Biofilm Populations Research Articles

Diabetic wound infection: A review on microbial population and infection control

A diabetic foot ulcer is one of the major complications of diabetes and it leads to lower extremity amputation in patients. This review explores the current research on microbial populations on diabetic wounds, and also treatment alternatives to combat the infection on chronic diabetic wounds. The microbial communities exist in diabetic wound infection are diverse. Microbes rarely survived in single species of planktonic cells. They usually exist in a complex polymicrobial biofilm population which consists of different types of microorganisms. Furthermore, the development of bacterial biofilm on the wound that usually consists of multidrug-resistant pathogens also delayed the wound healing. To overcome this problem, many types of modern wound dressing were developed including hydrocolloid, hydrogel, alginate and collagen wound dressing. Besides, modern biotechnological advancements such as cell therapy, bioengineered skin, dermal scaffolds, tissue-engineered artificial skin and growth factors were also employed to promote the recovery of the wound. In conclusion, diabetes mellitus is a major health care challenge worldwide. Diabetic patients are at risk for developing foot ulcer which ultimately leads to amputation; hence a safe and effective alternative treatment is required to improve diabetic patients’ quality life.

Detection of Biofilm Formation of Klebsiella Pneumoniae Isolated from Medical Devices at the University Hospital of Tlemcen, Algeria

Background: Klebsiella pneumoniae is a major cause of community-acquired and nosocomial infections. This germ is responsible for acute and chronic infections, most of which are due to its ability to adhere to medical implants and form a biofilm. The objective of this work is to study the interaction between clinical isolates of K. pneumoniae and abiotic surfaces (medical devices) and some factors influencing biofilm formation. Methods: Over a period of 2 years, 115 strains of K. pneumoniae were isolated from medical devices CHU Tlemcen, most of which had a high level of resistance to cephalosporins 1st, 2nd, and 3rd generation. Their capacity to form biofilm was assessed using two 3 techniques: TCP, TP, and RCA. We determined in vitro the effects of three antimicrobial agents against planktonic and biofilm forms of K. pneumoniae. The presence of MrkD genes was detected by polymerase chain reaction (PCR). Results: According to the studied (TCP, TP, RCA) strains of K .pneumoniae isolated from urinary catheters have proved very good, forming the biofilm to those isolated from other medical devices. 24 of 115 isolated strains showed a clear difference in antibiotic susceptibility between planktonic populations and biofilm populations. They were 10-20 times higher. All strains presented a highly hydrophilic character and adhesion 2-10 times greater in PVC with respect to glass support. The MrkD gene (detected by PCR) responsible for biofilm formation was found in 22 strains of K. pneumoniae, which may explain their adhesion and therefore their pathogenicity. Conclusion: Our results show the great ability of K.pneumoniae strains to form a biofilm on medical devices, and the isolates were at least 10 times more resistant than their planktonic counterparts. In addition, we showed that the presence of type 3-encoding gene mrkD was associated with high adhesion indexes.

Thermal and non-thermal treatment effects on Staphylococcus aureus biofilms formed at different temperatures and maturation periods

