Chip-calorimetry provides real time insights into the inactivation of biofilms by predatory bacteria

Control or removal of undesired biofilms has frequently been found to be quite difficult. In addition to biocidal or antibiotic chemicals or materials designed to prevent biofouling, biological control agents appear to be promising. Reports of bacterial predators eradicating biofilms or eliminating pathogens motivate a more systematic screening of biofilm-eliminating bacterial predators. Unfortunately, the analysis of the eradication process is demanding. In the present study, chip-calorimetry was applied to monitor the elimination of Pseudomonas sp. biofilms by Bdellovibrio bacteriovorus. The method uses metabolic heat as a real-time parameter for biofilm activity. The method is non-invasive, fast and convenient due to real-time data acquisition. In addition, heat-production data can reveal information about the energetics of the predator-prey interaction. The calorimetric results were validated by confocal laser scanning microscopy. The approach described may be useful for the screening of biofilm susceptibility to different predators.     

A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms

Aims: Research on biofilms requires validated quantitative models that focus both on matrix and viable bacterial mass. In this study, a new microplate model for the detection of Staphylococcus aureus biofilms was developed. Methods and Results: Dimethyl methylene blue (DMMB) dye was used to quantify biofilm matrix colorimetrically. Initially developed for the detection of glycosaminoglycans, the DMMB protocol was optimized for S. aureus biofilm research. In addition, the redox indicator resazurin was used to determine the viable bacterial biofilm burden. Conclusion: A new, simple and reproducible microplate test system based on DMMB and resazurin, offering a reliable differentiation between biofilm matrix and cellular activity, was developed and validated for the detection of S. aureus biofilms. Significance and Impact of the Study: The DMMB–resazurin microtitre plate model is a valuable tool for high capacity screening of biocides and for the development of synergistic mixtures of biocides, destroying both biofilm matrix and bacteria.     

Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry

The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study enzyme effects on the composition of extracellular polymeric substances (EPS) and biofilm removal in a Clean-in-Place (CIP) procedure. The screening of 7 proteases and polysaccharidases for removal of biofilms of 16 bacterial species was first evaluated using a microtiter plate assay. The alkaline pH buffer removed more biofilm biomass as well as affecting a larger range of bacterial species. The two serine proteases and α-amylase were the most efficient enzymes. Proteolytic enzymes promoted biofilm removal of a larger range of bacterial species than polysaccharidases. Using three isolates derived from two bacterial species widely found in food processing lines (Pseudomonas fluorescens and the Bacillus cereus group), biofilms were developed on stainless steel slides and enzymatic solutions were used to remove the biofilms using CIP procedure. Serine proteases were more efficient in removing cells of Bacillus biofilms than polysaccharidases. However, polysaccharidases were more efficient in removing P. fluorescens biofilms than serine proteases. Solubilization of enzymes with a buffer containing surfactants, and dispersing and chelating agents enhanced the efficiency of polysaccharidases and proteases respectively in removing biofilms of Bacillus and P. fluorescens. A combination of enzymes targeting several components of EPS, surfactants, dispersing and chelating agents would be an efficient alternative to chemical cleaning agents.     

Real-time in-situ electrochemical monitoring of Pseudomonas aeruginosa biofilms grown on air–liquid interface and its antibiotic susceptibility using a novel dual-chamber microfluidic device

Biofilms are communities of bacterial cells encased in a self-produced polymeric matrix that exhibit high tolerance toward environmental stress. Despite the plethora of research on biofilms, most P. aeruginosa biofilm models are cultured on a solid–liquid interface, and the longitudinal growth characteristics of P. aeruginosa biofilm are unclear. This study demonstrates the real-time and noninvasive monitoring of biofilm growth using a novel dual-chamber microfluidic device integrated with electrochemical detection capabilities to monitor pyocyanin (PYO). The growth of P. aeruginosa biofilms on the air–liquid interface (ALI) was monitored over 48 h, and its antibiotic susceptibility to 6 h exposure of 50, 400, and 1600 µg/ml of ciprofloxacin solutions was analyzed. The biofilm was treated directly on its surface and indirectly from the substratum by delivering the CIP solution to the top or bottom chamber of the microfluidic device. Results showed that P. aeruginosa biofilm developed on ALI produces PYO continuously, with the PYO production rate varying longitudinally and peak production observed between 24 and 30 h. In addition, this current study shows that the amount of PYO produced by the ALI biofilm is proportional to its viable cell numbers, which has not been previously demonstrated. Biofilm treated with ciprofloxacin solution above 400 µg/ml showed significant PYO reduction, with biofilms being killed more effectively when treatment was applied to their surfaces. The electrochemical measurement results have been verified with colony-forming unit count results, and the strong correlation between the PYO electrical signal and the viable cell number highlights the usefulness of this approach for fast and low-cost ALI biofilm study and antimicrobial tests.     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.     

