Journal Pre-proof

Staphylococcal Biofilm: Penetration and bioavailability of vancomycin

with or without rifampin

Kathryn E. Daffinee , Emily T. O’Neill , Callan R. Bleick ,

Geoff Williams , Valentin Antoci , Dioscaris Garcia ,
Kerry L. LaPlante

PII: S0732-8893(23)00057-3
DOI: https://doi.org/10.1016/j.diagmicrobio.2023.115947
Reference: DMB 115947

To appear in: Diagnostic Microbiology & Infectious Disease

Received date: 22 March 2022

Revised date: 14 March 2023
Accepted date: 25 March 2023

Please cite this article as: Kathryn E. Daffinee , Emily T. O’Neill , Callan R. Bleick , Geoff Williams ,
Valentin Antoci , Dioscaris Garcia , Kerry L. LaPlante , Staphylococcal Biofilm: Penetration and
bioavailability of vancomycin with or without rifampin, Diagnostic Microbiology & Infectious Disease
(2023), doi: https://doi.org/10.1016/j.diagmicrobio.2023.115947

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Highlights

 Rifampin does not improve humanized doses of vancomycin biofilm penetration

 S. aureus biofilm vancomycin bioavailability with Fluorescence Recovery After
Photobleaching
 Vancomycin quickly attaches to biofilm but its biofilm penetration is slow and incomplete
 Vancomycin’s penetration through a biofilm on medical device material
Staphylococcal Biofilm: Penetration and bioavailability of vancomycin with or without
rifampin

Authors: Kathryn E. Daffinee1; Emily T. O’Neill1,2; Callan R. Bleick2; Geoff Williams3; Valentin
Antoci4,5; Dioscaris Garcia4,5; Kerry L. LaPlante*1,2,6

Author Affiliations:
1. Infectious Diseases Research Program, Providence Veterans Affairs Medical Center,
Providence, RI
2. College of Pharmacy, University of Rhode Island, Kingston, RI
3. Leduc Bioimaging Facility, Brown University, Division of Biology and Medicine,
Providence, RI
4. Department of Orthopaedics, The Warren Alpert School of Medicine, Brown University,
Providence, USA.
5. The Diane N. Weiss Center for Orthopaedic Trauma Research, Rhode Island Hospital,
Providence, RI, 02906, USA.
6. Warren Alpert Medical School of Brown University, Division of Infectious Diseases,
Providence, RI

Principal Investigator: Kerry L. LaPlante, Pharm.D., FCCP, FIDSA, FIDP, University of Rhode
Island College of Pharmacy, 7 Greenhouse Road, Kingston, RI 02881 E-mail:
KerryLaPlante@uri.edu

Corresponding Author:
Kerry L. LaPlante, Pharm.D., FCCP, FIDSA, FIDP
University of Rhode Island College of Pharmacy
7 Greenhouse Road, Suite 255A-C
Kingston RI 02881
E-mail: KerryLaPlante@uri.edu Office: 401-874-5560

Keywords: Staphylococcus epidermidis, biofilm, vancomycin, rifampin, microscopy

Declarations of Interest: Kerry LaPlante receives research funding from Merck, Pfizer
Pharmaceuticals, Shionogi, Paratek, and Entasis. Callan A. Bleick, Emily T. O’Neill, Geoff
Williams, Valentin Antoci, and Kathryn E. Daffinee have no relevant financial relationships.
Dioscaris Garcia holds equity in BI Medical, LLC.,

Acknowledgements: The information provided in this note are those of the authors and do not
necessarily reflect the position or policy of the United States Department of Veterans Affairs.
This work was supported in part, by the Office of Academic Affiliations (OAA) at the Department
of Veterans Affairs.

Funding: This work was unfunded, and the authors received no outside support for this work.

Abstract:
We measured antibiotic penetration and bioavailability in staphylococcus biofilms using

simulated humanized concentrations of fluorescent vancomycin plus or minus rifampin.

Vancomycin percent recovery across biofilm layers was:upper=46%, middle=40%, and

lower=33%. Vancomycin plus rifampin was not significantly different (p= 0.65). Addition of

rifampin did not improve vancomycin penetration across biofilm layers.