The objective of this study was to investigate the effect of temperature and maturation period on the resistance of Staphylococcus aureus biofilms to thermal and non-thermal treatments. First, biofilm development was compared at three different temperatures (15, 25, and 37°C) for 5 days. The cell population at 15 and 25°C remained relatively consistent approximately at 6.3 log CFU/cm2, whereas 37°C resulted in the highest cell population on day 1 (7.6 log CFU/cm2) followed by a continual decline. Then, biofilm resistance to steam and sodium hypochlorite (NaOCl) treatments was evaluated. Obtained results highlighted that biofilms had different resistance to both treatments depending on development conditions. Specifically, steam treatment of 10 s eliminated 4.1 log CFU/cm2 of the biofilm formed at 25°C for 5 days. The same treatment inactivated over 5 log population of biofilms developed in other temperature and maturation period conditions. Treatment with NaOCl reduced approximately 1 log CFU/cm2 of biofilm cells developed at 25°C for 5 days. However, inactivation was found to be over 2 log CFU/cm2 under other development conditions. An extracellular polymeric substances (EPS) quantification using 96-well plates and stainless steel coupons was conducted. In the 96-well plate experiment, it was found that the highest amount of polysaccharide was secreted at 25°C (p < 0.05), while total biomass and protein contents were greatest at 37°C (p < 0.05). No significant difference in EPS content was observed for stainless steel, but the results displayed a similar trend to the 96-well plate. In particular, biofilms developed at 25°C tended to secret the highest amount of polysaccharide, which aligned with the current literature. This finding indicated that polysaccharide was the main contribution to the enhanced resistance of S. aureus biofilms. Overall, it was shown that biofilms formed at 25°C for 5 days exhibited the greatest resistance to thermal and nonthermal treatments due to the elevated exopolysaccharide secretion. This study demonstrates that temperature and maturation period significantly affect the resistance of S. aureus biofilms to thermal and non-thermal treatments.

Real-time monitoring of bacterial biofilms metabolic activity by a redox-reactive nanosensors array

BackgroundBacterial biofilms are communities of surface-associated microorganisms living in cellular clusters or micro-colonies, encapsulated in a complex matrix composed of an extracellular polymeric substance, separated by open water channels that act as a circulatory system that enable better diffusion of nutrients and easier removal of metabolic waste products. The monitoring of biofilms can provide important information on fundamental biofilm-related processes. That information can shed light on the bacterial processes and enable scientists to find ways of preventing future bacterial infections. Various approaches in use for biofilm analysis are based on microscopic, spectrochemical, electrochemical, and piezoelectrical methods. All these methods provide significant progress in understanding the bio-process related to biofilm formation and eradication, nevertheless, the development of novel approaches for the real-time monitoring of biochemical, in particular metabolic activity, of bacterial species during the formation, life and eradication of biofilms is of great potential importance.ResultsHere, detection and monitoring of the metabolic activity of bacterial biofilms in high-ionic-strength solutions were enabled as a result of novel surface modification by an active redox system, composed of 9,10-dihydroxyanthracene/9,10-anthraquinone, on the oxide layer of the SiNW, yielding a chemically-gated FET array. With the use of enzymatic reactions of oxidases, metabolites can be converted to H2O2 and monitored by the nanosensors. Here, the successful detection of glucose metabolites in high-ionic-strength solutions, such as bacterial media, without pre-processing of small volume samples under different conditions and treatments, has been demonstrated. The biofilms were treated with antibiotics differing in their mechanisms of action and were compared to untreated biofilms. Further examination of biofilms under antibiotic treatment with SiNW-FET devices could shed light on the bioprocess that occurs within the biofilm. Moreover, finding proper treatment that eliminates the biofilm could be examined by the novel nanosensor as a monitoring tool.ConclusionsTo summarize, the combination of redox-reactive SiNW-FET devices with micro-fluidic techniques enables the performance of rapid, automated, and real-time metabolite detection with the use of minimal sample size, noninvasively and label-free. This novel platform can be used as an extremely sensitive tool for detection and establishing medical solutions for bacterial-biofilm eradication and for finding a proper treatment to eliminate biofilm contaminations. Moreover, the sensing system can be used as a research tool for further understanding of the metabolic processes that occur within the bacterial biofilm population.

