Resident Biofilm Research Articles

Comparative analysis of commercial cleaning and disinfection formulations and protocols for effective eradication of biofilms formed by a Pseudomonas fluorescens strain isolated from a poultry meat plant

Pseudomonas spp. is the main genus of spoilage bacteria for meat stored under aerobic chilling conditions. Given their ability to adapt to various environmental conditions and to form biofilms, they are common among residential microbiota in the food industry. Biofilms are complex, structured microbial communities encased in a self-produced exopolysaccharide matrix. Resident biofilm bacteria show increased resistance against disinfectants and dynamic or hostile environments, and are therefore a persistent source of food contamination. Hence, there is a need to design and implement control strategies that are effective against biofilms in the food industry. In this study, we tested a series of commercial cleaning and disinfection agents applied by immersion or aerosolization against robust biofilms of a Pseudomonas fluorescens strain isolated from a poultry meat plant by comparing the sessile cell counts before and after the procedure. The single application of a) an alkaline or an enzymatic detergent, b) a disinfectant based on peracetic acid and hydrogen peroxide, or c) a detergent-disinfectant based on tertiary alkylamines or sodium hypochlorite, failed to completely eradicate the biofilm. However, two-step cleaning and disinfection procedures involving the application of a detergent (either alkaline or enzymatic at 50 °C) followed by the application of the disinfectant based on peracetic acid and hydrogen peroxide at the manufacturer's recommended concentrations did result in the complete eradication (p ≤ 0.05) of the robust biofilm. In addition, cleaning and disinfection procedures applied by aerosolization showed a notable efficacy (p ≤ 0.05) against surface-dried cells and sessile cells of P. fluorescens. Therefore, this approach could be regarded as a potentially viable alternative to commonly applied immersion techniques.

The Role of Living, Resident Bacterial Biofilm Populations in the Osseointegration of Oral Implants

The Role of Living, Resident Bacterial Biofilm Populations in the Osseointegration of Oral Implants

Matrix-trapped viruses can prevent invasion of bacterial biofilms by colonizing cells.

Bacteriophages can be trapped in the matrix of bacterial biofilms, such that the cells inside them are protected. It is not known whether these phages are still infectious and whether they pose a threat to newly arriving bacteria. Here, we address these questions using Escherichia coli and its lytic phage T7. Prior work has demonstrated that T7 phages are bound in the outermost curli polymer layers of the E. coli biofilm matrix. We show that these phages do remain viable and can kill colonizing cells that are T7-susceptible. If cells colonize a resident biofilm before phages do, we find that they can still be killed by phage exposure if it occurs soon thereafter. However, if colonizing cells are present on the biofilm long enough before phage exposure, they gain phage protection via envelopment within curli-producing clusters of the resident biofilm cells.

The role of inflammation and genetics in periodontal disease.

Periodontitis is a complex disease: (a) various causative factors play a role simultaneously and interact with each other; and (b) the disease is episodic in nature, and bursts of disease activity can be recognized, ie, the disease develops and cycles in a nonlinear fashion. We recognize that various causative factors determine the immune blueprint and, consequently, the immune fitness of a subject. Normally, the host lives in a state of homeostasis or symbiosis with the oral microbiome; however, disturbances in homeostatic balance can occur, because of an aberrant host response (inherited and/or acquired during life). This imbalance results from hyper‐ or hyporesponsiveness and/or lack of sufficient resolution of inflammation, which in turn is responsible for much of the disease destruction seen in periodontitis. The control of this destruction by anti‐inflammatory processes and proresolution processes limits the destruction to the tissues surrounding the teeth. The local inflammatory processes can also become systemic, which in turn affect organs such as the heart. Gingival inflammation also elicits changes in the ecology of the subgingival environment providing optimal conditions for the outgrowth of gram‐negative, anaerobic species, which become pathobionts and can propagate periodontal inflammation and can further negatively impact immune fitness. The factors that determine immune fitness are often the same factors that determine the response to the resident biofilm, and are clustered as follows: (a) genetic and epigenetic factors; (b) lifestyle factors, such as smoking, diet, and psychosocial conditions; (c) comorbidities, such as diabetes; and (d) local and dental factors, as well as randomly determined factors (stochasticity). Of critical importance are the pathobionts in a dysbiotic biofilm that drive the viscious cycle. Focusing on genetic factors, currently variants in at least 65 genes have been suggested as being associated with periodontitis based on genome‐wide association studies and candidate gene case control studies. These studies have found pleiotropy between periodontitis and cardiovascular diseases. Most of these studies point to potential pathways in the pathogenesis of periodontal disease. Also, most contribute to a small portion of the total risk profile of periodontitis, often limited to specific racial and ethnic groups. To date, 4 genetic loci are shared between atherosclerotic cardiovascular diseases and periodontitis, ie, CDKN2B‐AS1(ANRIL), a conserved noncoding element within CAMTA1 upstream of VAMP3, PLG, and a haplotype block at the VAMP 8 locus. The shared genes suggest that periodontitis is not causally related to atherosclerotic diseases, but rather both conditions are sequelae of similar (the same?) aberrant inflammatory pathways. In addition to variations in genomic sequences, epigenetic modifications of DNA can affect the genetic blueprint of the host responses. This emerging field will yield new valuable information about susceptibility to periodontitis and subsequent persisting inflammatory reactions in periodontitis. Further studies are required to verify and expand our knowledge base before final cause and effect conclusions about the role of inflammation and genetic factors in periodontitis can be made.

