https://orcid.org/0000-0001-7768-8972
Figures
Abstract
Laboratory grown biofilms are used to simulate bacterial growth in diverse environmental conditions and screen the effectiveness of anti-biofilm therapies. Recently, we developed a glass bead biofilm reactor that utilizes low broth volume to provide high-throughput biofilm growth for testing and translation across the research continuum (e.g., benchtop assays to preclinical models). Bioburden per mm2 surface area of Staphylococcus aureus and Pseudomonas aeruginosa biofilms were comparable on beads and CDC Biofilm Reactor® coupons. In this study, we hypothesized that biofilms grown on beads would be more susceptible to ertapenem, moxifloxacin, and tobramycin than those grown on coupons. Results indicated a significant reduction in S. aureus bioburden on glass beads compared to glass coupons following treatment with ertapenem (p = 0.005) and tobramycin (p = 0.014). P. aeruginosa biofilms had smaller differences in antibiotic response between the two systems. There was a significantly greater reduction in bead P. aeruginosa biofilm than coupon when treated with tobramycin (p = 0.035). This work offered insight into how the bead biofilm reactor could be used as a tool for antibiotic screening and translation across the continuum of in vitro to in vivo systems that support development of antimicrobial technology.
Citation: Gilmore AL, Vu H, Martinez T, Peniata L, Kawaguchi B, Armbruster DA, et al. (2025) In vitro antibiofilm efficacy of ertapenem, tobramycin, and moxifloxacin against biofilms grown in a glass bead or CDC Biofilm Reactor®. PLoS ONE 20(2): e0318487. https://doi.org/10.1371/journal.pone.0318487
Editor: Abdelwahab Omri, Laurentian University, CANADA
Received: November 7, 2024; Accepted: January 16, 2025; Published: February 10, 2025
Copyright: © 2025 Gilmore et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: This research was funded by Congressionally Directed Medical Research Programs (CDMRP), award W81XWH-20-1-0378 to DW and DA.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Researchers often use laboratory grown biofilms to predict the antimicrobial and clinical potential of anti-biofilm therapies [1,2]. There are myriad biofilm reactor systems to accomplish this; each provides unique properties to replicate biofilms in different environments. There are two broad categories of biofilm growth: static and dynamic. Static growth often occurs in closed, “batch” styled reactor environments that lack continuous replacement of nutrients. For example, the minimum biofilm eradication concentration (MBEC) titer plate (previously Calgary biofilm device) is a commonly used static system to determine the amount of antibiotic required to reduce bioburden of single-layered biofilms grown on pegs [3,4]. Dynamic, comparatively, has continuous nutrient replacement and often incorporates high shear via spinning or stirring the growth medium. The most common and widely validated dynamic growth system is the CDC Biofilm Reactor® in which biofilms can be grown on 24 coupons in a turbulent, annular-like reactor under batch and flow phase.
The CDC Biofilm Reactor® is widely published on, yet the system is limited by substrate number and bulk [5,6]. Translating the biofilm-ridden substrates from in vitro to in vivo applications is challenging [7,8]. We recently developed a bead biofilm reactor containing a custom stainless-steel insert with divots designed to hold fifty roughened bead substrates, allowing them to be exposed to low (40 rpm) shear force for biofilm growth [9]. Glass beads were selected to mimic environmental debris such as soil that often contaminates open wound sites following traumatic injury. The beads can be used for high-throughput in vitro screening or as in vivo inocula [10]. Staphylococcus aureus and Pseudomonas aeruginosa biofilms grown on glass beads develop comparable bioburden (colony forming units (CFUs)) per surface area [9]. However, we did not previously determine the antibiotic susceptibility of biofilms grown on glass beads, nor compare outcomes to biofilms grown in a standardized CDC Biofilm Reactor®. We hypothesized that, based on nuance differences in growth parameters, biofilms grown on glass beads would be less robust and thus more susceptible to antibiotics than biofilms grown on glass coupons in a CDC Biofilm Reactor®.
Three antibiotics were selected for comparison: ertapenem, moxifloxacin, and tobramycin. These are military-relevant and apply to our broader orthopedic research program. Ertapenem and moxifloxacin are broad-spectrum antibiotics administered quickly after US soldiers sustain front-line injuries [11]. Clinicians commonly deliver tobramycin on-label (i.e., intravenously (IV) or intramuscularly (IM)) to manage Gram-negative-related infections [12], or off-label as a local antibiotic powder sprinkled into wounds [13].
