Nanoscale Investigation of Pathogenic Microbial Adhesion to a Biomaterial

Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. In many cases, the device infections are caused by the adhesion of microbes to the implant, ensuing growth, pathogenesis, and dissemination. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an atomic force microscope (AFM) cantilever, and a biomaterial or biofilm substrate. The two model microbes used in this study, Candida parapsilosis (ATCC 90018) and Pseudomonas aeruginosa (ATCC 10145), were chosen for both their clinical relevance and their ease of acquisition and handling in the laboratory setting. Attractive interactions exist between C. parapsilosis and both unmodified silicone rubber and P. aeruginosa biofilms. Using C. parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive forces of 4.3 ± 0.25 nN in the approach portion of the force cycle. On P. aeruginosa biofilms, the magnitude of the attractive force decreases to 2.0 ± 0.40 nN and is preceded by a 2.0-nN repulsion at approximately 75 nm from the cell surface. These data suggest that C. parapsilosis may adhere to both silicone rubber and P. aeruginosa biofilms, possibly contributing to patient morbidity and mortality. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative link between the physicomechanical and physicochemical properties of implant materials and the nanoscale interactions leading to microbial colonization and infection.     

Interactions of Candida albicans with other Candida spp. and bacteria in the biofilms

AIMS: To study the interactions between Candida albicans and 12 other species of Candida and bacteria in biofilms. METHODS AND RESULTS: The number of cells within growing biofilms in a polystyrene tube model was measured after adding C. albicans to preformed biofilms of other micro-organisms and vice versa. It was also measured after simultaneous biofilm formation of C. albicans and other micro-organisms. The number of cells of C. albicans within the growing biofilms decreased significantly (P < 0.05) when the fungus was added to preformed biofilms of Candida spp. and bacteria except, with C. parapsilosis, Torulopsis glabrata and the glycocalyx producer Pseudomonas aeruginosa. When C. parapsilosis, Staphylococcus epidermidis (nonglycocalyx producer) or Serratia marcescens was added to preformed biofilms of C. albicans, the number of cells of these micro-organisms increased in the growing biofilms. CONCLUSIONS: Biofilms of C. albicans are capable of holding other micro-organisms and more likely to be heterogeneous with other bacteria and fungi in the environment and on medical devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of the heterogeneity of biofilm-associated organisms can influence treatment decisions, particularly in patients who do not respond to initial appropriate therapy.     

Analysis of DNA and Zinc finger interactions using mechanical force spectroscopy

The unbinding force of Zif268-DNA complex has been studied by atomic force microscopy (AFM). DNA and Zif268 were covalently immobilized on the surfaces of an AFM tip and glass substrate, respectively. Confocal microscopy was used to confirm the successful immobilization of DNA. Because of the complexity of the protein-DNA interaction, parallel experiments were designed to discriminate specific interactions. For such experiments, a typical unbinding force of a single Zif268-DNA complex (approx 550 pN at 40 nN/s force loading rate) was evaluated.     

Nanoscale investigation on adhesion of E. coli to surface modified silicone using atomic force microscopy

Bacterial adhesion on biomaterial surfaces is the initial step in establishing infections and leads to the formation of biofilms. In this study, silicone was modified with different biopolymers and silanes, including: heparin, hyaluronan, and self-assembled octadecyltrichlorosilane (OTS), and fluoroalkylsilane (FAS). The aim was to provide a stable and bacteria-resistant surface by varying the degree of hydrophobicity and the surface structure. The adhesion of Escherichia coli (JM 109) on different modified silicone surfaces was investigated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Mica, an ideal hydrophilic and smooth surface, was employed as a control specimen to study the effect of hydrophobicity and surfaces roughness on bacterial adhesion. AFM probes were coated with E. coli and the force measurements between the bacteria-immobilized tip and various materials surfaces were obtained while approaching to and retracting from the surfaces. A short-range repulsive force was observed between the FAS coated silicone and bacteria. The pull-off force of bacteria to FAS was the smallest among coated surfaces. On the other hand, heparin exhibited a long-range attractive force during approach and required a higher pull-off force in retraction. Both AFM and SEM results indicated that FAS reduced bacterial adhesion whereas heparin enhanced the adhesion compared to pure silicone. The work demonstrates that hydrophobicity cannot be used as a criterion to predict bacterial adhesion. Rather, both the native properties of the individual strain of bacteria and the specific functional structure of the surfaces determine the strength of force interaction, and thus the extent of adhesion.     

