Decomposition of Growth Curves into Growth Rate and Acceleration: a Novel Procedure To Monitor Bacterial Growth and the Time-Dependent Effect of Antimicrobials.

In this paper, a simple numerical procedure is presented to monitor the growth of Streptococcus sanguinis over time in the absence and presence of propolis, a natural antimicrobial. In particular, it is shown that the real-time decomposition of growth curves obtained through optical density measurements into growth rate and acceleration can be a powerful tool to precisely assess a large range of key parameters (i.e., lag time [t0], starting growth rate [γ0], initial acceleration of the growth [a0], maximum growth rate [γmax], maximum acceleration [amax], and deceleration [amin] of the growth and the total number of cells at the beginning of the saturation phase [Ns]) that can be readily used to fully describe growth over time. Consequently, the procedure presented provides precise data of the time course of the different growth phases and features, which is expected to be relevant, for instance, to thoroughly evaluate the effect of new antimicrobial agents. It further provides insight into predictive microbiology, likely having important implications for assumptions adopted in mathematical models to predict the progress of bacterial growth. IMPORTANCE The new and simple numerical procedure presented in this paper to analyze bacterial growth will possibly allow the identification of true differences in efficacy among antimicrobial drugs for their applications in human health, food security, and the environment, among others. It further provides insight into predictive microbiology, likely helping in the development of proper mathematical models to predict the course of bacterial growth under diverse circumstances.

Antimicrobial activity of a novel Spanish propolis against planktonic and sessile oral Streptococcus spp.

Increased bacterial resistance to traditional antimicrobial agents has prompted the use of natural products with antimicrobial properties such as propolis, extensively employed since ancient times. However, the chemical composition of propolis extracts is extremely complex and has been shown to vary depending on the region and season of collection, due to variations in the flora from which the pharmacological substances are obtained, being therefore essential for their antimicrobial activity to be checked before use. For this purpose, we evaluate the in vitro antimicrobial and anti-biofilm activity of a new and promising Spanish ethanolic extract of propolis (SEEP) on Streptococcus mutans and Streptococcus sanguinis, responsible, as dominant 'pioneer' species, for dental plaque. Results reveal that S. sanguinis is more sensitive to SEEP, slowing and retarding its growth considerably with lower concentrations than those needed to produce the same effect in S. mutans. SEEP presents concentration- and time-dependent killing activity and, furthermore, some of the subinhibitory concentrations employed increased biofilm formation even when bacterial growth decreased. Mono and dual-species biofilms were also inhibited by SEEP. Findings obtained clearly show the relevance of using biofilm and subinhibitory concentration models to determine optimal treatment concentrations.

A physico-chemical study of the interaction of ethanolic extracts of propolis with bacterial cells.

In the present study, an effort has been made to understand the interaction mode of propolis, a natural substance produced by honey bees, with gram-positive and gram-negative bacterial cells by measuring alterations in cell surface physico-chemical properties following the incubation of the cells with different sub-inhibitory concentrations of this antimicrobial agent. Electrophoretic mobility and surface hydrophobicity measurements revealed for the first time that propolis induced substantial changes in the volumetric charge density, electrophoretic softness and degree of hydrophobicity characterizing the outermost surface layer of cells. These changes, which appear to be dose-dependent, seem to be consistent with the increasing accumulation and penetration of the propolis antimicrobial components through the cells extracellular layer. Moreover, electron microscopy observation and the determination of the cell constituents' release demonstrated that propolis at sub-bactericidal concentrations already provoked (at least localized) cell wall damage and/or perturbations. These findings thus suggest that the initial mechanism of action of propolis is most likely structural, resulting from sufficient interaction between the different propolis components and bacterial cell wall structures.

Mechanically Induced Bacterial Death Imaged in Real Time: A Simultaneous Nanoindentation and Fluorescence Microscopy Study.