Does the primary deposit affect the biogeochemical transformation of placer gold and associated biofilms?

Placer gold particles derived from epithermal deposits display distinct morphological and compositional features compared with particles from mesothermal systems. Here, it is hypothesized that the chemical composition of primary gold derived from different deposit types is a principle factor affecting the composition of resident biofilm communities as well as the transformation of placer particles. Gold particles were collected from placers originating from the epithermal system at the Eisenberg, Germany's largest primary gold deposit. For comparison, placer gold from mesothermal sources was studied. Morphological differences due to mechanical transport and physical reshaping were not observed. Biogeochemical gold/silver dissolution and gold re-precipitation were evident on epithermal particles and they accentuate the silver-fabrics and gold-rich clusters. In contrast, on mesothermally derived gold particles these processes led to the development of gold-rich rims via the formation of nano-porous secondary gold. Microprobe- and laser ablation mass spectrometric analyses of polished whole particle mounts confirmed differences in gold/silver content/distribution and trace metal content between particles from epi- and mesothermal sources, respectively. On particles from all sites nano- and micro-particulate gold is associated with polymorphic layers. These are composed of microbial cells, extracellular polymeric substances (EPS) and clay-sized minerals. Multivariate statistical analyses shows a significant difference between biofilm communities from epi- vs. mesothermally derived particles, which is linked to the chemical composition of the primary gold. While a number of key-species capable of gold transformation, e.g., Cupriavidus sp., Geobacter sp. and Rhodoferax sp., were detected on particles from both sources, higher numbers of organisms with the potential for gold solubilization, precipitation and detoxification were associated with particles from the epithermal sources. A range of species involved in gold transformation, i.e., Arthrobacter spp., Delftia sp., Shewanella sp., and Stenotrophomonas spp., were detected only on epithermally derived placer gold. This indicates the communities are sensitive to differences in gold/silver and possibly trace metal-cycling, resulting from differences in their content, distribution and mobilization behaviour in epi- vs. mesothermally derived particles. Ultimately, this study shows that the chemical composition of the primary deposit strongly influences the biogeochemical transformation of placer gold and the composition of associated biofilms, whereas physical transformations appear to be largely unaffected.

Pyruvate-depleting conditions induce biofilm dispersion and enhance the efficacy of antibiotics in killing biofilms in vitro and in vivo

The formation of biofilms is a developmental process initiated by planktonic cells transitioning to the surface, which comes full circle when cells disperse from the biofilm and transition to the planktonic mode of growth. Considering that pyruvate has been previously demonstrated to be required for the formation of P. aeruginosa biofilms, we asked whether pyruvate likewise contributes to the maintenance of the biofilm structure, with depletion of pyruvate resulting in dispersion. Here, we demonstrate that the enzymatic depletion of pyruvate coincided with the dispersion of established biofilms by S. aureus and laboratory and clinical P. aeruginosa isolates. The dispersion response was dependent on pyruvate fermentation pathway components but independent of proteins previously described to contribute to P. aeruginosa biofilm dispersion. Using porcine second-degree burn wounds infected with P. aeruginosa biofilm cells, we furthermore demonstrated that pyruvate depletion resulted in a reduction of biofilm biomass in vivo. Pyruvate-depleting conditions enhanced the efficacy of tobramycin killing of the resident wound biofilms by up to 5-logs. Our findings strongly suggest the management of pyruvate availability to be a promising strategy to combat biofilm-related infections by two principal pathogens associated with wound and cystic fibrosis lung infections.