Treatment with disinfectants may induce an increase in viable but non culturable populations of Listeria monocytogenes in biofilms formed in smoked salmon processing environments

The objectives of this study were 1) to evaluate the impact of two industrial disinfectants on the viability of Listeria monocytogenes populations in biofilm and 2) to investigate the viability state of L. monocytogenes cells present on contact surfaces in the smoked salmon processing environment. In the first step, we cultured mono species and mixed species biofilms containing L. monocytogenes on stainless steel or polyvinyl chloride (PVC) at 8 °C for 48h. The biofilms were then exposed to quaternary ammonium- and hydrogen peroxide-based disinfectants. Residual total populations of L. monocytogenes were measured by qPCR, and viable culturable (VC) cell populations were quantified using standard microbiological culture-based techniques and by a quantitative PCR (qPCR) assay coupled with a propidium monoazide treatment. Decreases in VC populations and the appearance of viable but non culturable (VBNC) populations were observed in response to treatment with the disinfectants. An 8 month sampling campaign in 4 smoked salmon processing plants was also carried out to detect L. monocytogenes in environmental samples. VBNC cells were detected mainly after the cleaning and disinfection operations. This study showed that industrial disinfectants did not inactivate all L. monocytogenes cells on inert surfaces. The presence of VBNC populations of L. monocytogenes in the smoked salmon processing environment is a public health concern.

Metagenomic Insight Towards Vanillin-Mediated Membrane Biofouling Prevention: In Silico Docking Validation.

Biofouling leads to water quality deterioration and higher maintenance cost for cleaning of membranes. The present study has demonstrated the application of a biomolecule (vanillin) in scrubbing and destabilizing biofilms of drinking water reverse osmosis (RO) membrane module in lab scale reactor set-up. Reverse osmosis membrane reactor was connected with tap water supply and subjected with optimal concentration of vanillin. The pressure drop was delayed by 17-20days as compared to control reactor. Real-time PCR analysis of metagenome indicated the reduced copy number of functional biofilm-associated genes (bdlA, lasI, pgaC) in treated membrane. SEM and metagenome analysis revealed that the sticky biofilm communities shifted to loosely bound emboli after vanillin treatment. Metagenome sequence analysis revealed the inhibitory activity against major biofouling biota like members of Proteobacteria, Acidobacteria, Acnitobacteria, Bacteroidetes, Candidatus, Nitrospira, and Firmicutes. Biofouled membrane metagenome sequence was also compared with real-life (brackish water, waste water, domestic drinking water) biofouled membrane communities. In silico docking of vanillin to receptor proteins and chemical configuration simulation along with other phenolic derivatives were performed, which suggested that the autoiducer signal capability of vanillin was effective against representative broad spectrum biofilm population. Vanillin exhibited the quorum-quenching mode of action by virtue of docking towards similar amino acid (Thr 131, Ilu 214) responsible of autoinducer signal anchoring in the transcriptional regulatory proteins.

Growth Dynamics of Bacterial Populations in a Two-Compartment Biofilm Bioreactor Designed for Continuous Surfactin Biosynthesis.

Biofilm bioreactors are promising systems for continuous biosurfactant production since they provide process stability through cell immobilization and avoid foam formation. In this work, a two-compartment biofilm bioreactor was designed consisting of a stirred tank reactor and a trickle-bed reactor containing a structured metal packing for biofilm formation. A strong and poor biofilm forming B. subtilis 168 strain due to restored exopolysaccharides (EPS) production or not were cultivated in the system to study the growth behavior of the planktonic and biofilm population for the establishment of a growth model. A high dilution rate was used in order to promote biofilm formation on the packing and wash out unwanted planktonic cells. Biofilm development kinetics on the packing were assessed through a total organic carbon mass balance. The EPS+ strain showed a significantly improved performance in terms of adhesion capacity and surfactin production. The mean surfactin productivity of the EPS+ strain was about 37% higher during the continuous cultivation compared to the EPS- strain. The substrate consumption together with the planktonic cell and biofilm development were properly predicted by the model (α = 0.05). The results show the efficiency of the biofilm bioreactor for continuous surfactin production using an EPS producing strain.

Nutrient Level Determines Biofilm Characteristics and Subsequent Impact on Microbial Corrosion and Biocide Effectiveness.