Ertapenem is a beta-lactam that inhibits bacterial cell wall synthesis. Adding a carbapenem structure to the beta-lactam ring enhances its resistance to bacterial-produced beta-lactamases. Carbapenems, including ertapenem, are generally safe and well-tolerated. While ertapenem works effectively against a wide range of bacteria, certain Gram-negative pathogens like Klebsiella, Pseudomonas, and Acinetobacter often exhibit resistance. Further, sub-inhibitory concentrations of carbapenems may induce phenotypic changes in bacteria that promote biofilm formation [14]. Despite these limitations, civilians commonly receive carbapenems as prophylaxis following traumatic injury and ertapenem remains an antibiotic of choice amongst frontline US military personnel.
Moxifloxacin targets bacterial DNA synthesis, hindering both survival and reproduction of several anaerobic and mixed bacterial populations [15]. However, the risk-benefit profile of moxifloxacin must be carefully evaluated before use as serious adverse side effects such as tendinosis may occur in certain patient populations [16]. Moxifloxacin is the prophylactic antibiotic of choice for US soldiers to self-administer immediately following traumatic injury.
Tobramycin, an aminoglycoside, is commonly administered to treat serious infections and is rapidly absorbed by surrounding muscular tissue following administration [17]. By binding the 16S rRNA of the bacterial 30S ribosomal subunit, tobramycin inhibits protein synthesis and causes the accumulation of misfolded proteins to develop within the bacterial cell. Other researchers have found aminoglycosides to be primarily effective against metabolically active cells [18,19]. Nevertheless, Tobramycin is commonly used to treat orthopedic infections as it is generally well tolerated and effective against a broad spectrum of pathogens.
Determining the susceptibility profiles of biofilms against antibiotics like these can directly influence clinical decisions. Up to 75% of infections are biofilm related [20]. Such infections are often recalcitrant and require extensive regimens to remove bioburden [21]. Nutrient and/or oxygen gradients within a biofilm may influence persister or dormant cell populations at the core, which can directly affect susceptibility to antibiotics [22,23]. Notably, phenotypic characteristics of biofilms, such as antibiotic tolerance, can differ depending on the environment and available nutrients; S. aureus may form large multicellular plumes when cultured in a CDC biofilm reactor, or smaller aggregates when imaged in vivo.
Screening anti-infective therapies against biofilms grown in reactor systems can elucidate biofilm-specific characteristics and antibiotic responses. Biofilm susceptibility profiles are highly distinct from planktonic outcomes. While individual planktonic cells are susceptible to antibiotic therapy, biofilm communities of the same species often exhibit increased tolerance [23,24]. Likewise, biofilm antibiotic tolerance can differ from structure to structure, depending on the architecture, age, and strain [23]. Biofilm extracellular polymeric substances (EPS) can hinder antimicrobial penetration, enzymes can inactivate antibiotics, and cellular metabolic differences may reduce antibiotic uptake [25,26]. As new anti-biofilms technologies are developed, most require in vitro screening. Therefore, a unique biofilm reactor system that can be translated through the continuum of in vitro and in vivo testing may influence clinical decision making and offer another growth system more representative of biofilm wound contaminant.
Methods
Reagents and materials
Brain heart infusion (BHI) broth was sourced from Becton, Dickinson and Company (Franklin Lakes, NJ), cation-adjusted Mueller Hinton broth (CAMHB) from Hardy Diagnostics (Santa Maria, CA), and 10x phosphate buffered saline (PBS) from Thomas Scientific (Swedesboro, NJ). Ten x PBS was diluted to 1x with deionized (DI) water before being autoclaved. Tryptic soy agar (TSA) plates were prepared with a base from MilliporeSigma (Burlington, MA).
Different classes of antibiotics were selected for military and orthopedic relevance [11,27–29]. Ertapenem sodium was sourced from Research Products International (Mount Prospect, IL), moxifloxacin hydrochloride from Chem-Impex (Wood Dale, IL), and tobramycin from Medisca (Irving, TX).
Bacterial isolates
Isolates were purchased from the American Type Culture Collection (ATCC) and were selected because of their orthopedic infection prevalence [30–32] and biofilm forming potential [33,34]. Frozen stocks of Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 27853 were maintained in BHI with 30% glycerol. Isolates were subbed onto TSA plates and incubated at 37°C overnight 1–2 days before use.
Reactor design and materials
The bead reactor [9] (Fig 1A and B) was assembled with a 100 mm x 50 mm Pyrex crystalizing dish (Charleroi, PA), a custom-machined 304 stainless steel ring insert, and fifty 4 mm diameter borosilicate glass beads (Sigma Aldrich; St. Louis, MO).
