Eric S. Gilbert

Influences of Biofilm Structure and Antibiotic Resistance Mechanisms on Indirect Pathogenicity in a Model Polymicrobial Biofilm

ABSTRACT Indirect pathogenicity (IP), the commensal protection of antibiotic-sensitive pathogens by resistant microorganisms of low intrinsic virulence, can prevent the eradication of polymicrobial infections. The contributions of antibiotic resistance mechanisms and biofilm structure to IP within polymicrobial biofilms were investigated using a model two-member consortium. Escherichia coli ATCC 33456 was transformed with vectors conferring either ampicillin or spectinomycin resistance, creating two distinct populations with different resistance mechanisms. Each strain alone or the consortium was grown as biofilms in flow cells and planktonically in chemostats. Comparisons in survival and activity were made on the basis of MICs and minimum biofilm preventative concentrations, a newly introduced descriptor. In ampicillin-containing medium, commensal interactions were evident during both modes of cultivation, but the sensitive strain experienced a greater benefit in the chemostat, indicating that the biofilm environment limited the commensal interaction between the Ampr and Sptr strains. In spectinomycin-containing medium, growth of the sensitive strain in chemostats and biofilms was not aided by the resistant strain. However, green fluorescent protein expression by the sensitive strain was greater in mixed-population biofilms (9% ± 1%) than when the strain was grown alone (2% ± 0%). No comparable benefit was evident during growth in the chemostat, indicating that the biofilm structure contributed to enhanced activity of the sensitive strain.

Constructing multispecies biofilms with defined compositions by sequential deposition of bacteria

Rationally-assembled multispecies biofilms could benefit applied processes including mixed waste biodegradation and drug biosynthesis by combining complementary metabolic pathways into single functional communities. We hypothesized that the cellular composition of mature multispecies biofilms could be manipulated by controlling the number of each cell type present on newly colonized surfaces. To test this idea, we developed a method for attaching specific numbers of bacteria to a flow cell by recirculating cell suspensions. Initial work revealed a nonlinear relationship between suspension cell density and areal density when two strains of Escherichia coli were simultaneously recirculated; in contrast, sequential recirculation resulted in a predictable deposition of cell numbers. Quantitative analysis of cell distributions in 48-h biofilms comprised of the E. coli strains demonstrated a strong relationship between their distribution at the substratum and their presence in mature biofilms. Sequentially depositing E. coli with either Pseudomonas aeruginosa or Bacillus subtilis determined small but reproducible differences in the areal density of the second microorganism recirculated relative to its areal density when recirculated alone. Overall, the presented method offers a simple and reproducible way to construct multispecies biofilms with defined compositions for biocatalytic processes.

Inhibition of quorum sensing in Vibrio harveyi by boronic acids.

Bacterial quorum sensing refers to the ability of bacteria to control gene expression through the detection of a threshold concentration of certain chemicals called autoinducer(s), which are secreted by self and/or other bacteria. Quorum sensing is implicated in the regulation of pathologically relevant events such as biofilm formation, virulence, conjugation, sporulation, and swarming mobility. Inhibitors of bacterial quorum sensing are valuable research tools and potential antimicrobial agents. In this paper, we describe the discovery of several boronic acid inhibitors of bacterial quorum sensing in Vibrio harveyi with IC50 values in the low to sub‐micromolar range in whole cell assays.

Agarose stabilization of fragile biofilms for quantitative structure analysis.

Agarose was used to stabilize fragile biofilms cultivated in parallel plate flow cells prior to imaging by confocal laser scanning microscopy. An essential element to the success of the procedure was the application of a ceramic heat pad to the flow cell to maintain agarose fluidity until the biofilm was enveloped. Quantitative digital image analysis demonstrated the effectiveness of this technique for generating reproducible measurements of a three-dimensional biofilm structure. The described method will also benefit researchers who transport their flow cell-cultivated biofilms to a core facility for imaging.

Multi-resolution border segmentation for measuring spatial heterogeneity of mixed population biofilm bacteria

Multi-resolution image clustering and segmentation interactive system has been developed to analyze the interaction between clusters of heterogeneous microbial populations residing in biofilms. Biofilms are biological microorganisms attached to surfaces, which develop a complex heterogeneous three-dimensional structure. The hierarchical structural analysis concept underlying multi-resolution image segmentation is that the clusters will be more complex and noisy for higher-resolution while less complex and smoother for lower-resolution image. This hierarchical structure analysis can be used to simplify the image storage and retrieval in well-mixed populations. We are proposing an algorithm that combines Fuzzy C-Means, SOM and LVQ neural networks to segment and identify clusters. The outcome of the image segmentation is quantified by the number of cluster objects of each kind of microorganism within sections of the biofilm, and the centroid distances between the identified cluster objects. Experimental evaluations of the algorithm showed its effectiveness in enumerating cluster objects of bacteria in dual-species biofilms at the substratum and measuring the associated intercellular distances.

