A convenient sample preparation protocol for scanning electron microscope examination of xylem-occluding bacterial biofilm on cut flowers and foliage

Microbes and their exopolysaccharides (EPS) can block xylem vessels, thereby increasing the hydraulic resistance and decreasing the vase life of cut flowers and foliage. Scanning electron microscopy (SEM) provides a powerful tool for investigation of bacteria-induced xylem occlusion. However, conventional preparation protocols for SEM involving chemicals can cause loss of hydrated EPS material, and thereby damage the bacterial biofilms during dehydration. A modified chemical fixation protocol involving pre-fixation with 75 mM lysine plus 2.5% glutaraldehyde followed by the normal fixation in 3% glutaraldehyde was, therefore, tested for improved preservation of bacterial biofilm at the stem-ends of cut Acacia holosericea foliage stems. Stem-end segments with different stages of bacterial growth were obtained from stems stood into water. The lysine-based protocol was compared with four other processing protocols of critical point drying (CPD) without fixation (control), freeze-drying (FD), conventional chemical fixation followed by drying with hexamethyldisilazane (HMDS), and conventional chemical fixation with CPD. The non-fixed control. FD and the glutaraldehyde fixation with HMDS drying gave poor preservation of hydrated material, including bacterial EPS. Conventional glutaraldehyde fixation followed by CPD was superior to these three methods in terms of better preserving the EPS. However, this fourth method gave condensation of biofilms during dehydration. In contrast, the modified lysine-based protocol resulted in superior preservation of EPS and biofilm structure. Thus, this fifth method was the most appropriate for examination of bacterial stem-end blockage in cut ornamentals. (C) 2012 Elsevier B.V. All rights reserved.

Towards the identification of the common features of bacterial biofilm development

UPNa. Instituto de Agrobiotecnología. Laboratorio de Biofilms Microbianos

Incorporation of chitosan in acrylic bone cement: Effect on antibiotic release, bacterial biofilm formation and mechanical properties

Bacterial infection remains a significant problem following total joint replacement. Efforts to prevent recurrent implant infection, including the use of antibiotic-loaded bone cement for implant fixation at the time of revision surgery, are not always successful. In this in vitro study, we investigated whether the addition of chitosan to gentamicin-loaded Palacos® R bone cement increased antibiotic release and prevented bacterial adherence and biofilm formation by Staphylococcus spp. clinical isolates. Furthermore, mechanical tests were performed as a function of time post-polymerisation in pseudo-physiological conditions. The addition of chitosan to gentamicin-loaded Palacos® R bone cement significantly decreased gentamicin release and did not increase the efficacy of the bone cement at preventing bacterial colonisation and biofilm formation. Moreover, the mechanical performance of cement containing chitosan was significantly reduced after 28 days of saline degradation with the compressive and bending strengths not in compliance with the minimum requirements as stipulated by the ISO standard for PMMA bone cement. Therefore, incorporating chitosan into gentamicin-loaded Palacos® R bone cement for use in revision surgery has no clinical antimicrobial benefit and the detrimental effect on mechanical properties could adversely affect the longevity of the prosthetic joint.

EM evaluation of bacterial biofilm and microorganisms on the apical external root surface of human teeth

The aim of this study was to evaluate the presence of bacterial biofilm on the external surface of the root apex in teeth with pulp necrosis, with and without radiographically visible periapical lesions, and in teeth with a vital pulp. Twenty-one teeth were extracted, eight with pulp necrosis and periapical lesions, eight with pulp necrosis without radiographically visible periapical lesions, and five with a vital pulp. The roots were sectioned, and the root apexes (+/- 3 mm) were processed for scanning electron microscope evaluation. The surface of the apical root was evaluated for the presence of microorganisms, root resorption, and biofilm. There were no microorganisms on the apical root surface of either teeth with pulp vitality or with pulp necrosis with no radiographically visible periapical lesions. Microorganisms were always present in teeth with pulp necrosis and radiographically visible periapical lesions. These included cocci, bacilli, and filaments and the presence of an apical biofilm. Apical biofilm is clinically important because microbial biofilms are inherently resistant to antimicrobial agents and cannot be removed by biomechanical preparation alone. This may cause failure of endodontic treatment as a consequence of persistent infection.