(A) S. aureus biofilm growth in the bead reactor. (B) Fifty glass beads were stabilized by a 100 mm × 50 mm insert in a petri dish.
A CDC Biofilm Reactor® (Figs 2A and B) comprised of eight polypropylene reactor arms and 24 borosilicate glass coupons were sourced from BioSurface Technologies (Bozeman, MT). Tubing, barbed connectors, and a peristaltic pump to flow broth into the CDC Biofilm Reactor® were purchased from Cole-Palmer (Vernon Hills, IL).
(A) S. aureus biofilm grown in 100% BHI. (B) Image of empty CDC Biofilm Reactor® reprinted with permission from BioSurface Technologies.
Both the glass beads and borosilicate coupons were roughened by hand with 60-grit sandpaper (3M; Maplewood, MN). All glass parts were cleaned with Alconox (White Plains, NY) and DI water before autoclaving. Reactor parts were assembled prior to autoclaving. Beads were placed into divots in a biosafety cabinet prior to use.
Biofilm growth
A 0.5 McFarland standard (~7.5 × 107 colony forming units (CFUs)/mL) of each bacterium was prepared from a fresh culture. One mL of the 0.5 McFarland standard was added to 50 mL of 50% BHI in the bead reactor and 500 mL of 100% BHI in the CDC Biofilm Reactor®. The bead reactor was placed into a shaking incubator at 37° C and 40 rpm for 72 h. Every 24 h, broth was manually exchanged and replaced using a serological pipette in a biosafety cabinet (Fig 1). For the CDC Biofilm Reactor®, batch-phase growth occurred for 24 h on a hot plate at 37° C and baffle rotation of 130 rpm (Fig 2). A 10% BHI solution flowed through the reactor for another 24 h at 6.94 mL/min. The total CDC Biofilm Reactor® growth time was 48 h.
Scanning electron microscopy imaging
Coupon (n = 2) and bead substrates (n = 8) were removed immediately after reactor growth and fixed with modified Karnovksy’s fixative–2.5% glutaraldehyde and 4% formaldehyde in 0.2 M PBS—at room temperature for 24 h. Fixed substrates were then dehydrated in increasingly concentrated ethanol (EtOH) solutions (70%, 80%, and 90%) for 1–3 h each. The substrates were left in 100% EtOH overnight, then air dried. Substrates were placed onto a scanning electron microscopy (SEM) stage with carbon tape and gold coated with a Hummer 6.2 gold sputter coater (Anatech LTD). Biofilms dwelling on substrates were imaged in secondary electron mode with a JEOL JSM-6610 SEM (Tokyo, Japan).
Antibiotic treatment
The minimum inhibitory concentration (MIC) of each antibiotic was determined against both bacterial strains using the Clinical & Laboratory Standards Institute protocol [35]. In brief, 50 µL of CAMHB were added to columns 2–12 followed by 50 µL of 256 μg/mL antibiotic in columns 1–2. The solution was mixed with a multichannel pipette, and 50 µL was removed and added to wells in column 3. A 1:2 dilution was continued until column 11 for a final concentration range of 64 to 0.0125 μg/mL. A 0.5 McFarland standard was prepared from a fresh culture of bacteria and diluted 1:100; 50 µL of McFarland standard was added per well (~7.5 × 105 CFU/mL) in columns 2–12. Column 1 was reserved as a negative control and column 12 a positive bacterial control. Plates were incubated at 37°C overnight and MICs were determined the following day.
Ten mg/mL primary stock solutions were prepared in scintillation tubes from weighted antibiotic powder suspended in PBS. Primary stocks were diluted with 50% BHI to obtain 100 µg/mL treatment concentrations. To reflect an appropriate ratio of biofilm surface area to treatment volume (SA/V) between different substrate types (253.4 mm2 for CDC Biofilm Reactor® coupon and 50.3 mm2 for bead), 1 mL of treatment was used for beads and 5 mL for CDC Biofilm Reactor® coupons. Following preparation of conical treatment tubes (Kimex), biofilm containing coupons and beads (n = 6 per treatment, reactor type, and run) were aseptically removed from reactors and gently placed into capped tubes. Biofilms in treatment were incubated at 37°C and 40 rpm in a shaking incubator for 24 h.
