Here, we present a protocol to evaluate the antibacterial efficacy of an antibiotic-eluting polymer to simulate prophylactic clinical application by using a commercially available real-time ATP-based luminescent microbial viability assay. This method enables the monitoring of the longitudinal activity of drug-eluting materials and can be widely adapted to test anti-microbial drug delivery platforms.
Ultrahigh molecular weight polyethylene (UHMWPE) is widely used in total joint arthroplasties as a load-bearing surface. Periprosthetic joint infections, the majority of which occur shortly after joint replacement, constitute almost 25% of total knee revision surgeries, and the complete eradication of bacterial infection poses a major challenge. A promising way to tackle this problem is to ensure the local sustained delivery of antibiotic concentrations sufficient to inhibit the bacteria to support routine systemic antibiotic prophylaxis. There is increased research into the development of efficient local drug delivery devices. Although established antibacterial testing methods for drugs can be used to test the antibacterial efficacy of drug-eluting materials, they are lacking in terms of providing real-time and longitudinal antibacterial efficacy data that can be correlated to the elution profiles of antibiotics from these devices. Here, we report a direct and versatile methodology to determine the antibacterial efficacy of antibiotic-eluting UHMWPE implants. This methodology can be used as a platform to avoid bacterial culture at each time point of a lengthy experiment and can also be adapted to other local drug delivery devices.
Osteoarthritis is a significant degenerative condition that afflicts 500 million adults worldwide1. The gold standard in the treatment of end-stage arthritis is total joint replacement, which is projected to be performed in over 2 million patients annually in the United States alone by 20302, with global demand also increasing tremendously3,4. The procedure involves the replacement of the articulating cartilaginous surfaces of the joints with load-bearing synthetic materials made of metal/ceramic and highly crosslinked ultrahigh molecular weight polyethylene (UHMWPE). Total joint replacements significantly improve the quality of life for patients suffering from arthritis5, but a significant complication that endangers the performance of the implants and the satisfaction of the patients is periprosthetic joint infection (PJI), which accounts for 15%-25% of revision surgeries6. The cause of infection in most cases is believed to be the microbial contamination of the implant site during surgery7. The contaminating population then expands on the implant and tissue surfaces8. The host immune system is triggered in response, but the growth rate and biofilm formation of the contaminating organisms can enable them to evade the immune cells, which can lead to a heightened cytokine and chemokine storm without the eradication of the bacteria9,10,11. The resulting deep infection of the bone jeopardizes the fixation and performance of the implant as well as the health of the patient12.
Staphylococcus aureus and coagulase-negative staphylococci are the predominant causative organisms of PJI13. The severity of staphylococcal infections is high due to their increased resistance to antibiotics, biofilm formation, and ability to persist as small colony variants14,15,16. Implant-associated infections occur due to the adhesion of microorganisms onto the surface of the implant and the establishment of a complex biofilm matrix that enables the bacteria to evade deleterious host immune factors and effective concentrations of antibiotics14,15,16. Conventional treatment methods include intravenous and oral antibiotic combinations for a prolonged period17. A major drawback of this approach is the low bioavailability of the drug at the infection site and the difficulty of achieving sufficient concentrations of antibiotics to eradicate the bacteria consistently over the treatment period, which often results in poor outcomes18. To address these shortcomings, a fully functional localized drug-eluting UHMWPE implant was designed to ensure a sustained release of effective concentrations of antibiotics into the joint space19. Local elution from the drug-eluting implant is used as a complementary tool to prevent the growth and colonization of any bacteria remaining after the implant removal and debridement of the tissue. In vitro antibacterial testing of this antibiotic-eluting UHMWPE can be performed by incubating the material directly with the bacteria and quantifying the bacteria by the spread-plate method20,21. Alternatively, aliquots of eluent media can be tested against bacteria using methods such as the agar disk-diffusion method, broth dilution methods, and time-kill testing22. All these methods rely upon growth observation about 16-18 h after the collection of the aliquots by means of colony counting and turbidity measurements. The elution of antibiotics from such devices can be longitudinally quantified using these in vitro methods; however, to determine the translational value of these concentration profiles, a robust real-time in vitro method to assess antibacterial activity is needed.
