Pseudomonas aeruginosa biofilm formation and slime excretion on antibiotic-loaded bone cement

Background Infection is an infrequent but serious complication of prosthetic joint surgery. These infections will usually not clear until the implant is removed and re-implantation has a high failure rate, especially when Pseudomonas aeruginosa is involved. Material and methods We examined Pseudomonas aeruginosa biofilm formation on plain and gentamicin-loaded bone cement with confocal scanning laser microscopy (CSLM). Two different stains were applied in order to visualize and quantify the distribution of bacterial cells and extracellular polymeric substances (slime) from the bone cement surface to the top of the biofilm. Staining with LIVE/DEAD viability stain differentiated between live and dead bacteria within the biofilm, and slime production was evaluated after staining with Calcofluor white. Results CSLM showed that the biofilm was a nonuniform structure of variable thickness, with differences in local bacterial cell and slime densities. Incorporation of gentamicin in bone cement resulted in a 44% reduction in bacterial viability, while the slime density increased significantly. In addition, conventional plate counting showed the development of small-colony variants on gentamicin-loaded bone cement with a decreased sensitivity for gentamicin (MIC: 8 mg/L), as compared with normal-sized colonies taken from plain and gentamicin-loaded bone cement (MIC: 3 mg/L). The enhanced slime production on antibiotic-loaded bone cement, together with the formation of small-colony variants, resulted in decreased susceptibility to antibiotics-probably concomitant with the onset of persistent and relapsing infections. Interpretation In the clinical situation, our findings help to explain the frequent re-implantation failure of joint replacements infected with P. aeruginosa when the procedure has been performed using antibiotic-loaded bone cement

How Do Bacteria Know They Are on a Surface and Regulate Their Response to an Adhering State?

Bacteria adhere to virtually all natural and synthetic surfaces [1,2]. Although there are a number of different reasons as to why bacteria adhere to a surface, the summarizing answer is brief: ‘‘Adhesion to a surface is a survival mechanism for bacteria’’. Nutrients in aqueous environments have the tendency to accumulate at surfaces [1,3], giving adhering bacteria a benefit over free floating, so-called planktonic ones. This is why mountain creeks may contain crystal clear, drinkable water, while stepping stones underneath the water may be covered with a slippery film of adhering microbes. In the oral cavity, adhesion to dental hard and soft tissues is life-saving to the organisms, because microbes that do not manage to adhere and remain planktonic in saliva are swallowed with an almost certain death in the gastrointestinal tract. Bacterial adhesion is generally recognized as the first step in biofilm formation, and for the human host, the ability of

Surface thermodynamic homeostasis of salivary conditioning films through polar–apolar layering

Salivary conditioning films (SCFs) form on all surfaces exposed to the oral cavity and control diverse oral surface phenomena. Oral chemotherapeutics and dietary components present perturbations to SCFs. Here we determine the surface energetics of SCFs through contact angle measurements with various liquids on SCFs following perturbations with a variety of chemotherapeutics as well as after renewed SCF formation. Sixteen-hour SCFs on polished enamel surfaces were treated with a variety of chemotherapeutics, including toothpastes and mouthrinses. After treatment with chemotherapeutics, a SCF was applied again for 3 h. Contact angles with four different liquids on untreated and treated SCF-coated enamel surfaces were measured and surface free energies were calculated. Perturbations either caused the SCF to become more polar or more apolar, but in all cases, renewed SCF formation compensated these changes. Thus, a polar SCF attracts different salivary proteins or adsorbs proteins in a different conformation to create a more apolar SCF surface after renewed SCF formation and vice versa for apolar SCFs. This polar–apolar layering in SCF formation presents a powerful mechanism in the oral cavity to maintain surface thermodynamic homeostasis—defining oral surface properties within a narrow, biological range and influencing chemotherapeutic strategies. Surface chemical changes brought about by dietary or chemotherapeutic perturbations to SCFs make it more polar or apolar, but new SCFs are rapidly formed compensating for changes in surface energetics

Biofilm development in time on a silicone voice prosthesis:A case study

Voice prostheses from silicone elastomers become rapidly colonised by a mixed biofilm of bacteria and yeasts. In this study, microorganisms were isolated from biofilms on explanted prostheses after having been in place for various time intervals ranging from 1 to 67 d. The isolates were examined for their identity, adhesion to hexadecane and electrophoretic mobility. Bacteria from early (shorter than 8 d) and late (longer than 8 d) explants could not be classified according to their taxonomy, hydrophobicity or electrophoretic mobility. However, the yeasts clearly revealed a dominance of only hydrophilic Candida albicans isolates from early explants and only hydrophobic C. tropicalis isolates from late explants. These findings may be of significance for the development of strategies to control mixed biofilms on biomaterials.</p

