PacSurf2022 Session BI-ThM1: Bacteria Biomaterial Interactions

Many new antimicrobial biomaterials and coatings are being developed to address the need for implantable medical devices that prevent infections. Researchers, medical device manufacturers, and regulatory bodies are all interested in seeing “promising research results” translated to clinical use, but how are these claims evaluated? What experimental evidence is needed to improve translation of in vitro antimicrobial materials to the clinic?

It is illustrative to explore one of the most interesting classes of antimicrobial biomaterials: those which purport to have a contact-inhibition mechanism of action. These materials present covalently attached antimicrobial molecules from surfaces which inhibit or kill adhering microbes on contact. On the one hand, this surface design could open up new paradigms for antimicrobial therapy by virtue of making implants with potentially long-lasting antimicrobial activity. On the other hand, questions about whether these materials release antimicrobials and whether their surface activity would be nullified by protein fouling deserve serious investigation.

This presentation will delve into these issues by explaining why mechanism matters for antimicrobial biomaterials. It can also be viewed generally by biomaterials researchers from other fields who design bioactive surfaces using covalent surface immobilization and face questions about how to interpret the results of cellular assays. The presentation aims to raise awareness of the potential role of confirmation bias in antimicrobial susceptibility experiments which could lead researchers to unwittingly misinterpret antimicrobial mechanisms of action. It discusses ways to avoid this pitfall by proposing a methodological approach that emphasizes the importance of surface analysis. It is hoped that greater awareness of these issues will help “promising” in vitro antimicrobial surface technologies to have greater uptake in animal studies and clinical trials.

Multidrug-resistant (MDR) bacteria are a severe threat to public health and it is urgent to identify novel antibacterial compounds or pathogen-specific mixtures of antibiotics. In a first paper we reported the application of Droplet Microarrays developed by Aquarray as a cost effective high-throughput screening method for the evaluation of drug resistance of Pseudomonas aeruginosa, an opportunistic human pathogen /1/. The DMA consists of an array of hydrophilic spots divided by superhydrophobic borders to generate arrays of hundreds of nanoliter-sized droplets containing bacteria and different drugs for screening applications. A novel simple colorimetric readout method compatible with the nanoliter size of the droplets was established. Furthermore, the drug-resistance of Pseudomonas aeruginosa 49, a multi-resistant strain from an environmental isolate was investigated by screening of a small library containing 18 antibiotics. We were able to show that our methology reproduces the data obtained with a 96 well microplate.

In the study reported here the search for antibiotic compounds was extended to screen over 2000 compounds for their antimicrobial properties against carbapenem-resistant Klebsiella pneumoniae and and methicillin resistant Staphylococcus aureus (MRSA). A fast single-step detection method measured the inhibitory effect of the compounds on bacterial growth on the whole array. Six hit compounds, including coumarins and structurally simplified estrogen analogs are identified in the primary screening and validated with minimum inhibition concentration assay for their antibacterial effect. This study demonstrates that the DMA-based high-throughput screening system identifies potential antibiotics from novel synthetic compound libraries, and thus offering opportunities for development of new treatments against multidrug-resistant bacteria.Due to its simplicity, the method is suitable for rapid screens in personalized medicine. If time allows, further examples of rapid screens using DMA´s which habe been designed for personalized medicine will be presented.

/1/ W. Lei, K. Demir, J. Overhage, M. Grunze, T. Schwartz, P. A. Levkin, Droplet-Microarray: Miniaturized Platform for High-Throughput Screening of Antimicrobial Compounds, Adv. Biosys., 2000073 (1-9), 2020

A type of bacteria known as dissimilatory metal-reducing bacteria (DMRB) can “breathe metals” to reduce heavy metal ions as part of their metabolic process. Shewanella oneidensis MR-1 is a type of DMRB that was first discovered in Lake Oneida, New York for its ability to reduce manganese, but has since been shown to reduce a variety of different electron acceptors including Fe(III), As(V), Cr(VI), and thiosulfate. In previous literature, S. oneidensis MR-1 has been grown anaerobically in media enhanced with sulfur and metal ions in order to produce several types of nanoparticles, including molybdenum disulfide, zinc sulfide, and cadmium sulfide.

This work presents the bacterial nanofabrication of tungsten disulfide particles using S. oneidensis MR-1. Bacterial nanofabrication synthesis, where the bacteria is the main catalyst for the production of nanomaterials, has numerous advantages compared to traditional chemical synthesis methods in that it can be conducted at room temperature and requires less chemical reagents in the reaction. One of the primary reasons to produce nanomaterials using bacteria is for a low resource input alternative to material fabrication for use in electronic devices.

Tungsten disulfide (WS₂) is a two-dimensional transition metal dichalcogenide which has a bandgap transition from an indirect bandgap in the bulk material to a direct bandgap in its monolayer form. Tungsten disulfide has a range of potential applications including photodetection, biosensing, and chemical catalysis. These applications are further enhanced when control can be exerted over crystal growth and thickness.

An anaerobic batch culture of S. oneidensis MR-1 was incubated at room temperature in the presence of tungsten trioxide, resulting in the production of tungsten disulfide crystalline nanostructures of varied shapes and size. Several characterization techniques were employed to identify the material, including scanning electron microscopy, transmission electron microscopy, Raman spectroscopy, absorbance spectroscopy and X-ray diffraction. In addition to confirming that tungsten disulfide can be produced by Shewanella bacteria, the data collected using these methods provide insight on the size, morphology, and photoresponse of nanoparticles generated this way.