Mechano-bactericidal nanomaterials rely on their mechanical or physical interactions with bacteria and are promising antimicrobial strategies that overcome bacterial resistance. However, the real effect of mechanical versus chemical action on their activity is under debate. In this paper, we quantify the forces necessary to produce critical damage to the bacterial cell wall by performing simultaneous nanoindentation and fluorescence imaging of single bacterial cells. Our experimental setup allows puncturing the cell wall of an immobilized bacterium with the tip of an atomic force microscope (AFM) and following in real time the increase in the fluorescence signal from a cell membrane integrity marker. We correlate the forces exerted by the AFM tip with the fluorescence dynamics for tens of cells, and we find that forces above 20 nN are necessary to exert critical damage. Moreover, a similar experiment is performed in which bacterial viability is assessed through physiological activity, in order to gain a more complete view of the effect of mechanical forces on bacteria. Our results contribute to the quantitative understanding of the interaction between bacteria and nanomaterials.

Bacterial response to spatially organized microtopographic surface patterns with nanometer scale roughness.

In this study, the influence of nanometer scale roughness on bacterial adhesion and subsequent biofilm formation has been evaluated using spatially organized microtopographic surface patterns for four major opportunistic pathogens of the genus Staphylococcus (S. epidermidis and S. aureus) responsible for associated-biofilm infections on biomedical devices. The results presented demonstrated that regardless of the strain employed the initial adhesion events to these surfaces are directed by cell-surface contact points maximisation and thus, bacterial cells actively choose their position to settle based on that principle. Accordingly, bacterial cells were found to preferably adhere to the square corners and convex walls of recessed surface features rather than the flat or concave walls of equal protruding features. This finding reveals, for the first time, that the particular shape of the surfaces features employed potentially determined the initial location of the adhering cells on textured surfaces. It was further shown that all surfaces patterns investigated produce a significant reduction in bacterial adhesion (40-95%) and biofilm formation (22-58%). This important observation could not be related to physical constrains or increased solid surface hydrophobicity, as previously suggested by other authors using engineered topographies with microscale surface roughness. It is evident that other causes, such as nanoscale surface roughness-induced interaction energies, might be controlling the process of bacterial adhesion and biofilm formation on surfaces with well-defined nanoscale topography.

On the interactions of human bone cells with Ti6Al4V thermally oxidized by means of laser shock processing.

We investigated a Ti6Al4V alloy modified by means of laser peening in the absence of sacrificial coatings. As a consequence of the temperature rise during laser focusing, melting and ablation generated an undulated surface that exhibits an important increase in the content of titanium oxides and OH- ions. Human mesenchymal stem cells and osteoblasts cultured on the oxidized alloy develop noticeable filopodia and lamellipodia. Their paxillin-stained focal adhesions are smaller than in cells attached to the untreated alloy and exhibit a marked loss of colocalization with the ends of actin stress fibers. An important imbalance of phosphorylation and/or dephosphorylation of the focal adhesion kinase is detected in cells grown on the oxidized alloy. Although these mechanisms of adhesion are deeply altered, the surface treatment does not affect cell attachment or proliferation rates on the alloy. Human mesenchymal stem cells cultured on the treated alloy in media containing osteogenic inducers differentiate towards the osteoblastic phenotype to a higher extent than those on the untreated surface. Also, the specific functions of human osteoblasts cultured on these media are enhanced on the treated alloy. In summary, laser peening tailors the Ti6Al4V surface to yield an oxidized layer with increased roughness that allows the colonization and activities of bone-lineage cells.

Studying the influence of surface topography on bacterial adhesion using spatially organized microtopographic surface patterns.