The impact that nutrient level has on biofilm characteristics, biocide effectiveness, and the associated risk of microbiologically influenced corrosion (MIC) was assessed using multispecies biofilms from two different oilfield consortia. A range of microbiological, microscopy, and corrosion methods demonstrated that the continuous flow of nutrients for the microbial growth resulted in higher activity, thickness, and robustness of the biofilms formed on carbon steel, which induced greater localized corrosion compared to biofilms formed under batch, nutrient-depleted conditions. Despite of the differences in biofilm characteristics, biofilms displayed comparable susceptibilities to glutaraldehyde biocide, with similar log10 reductions and percent reductions of microorganisms under both nutrient conditions. Nevertheless, nutrient replenishment impacted the effectiveness of the biocide in controlling microbial populations; a higher concentration of cells survived the biocide treatment in biofilms formed under a continuous flow of nutrients. Complementary DNA-/RNA-based amplicon sequencing and bioinformatics analysis were used to discriminate the active within the total populations in biofilms established at the different nutrient conditions and allowed the identification of the microbial species that remained active despite nutrient depletion and biocide treatment. Detection of persistent active microorganisms after exposure to glutaraldehyde, regardless of biofilm structure, suggested the presence of microorganisms less susceptible to this biocide and highlighted the importance of monitoring active microbial species for the early detection of biocide resistance in oil production facilities.IMPORTANCE Microbiologically influenced corrosion (MIC) is a complex process that generates economic losses to the industry every year. Corrosion must be managed to prevent a loss of containment of produced fluids to the external environment. MIC management includes the identification of assets with higher MIC risk, which could be influenced by nutrient levels in the system. Assessing biofilms under different nutrient conditions is essential for understanding the impact of flow regime on microbial communities and the subsequent impact on microbial corrosion and on the effectiveness of biocide treatment. This investigation simulates closely oil production systems, which contain piping sections exposed to continuous flow and sections that remain stagnant for long periods. Therefore, the results reported here are useful for MIC management and prevention. Moreover, the complementary methodological approach applied in this investigation highlighted the importance of implementing RNA-based methods for better identification of active microorganisms that survive stress conditions in oil systems.

A rapid and simple method for routine determination of antibiotic sensitivity to biofilm populations of Pseudomonas aeruginosa

Treatment of infections by Pseudomonas aeruginosa forming biofilms after antimicrobial testing on planktonic bacteria can result in substantial failure. Therefore, we offer a robust and simple experimental platform to test the impact of antimicrobials on biofilms. Antibiotic response patterns varied uniquely within biofilm formation capacity and minimal biofilm eradication concentrations (MBECs) has a significantly better discriminatory power than minimum inhibitory concentrations (MICs) to differentiate the overall efficiency of antibiotics to eradicate biofilm. Our resazurin-based 96-well-plate platform is able to emulate bacterial responses to antibiotics under biofilm conditions in a fast, simple, and cost-effective screening method adaptable to automation, and warrants trials in the clinic.

Evolving Populations in Biofilms Contain More Persistent Plasmids.

Bacterial plasmids substantially contribute to the rapid spread of antibiotic resistance, which is a crisis in healthcare today. Coevolution of plasmids and their hosts promotes this spread of resistance by ameliorating the cost of plasmid carriage. However, our knowledge of plasmid–bacteria coevolution is solely based on studies done in well-mixed liquid cultures, even though biofilms represent the main way of bacterial life on Earth and are responsible for most infections. The spatial structure and the heterogeneity provided by biofilms are known to lead to increased genetic diversity as compared with well-mixed liquids. Therefore, we expect that growth in this complex environment could affect the evolutionary trajectories of plasmid–host dyads. We experimentally evolved Shewanella oneidensis MR-1 with plasmid pBP136Gm in biofilms and chemostats and sequenced the genomes of clones and populations. Biofilm populations not only maintained a higher diversity of mutations than chemostat populations but contained a few clones with markedly more persistent plasmids that evolved via multiple distinct trajectories. These included the acquisition of a putative toxin–antitoxin transposon by the plasmid and chromosomal mutations. Some of these genetic changes resulted in loss of plasmid transferability or decrease in plasmid cost. Growth in chemostats led to a higher proportion of variants with decreased plasmid persistence, a phenomenon not detected in biofilms. We suggest that the presence of more stable plasmid–host dyads in biofilms reflects higher genetic diversity and possibly unknown selection pressures. Overall, this study underscores the importance of the mode of growth in the evolution of antibiotic-resistant bacteria.

