affected the spatial organization of three binary biofilms. Biofilm surface-to-volume ratio increased from 0.8 μm2/μm3 for Lm5 in the monoculture to 2.1 μm2/μm3 for Lm5-Lp in the dual-species model (p < 0.05), and was characterized by a thicker structure with a largely increased surface area. Biofilm roughness increased from 0.2 for Lm7 to 1.0 for Lm7-Lb biofilms (p < 0.05), which appeared as interspecific segregation. Biofilm thickness increased from 34.2 μm for LmC to 46.3 μm for LmC–Lf (p < 0.05), which produced flat and compact structures that covered the entire surface available. The biomass of the extracellular matrix was higher in the case of some binary biofilms (p < 0.05); however, this effect was dependent upon the species pair. When treated with hypochlorite, Lm5 in binary biofilms had an approximately 1.5 log CFU/well greater survival than individually. The unique spatial organization and greater protein production may explain the protective effect of Lp after hypochlorite exposure.

A New Approach to Detection of the Protective Effect of Escherichia coli on Gram-Positive Bacteria in Binary Biofilms in the Presence of Antibiotics

Binary biofilms containing gram-positive bacteria Micrococcus thailandicus HB or Kocuria rhizophila strain 4A-2G and gram-negative bacteria Escherichia coli strain K-12 or strain ET12567 pRAG56 were obtained. Strain E. coli ET12567 contained a plasmid encoding aminoglycoside phosphotransferase, the enzyme which inactivates kanamycin. A new kinetic approach was developed for selective detection of metabolically active gram-positive bacteria in binary biofilms with E. coli, based on the differences in reduction rate of the electron acceptor, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). It was found that the studied E. coli strains did not exert a protective effect upon the action of antibiotics vancomycin and oxacillin on gram-positive bacteria in the binary biofilms under formation. However, a protective effect was found in the case of preformed binary biofilms: the growth of gram-positive bacteria in binary biofilms was observed at the concentrations of antibiotics oxacillin and kanamycin, which almost completely suppressed growth in the mono-species biofilms of these gram-positive bacteria. The presence of a protective effect was confirmed by several methods (dynamics of MTT reduction, densitometric evaluation of biofilm staining, measurement of the optical density of MTT extracts, and CFU enumeration).

Biofilm Formation by Monocultures and Mixed Cultures of Alcaligenes faecalis 2 and Rhodococcus ruber gt 1

Biofilm formation by monocultures and mixed cultures of nitrile-hydrolyzing bacteria Alcaligenes faecalis 2 and Rhodococcus ruber gt 1 was studied. Biofilm formation was assessed by staining with Crystal violet, by the energy state of the cells, and by the output of the polymer matrix. A quantitative method for assessment of the output of the polymer matrix by the fluorescence of konA-tetramethylrhodamine was developed. The medium used for obtaining mixed biofilms of Al. faecalis 2 and R. ruber gt 1 contained acetamide and glucose as carbon sources. In mixed biofilms Al. faecalis 2 was shown to act as a primary colonizer, while R. ruber gt 1 was a satellite microorganism. Compared to monoculture biofilms, mixed biofilms exhibited higher output of the polymer matrix and increased biofilm formation by Al. faecalis 2 and R. ruber gt 1 on addition of the culture liquid of the second strain. Daily addition of the toxic substrate acetonitrile to planktonic and biofilm cultures resulted in bacterial adaptation, as was indicated by higher numbers of surviving cells compared to the variants with acetonitrile addition every 10 min, mainly due to nitrile hydratase activity of R. ruber gt 1, which transformed acetonitrile into nontoxic acetamide. Our results show that binary biofilms of amidase-containing Al. faecalis 2 and R. ruber gt 1, a strain with high nitrile hydratase activity, are promising as biocatalysts for acrylic acid production and as the basis for a biofilter for nitrile removal from wastewater.

Regulation of Formation of Monospecies and Binary Biofilms by Human Skin Microbiota Components, Staphylococcus epidermidis and Staphylococcus aureus, by Human Natriuretic Peptides

Human natriuretic peptides (NUPs) types A and C were shown have different effects on the growth of monospecies and binary biofilms of human skin commensal microorganisms Staphylococcus epidermidis and Staphylococcus aureus. The effect of natriuretic peptides depended on cultivation conditions: at 37°С growth of monospecies biofilms formed by S. epidermidis and S. aureus was stimulated and inhibited, respectively, resulting in changed values of the biofilm average thickness and biomass in presence of NUPs. At 33°С effects of NUPs reversed: growth of S. epidermidis monospecies biofilms was inhibited. In binary biofilms at 37°С, NUPs were able to increase the competitiveness of S. epidermidis against S. aureus. NUPs affected predominantly biofilms and, to a lesser degree, planktonic cultures. This phenomenon makes it possible to consider NUPs as the molecules able to regulate the interactions between the human organism and skin microbiota.

