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. 2024 Dec 18;5(4):223–229. doi: 10.1089/phage.2024.0001

Bacteriophage Dosing and Its Effect on Bacterial Growth Suppression in a Staphylococcus epidermidis Model: An In Vitro Study

Jason Young 1,2,✉, Mohammad Javad Shariyate 2,3, Ahmad Hedayatzadeh Razavi 3,5, Ara Nazarian 2,3,4,5,6,*, Edward K Rodriguez 2,3,4,*
PMCID: PMC11876813  PMID: 40045941

Abstract

Background:

Phages are an emerging therapy in the treatment of prosthetic joint infections, though many challenges remain, including an incomplete understanding of optimal phage dosing.

Materials and Methods:

We performed an in vitro assessment of how phage dosing as measured by multiplicity of infection (MOI) impacts bacterial growth in planktonic and biofilm conditions using a Staphylococcus epidermidis model. Staphylococcus epidermidis ATCC 35984 was combined in planktonic and biofilm forms with phage vB_SepM_Alex at varying concentrations, and growth was monitored via spectrophotometry.

Results:

Planktonic bacterial growth was significantly higher when MOI ≤ 0.01 compared with MOI ≥ 10 (p < 0.05). Biofilms with phage dosing at ≤ 104 plaque-forming units (PFU)/mL had significantly greater spectrophotometer readings than those dosed at 1010 PFU/mL (p < 0.05).

Conclusions:

Our findings suggest lower, not higher, phage dosing is associated with greater bacterial persistence. Our study helps inform the dosing and delivery of this alternative form of antibiosis.

Keywords: biofilm, phage therapy, phage dosing, bacteriophage dosing, bacteriophages, Staphylococcus epidermidis

Introduction

Prosthetic joint infections (PJIs) cause significant morbidity in patients,1 with the presence of bacterial biofilms as a major barrier to treatment effectiveness, perpetuating infection persistence despite appropriate antibiotic therapy.2,3 Staphylococcus epidermidis is one of the most common causative organisms of biofilms on medical devices, including prosthetic joints.4 Staphylococcus epidermidis is known to form biofilms on implanted medical hardware that confer resistance to antibiotic treatment5 and promote recurrent infection.6 Consequently, in order to achieve infection eradication, the standard of care typically involves a surgical exchange of the infected prosthesis in either a one- or two-stage approach and long-term antibiotics,7 a process associated with significant patient morbidity and mortality.8 With an estimated incidence of up to 2.4% after primary arthroplasties9 and an economic burden exceeding $1.8 billion,10 PJIs thus remain a challenging clinical problem within orthopedic surgery, and there is an urgent need to develop more effective treatments than the standard of care.

Bacteriophages are being evaluated as an alternative form of antibacterial therapy for PJI management.11,12 Naturally occurring and highly selective, many phages have been observed to possess biofilm eradication potential,13,14 while being well-tolerated in humans.11,15–17

However, one of the principal challenges to the successful application of phage therapy is ongoing uncertainty regarding phage pharmacokinetics and pharmacodynamics,18 specifically in understanding optimal phage dosing thresholds.19 Prior mathematical modeling and preliminary clinical data have suggested that applied phage concentrations should be at least 108 plaque-forming units per milliliter (PFU/mL).19 From a mathematical standpoint, the administration of higher numbers of phage than there are bacteria (multiplicity of infection, or MOI > 1) has been posited as an important principle, as evidenced by Poisson distribution modeling of phage behavior, to achieve adequate adsorption to enable bacterial population eradication.20 Unfortunately, measuring and attempting to validate these thresholds clinically is difficult, and numerous other factors are also at play, including patient immune response, bacterial density, bacterial growth rate, phage infectivity, and burst sizes, among other factors.19

From the standpoint of empirical testing, there has also been controversy: prior studies have posited that delivering a higher ratio of bacteriophage host would be associated with lower bacterial resistance,21 while other studies have suggested that the extreme selective pressure accelerates the development of resistance.22 Therefore, there is a need for studies investigating how phage dosing might impact bacterial response and, ultimately, phage therapy treatment outcomes.

Consequently, we propose an initial in vitro study to assess how different applied phage concentrations impact bacterial growth in an in vitro model. We hypothesize that higher phage dosing is associated with greater bacterial growth suppression. Ultimately, this study aims to elucidate how phage concentration impacts its therapeutic potential to shed light on developing more informed phage delivery strategies.