Biofilm formation displays intrinsic offensive and defensive features of Bacillus cereus

Biofilm formation is a strategy of many bacterial species to adapt to a variety of stresses and has become a part of infections, contaminations, or beneficial interactions. In this study, we demonstrate that profound physiological changes permit Bacillus cereus to switch from a floating to a sessile lifestyle, to undergo further maturation of the biofilm and to differentiate into the offensive or defensive features. We report that floating and biofilm cells are populations that differentiate metabolically, with members of each subpopulation developing different branches of certain metabolic pathways. Secondly, biofilm populations rearrange nucleotides, sugars, amino acids, and energy metabolism. Thirdly, this metabolic rearrangement coexists with: the synthesis of the extracellular matrix, sporulation, reinforcement of the cell wall, activation of the ROS detoxification machinery and production of secondary metabolites. This strategy contributes to defend biofilm cells from competitors. However, floating cells maintain a fermentative metabolic status that ensures a higher aggressiveness against hosts, evidenced by the production of toxins. The maintenance of the two distinct subpopulations is an effective strategy to face different environmental conditions found in the life styles of B. cereus.

Ciprofloxacin- and gentamicin-mediated inhibition of Pseudomonas aeruginosa biofilms is enhanced when combined the volatile oil from Eucalyptus camaldulensis

Pseudomonas biofilms cause therapeutic failures in many clinical infections. This study investigated the combined actions of the volatile oil from Eucalyptus camaldulensis and antibiotics (ciprofloxacin and gentamicin) against planktonic and biofilm populations of Pseudomonas aeruginosa. Further, the quorum sensing genes of P. aeruginosa, grown as biofilms, were also investigated. The minimum inhibitory concentrations (MICs) and biofilm inhibitory concentrations (BICs) of gentamicin, ciprofloxacin, and/or the volatile oil of Eucalyptus camaldulensis were determined against planktonic and biofilm populations of clinical P. aeruginosa isolates. Further, the fractional inhibitory concentrations (FICs) were determined for the antibiotics and volatile oil using a modified checkerboard assay. The bacterial genomic DNA was extracted from biofilm-producing isolates and the target quorum sensing genes were amplified using polymerase chain reaction. Out of 96 biofilm-producing isolates, 43 (44.8%), 29 (30.2%), and 24 (25%) were, respectively, strong, intermediate, and weak biofilm producers. The ciprofloxacin BICs were 30–200-fold higher than the ciprofloxacin MICs. Ciprofloxacin and the volatile oil demonstrated a synergistic effect against both planktonic and biofilm populations. Although the volatile oil and gentamicin also demonstrated a synergistic effect against planktonic cells, only an additive effect was observed against biofilm cells. There was a significant relationship between the frequency of quorum sensing genes, rhlLR and lasLR, and the MICs of piperacillin, with P values of 0.043 and 0.032, respectively. The ciprofloxacin MIC was significantly correlated with the presence of lasLR (P = 0.031) and the ceftazidime MIC was correlated with the presence of lasLR (P = 0.023). The volatile oil from E. camaldulensis leaves has antibacterial effects on P. aeruginosa biofilms, alone and in combination with ciprofloxacin. There is a high correlation between the presence of quorum sensing genes (LaslR and rhlLR) and biofilm production, and with pipracillin resistance.