Interaction between chondroitin-6-sulfate and Entamoeba histolytica as revealed by force spectroscopy

We have observed by atomic force microscopy (AFM) the amoeba surface and probed the interaction force between Entamoeba histolytica and chondroitin-6-sulphate (C6S). We have used several substrates to adhere trophozoites. The best reproducibility in sample preparation was obtained with fibronectin-coated coverslips and when the cells were fixed with paraformaldehyde. The images obtained with the AFM showed that the trophozoite exhibits an irregular surface. Pseudopods and waving adhesion plaques could be observed. Force spectroscopy analysis showed that the trophozoite surface strongly interacts with C6S-functionalized tips. During cantilever retraction, attractive force peaks were observed at distances up to 1.3 microm above the trophozoite surface. Statistical analysis of the force distributions collected for five samples shown a reproducible 2.2 nN mean adhesion force. We observed a reduction of the adhesion force and of the interaction distance after addition of galactose to the buffer solution suggesting that the observed interaction is also Gal/GalNAc-lectin-mediated.     

Mutual influences of Pseudomonas aeruginosa and Desulfovibrio desulfuricans on their adhesion to stainless steel

The mutual influences of Pseudomonas aeruginosa PAO1 and Desulfovibrio desulfuricans subsp. desulfuricans (ATCC 29577) on their adhesion to stainless steel were investigated in batch and column experiments. It was found that P. aeruginosa promoted the adhesion of D. desulfuricans under conditions of turbulence, but not under quiescent conditions. The enhancement involved the alignment of most D. desulfuricans along P. aeruginosa cells and was attributed to the additional interaction surface area provided by adhered P. aeruginosa to aligning D. desulfuricans cells. A slightly positive effect of preadhered D. desulfuricans on the adhesion of P. aeruginosa was found. Under condition of laminar flow, substantially better adhesion of D. desulfuricans to confluent P. aeruginosa biofilms than to steel was observed. The mutual influences are discussed in terms of more favorable adhesion energies and the influence of changed hydraulic conditions due to the roughness of P. aeruginosa biofilms.     

Drosophila melanogaster as an animal model for the study of Pseudomonas aeruginosa biofilm infections in vivo

Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3) demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.     

Direct Probing of the Surface Ultrastructure and Molecular Interactions of Dormant and Germinating Spores of Phanerochaete chrysosporium

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus Phanerochaete chrysosporium. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 +/- 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 +/- 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.     

Effects of Contact Time, Pressure, Percent Relative Humidity (%RH), and Material Type on Listeria Biofilm Adhesive Strength at a Cellular Level Using Atomic Force Microscopy (AFM)

Whereas the transfer of Listeria from surfaces to foods and vice versa has been well documented, little is known about the mechanism of bacterial transfer. The objective of this work is to gain a better understanding of the forces involved in listerial biofilms adhesion using atomic force microscopy (AFM). L. monocytogenes Scott A was grown as biofilms on stainless steel surfaces by inoculating stainless steel coupons with Listeria and incubating the coupons for 48 h at 32 °C with a diluted 1:20 tryptic soy broth. After growth, biofilms were equilibrated over saturated salt solutions at a constant relative humidity (%RH) before measurement of adhesion forces using AFM. The effects of contact time, loading force, and biofilm relative humidity (%RH) suggested that neither contact time, loading force nor biofilm %RH had a significant effect on biofilm adhesiveness at a cellular level (P > 0.05). In a second set of experiments, the influence of material type on biofilm adhesiveness was evaluated using two different colloidal probes (SiO₂ and polyethylene). Results showed that the maximum pull-off force and retraction work needed to retract the cantilever for glass (−85.42 nN and 1.610⁻¹⁵ J, respectively) were significantly lower than those of polyethylene (−113.38 nN and 2.7 × 10^–15 J, respectively; P < 0.001). The results of this study suggest that Listeria biofilms adhere more strongly to hydrophobic surfaces than hydrophilic surfaces when measured at a cellular level. These results provide important insights that could lead to new ways to remediate and avoid listerial biofilm formation in the food industry.     

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4–0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.     