Methods

Bacterial strain

We used S. epidermidis strain ATCC 35984 (ATCC, Manassas, VA), a previously sequenced clinical isolate of S. epidermidis and a known aggressive biofilm former.23 Isolates were propagated in brain heart infusion (BHI) broth (AG Scientific Incorporated, San Diego, CA) for all experiments. Samples were stored in a BHI broth and 16.7% (v/v) of glycerol at −80°C.

Phage propagation and isolation

Phage vB_SepM_Alex (DSM 108061) was obtained from the Leibniz Institute-DSMZ (Braunschweig-Süd, Germany). The phage is a known obligate lytic phage originally isolated from wastewater that forms clear plaques on S. epidermidis lawns. The supplier did not report genome sequence information. Serial dilutions of all phages were propagated on lawns of ATCC 35984 via the double agar overlay method.24 The phage was observed to form distinct countable lytic plaques. Individual plaques of vB_SepM_Alex were then isolated, prepared in SM buffer, filtered through a 0.22 μm filter, and then propagated overnight in liquid BHI media containing exponential phase ATCC 35984. The next day, lysates were centrifuged at 3400 g for 5 min, and subsequently, the supernatant passed through a 0.22 μm filter. A serial dilution was repeated in triplicate to determine phage concentrations in PFU/mL. Phages were stored at 4°C.

Optical density measurement and calibration to bacterial concentration

Overnight cultures of ATCC 35984 were grown in BHI media at 37°C. Subsequently, serial dilutions were performed. Optical density (OD) measurements at a wavelength of 600 nm (OD600) were taken on a NanoDrop™ One/One C Microvolume UV-Vis Spectrophotometer (Thermo Fischer Scientific Inc., Waltham, MA). Dilutions at 100 μL were plated on BHI media with 1.5% agar plates and incubated overnight at 37°C. Colonies were counted, and colony-forming units per milliliter (CFU/mL) were determined. Concentrations were plotted against their corresponding OD600 measurements, and a linear regression was performed to calibrate OD600 measurements to bacterial concentrations in the exponential phase. Measurements and dilutions were repeated in triplicate.

Planktonic assay

An overnight culture of ATCC 35984 was diluted 1:100 in BHI media supplemented with 10 mM MgSO4. CFU/mL estimates of this dilution were performed using a spectrophotometer, and the replicates were normalized to an estimate of 1.66 × 106 CFU/mL. For the experiment, 100 μL of this bacterial culture was combined with 100 μL of appropriately diluted samples of vB_SepM_Alex at various multiplicities of infection (MOI 10,000, 1000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001) in polystyrene flat-bottom 96-well plates with low-evaporation lids (Corning™ Clear Polystyrene 96-Well Microplates, Corning Inc.) and subsequently incubated in an absorbance microplate reader (Sunrise™ Plate Reader, Tecan Group Ltd., Switzerland) at 37°C with readings taken every 5 min at OD600 and reported in optical density units (ODU). Subsequent analyses performed on the first 12 h of collected readings as stationary phase were noted in the bacterial control wells at approximately 10–12 h. A blank media control, bacterial control at approximately 8.30 × 105 CFU/mL, and a phage control at approximately 8.30 × 105 PFU/mL were also included on the plate. Four repeated experiments, each with four technical replicates, were performed. Data was collected through Tecan’s Magellan™ data analysis software and subsequently exported into Microsoft Excel (Microsoft Corporation, Redmond, WA). To confirm the accuracy of the estimated delivered bacterial load, bacterial samples were plated on agar, cultured overnight, and counted manually.

Biofilm assay

An overnight culture of ATCC 35984 was diluted in 1:100 BHI, and 200 μL of the dilutant was transferred to flat-bottom 96-well plates and grown for 48 h at 37°C without agitation. We chose an incubation period of 48 h based on prior reports documenting mature biofilm formation of ATCC 35984 after 48 h of incubation.23 Plates were subsequently gently rinsed three times with phosphate-buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA), and the remaining biofilms were incubated with vB_SepM_Alex at varying concentrations (1010 to 1 PFU/mL). A modified version of a previously described protocol was used to stain the biofilms with crystal violet25–28 at the following timepoints: 1, 2, 4, 8, and 12 h after phage administration. Briefly, assay plates were carefully washed three times in distilled water and subsequently stained for 15 min with 0.1% crystal violet solution (Thermo Fisher Scientific). Stained plates were dissolved with 95% ethanol, and absorbance was subsequently measured with a spectrophotometer at OD600. Due to known variability in biofilm mass formed in 96-well plates,29 eight replicates for each delivered phage concentration and four replicates for controls were performed.