Cell Ultrastructure in Azospirillum brasilense Biofilms

Due to the primary localization of both epiphytic and endophytic plant growth-promoting rhizobacteria on the surface of the plant root system, biofilm formation is an adaptive trait for these microorganisms. Under conditions of nitrogen limitation in liquid media, nitrogen-fixing Azospirillum brasilense strains switch mainly to the biofilm mode of growth. Overall ultrastructural similarities of the cells within A. brasilense biofilms were revealed, and their resistance to desiccation and oxidative stress was characterized. In strains Sp7, Cd, and Sp245, several types of single and undivided cells were revealed, as well as cystlike cells with pronounced morphological diversity. Resistance to desiccation and to oxidative stress was higher in the biofilm populations of these strains than in planktonic cultures. Dormant forms remained viable in dry biofilms of strains Sp245, Sp7, and Cd formed in a nitrogen-free medium after storage for 120 days. Viability of the biofilms of the same strains formed in the presence of nitrogen was retained for 120, 90, and 60 days, respectively. The minimal inhibitory concentration of H2O2 for biofilms was 1.0% for strains Sp7 and Cd and 0.1% for strain Sp245. Both the dormant forms and biofilms of strain Sp245 were more sensitive to H2O2 than those of strains Sp7 and Cd. Peroxidase activity was not previously reported in Azospirillum biofilms.

Breakdown of Vibrio cholerae biofilm architecture induced by antibiotics disrupts community barrier function.

Bacterial cells in nature are frequently exposed to changes in their chemical environment1,2. For such stimuli, the response mechanisms of isolated cells have been investigated in great detail. By contrast, little is known about the emergent multicellular responses to environmental changes, such as antibiotic exposure3–7, which may hold the key to understanding the structure and functions of the most common bacterial communities: biofilms. Here, by monitoring all individual cells in Vibrio cholerae biofilms during exposure to commonly administered antibiotics for cholera infections, we discovered that translational inhibitors cause strong effects on cell size and shape, as well as biofilm architectural properties. We identified that single-cell-level responses result from the metabolic consequences of protein synthesis inhibition, and that the community-level responses result from an interplay of matrix composition, matrix dissociation, and mechanical interactions between cells. We further discovered that the antibiotic-induced changes in biofilm architecture have substantial effects on biofilm population dynamics and community assembly, by enabling invasion of biofilms by bacteriophages and intruder cells of different species. These mechanistic causes and ecological consequences of biofilm exposure to antibiotics are an important step towards understanding collective bacterial responses to environmental changes, with implications for the effects of antimicrobial therapy on the ecological succession of biofilm communities.

Potential effects of Psidium sp., Mangifera sp., Mentha sp. and its mixture (PEM) in reducing bacterial populations in biofilms, adherence and acid production of S. sanguinis and S. mutans

ObjectivePsidium sp., Mangifera sp. and Mentha sp. and its mixture (PEM) are known to have antimicrobial and anti-adherence effects. DesignHere, we have investigated these individual plant extracts and its synergistic mixture (PEM) for its anti-cariogenic effect to reduce populations of single and mixed-species of Streptococcus sanguinis and Streptococcus mutans in a planktonic or/and biofilm and their others reduced virulence. Bacterial populations in the biofilm after 24 h, hydrophobic cell surface activity to n-hexadecane and pH changes at 5 min’ intervals until 90 min of incubation were recorded. Total phenolic content and bioactive compounds in the crude aqueous plant extracts were analysed. Regulatory gene expressions of S. mutans adhesins genes (gtfB, gtfC, gbpB and spaP) upon treatment with PEM were investigated in planktonic and biofilm conditions. ResultsAll plant extracts strongly reduced S. mutans in the biofilm compared to S. sanguinis in single and mixed-species. PEM reduced S. mutans by 84% with S. sanguinis 87% in the mixed population. Psidium sp. and PEM highly reduced cell-surface hydrophobicity of the two bacteria thus reducing adherence and biofilm formation. PEM and Mangifera sp. lowered initial pH change in the mixed populations of S. sanguinis and S. mutans. PEM downregulated the S. mutans gtfB gene expression in the single species planktonic and mixed-species biofilms. ConclusionsThe effectiveness of PEM in reducing S. mutans within the biofilm, cell-surface hydrophobicity, acid production and adhesin gene (gtfB) expression in mixed-species with S. sanguinis indicates its potential as an antibacterial agent against dental caries. This is attributed to the phenolic content in the PEM.