Staphylococcus epidermidis, a common biofilm forming bacteria, is a frequent cause of medical
1
device related infections. Health care-associated infections increase hospitalizations,

antimicrobial resistance, and cost the public health system 28 billion USD annually. 2 High rates

of antibiotic-resistant strains coupled with biofilm formation causes antibiotic treatment failure

and often requires device removal for infection source control. While in-vitro results demonstrate

that vancomycin suppresses bacterial numbers in biofilms, vancomycin alone does not

eradicate biofilms. Clinical outcomes for MRSA bacteremia remain poor with treatment failure as
3-9
vancomycin MICs increase. This may be due to a biofilm infection source with poor
10
antibacterial penetration and low bacterial metabolic activity. Therefore, empirical therapy
11
often requires rifampin adjunctive therapy. Rifampin is known for its ability to penetrate S.

epidermidis biofilms. Nevertheless, its use in biofilm infections is controversial as it fails to

suppress bacterial growth due to rapid resistance development.12,13 To address the controversy

of whether it is beneficial to add rifampin to vancomycin and to further understand the

mechanisms of medical device related infections, we built a model on the basis of a CDC biofilm

reactor to observe biofilm penetration and calculate antibiotic bioavailability of simulated

humanized vancomycin plus or minus rifampin within a S. epidermidis biofilm grown on

polyurethane, a common implant device material.14 Wild type slime producing MRSE (ATCC
15,16
35984™) was used due to its well characterized high biofilm forming properties. The

minimum inhibitory concentrations of vancomycin and rifampin were 2 µg/mL and 0.015 µg/mL

respectively and considered susceptible. They were determined via microbroth dilution

according to Clinical and Laboratory Standards guidelines.17,18Minimum biofilm eradication

concentrations for vancomycin and rifampin are published as >1024µg/mL and 128µg/mL
19
respectively. An overnight streak on Tryptic Soy Agar was used to make a starting inoculum

of 10^7 CFU/mL in Tryptic Soy Broth supplemented with 1.0% dextrose, 50 mg/L calcium, and

12.5 mg/L magnesium. Sterile polyurethane coupons (CA# RD128-PU), representative of

implanted medical devices, were placed in FluoroDish Cell Culture Dish and covered with 4mLs
of inoculum. Samples were incubated shaking 50RPMs at 35°C for 18-24 hrs. Afterwards,

biofilms were gently rinsed in triplicate with sterile water to remove planktonic bacteria. Biofilms

were then treated with red fluorescent FilmTracerTM Sypro® Biofilm Matrix Stain following

Thermo Fisher Scientific protocol.

Similar to previous work, biofilms were imaged with a 25 × dipping objective Olympus FV 1000

confocal microscope with 505-nm to 540-nm (green fluorescence representing BODIPY-FL-VAN

from Invitrogen™ V34850) and 575-nm to 620-nm (red fluorescence representing FilmTracerTM

Sypro® Biofilm Matrix Stain) emission filters. Z-slices were obtained every 2 microns, over a 60-

min period to observe antibiotic penetration and rate at which the drug traversed the biofilm

matrix. Images were analyzed using FIJI ImageJ Software at times 1, 5, 15, 30, and 60min at a

prechosen depth within the biofilm.20 Unique from previous work, each antibiotic or antibiotic

combination (with rifampin) was added gently in a singular 4mL final concentration mixture at

time zero before imaging commenced. Antibiotic concentrations were selected to simulate

maximum unbound free concentration of a humanized dose (ƒCmax): BODIPY-FL-VAN was

25µg/mL (IV 500mg Q12) and rifampin was 1.4µg/mL (oral single dose 600mg).21-23 A two-

person confirmation system was used to verify similar depths between biofilms to maintain

consistency with each analysis. Fluorescence intensity over time was quantified by color

histogram using FIJI ImageJ Software at each time period within the entire Z-slice and within a

specified region of interest (ROI) (measuring 505 mm x 89 mm). Experiments were performed in

triplicate.

Fluorescence Recovery After Photobleaching (FRAP) was performed as previously described

for other species, utilizing a high intensity light pulse applied to a defined ROI to irreversibly

photo-bleach an area for fluorescence recovery to be observed over time.24,25. If total

fluorescence is recovered, it is assumed that the molecule has high bioavailability and can move

freely inside the matrix. If partial or no fluorescence is recovered, it is assumed the molecule
interacted in some way with the matrix environment and is not freely diffusible or bioavailable.

FRAP was performed by acquiring three image scans at 3% of laser maximum intensity,

followed by a single bleach spot in a defined ROI with 488-nm and 458-nm at 100% laser

intensity. The bleached image size was fixed to 512x128 pixels with an 80-nm pixel size using

12-bit resolution recording. A series of 300 single-section images were then collected at 290-ms

intervals with the laser powered to its original setting. FRAP was performed at an upper, middle,

and lower layer of biofilm for comparison. Fluorescence recovery was analyzed using a

recovery curve to determine quantitative mobility of the molecule and subsequent bioavailability.

