MY  BIOFILM HISTORY: People and Places

1. Dr Bill Costerton.  “DR BIOFILM”. PIONEEAR. EXTRAODINARY SCIENTIST AND PERSON.  Had opportunity to work and talk with him for several years about our biofilm research. Kindred skiing spirit.  Creator of Center for Biofilm Engineering (CBE) at Montana State University. Over 660 publications.  I presented. PPT 1
2. University of Calgary. Linked initially with Dr Costerton, created CBD (Calgary Biofilm Device) practical, biofilm applications and clinical tools, establishing the MBEC Assay (Minimal Biofilm Elimination Concentration), enabling comparisons of antibiotic resistance between planktonic and biofilm phenotypes.
3. Dr David Williams, Cardiff University, Wales.   My mentor for 6 month Sabbatical in School of Dental and Restorative Medicine. Awesome scientist and friend.  Introduced me to ‘The Biofilm Club’, original group of UK scientists, famous for global introduction of biofilm concept. Held at Gregynog Hall, Powys, Wales. Published 9 volumes, I have 3. I presented.
4. WVU School of Dentistry. Established COHRA. (CENTER FOR ORAL HEALTH RESEARCH IN APPALACHIA.   Major Research Partnership funded by NIH between WVU, School of Dentistry and Univ Of Pittsburgh, School of Dental Medicine, addressing oral health disparities, focusing on the young and poor. Drs Dan McNiel and R. Crout. Here, also we created and integrated into daily dental practice the MOUNTAIN STATE ORAL FACIAL MICROBIOLOGY LABOTORATORY, only 1 of 7 dedicated to dental microbiology in US.
5.  Allegheny Health Network. Pittsburgh, CENTER OF EXCELLENCE IN BIOFILM ESEARCH. Application of advanced molecular methods to biofilm research with clinical outcomes. Expanded my oral principles to wound care, bio-bandage, integrating 3-D printing, combining strength of Carnegie Mellon University.  Real intervention options.  Clinical significance with diagnostic, microbiology inclusion.

BIOFILM OVERVIEW:  My perspective, using dental plaque as model

ORAL BIOFILMS: ARCHITECTS OF DISEASE, HEAD TO TOE

In the oral microbiota, there are greater than 770 different  microbial species; however, a select proportion will up-regulate from a planktonic phenotype  to  a biofilm phenotype , attached to a biotic surface  or non biotic surfaces  based on their stress response genes and ‘Quorum sensing’.  Eight key environmental/cultural characteristics magnify the selection of multi-species biofilm inhabitants during a 4 -stage life cycle (I-IV), enhance by metastasis to distal sights: gingivitis (micro-aerophilic microbes), periodontitis and endodontics (anaerobic microbes).

The unifying concept is the Ecological or Plaque Hypothesis , (PD Marsh, Wales)which recognizes that ecological pressure (including antibiotics) is necessary for low number  “Professional Pathogens” to outcompete resident flora , “Beneficial Microbes “ and achieve numerical dominance (ratio) associated with a biofilm phenotype .  In our 2 working BIOFILM models (VAP and Wounds), this was identified as ETT, a dynamic biofilm engine, and wound bed as a static biofilm reactor, respectively.

Architecturally, biofilms promote the theme of “Structure equals Function”, incorporating structural characteristics that redefine a biofilm, acting as a hydrated polymer, focusing on chemical features including Rheology (Movement/Stationary) and (Viscoelastic) dispersal of energy.  Biofilms include cross bridging for strength, nano-wires for communication, and HGT (Horizontal Gene Transfer), promoting antibiotic resistance.  Fungi, mostly Candida albicans, act as ‘universal co-aggregates’, promoting linking thru the outer most protective structures, EPS (Extra Polymeric Substance), highlighting prokaryotic and eukaryotic functionality.

Biofilms can also be described as a type of primitive developmental biology, in which spatial organization of cells within the matrix optimizes nutritional resources.  In this state it acts as an immobilized enzyme system, where stead state can be radically altered by applying physical forces, such as sheer.

Clinically, biofilms are markedly resistant to most antibiotic protocols designed to treat single cell associated infections, Planktonic phenotype.  Using biofilm designed tools like the CBD, an MBEC, Minimal Biofilm Elimination Concentration, can be established for antibiotic resistance, measuring a 100 fold increase to planktonic isolate treatment using the MIC, Minimal Inhibitory Concentration for single cells.  Multispecies are inherently resistant to antibiotic RX.

Hence, Therapeutic Modalities (sonication, silver ions/tobromycin, CMTs-chemically modified tetracyclines -and immune modulation) have refocused on multiple interventions, recognizing the properties of biofilms are similar to hydrated organic polymers, perhaps acting as tumors (multispecies communities) and the use of probiotics, Restorative Microbiology. We have combined these 2 pathways, recently, linking a “Disruptive:Reconstruction” concept  of biofilm intervention, followed by probiotic  reconstruction, recognizing the Plaque Hypothesis and Beneficial Microbes. (See Key Points, B. Biofilms and Oral health.)