Evolutionary pathways to antibiotic resistance are dependent upon environmental structure and bacterial lifestyle.

Bacterial populations vary in their stress tolerance and population structure depending upon whether growth occurs in well-mixed or structured environments. We hypothesized that evolution in biofilms would generate greater genetic diversity than well-mixed environments and lead to different pathways of antibiotic resistance. We used experimental evolution and whole genome sequencing to test how the biofilm lifestyle influenced the rate, genetic mechanisms, and pleiotropic effects of resistance to ciprofloxacin in Acinetobacter baumannii populations. Both evolutionary dynamics and the identities of mutations differed between lifestyle. Planktonic populations experienced selective sweeps of mutations including the primary topoisomerase drug targets, whereas biofilm-adapted populations acquired mutations in regulators of efflux pumps. An overall trade-off between fitness and resistance level emerged, wherein biofilm-adapted clones were less resistant than planktonic but more fit in the absence of drug. However, biofilm populations developed collateral sensitivity to cephalosporins, demonstrating the clinical relevance of lifestyle on the evolution of resistance.

Anti-Biofilm Effect of Selected Essential Oils and Main Components on Mono- and Polymicrobic Bacterial Cultures.

Biofilms are surface-associated microbial communities resistant to sanitizers and antimicrobials. Various interactions that can contribute to increased resistance occur between the populations in biofilms. These relationships are the focus of a range of studies dealing with biofilm-associated infections and food spoilage. The present study investigated the effects of cinnamon (Cinnamomum zeylanicum), marjoram (Origanum majorana), and thyme (Thymus vulgaris) essential oils (EOs) and their main components, i.e., trans-cinnamaldehyde, terpinen-4-ol, and thymol, respectively, on single- and dual-species biofilms of Escherichia coli, Listeria monocytogenes, Pseudomonas putida, and Staphylococcus aureus. In dual-species biofilms, L. monocytogenes was paired with each of the other three bacteria. Minimum inhibitory concentration (MIC) values for the individual bacteria ranged between 0.25 and 20 mg/mL, and trans-cinnamaldehyde and cinnamon showed the highest growth inhibitory effect. Single-species biofilms of L. monocytogenes, P. putida, and S. aureus were inhibited by the tested EOs and their components at sub-lethal concentrations. Scanning electron microscopy images showed that the three-dimensional structure of mature biofilms embedded in the exopolysaccharide matrix disappeared or was limited to micro-colonies with a simplified structure. In most dual-species biofilms, to eliminate living cells from the matrix, concentrations exceeding the MIC determined for individual bacteria were required.

Metabolomics Deciphered Metabolic Reprogramming Required for Biofilm Formation

Biofilm formation plays a key role in many bacteria causing infections, which mostly accounts for high-frequency infectious recurrence and antibiotics resistance. In this study, we sought to compare modified metabolism of biofilm and planktonic populations with UTI89, a predominant agent of urinary tract infection, by combining mass spectrometry based untargeted and targeted metabolomics methods, as well as cytological visualization, which enable us to identify the driven metabolites and associated metabolic pathways underlying biofilm formation. Surprisingly, our finding revealed distinct differences in both phenotypic morphology and metabolism between two patterns. Furthermore, we identified and characterized 38 differential metabolites and associated three metabolic pathways involving glycerolipid metabolism, amino acid metabolism and carbohydrate metabolism that were altered mostly during biofilm formation. This discovery in metabolic phenotyping permitted biofilm formation shall provide us a novel insight into the dissociation of biofilm, which enable to develop novel biofilm based treatments against pathogen causing infections, with lower antibiotic resistance.

Monitoring Biofilm Formation and Microbial Interactions that May Occur During a Salmonella Contamination Incident across the Network of a Water Bottling Plant.