Microbial quantification
Remaining bacteria was quantified both from treatment broth and on substrates. Treatment tubes were removed from the incubator and 200 µL of sterile glass sand was added to further disrupt adhered biofilm during a vortex-sonicate-vortex (VSV) protocol (1 min, 10 min, and 30 s). To reduce the effects of residual kill, 1 mL of the resulting solution was pipetted from the treatment tube into a microcentrifuge tube and pelleted by centrifugation for 3 min at 5000 rpm. Nine-hundred µL of supernatant was removed and replaced with 900 µL of fresh PBS. The centrifugation process was repeated three times to achieve sub-MIC concentrations of residual antibiotic. The VSV procedure was repeated with an additional 100 µL of sterile glass sand being added. Samples (n = 6 per group, both reactors) were then diluted 10-fold and plated on TSA plates for incubation overnight at 37°C. Colony forming units (CFUs) were counted the following day.
Statistical analysis
Three trials against S. aureus and P. aeruginosa were completed with n = 6 substrates per test group (n = 18 total samples). Bioburden per substrate area was log10 transformed. The average log10 CFU/mm2 of control, untreated substrates was calculated per trial (n = 3) and the remaining bioburden of antibiotic treated samples were subtracted from the average bioburden of the respective trial controls. The average bioburden reduction was then calculated per trial and standard deviation from the entire data set. A Student’s t-test using the average values of bioburden reduction of each trial was used to compare bioburden between CDC Biofilm Reactor® and bead reactor controls, and each treatment.
Results
Biofilm SEM imaging
SEM images of biofilms grown on coupons or beads revealed consistent, mature structures across each trial. However, the plumes of S. aureus grown in the CDC Biofilm Reactor® (Figs 3A–C) looked slightly thicker than those grown in the bead reactor (Figs 3D–F). On both substrate types, S. aureus biofilm exhibited larger 3-dimensional plumes around the roughened areas. Even, multilayered sheets of P. aeruginosa were visible in SEM images of both CDC Biofilm Reactor® coupons and beads (Fig 4). The overall thickness of coupon biofilm was not immediately evident (Figs 4A–C). Notably, flaking of P. aeruginosa biofilm following fixation and dehydration exposed sheet thickness of rounded glass beads (Figs 4D and E). Based on SEM images, we estimated bead biofilm thickness to be approximately 5 bacterial cells.
(A) 500x image of biofilm on a coupon. (B, C) at 1000x, coupon biofilm exhibited thick, multilayered plumes. (D) 500x image of bead biofilm. (E, F) 1000x images of bead biofilm also displayed multiple plumes that appeared slightly less dense than biofilm grown on coupons.
(A) 500x image of CDC Biofilm Reactor® coupon that, like (B and C) 1000x images, revealed even sheets of P. aeruginosa. (D) 500x image of bead biofilm with cracking due to fixation of biofilm onto rounded glass surfaces. (E,F) 1000x images of bead biofilm confirm multilayered sheets of P. aeruginosa growth.
Microbial quantification
CFU data can be accessed in the supplemental file ‘S1 CFU data.’ Quantification of substrate bioburden revealed similar S. aureus and P. aeruginosa CFU per mm2 on coupons and beads (Fig 5). CDC Biofilm Reactor® coupons had approximately 1 log10 CFU/mm2 more S. aureus bioburden than beads with an average of 6.88 ± 0.23 log10 CFU/mm2 compared to 5.65 ± 0.28 log10 CFU/mm2 per bead. There was not a statistically significant difference between P. aeruginosa bioburden between coupons (with an average of 6.39 ± 0.21 log10 CFU/mm2) or bead (6.41 ± 0.62 log10 CFU/mm2). Both reactors demonstrated repeatable, reliable S. aureus and P. aeruginosa biofilm growth.
The average (n = 18 bead, n = 17 coupon) of substrates from 3 trials are represented by grey and striped bars. Each black dot denotes an individual quantified substrate. There was a significant difference between coupon and bead S. aureus biofilm growth (p = 0.003). This was not the case for P. aeruginosa.
Treatment outcomes
S. aureus and P. aeruginosa were both susceptible (as determined by MIC) to all three antibiotics tested (Table 1). The highest MIC was that of ertapenem against P. aeruginosa, which was anticipated based on other studies and its known poor efficacy against that organism. All other MIC values indicated planktonic susceptibility of both organisms.