INFLUENCE OF BACTERIAL DIVERSITY AND INTERSPECIES INTERACTIONS ON DRINKING WATER-ASSOCIATED BIOFILMS OF YЕRSINIA ЕNTEROCOLITICA

The capability of the enteropathogenic Yersinia enterocolitica 8081 bio/serotype 1B/O:8 to form binary and multi-species biofilms with defined bacterial strains was studied. The interspecies interactions in the binary biofilms of the enteropathogen with three bacterial isolates from drinking water and water-associated biofilms were assessed. The effect of each individual partner strain for the Y. enterocolitica involvement in the four-species bacterial biofilm was evaluated by excluding one by one the isolates from the sessile community. It was found out that Y. enterocolitica and the tested bacterial strains interact each other in the binary biofilm formation. Moreover, the Y. enterocolitica involvement in the biofilms depends on the partner strain. In the multi-species biofilms, a synergetic effect of one of the bacterial partner strains on the Y. enterocolitica attachment was detected in contrast to the weak inhibitory effect of another one.

A Novel Binary Biofilm Coinfection Model for the Simultaneous Growth of Gram-Positive and Gram-Negative Bacterial Species

Objectives: The aim of this study was to determine if it is possible to establish and maintain a binary biofilm consisting of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria, which could be used as a multi-species model for antibiotic action studies. Materials and Methods: A medium controlled, continuous culture biofilm model was developed based upon the previously developed Sorbarod™ model. This new model was designed to enable the growth of B. subtilis and E. coli at the same time without either out-competing the other. Results: A pseudo-steady-state binary biofilm was established, which could be maintained for a total of 53 hours. This biofilm was tested to confirm the ability of the biofilm model to support the growth of a Gram-positive (Bacillus subtilis) and a Gram-negative (Escherichia coli) bacterial species under the same conditions of media throughput, aeration and temperature. Conclusions: This paper gives evidence of the ability to develop and control binary biofilm models to maintain the growth of two Gram-dissimilar species of bacteria. We believe this is a novel concept and will aid the future in vitro assessment of antibiotic activity in coinfection models.

Biofilm formation and interspecies interactions in mixed cultures of thermo-acidophilic archaea Acidianus spp. and Sulfolobus metallicus

The understanding of biofilm formation by bioleaching microorganisms is of great importance for influencing mineral dissolution rates and to prevent acid mine drainage (AMD). Thermo-acidophilic archaea such as Acidianus, Sulfolobus and Metallosphaera are of special interest due to their ability to perform leaching at high temperatures, thereby enhancing leaching rates. In this work, leaching experiments and visualization by microscopy of cell attachment and biofilm formation patterns of the crenarchaeotes Sulfolobus metallicus DSM 6482T and the Acidianus isolates DSM 29038 and DSM 29099 in pure and mixed cultures on sulfur or pyrite were studied. Confocal laser scanning microscopy (CLSM) combined with fluorescent dyes as well as fluorescently labeled lectins were used to visualize different components (e.g. DNA, proteins or glycoconjugates) of the aforementioned species. The data indicate that cell attachment and the subsequently formed biofilms were species- and substrate-dependent. Pyrite leaching experiments coupled with pre-colonization and further inoculation with a second species suggest that both species may negatively influence each other during pyrite leaching with respect to initial attachment and pyrite dissolution rates. In addition, the investigation of binary biofilms on pyrite showed that both species were heterogeneously distributed on pyrite surfaces in the form of individual cells or microcolonies. Physical contact between the two species seems to occur, as revealed by specific lectins able to specifically bind single species within mixed cultures.