Biofilm recruitment under nanofiltration conditions: the influence of resident biofilm structural parameters on planktonic cell invasion.

SummaryIt is now generally accepted that biofouling is inevitable in pressure‐driven membrane processes for water purification. A large number of published articles describe the development of novel membranes in an effort to address biofouling in such systems. It is reasonable to assume that such membranes, even those with antimicrobial properties, when applied in industrial‐scale systems will experience some degree of biofouling. In such a scenario, an understanding of the fate of planktonic cells, such as those entering with the feed water, has important implications with respect to contact killing particularly for membranes with antimicrobial properties. This study thus sought to investigate the fate of planktonic cells in a model nanofiltration biofouling system. Here, the interaction between auto‐fluorescent Pseudomonas putida planktonic cells and 7‐day‐old Pseudomonas fluorescens resident biofilms was studied under permeate flux conditions in a nanofiltration cross flow system. We demonstrate that biofilm cell recruitment during nanofiltration is affected by distinctive biofilm structural parameters such as biofilm depth.

Aminobacter sp. MSH1 invades sand filter community biofilms while retaining 2,6-dichlorobenzamide degradation functionality under C- and N-limiting conditions.

Aminobacter sp. MSH1 is of interest for bioaugmentation of biofiltration units in drinking water treatment plants (DWTPs) due to its ability to degrade the groundwater micropollutant 2,6-dichlorobenzamide (BAM). Using a continuous flow chamber biofilm model, MSH1 was previously shown to colonize surfaces and degrade BAM at trace concentrations as low as 1 μg/L under the oligotrophic conditions found in DWTPs. In DWTP filtration units, MSH1 has to compete with the resident biofilm microbiota for space and nutrients. Using the same model, we examined how a sand filter community (SFC) affects MSH1's BAM-degrading activity and biofilm formation under C- and N-limiting conditions when fed with trace concentrations of BAM. MSH1 was inoculated simultaneously with the SFC (co-colonization mode) or after the SFC formed a biofilm (invasion mode). MSH1 successfully established in the SFC biofilm showing growth and activity. In co-colonization mode, MSH1 decreased in number in the presence of the SFC and formed isolated colonies, while specific BAM-degradation activity increased. In the invasion mode, MSH1 also decreased in numbers in the presence of the SFC but formed mixed colonies, while specific BAM degradation was unaffected. Our results show that MSH1 invades and performs successfully in an SFC biofilm under the oligotrophic conditions of DWTPs.

Evaluating sulphate removal and identifying the bacterial community present in acid mine drainage treated with synthetic domestic wastewater sludge

Domestic wastewater sludge can serve as a carbon source in the passive biotic treatment of acid mine drainage (AMD) in microbial bioreactors to create anaerobic conditions for the removal of sulphate, chemical oxygen demand (COD) and pH neutralization. A synthetic medium simulating domestic wastewater sludge was used in AMD treatment in a ratio of 1:1 AMD: synthetic domestic wastewater sludge (SDWWS). Sulphate and COD removal were determined at different incubation temperatures and with and without a biofilm in the bioreactors. Sulphate and COD were removed by 60.8% and 96% within 26 d, after which a plateau was reached. Bacterial community analyses using next generation sequencing showed that Chlorobium spp. dominated at a relative percentage of 36% followed by Magnetospirillum spp. and Ornithobacterium spp. The effect of a resident biofilm in the bioreactors showed dominance of Chlorobium spp. at a relative percentage of 62% and removal of sulphates and COD at 96% and 58%, respectively, after 26 d. Incubation at 17–19°C reduced sulphates by only 10% and COD by 12% after 17 d, after which a plateau was reached. Magnetospirillum spp. was the dominate organism at the end of this trial. Keywords : acid mine drainage, domestic wastewater sludge, sulphates, COD, bacterial community

PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix.

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.

Invasibility of resident biofilms by allochthonous communities in bioreactors

Invasion of non-native species can drastically affect the community composition and diversity of engineered and natural ecosystems, biofilms included. In this study, a molecular community fingerprinting method was used to monitor the putative establishment and colonization of allochthonous consortia in resident multi-species biofilms. To do this, biofilms inoculated with tap water or activated sludge were grown for 10 days in bubble column reactors W1 and W2, and S, respectively, before being exposed to non-native microbial consortia. These consortia consisted of fresh activated sludge suspensions for the biofilms inoculated with tap water (reactors W1 and W2) and of transplanted mature tap water biofilm for the activated sludge biofilm (reactor S). The introduction of virgin, unoccupied coupons into W1 and W2 enabled us to additionally investigate the competition for new resources (space) among the resident biofilm and the allochthonous consortia. CE-SSCP revealed that after the invasion event changes were mostly observed in the abundance of the dominant species in the native biofilms rather than their composition. This suggests that the resident communities within a bioreactor immediately outcompete the allochthonous microbes and shape the microbial community assemblage on both new coupons and already colonized surfaces for the short term. However, with time, latent members of the allochthonous community might grow up affecting the diversity and composition of the original biofilms.