Each experiment was performed in triplicate and significance determined by Anova: Single

Factor.

Fluorescence microscopy revealed that while vancomycin quickly attached to the biofilm within

one minute, its penetration progression was slow and incomplete. After 60min, the BODIPY tag

could not be visualized in the lower layers of the biofilm (Figure 1). The addition of rifampin

showed no increased biofilm penetration of the vancomycin BODIPY tag (p= 0.65). FRAP

results showed only partial fluorescence recovery of BODIPY-vancomycin alone and the

addition of rifampin slowed fluorescence recovery of BODIPY-vancomycin in the lower and

middle layers. The reduced bioavailability of BODIPY-vancomycin by the addition of rifampin is

shown by the development of a FRAP Curve for each biofilm layer tested. (Figure 2) When

compared to BODIPY-vancomycin alone, addition of rifampin caused a -8% and -9% change in

fluorescence recovery (bioavailability) in the lower and middle biofilm layers respectively and a

4% increase in the upper layer.

We sought to determine vancomycin’s ability to penetrate and spread through a biofilm on

medical device material and whether the addition of rifampin could increase this activity.

Vancomycin failed to fully penetrate the biofilm and addition of rifampin did not increase
vancomycin penetration or bioavailability. Therefore, we can assume that BODIPY-vancomycin

interacted in some way with the matrix environment and was not freely diffusible or bioavailable

throughout the biofilm. Our data is consistent with clinical trials that have shown addition of
26,27
rifampin provided no additional benefit. Vancomycin’s lack of biofilm penetration has been
20
seen by others and while vancomycin is capable of biofilm eradication, it requires much
28
higher concentrations than systemic dosing can achieve. Our data may explain why recurrent

infections and stagnant mortality rates in implant-based and chronic infections are high.

However, while this study utilizes a ATCC representative quality control strain, we cannot

assume results to be applicable to all S. epidermidis and there is a continued need for

innovation in treating high inoculum, biofilm-forming Staphylococcal infections.

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AUTHOR CONTRIBUTIONS STATEMENT

Kerry LaPlante Conceptualization, Methodology, Funding acquisition, Writing- Reviewing and

Editing, Supervision
Kathryn Daffinee.: Data curation, Writing- Original draft preparation, Formal analysis,
Investigation, Visualization, Methodology, Project administration
Emily T. O’Neill: Data curation, Writing- Original draft preparation, Formal analysis,
investigation, Visualization
Callan R. Bleick: Writing- Original draft preparation
Geoff Williams: Data curation, Methodology
Valentin Antoci: Writing- Reviewing and Editing
Diocaris Garcia: Writing- Reviewing and Editing, Validation
Figure 1. S. epidermis ATCC® 35984 biofilm treated with FilmTracerTM Sypro® biofilm matrix
stain (red) first to show the top of the biofilm and then added BODIPY-Vancomycin (green) with
yellow coloring as the fluorescence signal overlap between the red biofilm stain and green
vancomycin. These are side-view images to highlight the slow rate of drug penetration into the
biofilm starting at T1min and at T60min after drug addition. A) Biofilm with 25µg/mL BODIPY-Van
(green) + 1.4 µg/mL Rifampin. B) Biofilm treated with 25µg/mL BODIPY-Vancomycin (green)
alone. Drug diffusion was incomplete and did not reach the bottom layers of the biofilm.
Figure 2. Fluorescence Recovery After Photobleaching (FRAP) Curves. Top: Graph depicts
fluorescence recovery of FL-BODIPY-Van with rifampin over 90 seconds within three distinct
layers in the biofilm. After 90 seconds, average percent recovered is as follows: lower=25%,
middle= 31%, and upper layer =50%). Bottom: Graph depicts fluorescence recovery of FL-
BODIPY-Van alone over 90 seconds within three distinct layers in the biofilm. After 90 seconds,
average percent recovered of vancomycin is: lower=33%, middle= 40%, and upper layer =46%.
AUTHOR CONTRIBUTIONS STATEMENT

Kerry LaPlante Conceptualization, Methodology, Funding acquisition, Writing- Reviewing and

Editing, Supervision
Kathryn Daffinee.: Data curation, Writing- Original draft preparation, Formal analysis,
Investigation, Visualization, Methodology, Project administration
Emily T. O’Neill: Data curation, Writing- Original draft preparation, Formal analysis,
investigation, Visualization
Callan R. Bleick: Writing- Original draft preparation
Geoff Williams: Data curation, Methodology
Valentin Antoci: Writing- Reviewing and Editing
Diocaris Garcia: Writing- Reviewing and Editing, Validation