A Comparison of the Adaptive Response of Staphylococcus aureus vs. Streptococcus mutans and the Development of Chlorhexidine Resistance.

Antimicrobials with nonselective antibacterial efficacy such as chlorhexidine can be effective in reducing biofilm, but bear the risk of inducing resistance in specific bacteria. In clinical practice, bacteria such as Staphylococcus aureus have been found resistant to chlorhexidine, but other bacteria, including Streptococcus mutans, have largely remained susceptible to chlorhexidine despite its widespread use in oral healthcare. Here, we aim to forward a possible reason as to why S. aureus can acquire resistance against chlorhexidine, while S. mutans remains susceptible to chlorhexidine. Measurement of surface-enhanced fluorescence indicated that chlorhexidine caused gradual, but irreversible deformation to adhering green fluorescent S. aureus due to irreparable damage to the cell wall. Concurrently, the metabolic activity of adhering staphylococci was higher than of planktonic bacteria, suggesting efflux mechanisms may have been activated upon cell wall deformation, impeding the buildup of a high chlorhexidine concentration in the cytoplasm and therewith stimulating the development of chlorhexidine resistance in S. aureus. Exposure of S. mutans to chlorhexidine caused immediate, but reversible deformation in adhering streptococci, indicative of rapid self-repair of cell wall damage done by chlorhexidine. Due to cell wall self-repair, S. mutans will be unable to effectively reduce the chlorhexidine concentration in the cytoplasm causing solidification of the cytoplasm. In line, no increased metabolic activity was observed in S. mutans during exposure to chlorhexidine. Therewith, self-repair is suicidal and prevents the development of a chlorhexidine-resistant progeny in S. mutans.

Role of adhesion forces in mechanosensitive channel gating in Staphylococcus aureus adhering to surfaces.

Mechanosensitive channels in bacterial membranes open or close in response to environmental changes to allow transmembrane transport, including antibiotic uptake and solute efflux. In this paper, we hypothesize that gating of mechanosensitive channels is stimulated by forces through which bacteria adhere to surfaces. Hereto, channel gating is related with adhesion forces to different surfaces of a Staphylococcus aureus strain and its isogenic ΔmscL mutant, deficient in MscL (large) channel gating. Staphylococci becoming fluorescent due to uptake of calcein, increased with adhesion force and were higher in the parent strain (66% when adhering with an adhesion force above 4.0 nN) than in the ΔmscL mutant (40% above 1.2 nN). This suggests that MscL channels open at a higher critical adhesion force than at which physically different, MscS (small) channels open and contribute to transmembrane transport. Uptake of the antibiotic dihydrostreptomycin was monitored by staphylococcal killing. The parent strain exposed to dihydrostreptomycin yielded a CFU reduction of 2.3 log-units when adhering with an adhesion force above 3.5 nN, but CFU reduction remained low (1.0 log-unit) in the mutant, independent of adhesion force. This confirms that large channels open at a higher critical adhesion-force than small channels, as also concluded from calcein transmembrane transport. Collectively, these observations support our hypothesis that adhesion forces to surfaces play an important role, next to other established driving forces, in staphylococcal channel gating. This provides an interesting extension of our understanding of transmembrane antibiotic uptake and solute efflux in infectious staphylococcal biofilms in which bacteria experience adhesion forces from a wide variety of surfaces, like those of other bacteria, tissue cells, or implanted biomaterials.

Physico-chemistry from initial bacterial adhesion to surface-programmed biofilm growth.