Emergent heterogeneous microenvironments in biofilms: substratum surface heterogeneity and bacterial adhesion force-sensing

Phenotypically-heterogeneous micro-environments emerge as biofilms mature across different environments. Phenotypic-heterogeneity in biofilm sub-populations not obeying quorum sensing-dictated, collective group-behavior, may be considered as a strategy allowing non-conformists to survive hostile conditions. Heterogeneous phenotype development has been amply studied with respect to gene expression and genotypic changes, but 'biofilm genes' responsible for pre-programmed development of heterogeneous micro-environments in biofilms have never been discovered. Moreover, the question of what triggers the development of phenotypically-heterogeneous micro-environments has never been addressed. The definition of biofilms as 'surface-adhering and surface-adapted' microbial communities contains the word 'surface' twice. This leads us to hypothesize that phenotypically-heterogeneous micro-environments in biofilms develop as an adaptive response of initial colonizers to their adhering state, governed by the forces through which they adhere to a substratum surface. No surface is entirely homogeneous, while adhering bacteria can substantially contribute to stochastically occurring surface heterogeneity. Accordingly, bacterial adhesion forces sensed by initial colonizers differ across a substratum surface, leading to differential mechanical deformation of the cell wall and membrane, where many environmental sensors are located. Bacteria directly adhering to heterogeneous substratum domains therewith formulate their own local responses to their adhering state and command non-conformist behavior, leading to phenotypically-heterogeneous micro-environments in biofilms

Comparative anatomical dimensions of the complete human and porcine spine

New spinal implants and surgical procedures are often tested pre-clinically on human cadaver spines. However, the availability of fresh frozen human cadaver material is very limited and alternative animal spines are more easily available in all desired age groups, and have more uniform geometrical and biomechanical properties. The porcine spine is said to be the most representative model for the human spine but a complete anatomical comparison is lacking. The goal of this descriptive study was to compare the anatomical dimensions of the cervical, thoracic, and lumbar vertebrae of the human and porcine spine in order to determine whether the porcine spine can be a representative model for the human spine. CT scans were made of 6 human and 6 porcine spines, and 16 anatomical dimensions were measured per individual vertebrae. Comparisons were made for the absolute values of the dimensions, for the patterns of the dimensions within four spinal regions, and normalised values of the dimensions within each individual vertebra. Similarities were found in vertebral body height, shape of the end-plates, shape of the spinal canal, and pedicle size. Furthermore, regional trends were comparable for all dimensions, except for spinal canal depth and spinous processus angle. The size of the end-plates increased more caudally in the human spine. Relating the dimensions to the size of the vertebral body, similarities were found in the size of the spinal canal, the transverse processus length, and size of the pedicles. Taking scaling differences into account, it is believed that the porcine spine can be a representative anatomical model for the human spine in specific research questions

A biodegradable gentamicin-hydroxyapatite-coating for infection prophylaxis in cementless hip prostheses

A degradable, poly (lactic-co-glycolic acid) (PLGA), gentamicin-loaded prophylactic coating for hydroxyapatite (HA)-coated cementless hip prostheses is developed with similar antibacterial efficacy as offered by gentamicin-loaded cements for fixing traditional, cemented prostheses in bone. We describe the development pathway, from in vitro investigation of antibiotic release and antibacterial properties of this PLGA-gentamicin-HA-coating in different in vitro models to an evaluation of its efficacy in preventing implant-related infection in rabbits. Bone in-growth in the absence and presence of the coating was investigated in a canine model. The PLGA-gentamicin-HA-coating showed high-burst release, with antibacterial efficacy in agar-assays completely disappearing after 4 days, minimising risk of inducing antibiotic resistance. Gentamicin-sensitive and gentamicin-resistant staphylococci were killed by the antibiotic-loaded coating, in a simulated prosthesis-related interfacial gap. PLGA-gentamicin-HA-coatings prevented growth of bioluminescent staphylococci around a miniature-stem mounted in bacterially contaminated agar, as observed using bio-optical imaging. PLGA-gentamicin-HA-coated pins inserted in bacterially contaminated medullary canals in rabbits caused a statistically significant reduction in infection rates compared to HA-coated pins without gentamicin. Bone ingrowth to PLGA-gentamicin-HA-coated pins, in condylar defects of Beagle dogs was not impaired by the presence of the degradable, gentamicin-loaded coating. In conclusion, the PLGA-gentamicin-HA-coating constitutes an effective strategy for infection prophylaxis in cementless prostheses

