Application of fluid mechanic and kinetic models to characterize mammalian cell detachment in a radial-flow chamber

The strength of adhesion and dynamics of detachment of murine 3T3 fibroblasts from self-assembled monolayers were measured in a radial-flow chamber (RFC) by applying models for fluid mechanics, adhesion strength probability distributions, and detachment kinetics. Four models for predicting fluid mechanics in a RFC were compared to evaluate the accuracy of each model and the significance of inlet effects. Analysis of these models indicated an outer region at large radial positions consistent with creeping flow, an intermediate region influenced by inertial dampening, and an inner region dominated by entrance effects from the axially-oriented inlet. In accompanying experiments patterns of the fraction of cells resisting detachment were constructed for individual surfaces as a function of the applied shear stress and evaluated by comparison with integrals of both a normal and a log-normal distribution function. The two functions were equally appropriate, yielding similar estimates of the mean strength of adhesion. Further, varying the Reynolds number in the inlet, Re(d), between 630 and 1480 (corresponding to volumetric flow rates between 0.9 and 2.1 mL/s) did not affect the mean strength of adhesion. For these same experiments, analysis of the dynamics of detachment revealed three temporal phases: 1) rapid detachment of cells at the onset of flow, consistent with a first-order homogeneous kinetic model; 2) time-dependent rate of detachment during the first 30 sec. of exposure to hydrodynamic shear, consistent with the first-order heterogeneous kinetic model proposed by Dickinson and Cooper (1995); and 3) negligible detachment, indicative of pseudo-steady state after 60 sec. of flow. Our results provide rigorous guidelines for the measurement of adhesive interactions between mammalian cells and prospective biomaterial surfaces using a RFC. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 616-629, 1997.     

Conspicuous ingestion of Staphylococcus aureus organisms by murine fibroblasts in vitro.

A conspicuous adhesion of Staphylococcus aureus organisms to murine cutaneous fibroblasts and NIH/3T3 cells cultured in vitro and subsequent ingestion of S. aureus organisms by these fibroblasts are described. In the present experimental system, only fibroblasts-adhering S. aureus organisms were efficiently ingested by fibroblasts unlike S. epidermidis and S. saprophyticus. These findings might suggest a correlation between the pathogenesis of S. aureus and its intracellular localization in non-professional phagocytes such as fibroblasts in a special reference to its higher pathogenicity than those of coagulase negative counterparts.     

Fibronectin binding protein A of Staphylococcus aureus can mediate human T lymphocyte adhesion and coactivation

The extracellular matrix protein fibronectin (FN) mediates the adhesion of bacteria as well as T lymphocytes. Mammalian cells express integrins alpha(4)beta(1) and alpha(5)beta(1) as the major FN-binding cell surface receptors. Bacteria such as Staphylococcus aureus, also express FN-binding receptors that are important for adherence to host tissue and initiation of infection. The S. aureus FN-binding protein, FnbpA, has been previously identified, and recombinant proteins that correspond to distinct functional regions of this protein have been made. Three recombinant truncated forms of FnbpA, rFnbpA(37-881), rFnbpA(37-605), and rFnbpA(620-881), were examined for effects on in vitro adhesion and coactivation of human T lymphocytes. These proteins, when coimmobilized with anti-CD3 mAb, activated T lymphocyte proliferation. The coactivation signal generated by the rFnbpA proteins required medium containing serum with FN. Furthermore, the costimulatory signal could be restored in FN-depleted serum when the rFnbpAs were preloaded with soluble FN. Monoclonal Ab blocking studies revealed that integrin alpha(5)beta(1) is the major receptor responsible for the rFnbpA costimulatory signal. Shear flow cell detachment assays confirmed that lymphocytes can bind to FN captured by the rFnbpA proteins. These results suggest that the S. aureus rFnbpA can interact with integrin alpha(5)beta(1) via an FN bridge to mediate adhesion and costimulatory signals to T lymphocytes.     

