The Allegheny Health Network Center for Excellence in Biofilm Research (AHN CEBR) is an innovative research environment. Working in close collaboration with Allegheny Health Network’s clinicians and Carnegie Mellon faculty, the CEBR fosters the development of surgeon-scientists, physician-scientists and biomedical researchers by actively working to develop partnerships among teams of individuals with diverse scientific, clinical, computational and engineering backgrounds.
The CEBR’s goal is to promote human health by providing a deeper understanding of the role of microbial biofilms in the disease process. Currently researchers are conducting pioneering studies in the specialties of orthopaedics, cardiology, oncology, obstetrics, and urology. The goal of these collaborations is to better understand the role bacteria, particularly biofilms, play in complex clinical situations.
Bacteria in biofilms tend to be more difficult to culture and more resistant to control strategies (antibiotics and biocides) and host defenses than when grown planktonically in the laboratory. Biofilms are communities of micro-organisms encased within an extracellular polymeric slime (EPS) matrix living on surfaces. Their resilience has been related to physiology and protection by the EPS slime matrix that they produce. Biofilms are dynamic and the composite microbial communities change over time, moving, shedding and re-growing adherent colonies. These phenomena may explain seemingly conflicting features of the disease when signs and symptoms are otherwise consistent with infection.
In order to study biofilms, state of the art molecular technologies are necessary. The CEBR employs a suite of advanced molecular technologies to study biofilm populations. The Pathogen Pipeline not only identifies the microbial composition of a sample but provides visual proof of the pathogen’s existence. The combination of the three technologies: PCR-ESI-TOF-MS, microbial sequencing, and FISH-confocal microscopy do not exist in combination at any other location. In addition the Center offers genomics, transcriptomics, proteomics, and bioinformatics to provide additional insights about the role of biofilms in the disease process and help us investigate our critical areas of interest.
Using cutting-edge DNA-based technology, PCR-ESI-TOF-MS, AHN CEBR scientists can identify specific strains of bacteria and fungi without the need for culture. The system combines three technologies that provide researchers with broad-based microbial diagnostics. First, this technology employs multiple PCR-based DNA amplifications targeted to highly conserved genes throughout the bacterial and fungal domains. Next, it employs a novel mass spectroscopic analysis termed ESI-TOF-MS that provides exact DNA base compositions for each of the PCR products. Finally, it utilizes a sophisticated mass look-up table and a computational triangulation approach to arrive at a strain-level diagnosis based on the presence/absence and weights of all of the amplified DNA targets.
In addition, microbial detection can be accomplished with the use of 16S ribosomal RNA (rRNA) sequencing available through the CEBR. 16S ribosomal RNA (rRNA) sequencing provides another avenue to identify and compare bacteria present within a given sample. Sample barcoding allows for the simultaneous sequencing of multiple samples in a single run. Although more expensive than the PCR-ESI-TOF-MS technology, 16S rRNA gene sequencing can be used independently or as a confirmatory technology. Data from 16S studies can be used to improve the accuracy of bacterial classification.
The imaging of biofilms is fundamental to biofilm research. High-resolution confocal images provided the first evidence for the structural heterogeneity of biofilm architecture. This evidence was used to establish models of biofilm growth and ultrastructure. The use of confocal scanning laser microscopy and florescent in situ hybridization (FISH) on in vivo clinical samples allows researchers the ability to detect and localize the presence or absence of specific nucleic acid sequences. Florescent probes bind to complementary sequences which can then be visualized using a confocal microscope. The technique is powerful not only in its ability to confirm the IBIS results but to visualize the location of the bacteria within a sample.
There are many other tools available to investigate biofilms. Here is a brief list of some of the other services we can provide.