Biofilm development at low temperatures enhances Listeria monocytogenes resistance to chitosan

The persistence of certain Listeria monocytogenes (Lm) strains in the food industry is a concerning food safety issue. Most of the studies that link persistence with other features, such as biofilm (BF) formation and tolerance to antimicrobials, are conducted at different temperatures from those generally used in food facilities. In this work, two Lm strains, one persistent and one sporadic, plus the reference strain Scott A were cultivated either alone or with Pseudomonas fluorescens (Pf) to form BF at 20 °C or 4 °C. In both systems, the ability of Lm cells to recover after chitosan treatment was evaluated. Structural changes after chitosan exposure and over the revitalization period were analysed using confocal laser scanning microscopy (CLSM). More Lm cells per surface unit were attained in binary BF formed at room temperature, though in those formed under refrigeration, higher tolerance to chitosan was observed. Whereas the persistent Lm strain recovered from chitosan damage at almost the same rate with or without Pf, the presence of this accompanying species was crucial for non-persistent Lm strain recovery, especially at those BF formed at 4 °C. The temperature at which BF were formed, influenced the morphology of the lesions caused by chitosan, as well as the time profile of Lm's viability recovery, suggesting that cold stress increases the tolerance to this antimicrobial, either through structural BF modification, or in some other way. These findings are relevant to the design of realistic BF control strategies in food industry, where most products are handled under refrigerated conditions.

Interspecies Interactions of Metal-Oxidizing Thermo-Acidophilic Archaea &lt;i&gt;Acidianus&lt;/i&gt; and &lt;i&gt;Sulfolobus&lt;/i&gt;

Biofilm formation of microorganisms on relevant surfaces is of great importance for biomining and acid mine drainage (AMD). Thermo-acidophilic archaea like Acidianus, Sulfolobus and Metallosphaera are of special interest due to their ability to enhance leaching rates. Visualization and investigation of microbial attachment and biofilm formation of metal-oxidizing organisms up to now has been done mostly with mesophilic or moderately thermophilic bacteria. In this study, attachment and biofilms by the crenarchaeota Sulfolobus metallicus DSM 6482T and a new isolate Acidianus sp. DSM 29099 on sulfur or pyrite were analyzed. Confocal laser scanning microscopy (CLSM) combined with fluorescent dyes specific for nucleic acids or glycoconjugates were used to monitor biofilm formation on surfaces. The data indicate that cell attachment and the subsequently formed biofilm structures were species and substrate dependent. The investigation of binary biofilms on pyrite showed that both species were heterogeneously distributed on pyrite surfaces in the form of individual cells or microcolonies. In addition, physical contact between the two species was visible, as revealed by specific lectins able to distinguish single species.

Cold Stress and Presence of Pseudomonas fluorescens Affect Listeria monocytogenes Biofilm Structure and Response to Chitosan

Life in biofilms (BF) provides microorganisms with protection against different adverse conditions and agents. In food industry, as they can host and transfer to food both pathogenic and spoilage microorganism, they have to be constantly kept under control. Many hygienic practices and disinfectants aim at preventing and/or destroying BF, and chitosan has a promising future in this respect. Listeria monocytogenes (Lm) is a dangerous foodborne pathogen that can live in BF and survive many restrictive conditions used to preserve foods, such as refrigeration. In this work, nine Lm strains, persistently or sporadically isolated from a meat processing plant, were cultured at 20°C and 4°C to obtain mature BF either in isolation or with Pseudomonas fluorescens (Pf), both species being simultaneously inoculated at similar low population levels. Pf was more compatible with the persistent Lm strains than with the rest, enhancing or maintaining their viable counts in the corresponding dual species BF. All dual species BF formed at 4°C were much thinner than those formed at 20°C, but contained more cells per cm3 of BF biomass. Chitosan damage was observed both as reduction of Lm viable cells and by confocal laser scanning microscopy (CLSM) with Live/Dead stains. In Lm monospecies BF, 1 h chitosan exposure reduced viable counts between 3 and 6 Log when cultured at 20°C and 2-4 Log when at 4°C. Both temperature of BF formation and Lm strain affected their susceptibility to chitosan in dual species BF. CLSM showed focalized chitosan injuries in binary BF, particularly in those with persistent Lm strains.

Microbial corrosion as a manifestation of technogenesis in biofilms formed on surfaces of underground structures

It is established that the place of formation of an aggressive microbial aggregate is a ferrosphere, which is a soil zone with a thickness to 3 mm contacting directly with the surface of an underground structure. In essence, it is a biofilm in which the biogeochemical activity of corrosion-inducing bacteria manifests itself and the development of electrochemical processes is accelerated. It is shown that, in a biofilm, population changes in bacteria of certain physiological groups, namely, sulfate-reducing, denitrifying, and iron-reducing bacteria, occur. Original data of the formation of sulfate-reducing bacteria in a biofilm at the place of contact with the metal of a binary layer in the form pyrotine and pyrite are obtained. We investigate the architectonics of mono- and binary biofilms formed by destructor bacteria of protective coatings. Using the method of confocal laser scanning microscopy, it is established that, in the basal part of the binary biofilm, on the surface of a butyl-rubber layer of the coating, bacteria of the genus Arthrobacter, which accelerate its degradation, are located. An efficient method of protection of materials from microbial damages is the introduction of biocide, which coagulates proteins and oxidizes sulfhydryl groups in structures of proteins, into their composition, which leads to the death of cells of microorganisms.