Additionally, to assess whether antibiotic co-administration impacts bacterial growth at different levels of applied phage doses, the biofilm experiments were repeated with the addition of vancomycin. Vancomycin was dosed at 15 mg/kg to mimic in vivo optimal intravenous concentrations for PJI treatment30 and to exceed previously documented minimum inhibitory concentrations needed for ATCC 35984.31 These assays were performed in quadruplicate.

Statistics

Descriptive statistics include median OD600 measurement and associated 95% confidence interval. Non-parametric statistics were used due to the non-normality of the data based on histogram assessment and Shapiro–Wilk testing (p < 0.05). Kruskal–Wallis analyses with Dunn’s test pairwise comparisons with Bonferroni correction were performed to assess between-group differences in spectrophotometer readings. Alpha was set at 0.05. All statistical analyses were performed in STATA (STATA 18.0, StataCorp, College Station, TX).

Results

Planktonic assays

In our planktonic assays, we observed the dose-dependent effects of phage application on bacterial regrowth (Fig. 1). At 12 h, greater bacterial growth was observed with lower phage doses compared with higher phage dosing (χ2 = 146.0, p < 0.0001) (Fig. 2). Specifically, we noted greater bacterial load when phage was dosed at MOI 0.1 compared with MOI 10,000 (p = 0.023). Additionally, when the phage was dosed at MOI 0.01, bacterial load was greater than when the phage was dosed at MOI 10 or higher (p < 0.005, all pairwise tests). Finally, when the phages were dosed at MOI 0.001, bacterial load was significantly greater than when the phage was dosed at MOI 0.1 or higher (p < 0.05, all pairwise tests). There was no evidence of significantly greater bacterial growth at higher MOI, with no differences in mean spectrophotometer readings between negative controls and groups dosed at MOI 1 or greater. Individual Dunn pairwise testing with Bonferroni corrections is available in Supplementary Appendix SA1.

FIG. 1.

FIG. 1.

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Median continuous OD600 spectrophotometer readings of planktonic Staphylococcus epidermidis (ATCC 35984) after exposure to phage vB_SepM_Alex at varying MOI. The interquartile range is depicted as a colored band. MOI, multiplicity of infection

FIG. 2.

FIG. 2.

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Dunn’s test pairwise comparisons of planktonic bacterial regrowth in assays receiving phage at varying MOI, as measured by mean OD600 spectrophotometer readings. *Bacterial growth was significantly higher at MOI 0.1 compared with MOI 10,000; **Bacterial growth was significantly higher at MOI 0.01 compared with phage control and phage dosed at MOI 10 or higher; ***Bacterial growth was significantly higher than negative controls and phage dosed at MOI 0.1 or higher.

Biofilm assays

At 12 h, our biofilm analysis also supported a dose-dependent effect of phage application on biofilm load (Supplementary Appendix SA2). Specifically, wells where phage was delivered at less than or equal to 104 PFU/mL demonstrated significantly higher bacterial load when compared with wells receiving the highest dose phage at 1010 PFU/mL or negative control wells (χ2 = 56.9, p < 0.0001; p < 0.05 for all pairwise tests) (Fig. 3). Individual Dunn pairwise testing with Bonferroni corrections is available in Supplementary Appendix SA3.

FIG. 3.

FIG. 3.

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Dunn’s test pairwise comparisons of biofilm bacterial regrowth in assays receiving phage at varying MOI, 12 h. *Bacterial growth was significantly higher in selected groups compared with media control; **Bacterial growth was significantly higher in selected groups compared with phage control; ***Bacterial growth was significantly higher in selected groups compared with the group receiving phage at 1010 PFU/mL.

Antibiotic co-administration

Biofilm assays were repeated with vancomycin co-administration (Supplementary Appendix SA2). At 12 h, while significant differences were detected between groups (χ2 = 41.6, p < 0.0001) (Fig. 4), no differences in median bacterial load were observed between phage treatment groups, either when compared with each other or when compared with vancomycin alone (Supplementary Appendix SA4). Significantly higher bacterial loads were detected among the following groups as compared with negative (media) control: biofilm control, biofilm with vancomycin control, vancomycin with phage administered at 106 PFU/mL, and vancomycin with phage administered at 100 PFU/mL (p < 0.05, all pairwise tests). Bacterial loads were significantly higher in the positive control group (biofilm alone) compared with both negative control groups (phage alone or vancomycin alone) (p < 0.05, all pairwise tests).