Microjet impingement followed by scanning electron microscopy as a qualitative technique to compare cellular adhesion to various biomaterials

Adhesion of cells to biomaterial surfaces is one of the major factors which mediates their biocompatibility. Quantitative or qualitative cell adhesion measurements would be useful for screening new implant materials. Microjet impingement has been evaluated by scanning electron microscopy, to determine to what extent it measures cell adhesion. The shear forces of the impingement, on the materials tested here, are seen to be greater than the cohesive strength of the cells in the impinged area, causing their rupture. The cell bodies are removed during impingement, leaving the sites of adhesion and other cellular material behind. Thus the method is shown not to provide quantification of cell adhesion forces for the metals and culture plastic tested. It is suggested that with highly adherent biomaterials, the distribution and patterns of these adhesion sites could be used for qualitative comparisons for screening of implant surfaces.     

Endothelial cell migration on RGD-peptide-containing PEG hydrogels in the presence of sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P) is a potent chemokinetic agent for endothelial cells that is released by activated platelets. We previously developed Arg-Gly-Asp (RGD)-containing polyethylene glycol biomaterials for the controlled delivery of S1P to promote endothelialization. Here, we studied the effects of cell adhesion strength on S1P-stimulated endothelial cell migration in the presence of arterial levels of fluid shear stress, since an upward shift in optimal cell adhesion strengths may be beneficial for promoting long-term cell adhesion to materials. Two RGD peptides with different integrin-binding specificities were added to the polyethylene glycol hydrogels. A linear RGD bound primarily to β₃ integrins, whereas a cyclic RGD bound through both β₁ and β₃ integrins. We observed increased focal adhesion formation and better long-term adhesion in flow with endothelial cells on linear RGD peptide, versus cyclic RGD, even though initial adhesion strengths were higher for cells on cyclic RGD. Addition of 100 nM S1P increased cell speed and random motility coefficients on both RGD peptides, with the largest increases found on cyclic RGD. For both peptides, much of the increase in cell migration speed was found for smaller cells (<1522 μm² projected area), although the large increases on cyclic RGD were also due to medium-sized cells (2288-3519 μm²). Overall, a compromise between high cell migration rates and long-term adhesion will be important in the design of materials that endothelialize after implantation.     

Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 x 10(6) cells cm(-2) was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s(-1)), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as "bacterial retention" rather than "bacterial adhesion".     

Cellular and bacterial toxicities of topical antimicrobials

Cellular and bacterial toxicities of four commonly used topical antimicrobials (1% povidone-iodine, 3% hydrogen peroxide, 0.25% acetic acid, and 0.5% sodium hypochlorite) were assayed in vitro using cultures of human fibroblasts and Staphylococcus aureus. All agents tested at full strength killed 100 percent of exposed fibroblasts. Fibroblast toxicity exceeded bacterial toxicity with serial dilutions of hydrogen peroxide and acetic acid. Dilutions of povidone-iodine (1:1000) and sodium hypochlorite (1:100) were identified where no fibroblast toxicity occurred while full bactericidal activity persisted.     

Platelets as a source of fibroblast growth-promoting activity

Evidence is presented that platelets are an enriched source of growth-promoting activity for 3T3 cells, an established line of mouse fibroblasts. Rat or human serum stimulates 3T3 cell growth more than autologous plasma; this increased activity is at least partly due to the release of a growth-promoting activity from platelets disrupted during the clotting process. Platelet extract stimulation of 3T3 cell growth is approx. 5 times greater than an equal amount of autologous serum. This platelet growth activity is (1) pH-stable; (2) pepsin-labile; (3) heat-stable at 56 °C; (4) non-dialysable, and (5) similar in size to the serum 3T3 growth-promoting molecule. 3T3 cells transformed by Simian virus 40 show an equivalent stimulation by the platelet solution and serum.     