The influence of surface topography on bacterial adhesion has been investigated using a range of spatially organized microtopographic surface patterns generated on polydimethylsiloxane (PDMS) and three unrelated bacterial strains. The results presented indicate that bacterial cells actively choose their position to settle, differentiating upper and lower areas in all the surface patterns evaluated. Such selective adhesion depends on the cells' size and shape relative to the dimensions of the surface topographical features and surface hydrophobicity/hydrophilicity. Moreover, it was found that all the topographies investigated provoke a significant reduction in bacterial adhesion (30-45%) relative to the smooth control samples regardless of surface hydrophobicity/hydrophilicity. This remarkable finding constitutes a general phenomenon, occurring in both Gram-positive and Gram-negative cells with spherical or rod shape, dictated by only surface topography. Collectively, the results presented in this study demonstrate that spatially organized microtopographic surface patterns represent a promising approach to controlling/inhibiting bacterial adhesion and biofilm formation.

Electrochemical analysis of the UV treated bactericidal Ti6Al4V surfaces.

This research investigates in detail the bactericidal effect exhibited by the surface of the biomaterial Ti6Al4V after being subjected to UV-C light. It has been recently hypothesized that small surface currents, occurring as a consequence of the electron-hole pair recombination taking place after the excitation process, are behind the bactericidal properties displayed by this UV-treated material. To corroborate this hypothesis we have used different electrochemical techniques, such as electrochemical impedance spectroscopy (EIS), potentiodynamic polarization plots and Mott-Schottky plots. EIS and Mott-Schottky plots have shown that UV-C treatment causes an initial increase on the surface electrical conduction of this material. In addition, EIS and polarization plots demonstrated that higher corrosion currents occur at the UV treated than at the non-irradiated samples. Despite this increase in the corrosion currents, EIS has also shown that such currents are not likely to affect the good stability of this material oxide film since the irradiated samples completely recovered the control values after being stored in dark conditions for a period not longer than 24h. These results agree with the already-published in vitro transitory behavior of the bactericidal effect, which was shown to be present at initial times after the biomaterial implantation, a crucial moment to avoid a large number of biomaterial associated infections.

Surface-dependent mechanical stability of adsorbed human plasma fibronectin on Ti6Al4V: domain unfolding and stepwise unraveling of single compact molecules.

In this study, the structure and mechanical stability of human plasma fibronectin (HFN), a major protein component of blood plasma, have been evaluated in detail upon adsorption on the nonirradiated and irradiated Ti6Al4V material through the use of atomic force microscopy. The results indicated that the material surface changes occurring after the irradiation process reduce the disulfide bonds that typically preclude the mechanical denaturation of individual HFN domains and interfere significantly with the intraionic interactions stabilizing the compact conformation of the adsorbed HFN molecules. In particular, upon adsorption on this material, the molecules adopt a more flexible conformation and become mechanically more compliant. Unexpected observations also indicated that, regardless the material surface, a single HFN molecule can be pulled into an extended conformation without the unfolding of its domains through a series of three unraveling steps. The forces involved in the unraveling process were found to be generally lower than the forces required to unfold the individual protein domains. This report is the first one to present the force displacement details associated to the straightening of a single compact protein at the molecular level.

Adsorption behavior of human plasma fibronectin on hydrophobic and hydrophilic Ti6Al4V substrata and its influence on bacterial adhesion and detachment.

Biomaterial implant-associated infections, a common cause of medical devices' failure, are initiated by bacterial adhesion to an adsorbed protein layer on the implant material surface. In this study, the influence of protein surface orientation on bacterial adhesion has been examined using three clinically relevant bacterial strains known to express specific binding sites for human plasma fibronectin (HFN). HFN was allowed to adsorb on hydrophobic Ti6Al4V and physically modified hydrophilic Ti6Al4V substrata. Ellipsometric data reveal that the characteristics of the adsorbed protein layers primary depend on solid surface tension and the initial protein concentration in solution. In particular, HFN molecules adopt a more extended conformation on hydrophobic than hydrophilic surfaces, an effect that is more pronounced at low than at high initial protein concentrations. Moreover, the extended conformation of the protein molecules on these surfaces likely facilitates the exposure of specific sites for adhesion, resulting in the higher bacterial-cell attachment observed regardless of the strain considered. Contact angle measurements and the analysis of the number of remaining adhering cells after being subjected to external forces further suggest that both specific and nonspecific (hydrophobic) interactions play an important role on bacterial attachment. This study is the first one to evaluate the influence of surface hydrophobicity on protein adsorption and its subsequent effect on bacterial adhesion using a material whose hydrophobicity was not modified using chemical treatments that potentially led to surface properties changes other than hydrophobicity.