Adhesive interactions between voice prosthetic yeast and bacteria on silicone rubber in the absence and presence of saliva

Biofilms on silicone rubber voice prostheses are the major cause for frequent failure and replacement of these devices. The presence of both bacterial strains and yeast has been suggested to be crucial for the development of voice prosthetic biofilms. Adhesive interactions between Candida albicans, Candida krusei, and Candida tropicalis with 14 bacterial strains, all isolated from explanted voice prostheses were investigated in a parallel plate flow chamber. Bacteria were first allowed to adhere to silicone rubber, after which the flow chamber was perfused with yeast, suspended either in saliva or buffer. Generally, when yeast were adhering from buffer and saliva, the presence of adhering bacteria suppressed adhesion of yeast. In saliva, Rothia dentocariosa and Staphylococcus aureus enhanced adhesion of yeast, especially of C. albicans. This study shows that bacterial adhesion mostly reduces subsequent adhesion of yeast, while only a few bacterial strains stimulate adhesion of yeast, provided salivary adhesion mediators are present. Interestingly, different clinical studies have identified R. dentocariosa and S. aureus in biofilms on explanted prostheses of patients needing most frequent replacement, while C. albicans is one of the yeast generally held responsible for silicone rubber deterioration.     

Pattern differentiation in co-culture biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa

Biofilm infections may not simply be the result of colonization by one bacterium, but rather the consequence of pathogenic contributions from several bacteria. Interspecies interactions of different organisms in mixed-species biofilms remain largely unexplained, but knowledge of these is very important for understanding of biofilm physiology and the treatment of biofilm-related infectious diseases. Here, we have investigated interactions of two of the major bacterial species of cystic fibrosis lung microbial communities -Pseudomonas aeruginosa and Staphylococcus aureus- when grown in co-culture biofilms. By growing co-culture biofilms of S. aureus with P. aeruginosa mutants in a flow-chamber system and observing them using confocal laser scanning microscopy, we show that wild-type P. aeruginosa PAO1 facilitates S. aureus microcolony formation. In contrast, P. aeruginosa mucA and rpoN mutants do not facilitate S. aureus microcolony formation and tend to outcompete S. aureus in co-culture biofilms. Further investigations reveal that extracellular DNA (eDNA) plays an important role in S. aureus microcolony formation and that P. aeruginosa type IV pili are required for this process, probably through their ability to bind to eDNA. Furthermore, P. aeruginosa is able to protect S. aureus against Dictyostelium discoideum phagocytosis in co-culture biofilms.     

Successive detection of insulin‐like growth factor‐II bound to receptors on a living cell surface using an AFM

In this study, we have developed a method of mechanical force detection for ligands bound to receptors on a cell surface, both of which are involved in a signal transduction pathway. This pathway is an autocrine pathway, involving the production of insulin‐like growth factor‐II (IGF‐II) and activation of the IGF‐I receptor, involved in myoblast differentiation induced by MyoD in C3H10T1/2 mouse mesenchymal stem cells. Differentiation of C3H10T1/2 was induced with the DNA demethylation agent 5‐azacytidine (5‐aza). The etched AFM tip used in the force detection had a flat surface of which about 10 µm² was in contact with a cell surface. The forces required to rupture the interactions of IGF‐IIs on a cell and anti mouse IGF‐II polyclonal antibody immobilized on an etched AFM tip were measured within 5 days of induction of differentiation. The mean unbinding force for a single paired antibody–ligand on a cell was about 81 pN, which was measured at a force loading rate of about 440 nN/s. The percentage of unbinding forces over 100 pN increased to 32% after 2 days from the addition of 5‐aza to the medium. This method could be used in non‐invasive and successive evaluation of a living cell's behavior. Copyright © 2009 John Wiley & Sons, Ltd.     

DKP对3株病原菌生物膜的抑制作用研究

生物膜的存在使一些由病原菌引发的疾病变得更加难以治疗。经研究发现一种环二肽物质DKP——cyclo(Pro-Phe)能够抑制这3株病原菌(Staphylococcus aureus,Pseudomonas aeruginosa,Candida albicans)生物膜的形成。通过对不同浓度DKP作用下所形成的生物膜进行结晶紫定量、菌落计数分析和结构显微分析表明:在DKP的浓度达到10 mg/ml时,S. aureus和P. aeruginosa的生物膜几乎消失;在DKP的浓度达到12 mg/ml时,C. albicans的生物膜被显著抑制。这一发现为寻找新型的生物膜抑制剂治愈顽固疾病带来了新的希望。     

Micro-cantilever method for measuring the tensile strength of biofilms and microbial flocs