Molecular fingerprinting and amplicon sequencing of the bacterial biofilm on settlement tiles deployed along natural pH gradients in Papua New Guinea

Bacterial biofilms provide cues for the settlement of marine invertebrates such as coral larvae, and are therefore important for the resilience and recovery of coral reefs. This study aimed to better understand how ocean acidification may affect the community composition and diversity of bacterial biofilms on surfaces under naturally reduced pH conditions. Settlement tiles were deployed at coral reefs in Papua New Guinea along pH gradients created by two CO2 seeps, and upper and lower tiles surfaces were sampled 5 and 13 months after deployment. Automated Ribosomal Intergenic Spacer Analysis were used to characterize more than 200 separate bacterial communities, complemented by amplicon sequencing of the bacterial 16S rRNA gene of 16 samples. The bacterial biofilm consisted predominantly of Alpha-, Gamma- and Deltaproteobacteria, as well as Cyanobacteria, Flavobacteriia and Cytophaga, whereas putative settlement-inducing taxa only accounted for a small fraction of the community. Bacterial biofilm composition was heterogeneous with approximately 25% shared operational taxonomic units between samples. Among the observed environmental parameters, pH only had a weak effect on community composition (R² ~ 1%) and did not affect community richness and evenness. In contrast, there were strong differences between upper and lower surfaces (contrasting in light exposure and grazing intensity). There also appeared to be a strong interaction between bacterial biofilm composition and the macroscopic components of the tile community. Our results suggest that on mature settlement surfaces in situ, pH does not have a strong impact on the composition of bacterial biofilms. Other abiotic and biotic factors such as light exposure and interactions with other organisms may be more important in shaping bacterial biofilms than changes in seawater pH.

Using new scanning electron microscopy (SEM) approaches to elucidate bacterial biofilm architecture

Healthcare-associated infections (HAI) are a major public health problem being Klebsiella pneumoniae and nontuberculous mycobacteria, both with high antibiotic resistance rates, among their etiological agent. Since biofilme assembly is pointed as one of the mechanisms involved in emergence of antibiotic resistance understanding bacteria organization within the biofilm and the identification of differences between planktonic and sessile forms of bacteria will be a step forward to fight HAI. In the present work we used SEM as a tool to characterize the internal structure of biofilm assembled on different surfaces. For SEM analysis, biofilms were allowed to form either on six-well cell culture plates, silicon or metallic disks placed inside the wells for different incubation periods at 37 °C. The biofilm assembled on the cell culture dish was for both secondary and backscattered electron analysis as described before. Biofilms assembled on silicon disks instead of being sectioned were prepared as metallographic samples, by grinding with grit SIC paper and polishing with diamond particles. Samples were cleaned (70% ethanol), dried with hot air, further coated and analysed. A preliminary study using FIB-SEM has been performed to access the ultrastructure of biofilms assembled on metallic surfaces. The results obtained showed that the same bacteria assembled biofilms with different ratios of biomass and extracellular matrix depending on the surface. SEM performed on thin sections of biofilms is a powerful tool to elucidate biofilm structure allowing the quantification of the major components. FIB-SEM is also a promising tool in this field.

Bacterial adherence and biofilm formation on medical implants : a review

Biofilms are a complex group of microbial cells that adhere to the exopolysaccharide matrix present on the surface of medical devices. Biofilm-associated infections in the medical devices pose a serious problem to the public health and adversely affect the function of the device. Medical implants used in oral and orthopedic surgery are fabricated using alloys such as stainless steel and titanium. The biological behavior, such as osseointegration and its antibacterial activity, essentially depends on both the chemical composition and the morphology of the surface of the device. Surface treatment of medical implants by various physical and chemical techniques are attempted in order to improve their surface properties so as to facilitate bio-integration and prevent bacterial adhesion. The potential source of infection of the surrounding tissue and antimicrobial strategies are from bacteria adherent to or in a biofilm on the implant which should prevent both biofilm formation and tissue colonization. This article provides an overview of bacterial biofilm formation and methods adopted for the inhibition of bacterial adhesion on medical implants