Presence of Extracellular DNA in the <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Matrix and its Contribution to Biofilms

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.     

Formation of biofilms under phage predation: considerations concerning a biofilm increase

Bacteriophages are emerging as strong candidates for combating bacterial biofilms. However, reports indicating that host populations can, in some cases, respond to phage predation by an increase in biofilm formation are of concern. This study investigates whether phage predation can enhance the formation of biofilm and if so, if this phenomenon is governed by the emergence of phage-resistance or by non-evolutionary mechanisms (eg spatial refuge). Single-species biofilms of three bacterial pathogens (Pseudomonas aeruginosa, Salmonella enterica serotype Typhimurium, and Staphylococcus aureus) were pretreated and post-treated with species-specific phages. Some of the phage treatments resulted in an increase in the levels of biofilm of their host. It is proposed that the phenotypic change brought about by acquiring phage resistance is the main reason for the increase in the level of biofilm of P. aeruginosa. For biofilms of S. aureus and S. enterica Typhimurium, although resistance was detected, increased formation of biofilm appeared to be a result of non-evolutionary mechanisms.     

Colorimetric assay for biofilms in wet processing conditions

Controlling bacterial biofilms is necessary for food safety and industrial processing in clean room environments. Our goal was to develop a method to quantitatively measure biofilm produced by pathogens under wet poultry production and processing conditions. Stainless steel and glass coupons were incubated in aqueous media containing reduced nutrients and exposed to Listeria monocytogenes under static temperature and humidity conditions. Samples were measured separately by biofilm assay and viable cell density, and then confirmed by spectrophotometry and microscopy. The biofilm assay resulted in different t groupings from the cell density. The mean from the biofilm assay was 0.50, and the error% was 0.595. The mean of the log₁₀ density (cfu/cm²) was 5.90, and the standard deviation ranged from 0.127 to 0.438 on 24 coupons. The typical sequence of biofilm development, followed by microscopy of biofilm grown on glass coupons, exhibited a change from dispersed single cells to an all-over pattern of clumps with few dispersed cells. L. monocytogenes formed biofilms on all of the substrata tested. Bacterial counts from planktonic cultures at 24, 48, 72, and 144 h confirmed that L. monocytogenes remained viable throughout the experiment and reached equilibrium between 6 and 24 h. The cell density log₁₀/ml was 8.01, 8.03, 7.69, and 6.66, respectively; and the standard deviation ranged from 0.156 to 0.394. The data will be used to grow stable biofilms of Listeria spp. collected from the food processing environment for further study. This is the first use of the crystal violet assay for measurement of bacterial biofilms on stainless steel under these conditions. The methods tested are applicable to other bacteria and substrata.     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Abstract

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

Development of a multispecies periodontal biofilm model within a stirred bioreactor

Abstract

The objective of this work was to develop a subgingival biofilm model using a stirred bioreactor. Discs of bovine teeth were adapted to a stirred bioreactor filled with a culture medium containing bacterial species associated with periodontal health or disease. After anaerobic incubation, the biofilms growing on the substratum surfaces were collected and analyzed. The mean number of Colony-forming Units (CFUs) varied, but with no difference between 3 and 7?days of biofilm formation (p?>?0.05). Scanning Electron Microscopy (SEM) analysis showed a uniform biofilm layer covering the cement layer of the root surface containing bacteria with diverse morphology. In checkerboard DNA-DNA hybridization, bacterial species were identified in both biofilms. In conclusion, a subgingival biofilm model was developed using a stirred bioreactor, allowing the in vitro reproduction of complex microbial communities. This is an advanced model that may be useful to mimic complex clinical periodontal biofilms.     