S. aureus bacteria survived on substrates and in treatment suspensions following antibiotic treatment at concentrations greater than 100x the determined MIC (Fig 6). Seven (of 18) S. aureus biofilm beads were eradicated beyond detectable levels by tobramycin. For both S. aureus and P. aeruginosa, tobramycin and moxifloxacin achieved greater reduction in bioburden than ertapenem. The average reduction in S. aureus bead biofilm burden was 1.77 ± 0.72 log10 CFU/mm2 for ertapenem, 3.65 ± 1.06 log10 CFU/mm2 for moxifloxacin, and 4.56 ± 1.06 log10 CFU/mm2 for tobramycin. CDC-grown S. aureus biofilm had lower reductions in bioburden with an average of 0.76 ± 0.23 log10 CFU/mm2 for ertapenem, 2.63 ± 0.56 log10 CFU/mm2 for moxifloxacin, and 1.72 ± 0.95 log10 CFU/mm2 for tobramycin.
Bead and coupon biofilms were treated for 24 h of in 100 µg/mL antibiotic suspended in 50% BHI broth. Each bar represents the average bioburden of 18 samples. Solid grey represents the CDC Biofilm Reactor® coupons and diagonal lines the bead reactor. Black dots denote the specific CFU of each bead quantified (n = 18 per bar). There was a significant difference (p < 0.05) between the average bioburden of CDC and bead biofilm treated with ertapenem (p = 0.005) and tobramycin (p = 0.014).
Reduction of P. aeruginosa coupon and bead bioburden was more comparable than S. aureus substrates (Fig 7). This could be a result of similarly thick biofilm structures. Moxifloxacin and tobramycin efficacy against bead P. aeruginosa biofilm varied and bioburden was reduced to below detectable levels on some coupons and beads. Of 18 coupons, biofilm of 10 were eradicated beyond detectable levels by moxifloxacin and on 14 (of 18) beads. Tobramycin eradicated biofilm from 2 (of 18) coupons and 13 (of 18) beads. The average reduction in P. aeruginosa bead biofilm burden was 1.77 ± 0.77 log10 CFU/mm2 for ertapenem, 5.85 ± 1.53 log10 CFU/mm2 for moxifloxacin, and 6.16 ± 0.63 log10 CFU/mm2 for tobramycin. CDC-grown P. aeruginosa biofilm had average reductions of 1.25 ± 0.68 log10 CFU/mm2 for ertapenem, 6.10 ± 0.46 log10 CFU/mm2 for moxifloxacin, and 5.50 ± 0.82 log10 CFU/mm2 for tobramycin. Overall, the degree of reduction appears proportional to the shear in which the biofilm was grown with the high shear CDC Biofilm Reactor® biofilm, generally, being more difficult to eradicate than bead.
Biofilms were treated for 24 h in 100 µg/mL antibiotic suspended in 50% BHI broth. Solid grey bars represent the average CFU per three trials for CDC Biofilm Reactor® coupons and diagonal lines the beads. Black dots denote the specific CFU of each bead quantified (n = 6 per trial). There was a significant difference (<0.05) between average bioburden reduction of tobramycin-treated coupons and beads (p = 0.035).
Discussion
Previously, we developed a reactor that consistently produced S. aureus and P. aeruginosa biofilms on glass bead substrates in a low-shear environment [9]. In the current study, we advanced the development of this reactor by testing the hypothesis that biofilms grown on glass beads would be more susceptible to antibiotics than those grown on glass coupons in a CDC Biofilm Reactor®. The hypothesis was supported with S. aureus biofilms, but unsupported with P. aeruginosa. Future work will be needed to clarify why these discrepancies occurred, but it may be attributed to nuance differences in growth conditions including shear forces and batch vs. flow phases that may have influenced biofilm densities and accompanying tolerance—factors that have been well-defined previously [23,36,37].
S. aureus biofilm density is known to be influenced by shear force and a dynamic nutrient profile, which the CDC Biofilm Reactor® provides [38–40]. Kostenko et al. (2010) found S. aureus biofilm morphology to correlate strongly with shear stress, increased biofilm tolerance, and architectural size [41]. In the turbulent environment of the CDC Biofilm Reactor®, it is possible S. aureus biofilms develop more compactly than those in the bead reactor system.
Low shear in the glass bead reactor appeared to produce less dense S. aureus biofilms than those grown in the high shear CDC Biofilm Reactor®. When viewed by SEM, the general S. aureus biofilm plume morphology appeared similar on glass beads and coupons, but there was a noticeable difference in plume height. Only the tops of coupon biofilm easily came into focus (Figs 3A–C), whereas most of the plume structures were visible on beads (Figs D–F). Based on literature, S. aureus plume height and density may have contributed to antibiotic susceptibility [38]. This could explain the discrepancy between coupon and bead log reduction following treatment with tobramycin (Fig 6), in particular, though future studies are needed to determine the ratios of bacterial cell populations.