The zeta potential of extended dielectrics and conductors in terms of streaming potential and streaming current measurements.

The electrical characterization of surfaces in terms of the zeta potential (ζ), i.e., the electric potential contributing to the interaction potential energy, is of major importance in a wide variety of industrial, environmental and biomedical applications in which the integration of any material with the surrounding media is initially mediated by the physico-chemical properties of its outer surface layer. Among the different existing electrokinetic techniques for obtaining ζ, streaming potential (V(str)) and streaming current (I(str)) are important when dealing with flat-extended samples. Mostly dielectric materials have been subjected to this type of analysis and only a few papers can be found in the literature regarding the electrokinetic characterization of conducting materials. Nevertheless, a standardized procedure is typically followed to calculate ζ from the measured data and, importantly, it is shown in this paper that such a procedure leads to incorrect zeta potential values when conductors are investigated. In any case, assessment of a reliable numerical value of ζ requires careful consideration of the origin of the input data and the characteristics of the experimental setup. In particular, it is shown that the cell resistance (R) typically obtained through a.c. signals (R(a.c.)), and needed for the calculations of ζ, always underestimates the zeta potential values obtained from streaming potential measurements. The consideration of R(EK), derived from the V(str)/I(str) ratio, leads to reliable values of ζ when dielectrics are investigated. For metals, the contribution of conductivity of the sample to the cell resistance provokes an underestimation of R(EK), which leads to unrealistic values of ζ. For the electrical characterization of conducting samples I(str) measurements constitute a better choice. In general, the findings gathered in this manuscript establish a measurement protocol for obtaining reliable zeta potentials of dielectrics and conductors based on the intrinsic electrokinetic behavior of both types of samples.

Insights into bacterial contact angles: difficulties in defining hydrophobicity and surface Gibbs energy.

One of the principal techniques for evaluating the surface hydrophobicity of biological samples is contact angle. This method, applied readily to flat-smooth-inert surfaces, gives rise to an important debate when implemented with microbial lawns. After its initial description, in 1984, several authors have carried out modifications of the technique but the results obtained have not been previously judged. This work focuses on the particularities of contact angle measurements on bacterial lawns and enhances the idea of the impossibility of using water contact angle as a universal measurement of bacterial hydrophobicity. Contact angles can only be used as relative indicators of hydrophobicity, in a similar way to tests based on microbial adhesion to solvents. The strong dependence of contact angles on dried bacterial lawns with measuring time and environmental conditions (e.g. pH of the media) preclude the estimation of their absolute values, and so, of the cells surface Gibbs energy. Yet, for a given measuring time, it is found that the hydrophobicity and the apparent bacterial surface Gibbs energy components are qualitatively related to the bacterial surface electrical potential. In particular, a hydrophobic increase is always accompanied by an increase of the cells Lifshitz-Van der Waals component and a decrease of their acid-base component and absolute zeta potential. Therefore, the present study shows that the physico-chemical surface properties that characterize bacteria are not independent, and one of them can be qualitatively described in terms of the others when measuring contact angles at a fixed time after the drying of the microbial beds.

In situ characterization of differences in the viscoelastic response of individual gram-negative and gram-positive bacterial cells.

We used a novel atomic force microscopy (AFM)-based technique to compare the local viscoelastic properties of individual gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacterial cells. We found that the viscoelastic properties of the bacterial cells are well described by a three-component mechanical model that combines an instantaneous elastic response and a delayed elastic response. These experiments have allowed us to investigate the relationship between the viscoelastic properties and the structure and composition of the cell envelope. In addition, this is the first report in which the mechanical role of Lpp, the major peptidoglycan-associated lipoprotein and one of the most abundant outer membrane proteins in E. coli cells, has been quantified. We expect that our findings will be helpful in increasing the understanding of the structure-property relationships of bacterial cell envelopes.