Diversity of bacteria contaminating paper machines

Formation of microbial biofilms and slimes is a general and serious problem in the operation of paper machines. Studies of microbial populations in paper machine-derived biofilms have been conducted using standard microbiological procedures; however, the bacterial genera present in this type of samples as well as their diversity are quite poorly known. Here, the bacterial diversity of 38 process water and 22 biofilm samples from four different Finnish paper machines were analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA (LH-PCR). In addition, sequencing of the amplified 16S rRNA gene from 69 clones was conducted for characterization of the bacterial genera present in biofilm and slime samples. The LH-PCR profiles of both the free-living (process waters) and immobilized (biofilms) bacteria were diverse at all stages of the papermaking process. Out of the 69 sequenced clones, 44 belonged to alpha-Proteobacteria, most of which were close to the nitrogen-fixing root nodule genera Sinorhizobium, Rhizobium and Azorhizobium. Other clones were assigned to beta- and gamma-Proteobacteria and the phylum Bacteroidetes. In addition, eight of the clones were assigned to a yet uncultivated phylum, TM7. Finally, epifluorescence microscopy revealed that Gram-negative bacteria were predominant in both the biofilm (65%) and process water (54%) samples and a small coccoid cell morphology was most common in all samples. Together, our results show that the analysis of microbial samples from paper machines using modern molecular biology techniques adds valuable information and should, therefore, be useful as a more specific and sensitive microbiological method for the paper industry. This information could further be applied, e.g., in the development of more specific and environmental friendly antimicrobial agents for paper mills.     

Aggregation of ammonia-oxidizing bacteria in microbial biofilm on oyster shell surface

Summary Formation and activity of bacterial nitrifying biofilms play an important role in the closed seawater systems for shrimp cultivation. The structure of microbial biofilm on empty oyster shells, used as a biofilm carrier in biofiltration of aquacultural water, was studied using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy. FISH was performed with specific oligonucleotide probes for Bacteria and ammonia-oxidizing Nitrosomonas spp. The bacterial cells were arranged within the biofilm as a layer of vertically elongated aggregates. Aggregates of ammonia-oxidizing bacteria were embedded within the matrix formed by other bacteria. Vertically elongated cell aggregates may be ecologically important in bacterial biofilms because they have a higher surface-to-volume ratio than that of laminated biofilms.     

Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor‐mediated quorum sensing

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor‐mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis‐2‐decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.     

Cryosectioning of biofilms for microscopic examination

A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.     

The elimination effects of lavender essential oil on Listeria monocytogenes biofilms developed at different temperatures and the induction of VBNC state

Listeria monocytogenes is a typical foodborne pathogen that causes hard-to-treat bacterial infections, mainly due to its ability to form biofilm and enter into a viable but non-culturable state (VBNC). In this study, we investigated the removal effects of four antimicrobial agents on L. monocytogenes biofilms formed at 32°C and 10°C, analysed the resistances of the mature biofilms to antimicrobial agents, and explored the VBNC state of cells in mature biofilms induced by lavender essential oil (LEO). The results showed that the growth of L. monocytogenes was completely inhibited when 1·6% (v/v) of the LEO was added. Meanwhile, the results of the crystal violet staining and XTT reduction method indicated that different concentrations of LEO significantly reduced L. monocytogenes biofilms biomass and metabolic activities, followed by sodium hypochlorite, lactic acid, and hydrogen peroxide. Moreover, the confocal laser scanning microscopy (CLSM) images confirmed that the treated biofilms became thinner, the structure was sparse, and the appearance was blurry. More interestingly, L. monocytogenes biofilms developed at 10°C were less susceptible to the sanitizers than those formed at 32°C. In addition, LEO presented a more significant dispersing effect on the biofilm cells, and 1/2 MIC to 4 MIC of LEO could induce fewer VBNC state cells in biofilm and plankton compared with sodium hypochlorite. This study indicated that the LEO could be considered as an ideal antibiofilm agent for controlling L. monocytogenes. But we should pay attention to the resistance of the biofilms developed at low temperatures.     

Determination of biofilm mechanical properties from tensile tests performed using a micro-cantilever method

Recently, a micro-cantilever method was introduced for measuring the ultimate tensile strength of intact bacterial biofilms. Herein, is reported the analysis of the video files from the testing of a 4-day-old Staphylococcus epidermidis biofilm to determine the elastic modulus, toughness, and failure strain. Elastic modulus (1270±280 Pa) was within the range of previously reported values (17–6000 Pa). The high failure strains (240±16%) indicate the substantial ductility of bacterial biofilms. In addition, the toughness of the biofilm sample was determined from the area under the stress–strain plot (2.8±0.44 kJ m^?3). Thus, it was demonstrated that the micro-cantilever test video files can be used for the determination of other mechanical property parameters besides ultimate tensile strength.     