Inhibition and Dispersal of Pseudomonas aeruginosa Biofilms by Combination Treatment with Escapin Intermediate Products and Hydrogen Peroxide

ABSTRACT Escapin is an l-amino acid oxidase that acts on lysine to produce hydrogen peroxide (H2O2), ammonia, and equilibrium mixtures of several organic acids collectively called escapin intermediate products (EIP). Previous work showed that the combination of synthetic EIP and H2O2 functions synergistically as an antimicrobial toward diverse planktonic bacteria. We initiated the present study to investigate how the combination of EIP and H2O2 affected bacterial biofilms, using Pseudomonas aeruginosa as a model. Specifically, we examined concentrations of EIP and H2O2 that inhibited biofilm formation or fostered disruption of established biofilms. High-throughput assays of biofilm formation using microtiter plates and crystal violet staining showed a significant effect from pairing EIP and H2O2, resulting in inhibition of biofilm formation relative to biofilm formation in untreated controls or with EIP or H2O2 alone. Similarly, flow cell analysis and confocal laser scanning microscopy revealed that the EIP and H2O2 combination reduced the biomass of established biofilms relative to that of the controls. Area layer analysis of biofilms posttreatment indicated that disruption of biomass occurs down to the substratum. Only nanomolar to micromolar concentrations of EIP and H2O2 were required to impact biofilm formation or disruption, and these concentrations are significantly lower than those causing bactericidal effects on planktonic bacteria. Micromolar concentrations of EIP and H2O2 combined enhanced P. aeruginosa swimming motility compared to the effect of either EIP or H2O2 alone. Collectively, our results suggest that the combination of EIP and H2O2 may affect biofilms by interfering with bacterial attachment and destabilizing the biofilm matrix.

Recurrent Aspergillus contamination in a biomedical research facility: a case study

Fungal contamination of biomedical processes and facilities can result in major revenue loss and product delay. A biomedical research facility (BRF) culturing human cell lines experienced recurring fungal contamination of clean room incubators over a 3-year period. In 2010, as part of the plan to mitigate contamination, 20 fungal specimens were isolated by air and swab samples at various locations within the BRF. Aspergillus niger and Aspergillus fumigatus were isolated from several clean-room incubators. A. niger and A. fumigatus were identified using sequence comparison of the 18S rRNA gene. To determine whether the contaminant strains isolated in 2010 were the same as or different from strains isolated between 2007 and 2009, a novel forensic approach to random amplified polymorphic DNA (RAPD) PCR was used. The phylogenetic relationship among isolates showed two main genotypic clusters, and indicated the continual presence of the same A. fumigatus strain in the clean room since 2007. Biofilms can serve as chronic sources of contamination; visual inspection of plugs within the incubators revealed fungal biofilms. Moreover, confocal microscopy imaging of flow cell-grown biofilms demonstrated that the strains isolated from the incubators formed dense biofilms relative to other environmental isolates from the BRF. Lastly, the efficacies of various disinfectants employed at the BRF were examined for their ability to prevent spore germination. Overall, the investigation found that the use of rubber plugs around thermometers in the tissue culture incubators provided a microenvironment where A. fumigatus could survive regular surface disinfection. A general lesson from this case study is that the presence of microenvironments harboring contaminants can undermine decontamination procedures and serve as a source of recurrent contamination.

The Molecules and Mechanisms Underlying the Antimicrobial Activity of Escapin, an L–Amino Acid Oxidase from the Ink of Sea Hares

Many marine animals use chemicals to defend themselves and their eggs from predators. 12 Beyond their ecologically relevant functions, these chemicals may also have properties that make 13 them beneficial for humans, including with biomedical and industrial applications. For example, 14 some chemical defenses are also powerful antimicrobial or anti-tumor agents with relevance to 15 human health and disease. One such chemical defense, Escapin, an L–amino acid oxidase in the 16 defensive ink of the sea hare Aplysia californica, and related proteins have been investigated for their 17 biomedical properties. This review details our current understanding of Escapin’s antimicrobial 18 activity, including the array of chemicals generated by Escapin’s oxidation of its major substrates, 19 L–lysine and L–arginine, and mechanisms underlying these molecules’ bactericidal and 20 bacteriostatic effects on planktonic cells and the prevention of formation and removal of bacterial 21 biofilms. Models of Escapin’s effects are presented, and future directions are proposed. 22

The effectiveness of multi resolution image segmentation for measuring spatial heterogeneity in mixed population biofilms

Multi resolution image clustering and segmentation tools has been developed to measure the distance between clusters of homogeneous microbial populations within two-dimensional sections of biofilms visualized by confocal laser scanning microscopy. The concept underlying multi resolution image segmentation is that the number of clusters are larger for higher resolution image and smaller for lower resolution image. This hierarchical structure analysis can be used to simplify the problem in well-mixed populations. The algorithm combines fuzzy C-means, SOM and LVQ neural networks to segment and identify clusters. The outcome of the segmentation is quantified by the number of clusters of each kind of microorganism within sections of the biofilm, and the centroid distances between the identified clusters. Experimental evaluations of the algorithm showed its effectiveness in analyzing biofilm mixed populations.