Oral biofilm models for mechanical plaque removal

In vitro plaque removal studies require biofilm models that resemble in vivo dental plaque. Here, we compare contact and non-contact removal of single and dual-species biofilms as well as of biofilms grown from human whole saliva in vitro using different biofilm models. Bacteria were adhered to a salivary pellicle for 2 h or grown after adhesion for 16 h, after which, their removal was evaluated. In a contact mode, no differences were observed between the manual, rotating, or sonic brushing; and removal was on average 39%, 84%, and 95% for Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, respectively, and 90% and 54% for the dual- and multi-species biofilms, respectively. However, in a non-contact mode, rotating and sonic brushes still removed considerable numbers of bacteria (24–40%), while the manual brush as a control (5–11%) did not. Single A. naeslundii and dual-species (A. naeslundii and S. oralis) biofilms were more difficult to remove after 16 h growth than after 2 h adhesion (on average, 62% and 93% for 16- and 2-h-old biofilms, respectively), while in contrast, biofilms grown from whole saliva were easier to remove (97% after 16 h and 54% after 2 h of growth). Considering the strong adhesion of dual-species biofilms and their easier more reproducible growth compared with biofilms grown from whole saliva, dual-species biofilms of A. naeslundii and S. oralis are suggested to be preferred for use in mechanical plaque removal studies in vitro

Attainment rate as a surrogate indicator of the intervertebral neutral zone length in lateral bending: An in vitro proof of concept study

Background Lumbar segmental instability is often considered to be a cause of chronic low back pain. However, defining its measurement has been largely limited to laboratory studies. These have characterised segmental stability as the intrinsic resistance of spine specimens to initial bending moments by quantifying the dynamic neutral zone. However these measurements have been impossible to obtain in vivo without invasive procedures, preventing the assessment of intervertebral stability in patients. Quantitative fluoroscopy (QF), measures the initial velocity of the attainment of intervertebral rotational motion in patients, which may to some extent be representative of the dynamic neutral zone. This study sought to explore the possible relationship between the dynamic neutral zone and intervertebral rotational attainment rate as measured with (QF) in an in vitro preparation. The purpose was to find out if further work into this concept is worth pursuing. Method This study used passive recumbent QF in a multi-segmental porcine model. This assessed the intrinsic intervertebral responses to a minimal coronal plane bending moment as measured with a digital force guage. Bending moments about each intervertebral joint were calculated and correlated with the rate at which global motion was attained at each intervertebral segment in the first 10° of global motion where the intervertebral joint was rotating. Results Unlike previous studies of single segment specimens, a neutral zone was found to exist during lateral bending. The initial attainment rates for left and right lateral flexion were comparable to previously published in vivo values for healthy controls. Substantial and highly significant levels of correlation between initial attainment rate and neutral zone were found for left (Rho = 0.75, P = 0.0002) and combined left-right bending (Rho = 0.72, P = 0.0001) and moderate ones for right alone (Rho = 0.55, P = 0.0012). Conclusions This study found good correlation between the initial intervertebral attainment rate and the dynamic neutral zone, thereby opening the possibility to detect segmental instability from clinical studies. However the results must be treated with caution. Further studies with multiple specimens and adding sagittal plane motion are warranted

Uptake of Hydrocarbon by Pseudomonas fluorescens (P1) and Pseudomonas putida (K1) Strains in the Presence of Surfactants: A Cell Surface Modification

The objective of this research was the evaluation of the effects of exogenous added surfactants on hydrocarbon biodegradation and on cell surface properties. Crude oil hydrocarbons are often difficult to remove from the environment because of their insolubility in water. The addition of surfactants enhances the removal of hydrocarbons by raising the solubility of these compounds. These surfactants cause them to become more vulnerable to degradation, thereby facilitating transportation across the cell membrane. The obtained results showed that the microorganism consortia of bacteria are useful biological agents within environmental bioremediation. The most effective amongst all, as regards biodegradation, were the consortia of Pseudomonas spp. and Bacillus spp. strains. The results indicated that the natural surfactants (rhamnolipides and saponins) are more effective surfactants in hydrocarbon biodegradation as compared to Triton X-100. The addition of natural surfactants enhanced the removal of hydrocarbon and diesel oil from the environment. Very promising was the use of saponins as a surfactant in hydrocarbon biodegradation. This surfactant significantly increases the organic compound biodegradation. In the case of those surfactants that could be easily adsorbed on cells of strains (e.g., rhamnolipides), a change of hydrophobicity to ca. 30–40% was noted. As the final result, an increase in hydrocarbon biodegradation was observed