Characterization of polymicrobial biofilms obtained from saliva or carious lesions in dentin

Abstract

This study aimed to compare the formation of polymicrobial biofilms using carious dentin or saliva as inoculum for application in in vitro microbiological studies on caries research. For biofilm growth, combined samples of infected dentin or saliva from three donors were used. The biofilms were grown on glass coverslips, under a regimen of intermittent exposure (6?h day^?1) to 1% sucrose for 4?days. Total bacterial loads, as well as specific aciduric bacteria and mutans streptococci loads were quantified and correlated with biofilm acidogenicity and susceptibility to chlorhexidine. The data were evaluated using the Student’s-t, Mann Whitney and Kruskal-Wallis tests. The two biofilms showed similar microbial loads (total bacteria, aciduric bacteria and mutans streptococci) on day 4, and high acidogenicity after 48?h and were susceptible to chlorhexidine at different time intervals. In conclusion, both dentin and saliva can be used as an inoculum in in vitro studies of processes related to biofilm formation.     

A refined technique for extraction of extracellular matrices from bacterial biofilms and its applicability

Biofilm‐forming bacteria embedded in polymeric extracellular matrices (ECMs) that consist of polysaccharides, proteins and/or extracellular DNAs (eDNAs) acquire high resistance to antimicrobial agents and host immune systems. To understand molecular mechanisms of biofilm formation and maintenance and to develop therapeutic countermeasures against chronic biofilm‐associated infections, reliable methods to isolate ECMs are inevitable. In this study, we refined the ECM extraction method recently reported and evaluated its applicability. Using three Staphylococcus aureus biofilms in which proteins, polysaccharides or eDNAs are major contributors to their integrity, ECMs were extracted using salts and detergents. We found that extraction with 1.5 M sodium chloride (NaCl) could be optimum for not only ECM proteins but also polysaccharides and eDNAs. In addition, long‐time incubation was not necessary for efficient ECM isolation. Lithium chloride (LiCl) was comparative to NaCl but is more expensive. In contrast to SDS, NaCl hardly caused leakage of intracellular proteins and did not affect viability of bacterial cells within biofilms. Furthermore, this method is applicable to other bacteria such as Gram‐positive Staphylococcus epidermidis and Gram‐negative Escherichia coli and Pseudomonas aeruginosa. Thus, this refined method is very simple, rapid, low cost and non‐invasive and could be used for a broad range of applications.     

An image-based 384-well high-throughput screening method for the discovery of biofilm inhibitors in Vibrio cholerae

Bacterial biofilms are assemblages of bacterial cells and extracellular matrix that result in the creation of surface-associated macrocolony formation. Most bacteria are capable of forming biofilms under suitable conditions. Biofilm formation by pathogenic bacteria on medical implant devices has been linked to implant rejection in up to 10% of cases, due to biofilm-related secondary infections. In addition, biofilm formation has been implicated in both bacterial persistence and antibiotic resistance. In this study, a method has been developed for the discovery of small molecule inhibitors of biofilm formation in Vibrio cholerae, through the use of high-throughput epifluorescence microscopy imaging. Adaptation of a strategy for the growth of bacterial biofilms in wellplates, and the subsequent quantification of biofilm coverage within these wells, provides the first example of an image-based 384-well format system for the evaluation of biofilm inhibition in V. cholerae. Application of this method to the high-throughput screening of small molecule libraries has lead to the discovery of 29 biofilm lead structures, many of which eliminate biofilm formation without altering bacterial cell viability.     

Modulation of <Emphasis Type="Italic">Escherichia coli</Emphasis> biofilm growth by cell-free spent cultures from lactobacilli

E. coli biofilms cause serious problems in medical practice by contaminating surfaces and indwelling catheters. Due to the rapid development of antibiotic resistance, alternative approaches to biofilm suppression are needed. This study addresses whether products released by antagonistic bacteria — Lactobacillus isolates from vaginal and dairy-product samples could be useful for controlling E. coli biofilms. The effects of diluted cell-free supernatants (CFS) from late-exponential Lactobacillus cultures on the growth and biofilm production of Escherichia coli were tested. Most of the CFS applied as 10⁻² had no impact on bacterial growth, biofilm development however was influenced even by 10⁻⁴ of CFS. Initial screening by crystal violet assay showed that biofilm modulation varied between different CFS and E. coli combinations from inhibition to activation; however three of the tested CFS showed consistency in biofilm suppression. This was not due to antibacterial activity since Live/Dead fluorescence labeling showed insignificant differences in the amount of dead cells in control and treated samples. Some E. coli strain-specific mechanisms of response to the three CFS included reduction in hydrophobicity and motility. Released exoploysaccharides isolated from the three CFS stimulated sessile growth, but proteinase K reduced their inhibitory activities implying participation of protein or peptide biofilm suppression factor(s).