Extracellular matrix structure governs invasion resistance in bacterial biofilms.

Many bacteria are highly adapted for life in communities, or biofilms. A defining feature of biofilms is the production of extracellular matrix that binds cells together. The biofilm matrix provides numerous fitness benefits, including protection from environmental stresses and enhanced nutrient availability. Here we investigate defense against biofilm invasion using the model bacterium Vibrio cholerae. We demonstrate that immotile cells, including those identical to the biofilm resident strain, are completely excluded from entry into resident biofilms. Motile cells can colonize and grow on the biofilm exterior, but are readily removed by shear forces. Protection from invasion into the biofilm interior is mediated by the secreted protein RbmA, which binds mother-daughter cell pairs to each other and to polysaccharide components of the matrix. RbmA, and the invasion protection it confers, strongly localize to the cell lineages that produce it.

Effects of physical disturbance on phosphorus uptake in temperate stream biofilms

Microbial biofilm nutrient uptake kinetics can provide insight into assimilative mechanisms that regulate stream primary productivity. While kinetic experiments are often performed, little work has estimated uptake in connection with disturbance (removal) or detailed the effects that common scour events may have on benthic microbes; therefore, the goal of this study was to evaluate physical disturbances on benthic stream biofilms to determine effects on phosphorus (P) uptake rate, physiological capability, and abiotic sorption. Artificial substrata were collected from 2 reaches along a temperate stream; resident biofilms were either removed via abrasion (disturbed) or left intact. A series of short-term radiotracer (H333PO4) experiments were then conducted to measure P uptake. In vivo autofluorescence was measured as a proxy of algal physiological condition. The experiments showed no difference in P-uptake rates (μgP/μgChl/d) between disturbed (x̄ = 0.77 ± 0.11 [SE] μgP/μgChl/d) and intact (x̄ = 0.91 ± 0.17 μgP/μgChl/d) biofilms (t = 0.69, p = 0.492, df = 33). Further, microbial physiology was not depressed by physical disturbance. While killed samples yielded significantly lower uptake compared to live biota (F = 17.51, p = 0.001), abiotic sorption still accounted for a moderate fraction (range = 0.021–0.038 μgP/μgChl/d) of total uptake and thus warrants estimation in metabolic studies. Overall, these findings lend credence to numerous experiments that investigate benthic microbial physiologic responses post-disturbance and highlight the importance of uptake following common physical disturbances that occur in turbulent environments. In addition, our work measured several kinetic constants across both disturbance and temporal gradients, and we discuss their significance within a physiological framework.

Self-produced exopolysaccharide is a signal that stimulates biofilm formation inPseudomonas aeruginosa

Bacteria have a tendency to attach to surfaces and grow as structured communities called biofilms. Chronic biofilm infections are a problem because they tend to resist antibiotic treatment and are difficult to eradicate. Bacterial biofilms have an extracellular matrix that is usually composed of a mixture of polysaccharides, proteins, and nucleic acids. This matrix has long been assumed to play a passive structural and protective role for resident biofilm cells. Here we show that this view is an oversimplification and that the biofilm matrix can play an active role in stimulating its own synthesis. Working with the model biofilm bacterium Pseudomonas aeruginosa, we found that Psl, a major biofilm matrix polysaccharide for this species, acts as a signal to stimulate two diguanylate cyclases, SiaD and SadC, to produce the intracellular secondary messenger molecule c-di-GMP. Elevated intracellular concentrations of c-di-GMP then lead to the increased production of Psl and other components of the biofilm. This mechanism represents a unique positive feedback regulatory circuit, where the expression of an extracellular polysaccharide promotes biofilm growth in a manner analogous to autocrine signaling in eukaryotes.

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.