BIOFILMS AS TUMORS, OR VICE VERSA.

BIOTUMORS or BIOFILMS: BENIGN TUMORS OR BIOFILMS AND TUMORS: A METASTATIC CONNECTION

Our passion for biofilm research also catalyzed ‘critical thinking’, thinking out-side the box. It became apparent to us that both human tumors, Eucaryotes, and biofilms, Procaryotes, were multi-system cellular organizations which shared at least 12 characteristics. It was apparent neoplasia’s, human, and biofilms, microbial, addressed multi-cellularity where metastasis was a common means of distribution, Stage II to Stage III. Together with Dr S. Shabahang, we then developed 2 hypotheses highlighting this link:

HYPOTHESIS I.  Biofilms are prokaryotic Tumors (BIOTUMOR) where communities of linked microbes masquerading as a biofilm are really Procariotic tumors.

HYPOTHESIS II.  Tumors are Eucaryotic Bioflms , where communities of similar linked cells masquerading as a tumor as really Eucaryotic Biofilms.

We developed a   unique  testing strategy, recognizing  these similarities, linking Prokaryotic and Eucaryotic Therapy, predicting cancer therapy on biofilm response, comparing multiple tumor therapies. The evolution followed designing a concept, proof of concept with ultimately the creation of a BioTumor classification dependent upon biofilm response of 12 cancer drugs, emphasizing synergy.

Our evolution could be traced by abstract and poster presentations at international meetings:

1. Extrapolating Tumor Therapy to Manage oral Biofilms simultaneously: A comparative Model
2. Effects of Anti-cancer drugs on Biofilms: Benign, Procaryotic  Tumors
3. A Model and Strategy to Compare Anti-tumor and Anti-biofilm Efficacy utilizing a Distinctive BioTyping Classification System
4. Biofilm (prokaryote)Communities predicting Tumor (Eucaryote ) Therapy

D  SPECIFIC DISEASES, My Biofilm Translational Research

1.  LUNG. Ventilator Associated Pneumonia (VAP) and EndoTrach Tubes (ETT).  Because of wife, Penny, became interested in ETT and nurse suctioning. Why? Began LONG adventure of ETT biofilms and association with Respiratory Therapy (RT). Designed and built VEL Model Simulator. Studied Silver (Ag) and others as means of lung protection from ETT biofilm luminal restriction.  Connected  inoculation with oral microbes, first, then hospital microflora, second, of ETT lumen. CO-BIOFILM   Most recently, linked with MGH, Boston, Critical Care and Anesthesia, Dr Lorenzo Berra, Harvard addressing  ETT and Nitrous Oxide use in covid 19 infected patients.
2. MOUTH.  Oral/Dental.  Given that both parents were dental professionals, and encouraged me to do same, (which I refused), remarkable transition for clinical microbiology to include oral microbiology and dental students, professionals.  Research involved plaque reduction (Biofilm) and use of oral rinses, a variety of mouth RX, which catalyzed interest and passion for probiotics as Restorative Microbiology (PerioBALANCE). Antibiotic research started with Low Dose Doxycycline  (LDD) latter changed to Sub Inhibitory Dose DOXY (FDA) with  multiple studies, and presentations to FDA. Finally, release of PERrioSTAT.  Periodontics, first followed by Endodontics, highlighted probiotics  as non-antibiotic intervention. NO RESISTANCE. MI. MINIMAL INTERVENTION. To assist in use of pre and probiotics, given a variety of options, e designed and integrated a COMPUTER ASSISTED DECESION TREE   ANALYSIS FOR PROBIOTIC OPTIONS,  based on published probiotic research emphasizing Bac-2-Health for disease specific targeting and Partners-4-Life for long term diet supported probiotics.  We focused initially on oral and dental, but added wound and aging diseases to the Decision Tree.
3. SKIN.  Wound care. Early, I drew similarities to wound architecture and periodontology, open wounds. Further catalyzed by LDD management in skin infections, and recognition of wound biofilms. Developed SMART Gauze concept and biphasic management, DISRUPTION:RECONSTRUCTION. Used Beneficial Microbes to create beneficial biofilm, integrating 3-D-printing to reconstruct gauze engineered to wound architecture. Carnegie Mellon University scientists.
4. GIT.  Cancer and Microbes.  We wanted to connect Microbial Clocks and Biologic Clocks in common pathway of multiple metabolic diseases over time.  We recognized that the loss of our microbiota over time promoted selected microbes to address a different phenotype, often associated with neoplasia, and metastasis to distal locations. It also prompted our work in biofilms predicting chemotherapy interventions (See above, Section BIOFILMS AS TUMORS, OR VICE VERSA)

SOCIO-MICROBIOLGY.  BIOFILMS IN NEW POLITICAL WORLD and Older Audiences. (OLLI)