The present study aims to monitor the ability of Salmonella to colonize and compete as a member of the mixed species biofilm within key points at a water bottling plant, in case of a contamination incident with this major foodborne pathogen. To achieve this goal, bacterial communities throughout the production line were collected and their identities were investigated by microbial counts and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). These bacterial communities alone or along with constructed Salmonella enterica serovar Typhimurium (ST) fluorescence-based bioreporters were left to form a biofilm on stainless steel for 6 days at 20 °C. ST bioreporters were constructed by introducing plasmids expressing EYFP (enhanced yellow fluorescent protein) fusions of the genes csgB, csrA, sspH2, and fliD into ST 14028S. The bead vortexing-plate counting method was applied for the enumeration of the biofilm population, while the behavior of the bioreporters was evaluated by fluorescence microscopy. From a set of 16 samples that were collected from the plant, species of Citrobacter, Staphylococcus, Pseudomonas, Bacillus, and Exiguobacterium were identified. The presence of these indigenous bacteria neither inhibited nor enhanced the biofilm formation of ST in mixed bacterial communities (p > 0.05). Furthermore, the csrA-based bioreporter was shown to be induced in multispecies biofilms with Citrobacter. In conclusion, this study enhanced our knowledge of bacterial interactions occurring within a biofilm in a water bottling plant.

Pseudomonas aeruginosa Increases the Sensitivity of Biofilm-Grown Staphylococcus aureus to Membrane-Targeting Antiseptics and Antibiotics.

Pseudomonas aeruginosa and Staphylococcus aureus often cause chronic, recalcitrant infections in large part due to their ability to form biofilms. The biofilm mode of growth enables these organisms to withstand antibacterial insults that would effectively eliminate their planktonic counterparts. We found that P. aeruginosa supernatant increased the sensitivity of S. aureus biofilms to multiple antimicrobial compounds, including fluoroquinolones and membrane-targeting antibacterial agents, including the antiseptic chloroxylenol. Treatment of S. aureus with the antiseptic chloroxylenol alone did not decrease biofilm cell viability; however, the combination of chloroxylenol and P. aeruginosa supernatant led to a 4-log reduction in S. aureus biofilm viability compared to exposure to chloroxylenol alone. We found that the P. aeruginosa-produced small molecule 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) is responsible for the observed heightened sensitivity of S. aureus to chloroxylenol. Similarly, HQNO increased the susceptibility of S. aureus biofilms to other compounds, including both traditional and nontraditional antibiotics, which permeabilize bacterial membranes. Genetic and phenotypic studies support a model whereby HQNO causes an increase in S. aureus membrane fluidity, thereby improving the efficacy of membrane-targeting antiseptics and antibiotics. Importantly, our data show that P. aeruginosa exoproducts can enhance the ability of various antimicrobial agents to kill biofilm populations of S. aureus that are typically difficult to eradicate. Finally, our discovery that altering membrane fluidity shifts antimicrobial sensitivity profiles of bacterial biofilms may guide new approaches to target persistent infections, such as those commonly found in respiratory tract infections and in chronic wounds.IMPORTANCE The thick mucus in the airways of cystic fibrosis (CF) patients predisposes them to frequent, polymicrobial respiratory infections. Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from the airways of individuals with CF, as well as from diabetic foot ulcers and other wounds. Both organisms form biofilms, which are notoriously difficult to eradicate and promote chronic infection. In this study, we have shown that P. aeruginosa-secreted factors can increase the efficacy of compounds that alone have little or no bactericidal activity against S. aureus biofilms. In particular, we discovered that P. aeruginosa exoproducts can potentiate the antistaphylococcal activity of phenol-based antiseptics and other membrane-active drugs. Our findings illustrate that polymicrobial interactions can dramatically increase antibacterial efficacy in vitro and suggest that altering membrane physiology promotes the ability of certain drugs to kill bacterial biofilms-knowledge that may provide a path for the discovery of new biofilm-targeting antimicrobial strategies.