The microbial viability assay used in this study was developed for the quantification of viable bacteria by measuring the luminescence corresponding to adenosine triphosphate (ATP) released from a live bacterial cell using luciferin-luciferase-based detection technology. ATP, as the energy currency of living cells, serves as a direct indicator of physiologically viable cells. The reagent performs cell lysis, which releases ATP molecules from viable cells that are then detected in the form of luminescence. This luminescence has a direct correlation to the proportion of viable bacterial cells within the sample23. This is a real-time, simple, versatile, fast, single reagent-based assay that can be adapted into a variety of assay designs and conditions with both gram-positive and gram-negative microorganisms. Here we report a method to determine the real-time antibacterial activity of antibiotic-eluting UHMWPE incubated with laboratory and clinical strains of S. aureus using a modified time-kill assay based on the microbial viability assay (e.g., BacTiter Glo). While the methodology is described with a specific implant material and a specific orthopedic application, the method can provide a platform for testing other antibiotic-eluting delivery devices with clinical applications.
1. Preparation of reagents
2. Preparation of virgin and drug-loaded UHMWPE
Supplementary Figure 1: Parts of the mold used for molding the UHMWPE samples. (A) Stainless steel insert plate; (B) mold cavity; (C) plunger; (D) backplate Please click here to download this File.
3. Determining the elution kinetics of drug-eluting UHMWPE
4. Determination of the vancomycin concentration
5. Determination of the gentamicin concentration by the OPA tagging method 27
6. Bacteria preparation
NOTE: The following S. aureus strains were used in this study: type strain 12600, clinical strains L1101 and L1163 (obtained from Dr. Kerry LaPlante at the University of Rhode Island). The susceptibility profiles of these strains are presented in Table 1.
Bacterial strains | Gentamicin MIC | Vancomycin MIC | |
ATCC 12600 | ≤1 µg/mL (Sensitive) | ≤0.5 µg/mL (Sensitive) | |
L1101 (Clinical strain) | ≥16 µg/mL (Resistant) | 8 µg/mL (Intermediate) | |
L1163 (Clinical strain) | ≤1 µg/mL (Sensitive) | 8 µg/mL (Intermediate) |
Table 1: Antibiotic susceptibility profiles of control and clinical S. aureus strains.
CAUTION: All the steps involving handling bacterial cultures and suspensions were performed in a BSL-2 lab space within a Class II, Type A2 Biosafety Cabinet.
7. Performing the real-time microbial viability assay
8. Generating a luminescence versus viable count standard curve for each bacterial strain
NOTE: Culture and enumerate all the S. aureus strains according to the methodology described in section 6.
9. Time-dependent antibacterial activity study setup
Figure 1: A schematic representation of the experimental setup. Please click here to view a larger version of this figure.
10. Quantification of adherent bacteria on the UHMWPE surface
Following the protocol, UHMWPE was molded with gentamicin and vancomycin at 7% w/w and eluted into deionized water. The drug concentration in the eluent from the material was determined at 6 h, 1 day, 2 days, 3 days, and 1 week. The drug release from vancomycin and gentamicin-loaded UHMWPE demonstrated a burst release at 6 h followed by a steady release rate with a release concentration greater than the minimum inhibitory concentration (MIC) until 7 days (Figure 2, Table 2).
Prior to the antibacterial study, a standard curve was generated to correlate the luminescence units to the CFU/mL of the bacteria for each S. aureus strain (ATCC 12600, L1101, and L1163). The corresponding log (luminescence) values were plotted against the log (CFU/mL) to generate a standard curve (Figure 3). The R2 values calculated were 0.997, 0.996, and 0.994 for ATCC 12600, L1101, and L1163, respectively, indicating a strong fit for the concentration range. The equation derived was subsequently used to calculate the bacterial viability across all the experiments. ATCC 12600 and L1101 demonstrated a limit of detection within a range of 1 x 102-1 x 103 CFU/mL. On the other hand, the limit of detection for the L1163 strain was shown to be 1 x 104 CFU/mL.