On the use of ultrasounds to quantify the longitudinal threshold force to detach osteoblastic cells from a conditioned glass substrate

Cell adhesion on a biomaterial is an important phase of the cell-material interactions and the quality of this phase governs the success of the biomaterial integration. Understanding of the phenomena of cell adhesion and in particular understanding of cell adhesion on biomaterials is of crucial importance for the development of new biomaterials with excellent biocompatibility. One of the physical quantitative indexes to evaluate the quality of cell-material adhesion is its strength. Determining the strength of adhesive bonds requires applying external forces to the cells. Thus, a few methods have been developed to evaluate the strength of cell-material adhesion (micropipette, microplates, microcantilever, ...). These methods apply shear forces on adherent cells. The aim of our work is the development of a new ultrasonic characterization method of cellular adhesion on substrates. With our method, longitudinal acoustic waves are applied on cell culture to impose a longitudinal strain on cells. Only the cells subjected to a sufficient level of strain will be detached from the substrate. The idea is to correlate cell detachment rate to the longitudinal strain threshold supported by cells. From this result, we can deduce the critical force just sufficient to detach the cell. This global method can be adapted for different cell types and for different substrates. This method can provide an evaluation of the effect of functionalization on substrates. The technique is investigated for the 200 kHz ultrasound frequency. An insonificator adapted to the use of cell culture boxes was developed and calibrated. Tests were carried out on a glass substrate with or without biological conditioning. We used the MC3T3-E1 osteoblastic cell line. Our results to date provide the value of the necessary force to detach with reproducibility osteoblastic cells from glass.     

The combined effects of plasma and hydrogel coating on adhesion of Staphylococcus epidermidis and Staphylococcus aureus to polyurethane catheters

Abstract The adhesion of three Staphylococcus epidermidis and three S. aureus clinical isolates, to uncoated and hydrogel-coated polyurethane catheters was tested, following pretreatment of catheters with human plasma. Plasma significantly decreased the adhesion of S. epidermidis strains to uncoated polyurethane catheters, but had no significant effect on the adhesion to hydrogel-coated catheters. The influence of plasma on adhesion of S. aureus strains to catheters was strain dependent. Plasma significantly increased the adhesion of one strain (SA6) to uncoated catheters. For two other strains (SA3 and SA14) plasma produced no clear effect on their adhesion to uncoated catheters; adhesion values for each strain showed either a small but significant increase or a replicate-dependent increase or decrease. However, plasma significantly increased the adhesion of all S. aureus strains to hydrogel-coated polyurethane catheters. Overall, with the exception of one batch culture of S. epidermidis strain SE3 tested, attachment to plasma-treated hydrogel-coated catheters was statistically significantly lower, by up to 85%, than attachment to plasma-treated uncoated catheters for both S. epidermidis and S. aureus .     

The effect of cross-bridge clustering and head-head competition on the mechanical response of skeletal muscle under equilibrium conditions.

The effect of cross-bridge clustering and head-head competition on the mechanical response of skeletal muscle under equilibrium conditions is considered. For this purpose, the recent multiple site equilibrium cross-bridge model of Schoenberg (Schoenberg, M., 1985, Biophys. J., 48:467-475) is extended in accordance with the formalism of T.L. Hill (1974, Prog. Biophys, Mol. Biol., 28:267-340) to consider the case where groups of independent cross-bridge heads compete with each other for binding to multiple actin sites. Cooperative behavior between heads is not allowed. Computations indicate that for the double-headed cross-bridge with two independent equivalent heads, the time course of force decay after a stretch is similar to that for the single-headed cross-bridge; that is, the rate constant for force decay is approximately equal to the cross-bridge head detachment rate constant. The results also show that the force decay after a stretch becomes slower than the detachment rate constant of a single head when cross-bridge heads bind adjacently in clusters so that competition between heads for binding to the available actin sites increases. However, if one assumes that the detachment rate constant of an unstrained head in a fiber is comparable to that of an S1 molecule in solution, this effect is not large enough to explain why some of the rate constants for force decay after a stretch in rigor, or in the presence of ATP analogues such as adenyl-5'-yl imidodiphosphate, appear to be significantly slower than the detachment rate constant of S1 from actin in solution.     