FIG. 4.

FIG. 4.

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Dunn’s test pairwise comparisons of biofilm bacterial regrowth in assays receiving both phages at varying MOIs and Vancomycin. *Bacterial growth was significantly higher in selected groups compared with media control; **Bacterial growth was significantly higher in the biofilm control compared with phage and Vancomycin controls.

Discussion

The difficulty of treating PJIs with conventional therapies has led to research into alternative forms of antibiosis, including phage therapy. However, the development of phage therapy as a treatment modality for PJIs has been hampered by a lack of understanding of the administration,18 notable in dosing and delivery.

This analysis represents one of the first dedicated assessments of phage dosing on bacterial load and one of the first to examine phage therapy against S. epidermidis in PJI treatment. In our planktonic assay, we observed significantly higher bacterial loads at 12 h when MOI ≤ 0.01 compared with MOI ≥ 10, while in our biofilm assay, phage dosing at ≤ 104 PFU/mL had significantly greater spectrophotometer readings than those dosed at 1010 PFU/mL at 12 h. No differences in mean spectrophotometer readings were observed between various phage doses when given with vancomycin.

Our planktonic and biofilm assays support the idea that higher applied phage doses are associated with lower levels of detectable bacteria at 12 h compared with lower doses. These findings support dose-dependency between administered phage and suppression of bacterial growth, at least in Staphylococcal assays, aligning with those in previous studies. In their work assessing phage-antibiotic pairs in vitro on Staphylococcus aureus biofilms, Kebriaei et al. report that for certain phage-bacterial pairs, an MOI of 0.1 yielded greater suppression of bacterial growth as compared with an MOI of 0.01.32 Similarly, Morris et al. observed that their anti-Staphylococcal phage cocktail reduced bacterial growth dose-dependently against two S. aureus strains.33 In their assessment of phage 191219 against S. aureus, the authors report dose-dependent antibiofilm activity of their phage with delivering increasing concentrations and increased antibacterial activity against planktonic bacteria when the phage was delivered at MOIs of 10 and 1 but not at MOIs of 0.1 or 0.001.34

Others have previously suggested that phages delivered at higher MOI may be associated with greater resistance emergence by promoting favorable mutations for survival.22 Our results do not support this hypothesis. Certainly, our 12-h time course may be too short to observe evidence of bacterial resistance and breakthrough growth, and it is likely true that any pre-existing resistant bacteria within a population would be selected through successful phage therapy application and eradication of a majority of bacteria within a population. However, conceptually, it is unclear how phage administration could promote mutations that confer phage resistance within targeted bacterial colonies. Additional work is needed to understand whether such a resistance pathway exists.

Interestingly, while our planktonic assay suggests even low phage ratios (MOI 0.1) can lead to significant reductions in bacterial load compared with control, our biofilm assay results suggest adequate reductions in bacterial load were only achieved with the highest concentration of delivered phage in the study (1010 PFU/mL). Unfortunately, obtaining estimates of approximate CFUs of biofilms is inaccurate based on standard plating techniques alone;29 consequently, accurate estimates of applied phage MOIs could not be reliably obtained. However, our administered phage concentration exceeds many dosing schemes used for the PJI treatment.35–37 While specific phage-host interactions and patient factors will influence the minimum necessary phage required to achieve therapeutic effect, our results support that successful phage therapy in treating biofilms may require higher doses than those needed for planktonic bacterial growth suppression. The reasons for this phenomenon remain unclear and require future investigation, but may relate to properties of the biofilm itself, including reduced phage adsorption rates from the extracellular matrix, decreased phage diffusion through the biofilm, or a downregulated metabolic state of bacterial cells in the biofilm limiting local phage proliferation.38

Finally, in our antibiotic co-administration assay, no differences in mean spectrophotometer readings were observed between various phage doses when vancomycin was administered. Certainly, multiple prior works have documented additive and synergistic effects of antibiotics and phage when co-administered across a variety of bacterial species.39–41 Unfortunately, due to high variability in biofilm formation using plating techniques,29 the variance associated with mean spectrophotometer readings was high in many of our treatment groups. Our analysis was likely underpowered to detect such effects. Additionally, the suppression of bacterial growth by vancomycin at our therapeutic-level vancomycin dosing limits our ability to interpret the dosing effects of antibiotic and phage co-administration in this study. In order to better elucidate any impacts co-administered antibiotics may have on bacterial growth from different applied phage doses, future work should consider alternative assay techniques, using sub-minimum inhibitory concentrations of antibiotics, and examining other antibiotic-phage combinations, including the use of antibiotics for which the bacteria have documented resistance.