The time-dependent antibacterial activity assay was performed using 1 x 105 CFU/mL as the starting inoculum for 12600, L1163, and L1101, which were exposed to 7% w/w drug-eluting materials for a period of 1 week. At each time point (6 h, 1 day, 2 days, 3 days, 1 week), the medium was refreshed, and the bacterial population was re-suspended. The exposure of the bacteria to the subsequent release of drugs from the material was continued until the next time point. UHMWPE with 7% w/w vancomycin and UHMWPE with 7% w/w gentamycin demonstrated >3log reduction for susceptible ATCC 12600 starting at 6 h, and complete eradication (no colony growth) was observed at the end of 3 days (Figure 4A). For the gentamicin-susceptible and vancomycin-intermediate strain L1163, both drug-eluting materials caused >3log reduction at 6 h, and complete eradication (no colony growth) was observed on day 1 of the experiment (Figure 4B). For the gentamicin-resistant and vancomycin-intermediate strain L1101, gentamicin elution from UHMWPE did not affect the bacterial viability of L1101 (Figure 4C). The bacteria proliferated, and the population stabilized within 6 h in the presence of virgin UHMWPE without antibiotic elution. In the presence of gentamicin-eluting UHMWPE, the population reached a similar growth level on day 2. On the contrary, vancomycin elution from UHMWPE significantly reduced the bacterial viability (>3log) at 6 h, and complete viability loss (no colony growth) was demonstrated by day 4.
The surfaces of both gentamicin-eluting and vancomycin-eluting UHMWPE showed no viable adherent bacteria when exposed to susceptible and intermediate-resistant strains after day 7 or complete eradication, whichever came first. Some viable bacteria (1 x 105 CFU/mL) were present on gentamicin-eluting UHMWPE exposed to gentamicin-resistant L1101. Similarly, approximately 1 x 105 CFU/mL of viable adherent bacteria were recovered from the control virgin PE (Figure 5).
Figure 2: Time-dependent average antibiotic release from 7% w/w antibiotic-loaded UHMWPE strip. The average antibiotic release between time points from one 7% w/w gentamicin and vancomycin-loaded UHMWPE strip (3 mm3 x 5 mm3 x 10 mm3 ~ 2 cm2 surface area). The MIC against control strain ATCC 12600 is shown as a dotted line for gentamicin and a solid line for vancomycin. The error bars represent the standard deviation of the mean from six replicates (n = 6). Please click here to view a larger version of this figure.
Figure 3: Real-time luminescent assay standard curve for all the S. aureus strains. Log (luminescence) was plotted against log (CFU/mL) to generate standard curves for control, (A) ATCC 12600, and clinical strains, (B) L1101 and (C) L1163. The equations describing the line of best fit and corresponding R2 values are indicated on the plots. Please click here to view a larger version of this figure.
Figure 4: Bacterial viability determined using a luminescent assay for 7% w/w gentamicin-eluting and 7% w/w vancomycin-eluting UHMWPE. The time-dependent antibacterial activity of gentamicin and vancomycin eluted from UHMWPE against control strains, (A) ATCC 12600, and clinical strains, (B) L1163 and (C) L1101, are shown. Virgin 1020 PE served as a control for the experiment. The yellow line in the plots indicates the limit of detection for the respective S. aureus strain. Data are shown as mean ± standard deviation (n = 3). Please click here to view a larger version of this figure.
Figure 5: Adherent bacteria viability determined using the luminescent assay Glo assay for 7% w/w gentamicin-eluting and 7% w/w vancomycin-eluting UHMWPE against all S. aureus strains. The bar chart indicates adherent bacteria (CFU) recovered per centimeter squared (cm2) of 7% gentamicin-loaded and 7% vancomycin-loaded UHMWPE at the end of the study period for all the strains tested. The bars show data as mean ± standard deviation (n = 3). Please click here to view a larger version of this figure.
Time points | Vancomycin | Gentamicin |
Peak concentration (µg/mL) | Peak concentration (µg/mL) | |
0 - 6 h | 336 ± 72 | 263 ± 24 |
6 h -1 day | 57 ± 18 | 16 ± 2 |
1 day - 2 day | 60 ± 18 | 7 ± 1 |
2 day - 3 day | 23 ± 6 | 5 ± 0.4 |
3 day - 7 day | 49 ± 20 | 15 ± 1 |
Table 2: Peak drug concentration (μg/mL) at different time points. Data are shown as mean ± standard deviation (n = 6).