Morphology and density did not appear to differ between P. aeruginosa biofilms on beads or coupons. In both cases, sheet-like morphologies were present. Cracking of bead biofilm allowed for visualization of sheet thickness (Figs 4D–F). Future work could aim to image cross sections of biofilm grown on coupons for a direct comparison of sheet thickness. We anticipated P. aeruginosa biofilm grown on beads and coupons to be similarly dense because quantification of control substrates indicated similar bioburden per surface area of bead and coupons (Fig 5), which may have correlated to equal antibiotic susceptibility profiles of each antibiotic tested (Fig 7).
Several factors may have contributed to denser S. aureus yet not P. aeruginosa coupon biofilms. First, the growth conditions varied between the CDC Biofilm Reactor® and bead reactor growth protocols. These differences included growth times (48 vs 72 h total), nutrient availability (continuous vs. batch phase), and applied shear force (130 vs 40 rpm). It is well established that growth protocols can significantly impact biofilm development. Shear force and variation in nutrient type and concentration are known alter S. aureus biofilm thickness [38,39,41,42]. Second, maintenance of the incubation temperature varied between systems. Hotplates set to 37°C maintained temperature in the CDC Biofilm Reactor® while a closed 37°C incubator likely keep a more consistent temperature within the bead reactor. Lower temperatures tend to promote biofilm formation [43]. Thus, biofilm growth within CDC Biofilm Reactor® might have been advantaged by partial exposure of the system to a lower ambient room temperature. Third, S. aureus cells may have generated a different plume base between substrate geometries. Coupons and beads were roughened with the same sanding protocol, however, fluid dynamics surrounding a flat coupon, and a spherical bead likely differed. Bead curvature might encourage different S. aureus biofilm adhesion affecting both colonization rate and overall biofilm density [36]. The CDC Biofilm Reactor® and bead reactor procedures differ significantly, yet both produce comparable bioburden per substrate surface area. Evaluating how stress, temperature, and nutrient availability vary has enhanced our understanding of resulting tolerance.
While MIC assays confirmed S. aureus and P. aeruginosa susceptibility to ertapenem, moxifloxacin, and tobramycin, biofilms of the same organisms were far less susceptible to antibiotic reduction. The mechanism of action of moxifloxacin is inhibits bacterial DNA replication; tobramycin inhibits bacterial protein translation. These antibiotics likely avoid common biofilm tolerance mechanisms such as creation of efflux pumps. Future studies should compare and aim to quantify cell populations (e.g., metabolically active, or dormant) throughout each biofilm. Dormant cells might not be as susceptible to antibiotic killing. Ertapenem had broad spectrum activity against many Gram-negative pathogens, and was effective against planktonic P. aeruginosa. Yet ertapenem achieved less than a 3 log10 reduction in both S. aureus and P. aeruginosa bioburden, and in the case of P. aeruginosa, appeared to promote biofilm growth. This finding motivates more challenging biofilm-related research when developing and screening anti-infective technologies.
Future research should consider screening against additional pathogens, including polymicrobial biofilms, and treating with lower concentrations of tobramycin and moxifloxacin. Many substrates had no detectable growth, despite utilization of an antibiotic wash method to mitigate false negative results. Additional concentrations and treatment timepoints could help identify further discrepancies between bead and coupon biofilm.
Conclusions
The bead and CDC Biofilm Reactor® allow for the consistent growth of S. aureus and P. aeruginosa biofilm advantageous to the testing and preclinical development of anti-biofilm products. This work offers insight into the antibiotic tolerance of bead-grown biofilm, further defining characteristics of this relatively new system in the context of a standardized method. Future work and expanded testing will be needed to validate the system but the data shared suggest the bead reactor may be a worthwhile tool for antibiotic screening and translational research applications.
Acknowledgments
The authors wish to thank Shawn Colby and the University of Utah School of Medicine Machine Shop for manufacturing and assisting in the refinement of metal plates for use in the bead reactor and Albert Parker, PhD for his guidance with statistical analysis.