Biofilm formation is initiated by adhesion of individual bacteria to a surface. However, surface adhesion alone is not sufficient to form the complex community architecture of a biofilm. Surface-sensing creates bacterial awareness of their adhering state on the surface and is essential to initiate the phenotypic and genotypic changes that characterize the transition from initial bacterial adhesion to a biofilm. Physico-chemistry has been frequently applied to explain initial bacterial adhesion phenomena, including bacterial mass transport, role of substratum surface properties in initial adhesion and the transition from reversible to irreversible adhesion. However, also emergent biofilm properties, such as production of extracellular-polymeric-substances (EPS), can be surface-programmed. This review presents a four-step, comprehensive description of the role of physico-chemistry from initial bacterial adhesion to surface-programmed biofilm growth: (1) bacterial mass transport towards a surface, (2) reversible bacterial adhesion and (3) transition to irreversible adhesion and (4) cell wall deformation and associated emergent properties. Bacterial transport mostly occurs from sedimentation or convective-diffusion, while initial bacterial adhesion can be described by surface thermodynamic and Derjaguin-Landau-Verwey-Overbeek (DLVO)-analyses, considering bacteria as smooth, inert colloidal particles. DLVO-analyses however, require precise indication of the bacterial cell surface, which is impossible due to the presence of bacterial surface tethers, creating a multi-scale roughness that impedes proper definition of the interaction distance in DLVO-analyses. Application of surface thermodynamics is also difficult, because initial bacterial adhesion is only an equilibrium phenomenon for a short period of time, when bacteria are attached to a substratum surface through few surface tethers. Physico-chemical bond-strengthening occurs in several minutes leading to irreversible adhesion due to progressive removal of interfacial water, conformational changes in cell surface proteins, re-orientation of bacteria on a surface and the progressive involvement of more tethers in adhesion. After initial bond-strengthening, adhesion forces arising from a substratum surface cause nanoscopic deformation of the bacterial cell wall against the elasticity of the rigid peptidoglycan layer positioned in the cell wall and the intracellular pressure of the cytoplasm. Cell wall deformation not only increases the contact area with a substratum surface, presenting another physico-chemical bond-strengthening mechanism, but is also accompanied by membrane surface tension changes. Membrane-located sensor molecules subsequently react to control emergent phenotypic and genotypic properties in biofilms, most notably adhesion-associated ones like EPS production. Moreover, also bacterial efflux pump systems may be activated or mechano-sensitive channels may be opened upon adhesion-induced cell wall deformation. The physico-chemical properties of the substratum surface thus control the response of initially adhering bacteria and through excretion of autoinducer molecules extend the awareness of their adhering state to other biofilm inhabitants who subsequently respond with similar emergent properties. Herewith, physico-chemistry is not only involved in initial bacterial adhesion to surfaces but also in what we here propose to call "surface-programmed" biofilm growth. This conclusion is pivotal for the development of new strategies to control biofilm formation on substratum surfaces, that have hitherto been largely confined to the initial bacterial adhesion phenomena.

Adhesion force sensing and activation of a membrane-bound sensor to activate nisin efflux pumps in Staphylococcus aureus under mechanical and chemical stresses.

Nisin-associated-sensitivity-response-regulator (NsaRS) in Staphylococcus aureus is important for its adhesion to surfaces and resistance against antibiotics, like nisin. NsaRS consists of an intra-membrane-located sensor NsaS and a cytoplasmatically-located response-regulator NsaR, which becomes activated upon receiving phosphate groups from the intra-membrane-located sensor. HYPOTHESIS: The intra-membrane location of the NsaS sensor leads us to hypothesize that the two-component NsaRS system not only senses "chemical" (nisin) but also "mechanical" (adhesion) stresses to modulate efflux of antibiotics from the cytoplasm. EXPERIMENTS: NsaS sensor and NsaAB efflux pump transcript levels in S. aureus SH1000 adhering to surfaces exerting different adhesion forces were compared, in presence and absence of nisin. Adhesion forces were measured using single-bacterial contact probe atomic force microscopy. FINDINGS: Gene expression became largest when staphylococci experienced strong adhesion forces combined with nisin-presence and the two-component NsaRS response to antibiotics was enhanced at a stronger adhesion force. This confirms that the intra-membrane-located sensor NsaS senses both chemical and mechanical stresses to modulate antibiotic clearance through the NsaAB efflux pump. This finding creates better understanding of the antibiotic resistance of bacteria adhering to surfaces and, in the fight against antibiotic-resistant pathogens, may aid development of advanced biomaterials on which bacterial efflux pumps are not activated.

Surface enhanced fluorescence and nanoscopic cell wall deformation in adhering Staphylococcus aureus upon exposure to cell wall active and non-active antibiotics.

In infections, bacteria often adhere to surfaces and become deformed by the forces with which they adhere. Nanoscopic cell wall deformation defines bacterial responses to environmental conditions and is likely influenced by antibiotics. Here, staphylococcal cell wall deformation upon exposure to cell wall active and non-active antibiotics or their combinations is compared for two green-fluorescent (GFP) isogenic Staphylococcus aureus strains adhering to a gold surface, of which one lacks peptidoglycan cross-linking. Exposure to cell wall active antibiotics caused greater cell wall deformation than a buffer control in the GFP parent and in the Δpbp4GFP isogenic mutant, as measured by surface-enhanced-fluorescence. Cell wall non-active antibiotics only yielded greater deformation than a buffer control in the parent strain, while combinations of cell wall active and non-active antibiotics did not cause greater cell wall deformation. 3D-analysis of the impact of adhesion forces and Young's moduli of the cell wall, both measured using atomic force microscopy, led to the conclusion that increased deformation was mainly due to cell wall weakening and not due to the effects of antibiotics on adhesion forces. Interactions between bacteria and antibiotics are mostly studied using planktonic bacteria, while during infection, bacteria are in an adhering state that deforms their cell wall and therewith influences their adaptive responses. We anticipate that the demonstration of cell wall weakening in adhering bacteria under the influence of antibiotics and the role of peptidoglycan herein will aid in the development of new antibiotics. Surface-enhanced-fluorescence may accordingly develop into a new, highly-sensitive method for diagnosing antibiotic-resistant bacteria.