Supplementary Figure 2: Luminescence signal decay over a period of 10 min from the addition of assay reagent to the sample. A ±5% difference in the signal is shown as a dotted line Please click here to download this File.
The localized sustained delivery of antibiotics is a necessary tool in the management of periprosthetic joint infections. Systemic antibiotics are the primary strategy in eradicating bacterial infection, and the local elution is used as a complementary tool to prevent the growth and colonization of any bacteria remaining after the implant removal and debridement of the tissue. The goal for the effective area under the curve (concentration over a period) for antibiotics with local administration is not well understood. The elution of antibiotics from such devices can be longitudinally quantified in vitro; however, to determine the translational value of these concentration profiles, a robust in vitro method to assess antibacterial activity is needed. In this paper, one such real-time method is described to determine the antibacterial activity of drug-eluting UHMWPE to be used as a sustained delivery device in joint replacements.
The real-time monitoring of bacterial viability is a crucial parameter of interest, and conventional microbiological methods lack the framework to accommodate this specific aspect of the study. The microbial viability assay used in this study was developed for the quantification of viable bacteria by measuring the luminescence corresponding to adenosine triphosphate (ATP). To directly investigate the time-dependent activity of the antibiotics eluted from the implant materials, three different strains with distinct antibiotic susceptibility profiles were incubated with them. The rationale for using laboratory and clinical strains with varying resistances to gentamicin and vancomycin was to understand the range of activity for a given implant formulation. Further, the antibacterial activity and efficacy against these distinct populations are dependent on the timing of administration. The method focuses on the feasibility of prophylaxis against these strains based on >70% of periprosthetic joint infections being caused by the contamination of the wound at the time of surgery24.
As a starting inoculum to develop this method, 1 x 105 CFU/mL was used. Different contaminating concentrations have been used for animal models although not much is known about the clinically relevant infection load for PJI. Animal infection models for PJI have been routinely established using 1 x 105 CFU, and a similar range is widely used in standardized methods (CLSI) to determine antibacterial activity25,26,27. Using 1 x 105 CFU/mL as an initial contaminating concentration allowed us to evaluate both the growth and eradication parameters at the same time.
Conventionally, MIC values are determined for a constant antibiotic concentration for a specific number of bacteria, and they fail to demonstrate the rate of antibacterial action. Due to this aspect, MIC values do not provide a quantitative differentiation to describe the antimicrobial activity profile28. The data from the current method emphasize the advantage of evaluating the strain-dependent killing kinetics of antibiotics rather than using the MIC to make dosing decisions. Using this method, it was possible to differentiate both the extent and the rate at which the implant materials affected the different strains. Gentamicin eluted from the implant material strips was effective in eradicating L1163 in 1 day and eradicating ATCC 12600 in 2-3 days, but it was ineffective in eradicating L1101 (Figure 4). In addition to the expected lack of activity of gentamicin elution against L1101 (MIC >32 µg/mL) due to its inherent gentamicin resistance (Figure 4C), the persistence of subpopulations was observed when exposed to vancomycin, for which L1101 exhibits intermediate resistance. In contrast, L1163 was definitively eradicated in the presence of vancomycin-eluting UHMWPE despite exhibiting similar intermediate resistance to vancomycin as L1101 (an MIC of 8 µg/mL has been observed for both strains).
The observations that the rate of activity of gentamicin against 12600 and L1163, which are gentamicin-susceptible with similar MIC values (an MIC of 1µg/mL has been observed for both strains), was different, as well as that the extent of activity of vancomycin against intermediate-resistant L1101 and L1163 was different (Figure 4A,B), supported the hypothesis that this real-time method in the presence of the eluting material could differentiate longitudinal differences in the activity.