Quantifying the tensile strength of microbial mats grown over noncohesive sediments

Biofilms in marine and fluvial environments can comprise strong bacterial and diatom mats covering large areas of the bed and act to bind sediments. In this case the bed material becomes highly resistant to shear stresses applied by the overlying fluid motion and detachment, when it does occur, is manifest in patches of biofilm of the order cm(2) being entrained into the flow. This article is the first to report tensile test data specific to the centimeter scale using moist biofilm/sediment composite materials; the strain (ε)-stress (σ) relationships permit quantification of the elasticity (Young's modulus, E) and cohesive strength of each specimen. Specifically, we compare the mechanical strength of cyanobacterial biofilm-only samples to that of biofilm cultured over sediment samples (glass beads or natural sands of d ~ 1 mm) for up to 8 weeks. The range of tensile strength (1,288-3,283 Pa) for composite materials was up to three times higher than previous tensile tests conducted at smaller scale on mixed culture biofilm [Ohashi et al. (1999) Water Sci Technol 39:261-268], yet of similar range to cohesive strength values recorded on return activated sludge flocs [RAS; Poppele and Hozalski (2003) J Microbiol Methods 55:607-615]. Composite materials were 3-6 times weaker than biofilm-only samples, indicating that adhesion to sediment grains is weaker than cohesion within the biofilm. Furthermore, in order to relate the tensile test results to the more common in-situ failure of bio-mats due to shear flow, controlled erosion experiments were conducted in a hydraulic flume with live fluid flow. Here, the fluid shear stress causing erosion was 3 orders of magnitude lower than tensile stress; this highlights both the problem of interpreting material properties measured ex-situ and the need for a better mechanistic model of bio-mat detachment.     

Evaluation of biocompatibility of apatite-wollastonite ceramics in fibroblast cultures

The aim of this work has been to test the biocompatibility of four bioactive, gel derived glass-ceramic materials of CaO-PO2-SiO2 system, modified by addition of boron, aluminum and magnesium compounds. We have examined the growth, collagen synthesis, adhesion and morphology of NRK rat fibroblasts cultured in direct and indirect contact with biomaterials. The growth of cells cultures has been quantified by two methods: [3H]thymidine incorporation and direct counting of cells. The level of collagen synthesis has been used as a parameter describing metabolic activity of cells. Cellular morphology has been assessed following 24 h and 4 days of culturing cells on biomaterials by using SEM and confocal microscopy, respectively. Additionally, in order to obtain information about the attachment of cells to substratum the presence of focal contacts has been examined. The results of all the experiments have demonstrated that none of the materials under study significantly altered cellular functions that were tested. This indicates that additions of MgO, Al2O3 and B2O3 have not induced cytotoxicity of the materials under study. This qualifies them for further in vivo experiments.     

Sensing surfaces: Challenges in studying the cell adhesion process and the cell adhesion forces on biomaterials

A major turning point in the biomaterials field would be to develop tools that can offer greater insight into cell behaviour on material surfaces. Obtaining this information is very important for the development of long-term implantable materials because it can aid in improving cell adhesion and proliferation properties. The amalgamation of multiple disciplines has already produced many interesting techniques and approaches for the characterisation of cell adhesion processes and force adhesion strength determination on biomaterials. In this review, the authors provide an overview of the recent techniques developed for the noninvasive in situ study of the adhesion process as well as systems that allow the measurement of adhesion force strengths over biomaterials. Techniques based on light internal reflection, electrochemical impedance spectroscopy, and the quartz crystal microbalance (QCM) are discussed for their capabilities in investigating the cell adhesion process. Conversely, techniques such as flow cells, centrifugation, and cytodetachers are presented for the adhesion force measurement. An emphasis on atomic force microscopy (AFM) will demonstrate its ability to probe both the cell adhesion process and cell adhesion force, depending on the approach used. A discussion is followed on the strengths and/or weaknesses of these techniques. Finally, new trends and possible long-term directions for determining both adhesion process and force are highlighted.     