Growth dependence of conjugation explains limited plasmid invasion in biofilms: an individual‐based modelling study

Plasmid invasion in biofilms is often surprisingly limited in spite of the close contact of cells in a biofilm. We hypothesized that this poor plasmid spread into deeper biofilm layers is caused by a dependence of conjugation on the growth rate (relative to the maximum growth rate) of the donor. By extending an individual-based model of microbial growth and interactions to include the dynamics of plasmid carriage and transfer by individual cells, we were able to conduct in silico tests of this and other hypotheses on the dynamics of conjugal plasmid transfer in biofilms. For a generic model plasmid, we find that invasion of a resident biofilm is indeed limited when plasmid transfer depends on growth, but not so in the absence of growth dependence. Using sensitivity analysis we also find that parameters related to timing (i.e. a lag before the transconjugant can transfer, transfer proficiency and scan speed) and spatial reach (EPS yield, conjugal pilus length) are more important for successful plasmid invasion than the recipients' growth rate or the probability of segregational loss. While this study identifies one factor that can limit plasmid invasion in biofilms, the new individual-based framework introduced in this work is a powerful tool that enables one to test additional hypotheses on the spread and role of plasmids in microbial biofilms.

Inhibition of Listeria monocytogenes by resident biofilms present on wooden shelves used for cheese ripening

The purpose of the present study was to characterize the development of Listeria monocytogenes on wooden shelves used for cheese ripening. The fate of two L. monocytogenes strains was analysed over time as a function of the presence of a native biofilm, the farmhouse origin of cheeses, and the wooden shelves properties. In presence of a native microbial flora on the shelves, deposited populations of L. monocytogenes remained stable or even decreased by up to 2 log10(CFU/cm2) after 12 days of incubation at 15 °C in all tested conditions. By contrast, L. monocytogenes populations increased by up to 4 log10(CFU/cm2) when the resident biofilm was thermally inactivated, suggesting a microbial origin of the observed inhibitory effect. All together, our results suggest that the biocontrol of pathogens multiplication on wooden shelves by resident biofilms should be considered for the microbiological safety of traditional ripened cheeses.

Genetic Features of Resident Biofilms Determine Attachment of Listeria monocytogenes

Planktonic Listeria monocytogenes cells in food-processing environments tend most frequently to adhere to solid surfaces. Under these conditions, they are likely to encounter resident biofilms rather than a raw solid surface. Although metabolic interactions between L. monocytogenes and resident microflora have been widely studied, little is known about the biofilm properties that influence the initial fixation of L. monocytogenes to the biofilm interface. To study these properties, we created a set of model resident Lactococcus lactis biofilms with various architectures, types of matrices, and individual cell surface properties. This was achieved using cell wall mutants that affect bacterial chain formation, exopolysaccharide (EPS) synthesis and surface hydrophobicity. The dynamics of the formation of these biofilm structures were analyzed in flow cell chambers using in situ time course confocal laser scanning microscopy imaging. All the L. lactis biofilms tested reduced the initial immobilization of L. monocytogenes compared to the glass substratum of the flow cell. Significant differences were seen in L. monocytogenes settlement as a function of the genetic background of resident lactococcal biofilm cells. In particular, biofilms of the L. lactis chain-forming mutant resulted in a marked increase in L. monocytogenes settlement, while biofilms of the EPS-secreting mutant efficiently prevented pathogen fixation. These results offer new insights into the role of resident biofilms in governing the settlement of pathogens on food chain surfaces and could be of relevance in the field of food safety controls.

Host–parasite interface and preventive dentistry

Current understanding of the pathogenic etiology of supra- and subgingival plaque assumes dynamic changes of pathogenic potential depending upon the crosstalk among resident biofilm, as well as that among microbial communities and host tissues, called the ecological plaque hypothesis. Manipulation of bacterial and environmental factors, which affect the proportion and virulence potential, has potential for the control of the pathogenicity of biofilm. Most periodontal pathogens, except Actinobacillus actinomycetemcomitans, are oxygen-sensitive, proteolytic species and are thus unable to survive inside the plaque, which is sufficiently thin to allow oxygen exposure, and also lacks a nutritional supply of protein and some other essential factors. A preventive strategy against these pathogenic microbes would be an ecological approach to maintain health-compatible microflora on the surface of teeth. On the other hand, the strategy against A. acinomycetemcomitans, which is the microbe to which Koch's postulate is most applicable, would be employed in an approach to eliminate this pathogen from the microbial community by use of antibiotics or the humoral immune response of the host. For accomplishing both strategies, we discuss the host–parasite interface, not only the interaction but also the ecological and structural factors, for the purpose of preventing the onset and progression of periodontal diseases.

Resident parking only: rhamnolipids maintain fluid channels in biofilms.

Resident parking only: rhamnolipids maintain fluid channels in biofilms.