Surface viscoelasticity of individual gram-negative bacterial cells measured using atomic force microscopy.

The cell envelope of gram-negative bacteria is responsible for many important biological functions: it plays a structural role, it accommodates the selective transfer of material across the cell wall, it undergoes changes made necessary by growth and division, and it transfers information about the environment into the cell. Thus, an accurate quantification of cell mechanical properties is required not only to understand physiological processes but also to help elucidate the relationship between cell surface structure and function. We have used a novel, atomic force microscopy (AFM)-based approach to probe the mechanical properties of single bacterial cells by applying a constant compressive force to the cell under fluid conditions while measuring the time-dependent displacement (creep) of the AFM tip due to the viscoelastic properties of the cell. For these experiments, we chose a representative gram-negative bacterium, Pseudomonas aeruginosa PAO1, and we used regular V-shaped AFM cantilevers with pyramid-shaped and colloidal tips. We find that the cell response is well described by a three-element mechanical model which describes an effective cell spring constant, k(1), and an effective time constant, tau, for the creep deformation. Adding glutaraldehyde, an agent that increases the covalent bonding of the cell surface, produced a significant increase in k(1) together with a significant decrease in tau. This work represents a new attempt toward the understanding of the nanomechanical properties of single bacteria while they are under fluid conditions, which could be of practical value for elucidating, for instance, the biomechanical effects of drugs (such as antibiotics) on pathogens.

Localized attraction correlates with bacterial adhesion to glass and metal oxide substrata.

Bacterial adhesion to surfaces does not always proceed according to theoretical expectations. Discrepancies are often attributed to surface heterogeneities that provide localized, favorable sites for bacterial attachment. The presence of these favorable deposition sites for bacteria, however, has never been directly measured. Atomic force microscopy (AFM) was used to quantify the distribution of attractive sites on clean substrata. Surfaces of silica and three different metal oxides mapped by adhesion force with regular or colloidal AFM tips showed a heterogeneous distribution of adhesion forces. Adhesion forces were normally distributed based on a colloid probe, but regular tips revealed a proportionately larger number of relatively more adhesive sites. No correlation was found between the average adhesion force (tip or colloid) and macroscopic adhesion tests using five strains of bacteria. However, when AFM tip results were compared to bacterial adhesion data on the basis of only the stickiest sites (the 5% of sites with the largest adhesion force), there was a good correlation of AFM data with adhesion data. These results demonstrate for the first time how overall bacterial adhesion to a surface effectively correlates with a relatively small fraction of highly adhesive sites rather than averaged adhesion force as detected using AFM.

Residence time, loading force, pH, and ionic strength affect adhesion forces between colloids and biopolymer-coated surfaces.

Exopolymers are thought to influence bacterial adhesion to surfaces, but the time-dependent nature of molecular-scale interactions of biopolymers with a surface are poorly understood. In this study, the adhesion forces between two proteins and a polysaccharide [Bovine serum albumin (BSA), lysozyme, or dextran] and colloids (uncoated or BSA-coated carboxylated latex microspheres) were analyzed using colloid probe atomic force microscopy (AFM). Increasing the residence time of an uncoated or BSA-coated microsphere on a surface consistently increased the adhesion force measured during retraction of the colloid from the surface, demonstrating the important contribution of polymer rearrangement to increased adhesion force. Increasing the force applied on the colloid (loading force) also increased the adhesion force. For example, at a lower loading force of approximately 0.6 nN there was little adhesion (less than -0.47 nN) measured between a microsphere and the BSA surface for an exposure time up to 10 s. Increasing the loading force to 5.4 nN increased the adhesion force to -4.1 nN for an uncoated microsphere to a BSA surface and to as much as -7.5 nN for a BSA-coated microsphere to a BSA-coated glass surface for a residence time of 10 s. Adhesion forces between colloids and biopolymer surfaces decreased inversely with pH over a pH range of 4.5-10.6, suggesting that hydrogen bonding and a reduction of electrostatic repulsion were dominant mechanisms of adhesion in lower pH solutions. Larger adhesion forces were observed at low (1 mM) versus high ionic strength (100 mM), consistent with previous AFM findings. These results show the importance of polymers for colloid adhesion to surfaces by demonstrating that adhesion forces increase with applied force and detention time, and that changes in the adhesion forces reflect changes in solution chemistry.