Role of planktonic and sessile extracellular metabolic byproducts on Pseudomonas aeruginosa and Escherichia coli intra and interspecies relationships

Bacterial species are found primarily as residents of complex surface-associated communities, known as biofilms. Although these structures prevail in nature, bacteria still exist in planktonic lifestyle and differ from those in morphology, physiology, and metabolism. This study aimed to investigate the influence of physiological states of Pseudomonas aeruginosa and Escherichia coli in cell-to-cell interactions. Filtered supernatants obtained under planktonic and biofilm cultures of each single species were supplemented with tryptic soy broth (TSB) and used as the growth media (conditioned media) to planktonic and sessile growth of both single- and two-species cultures. Planktonic bacterial growth was examined through OD(640) measurement. One-day-old biofilms were evaluated in terms of biofilm biomass (CV), respiratory activity (XTT), and CFU number. Conditioned media obtained either in biofilm or in planktonic mode of life triggered a synergistic effect on planktonic growth, mainly for E. coli single cultures growing in P. aeruginosa supernatants. Biofilms grown in the presence of P. aeruginosa biofilms-derived metabolites presented less mass and activity. These events highlight that, when developed in biofilm, P. aeruginosa release signals or metabolites able to prejudice single and binary biofilm growth of others species and of their own species. However, products released by their planktonic counterparts did not impair biofilm growth or activity. E. coli, living as planktonic or sessile cultures, released signals and metabolites or removed un-beneficial compounds which promoted the growth and activity of all the species. Our findings revealed that inter and intraspecies behaviors depend on the involved bacteria and their adopted mode of life.

Resistance of the oil-oxidizing microorganism Dietzia sp. to hyperosmotic shock in reconstituted biofilms

A number of halotolerant and halophilic bacterial strains were isolated from the Romashkinskoe oil field (Tatarstan) stratal waters having a salinity of up to 100 g/l. The isolation of pure cultures involved biofilm reconstitution on M9 medium with paraffins. The associations obtained were dispersed and reinoculated onto solid media that contained either peptone and yeast extract (PY) or paraffins. It was shown that such associations included both oil-oxidizing bacteria and accompanying chemoheterotrophic bacteria incapable of oil oxidation. The pure cultures that were isolated were used for creating binary biofilms. In these biofilms, interactions between halophilic and nonhalophilic bacteria under hypo- and hyperosmotic shocks were investigated. We conducted a detailed study of a biofilm obtained from an oil-oxidizing halotolerant species (with an upper growth limit of 10-12% NaCl) identified as Dietzia sp. and an extremely halophilic gram-negative bacterium (growing within the 5-20% NaCl concentration range) of the genus Chromohalobacter that did not oxidize paraffins. If these microorganisms were grown in a mixed suspension (planktonic) culture that was not supplemented with an additional amount of NaCl, no viable cells of the halophilic microorganism were detected after reinoculation. In contrast, only halophilic cells were detected at a NaCl concentration of 15%. Thus, no mutual protective influence of the microorganisms manifested itself in suspension culture, either under hypo- or under hyperosmotic shock. Neither could the halophile cells be detected after reinoculating a biofilm obtained on a peptone medium without addition of NaCl. However, biofilms produced at a NaCl concentration of 15% contained approximately equal numbers of cells of the halophilic and halotolerant organisms. Thus, the halophile in biofilms sustaining a hyperosmotic shock exerts a protective influence on the halotolerant microorganism. Preliminary data suggest that this effect is due to release by the halophile of osmoprotective substances (ectoine and glutamate), which are taken up by the halotolerant species. Such substances are diluted by a large medium volume in suspension cultures, whereas, in biofilms, their diffusion into the medium is apparently hampered by their interaction with the intercellular polymer matrix.