References
- 1. Thieme L, Hartung A, Tramm K, Klinger-Strobel M, Jandt KD, Makarewicz O, et al. MBEC versus MBIC: the lack of differentiation between biofilm reducing and inhibitory effects as a current problem in biofilm methodology. Biol Proced Online. 2019;21:18. pmid:31528123
- 2. Gomes IB, Meireles A, Gonçalves AL, Goeres DM, Sjollema J, Simões LC, et al. Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications. Crit Rev Biotechnol. 2018;38(5):657–70. pmid:28954541
- 3. Ceri H, Olson M, Morck D, Storey D, Read R, Buret A, et al. The MBEC assay system: multiple equivalent biofilms for antibiotic and biocide susceptibility testing. Methods Enzymol. 2001;337:377–85. pmid:11398443
- 4. Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A. The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol. 1999;37(6):1771–6. pmid:10325322
- 5. Gilmore BF, Hamill TM, Jones DS, Gorman SP. Validation of the CDC biofilm reactor as a dynamic model for assessment of encrustation formation on urological device materials. J Biomed Mater Res B Appl Biomater. 2010;93(1):128–40. pmid:20091909
- 6. Buckingham-Meyer K, Goeres DM, Hamilton MA. Comparative evaluation of biofilm disinfectant efficacy tests. J Microbiol Methods. 2007;70(2):236–44. pmid:17524505
- 7. Kay W, Hunt C, Nehring L, Barnum B, Ashton N, Williams D. Biofilm growth on simulated fracture fixation plates using a customized CDC biofilm reactor for a sheep model of biofilm-related infection. Microorganisms. 2022;10(4):759. pmid:35456808
- 8. Williams DL, Woodbury KL, Haymond BS, Parker AE, Bloebaum RD. A modified CDC biofilm reactor to produce mature biofilms on the surface of peek membranes for an in vivo animal model application. Curr Microbiol. 2011;62(6):1657–63. pmid:21437591
- 9. Gilmore A, Badham M, Rudisin W, Ashton N, Williams D. A bead biofilm reactor for high-throughput growth and translational applications. Microorganisms. 2024;12(8):1588. pmid:39203430
- 10. Epperson RT, Isaacson BM, Rothberg DL, Olsen RE, Kawaguchi B, Maxwell JM, et al. Developing a combat-relevant translatable large animal model of heterotopic ossification. Bone Rep. 2021;15:101127. pmid:34584904
- 11.
Government US. Tactical combat casualty care quick reference guide. 2017.
- 12.
Gilbert D, et al. The sanford guide to antimicrobial therapy. 2015.
- 13. Lee Y-P, Farhan S-D, Pendi A, Cunningham TJ, Kiester PD, Hahn P, et al. Does addition of tobramycin powder reduce infection rates after spine surgery? Global Spine J. 2018;8(8):816–20. pmid:30560033
- 14. Bagge N, Schuster M, Hentzer M, Ciofu O, Givskov M, Greenberg EP, et al. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production. Antimicrob Agents Chemother. 2004;48(4):1175–87. pmid:15047518
- 15. Tsaousoglou P, Nietzsche S, Cachovan G, Sculean A, Eick S. Antibacterial activity of moxifloxacin on bacteria associated with periodontitis within a biofilm. J Med Microbiol. 2014;63(Pt 2):284–92. pmid:24217128
- 16. Majalekar PP, Shirote PJ. Fluoroquinolones: blessings or curses. Curr Drug Targets. 2020;21(13):1354–70. pmid:32564750
- 17.
Hospira. Tobramycin for injection, USP. U.S. Food and Drug Administration Website. 2011.
- 18. Hall CW, Mah T-F. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol Rev. 2017;41(3):276–301. pmid:28369412
- 19. Mah T-F, Pitts B, Pellock B, Walker GC, Stewart PS, O’Toole GA. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature. 2003;426(6964):306–10. pmid:14628055
- 20.
Yadav MK, Song JJ, Singh BP, Vidal JE. Chapter 1. Microbial biofilms and human disease: a concise review. new and future developments in microbial biotechnology and bioengineering: microbial biofilms. Elsevier; 2020. p. 1–13. https://doi.org/10.1016/B978-0-444-64279-0.00001-3
- 21. Høiby N, Bjarnsholt T, Moser C, Bassi GL, Coenye T, Donelli G, et al. ESCMID guideline for the diagnosis and treatment of biofilm infections 2014. Clin Microbiol Infect. 2015;21 Suppl 1:S1–25. pmid:25596784
- 22. Lewis K. Persister cells: molecular mechanisms related to antibiotic tolerance. Antibiot Resist. 2012:121–33.