Role of lactobacillus cell surface hydrophobicity as probed by AFM in adhesion to surfaces at low and high ionic strength.

The S-layer present at the outermost cell surface of some lactobacillus species is known to convey hydrophobicity to the lactobacillus cell surface. Yet, it is commonly found that adhesion of lactobacilli to solid substrata does not proceed according to expectations based on cell surface hydrophobicity. In this paper, the role of cell surface hydrophobicity of two lactobacillus strains with and without a surface layer protein (SLP) layer has been investigated with regard to their adhesion to hydrophobically or hydrophilically functionalized glass surfaces under well-defined flow conditions and in low and high ionic strength suspensions. Similarly, the interaction of the lactobacilli with similarly functionalized atomic force microscope (AFM) tips was measured. In a low ionic strength suspension, both lactobacillus strains show higher initial deposition rates to hydrophobic glass than to hydrophilic glass, whereas in a high ionic strength suspension no clear influence of cell surface hydrophobicity on adhesion is observed. Independent of ionic strength, however, AFM detects stronger interaction forces when both bacteria and tip are hydrophobic or hydrophilic than when bacteria and tip have opposite hydrophobicities. This suggest that the interaction develops in a different way when a bacterium is forced into contact with the tip surface, like in AFM, as compared with contacts developing between a cell surface and a macroscopic substratum under flow. In addition, the distance dependence of the total Gibbs energy of interaction could only be qualitatively correlated with bacterial deposition and desorption in the parallel plate flow chamber.

Dynamic cell surface hydrophobicity of Lactobacillus strains with and without surface layer proteins.

Variations in surface hydrophobicity of six Lactobacillus strains with and without an S-layer upon changes in ionic strength are derived from contact angle measurements with low- and high-ionic-strength aqueous solutions. Cell surface hydrophobicity changed in response to changes in ionic strength in three out of the six strains, offering these strains a versatile mechanism to adhere to different surfaces. The dynamic behavior of the cell surface hydrophobicity could be confirmed for two selected strains by measuring the interaction force between hydrophobic and hydrophilic tips with use of atomic force microscopy.

Comparison of atomic force microscopy interaction forces between bacteria and silicon nitride substrata for three commonly used immobilization methods.

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.

Atomic force microscopic corroboration of bond aging for adhesion of Streptococcus thermophilus to solid substrata.

Initial bacterial adhesion is considered to be reversible, but over time the adhesive bond between a bacterium and a substratum surface may strengthen, turning the process into an irreversible state. Microbial desorption has been studied in situ in controlled flow devices as a function of the organisms resident time on the surface (J. Colloid Interface Sci. 164 (1994) 355). It appeared that desorption of Streptococcus thermophilus decreased strongly within approximately 50 s after initial adhesion due to bond aging. In this paper, bond aging between the S. thermophilus cell surface and the silicon nitride tip of an AFM (atomic force microscope) is corroborated microscopically and related to the macroscopic, residence time-dependent desorption of the organism under flow. AFM indicated bond strengthening between the tip and the cell surface within 100 s of contact, which is on the same order of magnitude as bond aging inferred from residence time-dependent desorption. Comparison of the interaction energies derived from AFM and macroscopic desorption indicate that bond strengthening arises as a result of multiple attachments of extracellular polymeric substances to a substratum surface.