Detachment characteristics and oxacillin resistance of Staphyloccocus aureus biofilm emboli in an in vitro catheter infection model

Catheter-related bloodstream infections due to Staphylococcus aureus are of increasing clinical importance. The pathophysiological steps leading to colonization and infection, however, are still incompletely defined. We observed growth and detachment of S. aureus biofilms in an in vitro catheter-infection model by using time-lapse microscopy. Biofilm emboli were characterized by their size and their susceptibility for oxacillin. Biofilm dispersal was found to be a dynamic process in which clumps of a wide range of diameters detach. Large detached clumps were highly tolerant to oxacillin compared with exponential-phase planktonic cultures. Interestingly, the degree of antibiotic tolerance in stationary-phase planktonic cultures was equal to that in the large clumps. The mechanical disruption of large clumps reduced the minimal bactericidal concentration (MBC) by more than 1,000 times. The MBC for whole biofilm effluent, consisting of particles with an average number of 20 bacteria was 3.5 times higher than the MBC for planktonic cultures. We conclude that the antibiotic resistance of detached biofilm particles depends on the embolus size and could be attributed to nutrient-limited stationary-phase physiology of cells within the clumps. We hypothesize that the detachment of multicellular clumps may explain the high rate of symptomatic metastatic infections seen with S. aureus.     

Cell cycle perturbations in normal and transformed fibroblasts caused by detachment from the substratum

Exponentially growing, anchorage-dependent fibroblasts were impeded in their progress through the cell cycle as a result of brief trypsinization from the substratum followed by replating. Untransformed mouse (3T3, clone A31), hamster (CHEF/18-1) and human (FS2) fibroblasts were partially inhibited from entering the DNA synthetic (S) phase of the cell cycle for 8 or 12 hours after detachment, even though the cells reattached within an hour of replating and attained a spread morphology 5 or 8 hours later. The decline in the proportion of cells in S phase was accompanied accumulation of cells in G1 as measured by autoradiography and flow microfluorimetry. Cells removed from the substratum by EDTA alone showed identical disturbances of exponential growth. These cell cycle perturbations could be a result of the detachment per se, as opposed to the rounded morphology. Synchronized A31 cells, exposed to colcemid or cytochalasin B for two hours, were not delayed in their entry into S, whereas trypsinization delayed S phase entry by 4 to 5 hours. These drugs disrupt the cytoskeleton without causing detachment. Isotope incorporation experiments revealed no decreases in the rates of protein or RNA synthesis following replating. However, exponentially growing A31 cells, treated for 2 hours with an inhibitor of protein synthesis behaved similarly to those briefly detached from their substratum: 7 hours after treatment, there were fewer cells in S and more cells in G1 relative to untreated cells. Brief treatment with an inhibitor of hn-RNA synthesis did not alter the cell cycle distribution of these fibroblasts. Three tumorogenic A31 derivatives were less affected by brief detachment from the substratum than were the untransformed cells. The derivative exhibiting the least in vitro growth control (an SV-40 transformant) showed the least sensitivity to trypsinization, while that derivative having the most in vitro growth control (a Moloney sarcoma virus transformant) was most sensitive. A chemically [benzo(a)pyrene] transformed derivative gave intermediate results with respect to both growth control and sensitivity to detachment from the substratum. The results suggest that as yet unidentified protein(s) necessary for the normal transit through G1 may be quite sensitive to the presence of an anchoring substratum.     

The Cytoskeleton Regulates Cell Attachment Strength

Quantitative information about adhesion strength is a fundamental part of our understanding of cell-extracellular matrix (ECM) interactions. Adhesion assays should measure integrin-ECM bond strength, but reports now suggest that cell components remain behind after exposure to acute force for radial shear assays in the presence of divalent cations that increase integrin-ECM affinity. Here, we show that focal adhesion proteins FAK, paxillin, and vinculin but not the cytoskeletal protein actin remain behind after shear-induced detachment of HT1080 fibrosarcoma cells. Cytoskeletal stabilization increased attachment strength by eightfold, whereas cross-linking integrins to the substrate only caused a 1.5-fold increase. Reducing temperature—only during shear application—also increased attachment strength eightfold, with detachment again occurring between focal adhesion proteins and actin. Detachment at the focal adhesion-cytoskeleton interface was also observed in mouse and human fibroblasts and was ligand-independent, highlighting the ubiquity of this mode of detachment in the presence of divalent cations. These data show that the cytoskeleton and its dynamic coupling to focal adhesions are critically important for cell adhesion in niche with divalent cations.     