- 23. Stewart PS, Parker AE. Measuring antimicrobial efficacy against biofilms: a meta-analysis. Antimicrob Agents Chemother. 2019;63(5):e00020-19. pmid:30803974
- 24. Nickel JC, Ruseska I, Wright JB, Costerton JW. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob Agents Chemother. 1985;27(4):619–24. pmid:3923925
- 25. Chiang W-C, Nilsson M, Jensen PØ, Høiby N, Nielsen TE, Givskov M, et al. Extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2013;57(5):2352–61. pmid:23478967
- 26. Wang S, Zhao Y, Breslawec AP, Liang T, Deng Z, Kuperman LL, et al. Strategy to combat biofilms: a focus on biofilm dispersal enzymes. NPJ Biofilms Microbiomes. 2023;9(1):63. pmid:37679355
- 27. Anderson A, Miller AD, Brandon Bookstaver P. Antimicrobial prophylaxis in open lower extremity fractures. Open Access Emerg Med. 2011;3:7–11. pmid:27147846
- 28. Garner MR, Sethuraman SA, Schade MA, Boateng H. Antibiotic prophylaxis in open fractures: evidence, evolving issues, and recommendations. J Am Acad Orthop Surg. 2020;28(8):309–15. pmid:31851021
- 29. Petfield JL, Lewandowski LR, Stewart L, Murray CK, Tribble DR. IDCRP combat-related extremity wound infection research. Mil Med. 2022;187(Suppl 2):25–33. pmid:35512376
- 30. Tuon FF, Cieslinski J, Ono AFM, Goto FL, Machinski JM, Mantovani LK, et al. Microbiological profile and susceptibility pattern of surgical site infections related to orthopaedic trauma. Int Orthop. 2019;43(6):1309–13. pmid:30069593
- 31. Murray CK. Epidemiology of infections associated with combat-related injuries in Iraq and Afghanistan. J Trauma. 2008;64(3 Suppl):S232–8. pmid:18316967
- 32. Gries CM, Kielian T. Staphylococcal biofilms and immune polarization during prosthetic joint infection. J Am Acad Orthop Surg. 2017;25 Suppl 1(Suppl 1):S20–4. pmid:27922945
- 33. Chen Q, Xie S, Lou X, Cheng S, Liu X, Zheng W, et al. Biofilm formation and prevalence of adhesion genes among Staphylococcus aureus isolates from different food sources. Microbiologyopen. 2020;9(1):e00946. pmid:31769202
- 34. Cao H, Lai Y, Bougouffa S, Xu Z, Yan A. Comparative genome and transcriptome analysis reveals distinctive surface characteristics and unique physiological potentials of Pseudomonas aeruginosa ATCC 27853. BMC Genomics. 2017;18(1):459. pmid:28606056
- 35.
CLSI. Performance standards for antimicrobial susceptibility testing -- 30th Edition. CLSI document M100; 2020.
- 36. Sauer K, Stoodley P, Goeres DM, Hall-Stoodley L, Burmølle M, Stewart PS, et al. The biofilm life cycle: expanding the conceptual model of biofilm formation. Nat Rev Microbiol. 2022;20(10):608–20. pmid:35922483
- 37. Ciofu O, Moser C, Jensen PØ, Høiby N. Tolerance and resistance of microbial biofilms. Nat Rev Microbiol. 2022;20(10):621–35. pmid:35115704
- 38. Sherman E, Bayles K, Moormeier D, Endres J, Wei T. Observations of shear stress effects on Staphylococcus aureus biofilm formation. mSphere. 2019;4(4):e00372-19. pmid:31315967
- 39. Schilcher K, Horswill AR. Staphylococcal biofilm development: structure, regulation, and treatment strategies. Microbiol Mol Biol Rev. 2020;84(3):e00026-19. pmid:32792334
- 40.
Technologies B. CDC biofilm reactor.
- 41. Kostenko V, Salek MM, Sattari P, Martinuzzi RJ. Staphylococcus aureus biofilm formation and tolerance to antibiotics in response to oscillatory shear stresses of physiological levels. FEMS Immunol Med Microbiol. 2010;59(3):421–31. pmid:20528928
- 42. Rasmussen RM, Epperson RT, Taylor NB, Williams DL. Plume height and surface coverage analysis of methicillin-resistant Staphylococcus aureus isolates grown in a CDC biofilm reactor. Biofouling. 2019;35(4):463–71. pmid:31144524
- 43. Eze EC, El Zowalaty ME. Combined effects of low incubation temperature, minimal growth medium, and low hydrodynamics optimize Acinetobacter baumannii biofilm formation. Infect Drug Resist. 2019;12:3523–36. pmid:31814741