Limitations

Our study is not without limitations. First, from a technical standpoint, there are known limitations to assessing biofilm formation using polystyrene plates and variability in biofilm dislodgement during the washing and staining process.29 While plate biofilm assays remain widely utilized, these limitations can generate variability in measured biofilm, in addition to having limited sensitivity to live bacteria compared with other assays.42 We attempted to mitigate these effects by adhering to a strict protocol, limiting inter-plate measurements, and increasing the number of replicates performed. However, future work should consider alternative assay approaches to investigate this topic. Second, we assessed a single phage-microbe pair in this analysis. While our results are corroborated by findings from other authors whose investigations use different bacteria and phages, a more rigorous analysis involving multiple phages and microbes may be warranted. Third, in addition to possibly being underpowered, the delivered dose of vancomycin, while mimicking clinical dosing for PJI, was likely too high for an in vitro co-administration assay with our phage strain, and measured spectrophotometer readings were likely near the detection limit for the assay. Future work should consider alternative approaches, such as assessing antibiotic co-administration at sub-minimum inhibitory concentrations or using antibiotic-resistant strains.43 Fourth, while we did not observe the emergence of bacterial resistance at 12 h after phage delivery, future studies should consider assays that enable longer-term observations to assess for the potential emergence of late phage resistance. Fifth, while spectrophotometry is commonly used to estimate bacterial density,44 it does not always accurately reflect true underlying bacterial density. Future work can consider corroboration of findings with flow cytometry, scanning electron microscopy, or other modalities to more accurately assess bacterial load and MOI. Despite these limitations, this study provides preliminary insights into phage dosing for PJI treatment and generates hypotheses about dosing, which should be further explored in other model systems and organisms.

Conclusions

While phage therapy is being explored as an alternative form of antibiosis for PJI treatment, questions remain regarding optimal dosing and delivery strategies. Our findings suggest higher phage dosing is associated with greater bacterial suppression in vitro at 12 h. Our findings also raise the hypothesis that achieving therapeutic biofilm eradication may require higher phage doses as compared with treating planktonic bacteria. Our study helps inform the dosing and delivery of this experimental form of antibiosis.

Acknowledgments

We would like to thank the Baym Lab for their generosity and expertise in aiding with study methadology.

Data Availability

All reasonable requests for datasets and code can be made available by contacting the corresponding author.

Ethical Statement

No animals or human subjects were involved in the conduct of the presented research. The research presented in this article was approved by the Beth Israel Deaconess Medical Center Institutional Biosafety Committee (ID 23-0025).

Authors’ Contributions

J.Y.: conceptualization, study design, data collection, analysis, article writing and editing, revisions; M.J.S.: data collection, article writing, and editing; A.H.R.: article editing, data visualization; A.V.R.: data analysis and presentation, revisions; A.N.: supervision, article writing, and editing; E.K.R.: supervision, article writing, and editing.

Author Disclosure Statement

The authors declare that they have no competing interests.

Funding Information

The Darwin Project generously supported this work. Additionally, this work was supported by the UL1TR002541 award through Harvard Catalyst, the Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, National Institutes of Health), and financial contributions from Harvard University and its affiliated academic healthcare centers. The content is solely the authors’ responsibility. It does not necessarily represent the official views of Harvard Catalyst, Harvard University, its affiliated academic healthcare centers, or the National Institutes of Health.

Supplementary Appendix SA1
Supplementary Appendix SA2
Supplementary Appendix SA3
Supplementary Appendix SA4

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Appendix SA1
phage.2024.0001_supplementary_appendix_sa1.pdf (152.8KB, pdf)
Supplementary Appendix SA2
phage.2024.0001_supplementary_appendix_sa2.pdf (302KB, pdf)
Supplementary Appendix SA3
phage.2024.0001_supplementary_appendix_sa3.pdf (145.3KB, pdf)
Supplementary Appendix SA4
phage.2024.0001_supplementary_appendix_sa4.pdf (152.6KB, pdf)

Data Availability Statement

All reasonable requests for datasets and code can be made available by contacting the corresponding author.

Articles from PHAGE: Therapy, Applications, and Research are provided here courtesy of SAGE Publications

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