1. HOLOGENOMIC THEORY  and genetics. I integrated ‘dual citizenship’ as co evolution and importance of microbes as ‘universal contributors/donors’ of genetic information, fast or slow almost MICROCHIP-like.  Created the Center for Hologenomic  Studies,  focusing on Aging and age related diseases.   Recognized that in biofilms genetic strength of whole much greater than individual, alone.  (Garth) We are not alone in who we are.
2. Microbial politics.  Bernie/Vermont.   In political climate of 2020, compared organization of biofilms to society, biofilms reflection a socio-political organization and function. Biofilms are more Left than Right, politically; Planktonic microbes, are Right. My state, Vermont and Bernie Saunders, Left.  Recognized microbial communities and human populations share schemes for survival and benefit of community. Created political buttons to include: “Microbes Mater, Don’t Trash your Microbiota”. There is a Microbial United Nations.

Center of Excellence in Biofilm Research

The Center of Excellence in Biofilm Research (CEBR) is an innovative research environment. Working in close collaboration with 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.

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 extracellular polymeric substances (EPS) living on surfaces. Their resilience has been related to physiology and protection by the EPS 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.

CEBR's Critical Areas of Interest:

• Characterize the microbiome in chronicdisease.
• Correlate bacterial genes and pathways to themolecular behavior of microbes and their hosts.
• Identify and functionally characterizemolecules involved in multiple stages of biofilm development.
• Describe the genomic variability within singlebacterial species.
• Capture strain plasticity during chronic andpolyclonal infections.
• Investigate strain evolution in the post-antibiotic era.
• Develop and test molecules and materials foranti-biofilm properties.

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.

Pathogen Pipeline

Step One: Microbial Detection

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 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.

Step Two: Microbial Visualization

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 ex vivo clinical samples allows researchers the ability to detect and localize the presence or absence of specific nucleic acid sequences. Fluorescent 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.

Step Three: Microbial Exploration

There are many other tools available to investigate biofilms. Here is a brief list of some of the other services we can provide.

• Microbial whole genome sequencing:de novo sequencing and resequencing available.
• RNA sequencing: identify isoforms,novel transcripts, gene fusions and non-coding RNAs.
• In vitro and ex vivo testing of anti-biofilm molecules and surfaces.
• Comparative Genomics
• Phylogenetics
• Transcriptomics
• Bioinformatics

Highlighted publications

1)Nickel JC, Stephens A, Landis JR, Chen J, Mullins C, van Bokhoven A, Lucia MS, Melton-Kreft R, Ehrlich GD; The MAPP Research Network. (2015) Search for Microorganisms in Men with Urologic Chronic Pelvic Pain Syndrome: A Culture-Independent Analysis in the MAPP Research Network. J Urol. Jul;194(1):127-35.

2)Kathju S, Nistico L, Melton-Kreft R, Lasko LA, Stoodley P.Direct demonstration of bacterial biofilms on prosthetic mesh after ventral herniorrhaphy. Surg Infect (Larchmt). 2015 Feb;16(1):45-53.

3)Palmer MP, Altman DT, Altman GT, Sewecke JJ, Ehrlich GD, Hu FZ, Nistico L, Melton-Kreft R, Gause TM 3rd, Costerton JW. Can we trust intraoperative culture results in nonunions? J Orthop Trauma. 2014 Jul;28(7):384-90.

4)Jacovides CL, Kreft R, Adeli B, Hozack B, Ehrlich GD,Parvizi J. Successful identification of pathogens by polymerase chain reaction (PCR)-based electron spray ionization time-of-flight mass spectrometry (ESI-TOF-MS) in culture-negative periprosthetic joint infection. J Bone Joint Surg Am. 2012 Dec 19;94(24):2247-54.

5)Yun HC, Kreft RE, Castillo MA, Ehrlich GD, Guymon CH,Crouch HK, Chung KK, Wenke JC, Hsu JR, Spirk TL, Costerton JW, Mende K, Murray CK. Comparison of PCR/electron spray ionization-time-of-flight-mass spectrometry versus traditional clinical microbiology for active surveillance of organisms contaminating high-use surfaces in a burn intensive care unit, an orthopedic ward and healthcare workers. BMC Infect Dis. 2012 Oct 10;12:252.

6)Tuttle MS, Mostow E, Mukherjee P, Hu FZ, Melton-KreftR, Ehrlich GD, Dowd SE, Ghannoum MA. Characterization of bacterial communities in venous insufficiency wounds by use of conventional culture and molecular diagnostic methods. J Clin Microbiol. 2011 Nov;49(11):3812-9.

7)Kreft, R., Costerton, J. W., & Ehrlich, G. D. . PCR ischanging clinical diagnostics. Microbe 2010; 8(1):15-20.

8)Hall-Stoodley L, Hu FZ, Gieseke A, Nistico L, Nguyen D,Hayes J, Forbes M, Greenberg DP, Dice B, Burrows A, Wackym PA, Stoodley P, Post JC, Ehrlich GD, Kerschner JE. Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media. JAMA. 2006 Jul 12;296(2):202-11.

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