In addition to the translational value of the results in interpreting how effective a given eluted concentration can be against these bacteria, there are several experimental methodological advantages. (1) The bacterial concentration is determined instantaneously at a given time, contrary to conventional methods in which the bacteria are incubated in broth or on agar for 18-24 h to determine viability. This period of growth can provide additional time for the bacteria to recover from antibiotic stress, introducing an additional possible source of error/variability. (2) The media is continually replaced while retaining the bacteria, which more closely resembles in vivo conditions than static conditions. (3) This assay inherently includes the drug release kinetics from the implant, which allows for better performance prediction. (4) The method has been developed using commonly available consumables without the need of any specific or expensive machinery.
Robust in vitro testing methods to evaluate drug-delivery applications are necessary before proceeding to in vivo animal studies and clinical trials. This assay can be modified and adapted to accommodate various approaches and drug delivery platforms such as particles, gels, films, and other drug-eluting materials to determine the efficiency of bacterial eradication in a simulated in vitro setup. Modifications can be performed for the sample setup by changing to a suitable in vitro medium environment, which has been shown to influence the activity of several antibiotics29,30.
The method also facilitates viable adherent bacteria determination, which is promising, as conventional methods to determine minimum biofilm eradication concentration is time-consuming and delivers inconsistent results. However, the method is to be rigorously tested on biofilms to develop a reliable and robust methodology to determine its sensitivity. The ATP-based luminescent method could be sensitive enough to detect viable forms of bacteria in biofilms including persisters, which may or may not be detected on an agar plate as visible colonies. Taken together, this versatile platform has the potential to incorporate relevant parameters to record real-time observations on the anti-bacterial and anti-biofilm activity of drugs of interest.
The efficacy of this method is governed by the following aspects:
Pre-determined elution characteristics and sample size
The elution profile of the antibiotic-eluting material can be identified in a separate experiment prior to this antibacterial activity measurement such that amount of material required to actively conduct the experiment within a stipulated time can be determined.
Container and volume determination
It is important to devise a setup in which the media volume of the experiment can accommodate the entire surface area of the same material and to ensure sufficient volume for the unobstructed release of the drug from the drug-loaded surface. The setup used was based on previous experimentation, ensuring "perfect sink" conditions for these hydrophilic drugs such that their diffusion is not hindered by solubility limitations.
Growth media characteristics
Growth media selection should be investigated to ensure the stability and the optimal performance of the selected drug(s)29. Cation-adjusted Mueller Hinton broth (CAMHB), which is widely used in the broth dilution method, was used to determine the MIC of known antibiotics. The medium enables optimal drug activity without the interference of toxic secondary metabolite accumulation31. The assay reagent has been tested and reported stable in different types of media, including those with serum components23,28. Although the relative luminescence unit values may vary across different media, the components of the media have been demonstrated to not interfere with the assay32,33. The experimental volume was further optimized to 1.5 mL, which is close to the synovial fluid volume present in an adult knee joint space34.
Temperature stability for the assay
The handling and addition of the luminescent reagent to the assay are to be performed in a consistent manner across experiments. Temperature changes alter the sensitivity of the assay, so it is important to incubate the reagent at room temperature for 2 h before adding it to the bacteria23.
Reagent incubation time
The luminescence from the reagent decays with time. The luminescent signal has a half-life of over 30 min, which is largely dependent on the medium and the type of bacterium used in the experiment23. Additionally, any differences in incubation time (i.e., the time between adding the reagent to the bacteria and reading the luminescence) will result in inconsistent readings for the same concentration of bacteria. A 5% difference was observed in the luminescent signal when taken within 1 min following the 5 min incubation time according to the manufacturer's instructions (Supplementary Figure 2). Taking this data into account, the luminescence readings were recorded within 1 min throughout the study to ensure the signal loss was not more than 5%. Further, it is important to limit the number of samples per plate to reduce the error introduced due to luminescence decay from the first well to the last well.
Maintenance of the bacterial population throughout the study
The method attempted to model the drug clearance and synovial volume turnover by continually separating the spent media from the bacterial population at each time point using high-speed centrifugation at 10,000 x g for 10 min35. This critical step ensures the sedimentation of all the viable and non-viable bacterial cells. Further to this, the sedimented bacteria are uniformly reconstituted in fresh MHB and transferred to the syringe setup, facilitating the complete carryover of the affected microbial population back to the experimental setup. The reproducibility and reliability of this method heavily rely upon simulating the sustained exposure of antibiotics to the microbial population derived from the initial inoculum.