Cation Type Specific Cell Remodeling Regulates Attachment Strength

Single-molecule experiments indicate that integrin affinity is cation-type-dependent, but in spread cells integrins are engaged in complex focal adhesions (FAs), which can also regulate affinity. To better understand cation-type-dependent adhesion in fully spread cells, we investigated attachment strength by application of external shear. While cell attachment strength is indeed modulated by cations, the regulation of integrin-mediated adhesion is also exceedingly complex, cell specific, and niche dependent. In the presence of magnesium only, fibroblasts and fibrosarcoma cells remodel their cytoskeleton to align in the direction of applied shear in an α₅-integrin/fibronectin-dependent manner, which allows them to withstand higher shear. In the presence of calcium or on collagen in modest shear, fibroblasts undergo piecewise detachment but fibrosarcoma cells exhibit increased attachment strength. These data augment the current understanding of force-mediated detachment by suggesting a dynamic interplay in situ between cell adhesion and integrins depending on local niche cation conditions.     

Adhesion dynamics, morphology, and organization of 3T3 fibroblast on chitosan and its derivative: the effect of O-carboxymethylation

Chitosan and O-carboxymethylchitosan (OCMCS) have been proved to have biocompatibility and have been extensively researched in the field of biomaterials. In this study, Confocal-reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast imaging was used to investigate the adhesion contact dynamics of 3T3 fibroblasts on chitoan and OCMCS surface-modified silica coverslips. The C-RICM results demonstrate that the weak cell contact forms on OCMCS surface while a much stronger contact area forms on the chitosan surface. 3T3 fibroblasts are found to spread randomly with spindlelike morphology on the chitosan surface, while they exhibit elongated morphology and align on the OCMCS surface. It is believed that fibroblast behaviors such as migration, spreading with an elongated morphology, and alignment on the OCMCS surface are correlated with the weak cell contact. The mechanisms to form cell adhesion contact on chitosan and OCMCS were discussed.     

Characteristics of Staphylococcus aureus, isolated from airways of cystic fibrosis patients, and their small colony variants

The colonization of respiratory tract by Staphylococcus aureus is a frequent feature of cystic fibrosis (CF), especially in pediatric patients. The formation of small colony variants (SCVs), which produce reduced amounts of alpha-toxin, is one of the proposed ways of staphylococcal accommodation in an intracellular niche. The aim of the present study was to compare some properties of S. aureus SCVs and their parent strains. A site-directed S. aureus hemB mutant and parent strain 8325-4 were included in the study (control pair). Normal and SCV strain pairs from CF patients as well as control strains were tested for the susceptibility to defensins, killing activity of professional phagocytes and adhesion to A549 cell line. Because S. aureus are exposed to many cationic proteins in the host, we challenged a clinical isolate with minimal subinhibitory concentration (subMIC) of protamine and found that hemin and menadione auxotrophic SCVs emerged. SCVs were more resistant than normal strains to protamine but not to dermaseptin. The susceptibility to the bactericidal activity of magainin was the same for normal and SCV strains. The protamine resistance of normal as well as SCVs was strongly enhanced by high salt concentration. The adhesion of some SCVs to A549 cells was higher than adhesion of parental strains. However, the number of adherent bacteria (SCVs) was diminished in the presence of hemin for hemin auxotrophs. The uptake of SCVs by granulocytes was lower than ingestion of normal strains, but SCVs were killed with equal or greater potency. SCVs are adapted to intracellular survival and persistence in the host under certain circumstances. The ability to form a variant subpopulation affords S. aureus additional survival options.