The role of hydrodynamic stress on the phenotypic characteristics of single and binary biofilms of Pseudomonas fluorescens

This study investigates the phenotype of turbulent (Re = 5,200) and laminar (Re = 2,000) flow-generated Pseudomonas fluorescens biofilms. Three P. fluorescens strains, the type strain ATCC 13525 and two strains isolated from an industrial processing plant, D3-348 and D3-350, were used throughout this study. The isolated strains were used to form single and binary biofilms. The biofilm physiology (metabolic activity, cellular density, mass, extracellular polymeric substances, structural characteristics and outer membrane proteins [OMP] expression) was compared. The results indicate that, for every situation, turbulent flow-generated biofilms were more active (p < 0.05), had more mass per cm(2) (p < 0.05), a higher cellular density (p < 0.05), distinct morphology, similar matrix proteins (p > 0.1) and identical (isolated strains -single and binary biofilms) and higher (type strain) matrix polysaccharides contents (p < 0.05) than laminar flow-generated biofilms. Flow-generated biofilms formed by the type strain revealed a considerably higher cellular density and amount of matrix polysaccharides than single and binary biofilms formed by the isolated strains (p < 0.05). Similar OMP expression was detected for the several single strains and for the binary situation, not dependent on the hydrodynamic conditions. Binary biofilms revealed an equal coexistence of the isolated strains with apparent neutral interactions. In summary, the biofilms formed by the type strain represent, apparently, the worst situation in a context of control. The results obtained clearly illustrate the importance of considering strain variation and hydrodynamics in biofilm development, and complement previous studies which have focused on physical aspects of structural and density differences.

Influence of gentamicin and tobramycin on binary biofilm formation by co-cultures ofBurkholderia cepacia andPseudomonas aeruginosa

The aim of this study was to investigate the stability and dynamics of binary culture biofilm formation following antibiotic treatment. Pseudo steady-state biofilm cultures of clinical strains of P. aeruginosa and B. cepacia concurrently isolated from a single CF patient were established in separate Constant Depth Film Fermenters (CDFFs). Pans, containing established biofilms, were swapped between CDFFs. Biofilms were treated either for 5 days with tobramycin (0.3 mg/ml) prior to pan-swapping, or with gentamicin (1 mg/ml) immediately following pan-swapping. In both instances stable binary biofilms were formed. In addition, fresh un-colonised surfaces added at the time of pan-swapping and exposed simultaneously to biofilm derived P. aeruginosa and B. cepacia and subjected to antibiotic challenge also established stable binary communities. Treatment of P. aeruginosa and B. cepacia with tobramycin or gentamicin, either prior to or immediately after surface colonisation had little effect on the establishment of stable binary biofilms. Such treatment also had little effect on the immigration of one organism into an established biofilm of a second organism.

Interactions in biofilms between Listeria monocytogenes and resident microorganisms from food industry premises

Twenty nine bacterial strains were grown as binary culture biofilms with Listeria monocytogenes to assess their influence on the settlement of the latter on stainless steel coupons. Most of the strains had been isolated from food processing plants after cleaning and disinfection and were tentatively identified by the APILAB Plus 3.3.3 database (bioMérieux). Sixteen of them decreased L. monocytogenes biofilm colony forming units (CFU) counts. Three strains, Bacillus sp. CCL 9 an unidentified Gram-positive strain CCL 59 and Pseudomonas fluorescens E9. 1, led to a 3-log difference in CFU counts between the pure L. monocytogenes biofilms and the mixed biofilms. Eleven strains had no effect and only four, Kocuria varians CCL 73, Staphylococcus capitis CCL 54, Stenotrophomonas maltophilia CCL 47 and Comamonas testosteroni CCL 24, had a positive effect, with a 0.5- to 1.0-log increase in the L. monocytogenes biofilm CFU counts. On its own, L. monocytogenes settled as single cells, but in binary biofilms, different spatial arrangements were observed: (i) with K. varians CCL 73, K. varians CCL 56 and S. capitis CCL 54, L. monocytogenes cells gathered around the microcolonies of the partner strain; (ii) with the two Gram-negative strains, C. testosteroni CCL 24 and CCL 25, L. monocytogenes cells formed its own microcolonies. No link could be found between the exopolysaccharide production capacity of the bacterial strains in pure-culture biofilms and their effect on the L. monocytogenes population in mixed biofilms. With one strain, C. testosteroni CCL 24, adding filter-sterilized supernatant from a pure-culture biofilm to a pure culture of L. monocytogenes increased the number of L. monocytogenes cells adhering to the stainless steel coupons and forming microcolonies. This study suggests that the “house flora” can have a strong effect on the likelihood of finding L. monocytogenes on inert surfaces.