A key limitation of this method is that it is a semi-static assay that does not accurately simulate drug half-lives and continuous synovial turnover. However, continual medium replacement partially compensates for this limitation. The sensitivity of the microbial viability assay was strain-dependent, ranging from 1 x 102-1 x 104 CFU/mL, which limits the detection capability. Furthermore, a standard curve needs to be plotted for each organism as the strain type contributes to the sensitivity and performance of the luminescent reagent. Both the bacteria growth dynamics and the activity of the used drug compound may be affected by the medium components, which should be further investigated.
This work was funded partially by National Institutes of Health Grant No. AR077023 (R01) and by the Harris Orthopaedic Laboratory. The authors thank Dr. Kerry Laplante and her team at the University of Rhode Island for providing the clinical strains L1101 and L1163.
Name | Company | Catalog Number | Comments |
 96 well plates - polystyrene, High Bind, white flat bottom wells, non-sterile, white, 100/cs | Corning, NY, USA | CLS3922-100EA | |
2-mercaptoethanol | Sigma Aldrich, Germany | ||
ATCC 12600 | American Type culture Collection, VA, USA | ||
BacTiter-Glo Microbial Cell Viability Assay | Promega Corporation, USA | G8231 | |
BD Bacto Tryptic Soy Broth (Soybean-Casein Digest Medium) | Becton-Dickinson, USA | BD 211825 | purchased from Fisher Scientific, USA |
BD Luer-Lok Syringe sterile, single use, 3 mL | BD, USA | 309657 | |
BD Needle 5/8 in. single use, sterile, 25 G | BD, USA | 305122 | |
BDÂ BBL Dehydrated Culture Media: Mueller Hinton II Broth (Cation-Adjusted) | Becton-Dickinson, USA | B12322 | purchased from Fisher Scientific, USA |
BDÂ Difco Dehydrated Culture Media: Tryptic Soy Agar (Soybean-Casein Digest Agar) | Becton-Dickinson, USA | DF0369-17-6 | purchased from Fisher Scientific, USA |
Boric Acid | Fisher Chemical, NJ, USA | ||
Branson 1800 ultrasonic bath | Emerson, MO, USA | ||
Corning Falcon Bacteriological Petri Dishes with Lid | Fisher Scientific, USA | 08-757-100D | |
Gentamicin Sulfate | Fujian Fukang Pharmaceutical Co., Fuzhou, China | ||
Greiner UV-Star 96 well plates | Sigma Aldrich, Germany | M3812-40EA | |
Hydraulic press | Carver, Inc. Wabash, IN, USA | ||
L1101Â | Clinical strain from Dr Kerry Laplante, University of Rhode Island | ||
L1163Â | Clinical strain from Dr Kerry Laplante, University of Rhode Island | ||
LSE benchtop shaking incubator | Corning, NY, USA | ||
Methanol, Optima for HPLC, Fisher Chemical | Fisher Scientific, NJ, USA | A454-1 | |
Napco CO2 6000 | Thermo Scientific, MA, USA | ||
PBS, Phosphate Buffered Saline, 1X Solution, pH 7.4, Fisher BioReagents | Fisher Scientific, USA | BP24384 | |
Phthaldiadehyde ≥97% (HPLC) | Sigma Aldrich, Germany | P1378-5g | |
Plate reader (Synergy H1 | Biotek, VT, USA | ||
press | Carver, Inc. Wabash, IN, USA | ||
shaker Innova 2100 | New Brunswick Scientific, NJ, USA | ||
ShopBot D2418 | ShopBot Tools, Inc., NC, USA | ||
Sodium Hydroxide | Sigma Aldrich, Germany | ||
Thermo Scientific Reagent Grade Deionized Water | Fisher Scientific, USA | 23-751628 | |
UHMWPE | GUR1020, Celanese, TX, USA | ||
Vancomycin Hydrochloride | Fagron, The Netherlands | 804148 | |
WAB Turbula | WAB Turbula, Switzerland |
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