Cohesive strength is an important factor in determining the structure and function of biofilm systems, and cohesive strength plays a key role in our ability to remove or control biofilms in engineered systems. A micro-mechanical device has been developed to directly measure the tensile strength of biofilms and other microbial aggregates. An important feature of this method is the combination of a direct measurement of force with particle separations that occur at a scale comparable to that observed for biofilm systems. The force required to separate an aggregate is determined directly from the deflection of cantilevered glass micropipettes with a 20-40-microm diameter. Combined with an estimate of the cross-sectional area of the aggregate at the point of separation this measurement indicates the cohesive strength of the aggregate. Samples of return activated sludge (RAS) and a Pseudomonas aeruginosa biofilm were tested using the device. The measured cohesive strengths of the RAS flocs ranged from 419 to 206,400 N/m(2), while many of the flocs exceeded the range of measurement of the device. Fragments of P. aeruginosa biofilm had cohesive strengths ranging from 395 to 15,640 N/m(2), with a median value of 3020 N/m(2). The median equivalent diameters of the particles detached from the aggregates were 32 microm for RAS and 30 microm for the P. aeruginosa biofilms.     

Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H(2)O(2) accumulation was transient, reaching a maximal measured value of 1 micro M. Following contact, stimulated degranulation was 20-40% (myeloperoxidase, beta-glucuronidase) and 40-80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H(2)O(2). Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.     

The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment

Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.     

Production of destruxins by coprotrophic Beauveria feline no. 7 strain

Beauveria feline No. 7 strain was isolated from the shrewmouse wool and characterized by production of a complex of destruxins cyclodepsipeptides. The strain was shown to produce significant quatities of destruxin B and pseudodestruxin C. The destruxins were found to be able to inhibit formation of biofilms by Pseudomonas aeruginosa ATCC 27833.     

Observations of binary population biofilms

Biofilm research has focused on studies of undefined mixed microbial populations and, more recently, on investigations of monopopulation biofilms. In the first case, the biofilm is considered a homogeneous mass, ignoring the properties of individual species. The second case concentrates on the properties and processes of one microbial species in the biofilm. This article describes biofilm experiments conducted with monopopulations of Klebsiella pneumoniae and Pseudomonas aeruginosa and with binary populations of K. pneumoniae and P. aeruginosa. Process rates and stoichiometric coefficients were determined for the monopopulation and for the binary population biofilms and evaluated in light of the species distribution in the latter. Results indicate that neither the specific cellular product formation rate nor the glucose-oxygen stoichiometric ratio of K. pneumoniae or P. aeruginosa in the binary biofilm is affected by the presence of the other species. Consequently, species interaction was not observed. Although the specific cellular growth rate of K. pneumoniae is five times that of P. aeruginosa, the former species did not dominate the microbial population in the biofilm. Possible reasons for this unexpected behavior are discussed.     

Adhesion of Aureobasidium pullulans is controlled by uronic acid based polymers and pullulan

Aureobasidium pullulans is a potentially pathogenic microfungus that produces and secretes the polysaccharide pullulan and other biomacromolecules, depending on the microbe's physiological state. The role of these macromolecules in mediating adhesion and attachment were examined. Interfacial forces and adhesion affinities of A. pullulans were probed for early-exponential phase (EEP) and late-exponential phase (LEP) cells, using atomic force microscopy (AFM). Biochemical assays showed that A. pullulans produces both pullulan and a uronic acid based polymer. The pullulan is not produced until the LEP, and it can be removed by treatment with pullulanase. Both adhesion forces between the microbe and the AFM tip (silicon nitride) and attachment of the cells to quartz sand grains were controlled by the density of the uronic acid polymer. Uronic acid polymers doubled in density between the EEP and the LEP and were unaffected by the enzyme pullulanase. Retention to quartz in a packed column was quantified using the collision efficiency (alpha), the fraction of collisions between the microbes, and the sand grains, that result in attachment. Adhesion forces and retention on glass were well correlated, with these values being higher for EEP cells (F(adh) = 7.65 +/-4.67 nN; alpha = 1.15) than LEP (F(adh) = 2.94 +/- 0.75; alpha = 0.49) and LEP + pullulanase cells (F(adh) = 2.33 +/-2.01 nN; alpha = 0.43). Steric interactions alone do not describe the adhesion behavior of this fungus, but they do provide information regarding the length and density of the macromolecules studied.