Calcium sulfate beads made with antibacterial essential oil-water emulsions exhibit growth inhibition against Staphylococcus aureus in agar pour plates.

Calcium sulfate bone void filler beads are fully absorbable in the body, and are often used in complicated orthopedic infection cases to release a relatively high dose of antibiotics locally to the body site over time. However, the antibiotic resistance crisis and/or inability to treat chronic biofilm infections remains to be a formidable and increasing health threat. In this report, we tested the hypothesis that plant essential oils (PEOs) with anti-staphylococcal qualities could inhibit the growth of Staphylococcus aureus (a major etiological agent of periprosthetic joint infection) in agar pour plates when infused in calcium sulfate beads. To begin, we conducted a screen of 57 single plant PEOs for anti-staphylococcal activity via disk diffusions assays. We observed that 55/57 of the PEOs had significant growth inhibitory activity compared to the null hypothesis, and 41/57 PEOs exhibited activity similar-to-or-higher-than a vancomycin minimum inhibitory control. When PEOs were infused in beads, we observed that 17/57 PEOs tested exhibited significant bacterial growth inhibition when encased in S. aureus-seeded agar compared to a null hypothesis of six millimeters (bead size). However, none of the PEO-beads had activity similar to a vancomycin bead control made according to a clinically relevant formula. To the best of our knowledge, this is the first report and screen of PEOs for growth inhibitory activity when infused in lab-made calcium sulfate beads. These data indicate that antibacterial PEOs warrant further investigations, and may be useful in developing new treatment strategies for periprosthetic joint infection.

Introduction

Staphylococcus aureus is a main causative agent of bone and joint infections in orthopedic medicine [1], and is a formidable Gram positive pathogen. S. aureus is also responsible for numerous other types of bacterial diseases including skin and soft tissue infections [2], sepsis [3], and respiratory illnesses [4]. In addition to the arsenal of virulence factors that S. aureus produces during infection [5], S. aureus also forms biofilms that resist both host defenses and chemical/antibiotic attack [5, 6]. What is more, a large portion of S. aureus infections are difficult to treat because they are caused by methicillin-resistant S. aureus (MRSA) [7], whereas, methicillin is a first line of treatment for S. aureus infections [7]. For MRSA infections, vancomycin is often used as the next line of treatment [8], however, vancomycin-resistant S. aureus (aka VRSA) isolates have also emerged in which case daptomycin may be implemented [9].

In orthopedics, the use of vancomycin-loaded fully resorbable therapeutic-grade calcium sulfate (CaSO4) bone void filler beads (beads), and/or non-absorbable poly(methylmethacrylate) (PMMA) is a common tactic for intractable enterococci infections, including MRSA [10]. In infection cases where an orthopedist elects to use antibiotic-loaded CaSO4 beads, pharmaceutical-grade antibiotic powders are hand-blended with purified CaSO4 powder, which is then mixed with a proprietary water-based solvent (which accelerates bead-hardening). The resulting paste is spackled into an elastomer bead mold (the beads solidify in minutes), and they are then expressed out of the mold for use in the body site.

The mixing of antibiotics into CaSO4 beads (or PMMA) occurs primarily via “off-label” use in U.S. operatories, and at the discretion of the surgeon [10, 11]. The threat of antibiotic resistance in infection cases however, especially in orthopedics, is a modern critical concern [10, 12], and the discovery and/or development of new or novel antimicrobials is crucial to treating modern and future infection cases. One potentially good, abundant, and affordable source of natural antimicrobials are plant distillates (aka plant essential oils, PEOs). Indeed, a wealth of literature exists reporting and reviewing the antimicrobial efficacy of PEOs against important microbes, including S. aureus, as well as the suspected mechanism(s) by which PEOs elicit antimicrobial effects [13].

Existing reports have modeled and researched the antimicrobial qualities and kinetics of lab-made antibiotic-loaded CaSO4 beads against both Gram positive and Gram negative orthopedic pathogens, including S. aureus [10, 14–16]. In this report, however, we tested the hypothesis that PEOs with anti-staphylococcal properties would elicit growth inhibition against S. aureus in vitro when infused in CaSO4 beads that mimic those made for use in orthopedic infection cases. To test this, we loaded PEOs into lab-made CaSO4 beads and observed if PEOs could elicit S. aureus growth inhibition in agar pour plates. In addition to identifying a number of PEOs that elicit anti-staphylococcal activity, we also note that initial disk diffusion assays were not reliably predictive of which PEOs would also elicit growth inhibition when infused in beads.

Materials and methods

Strains, media, growth conditions, and plant essential oils tested

This study used S. aureus ATCC® 25923™ (Rosenbauch) as model (orthopedic) pathogenic organism. For general culturing purposes, S. aureus was grown and maintained using BD Bacto™ tryptic soy broth (TSB) or TSB-agar (Fisher Scientific, Hampton, NH). Prior to performing disk diffusion assays, bacterial strains were grown to stationary phase overnight in TSB with shaking aeration at 37°C. For susceptibility testing, BD™ BBL™ MHII agar (Fisher Scientific) was prepared fresh according to the manufacturer’s instructions and dried at ambient room temperature for 24 hours. All susceptibility tests (see below) were carried out at 37°C in a humidified chamber with 5% CO2 atmospheric buffering. PEOs tested include Arborvitae, Balsam Fir, Basil, Bergamot, Black Pepper, Black Spruce, Blue Tansy, Cardamom, Cassia, Cedarwood, Roman Chamomile, Cilantro, Cinnamon Bark, Clary Sage, Clove, Copaiba, Coriander, Cypress, Douglass Fir, Eucalyptus, Fennel, Frankincense, Geranium, Ginger, Grapefruit, Green Mandarin, Helichrysum, Jasmine, Juniper Berry, Lavendar, Lemon, Lemongrass, Lime, Marjoram, Melaleuca, Melissa, Myrrh, Neroli, Oregano, Patchhouli, Peppermint, Petitgrain, Pink Pepper, Rose, Rosemary, Sandalwood, Hawaiian Sandalwood, Siberian Fir, Spearmint, Spikenard, Tangerine, Thyme, Turmeric, Vetiver, Wild Orange, Wintergreen, and Ylang Ylang.

Determining the growth inhibitory activity of 57 essential oils via disk diffusion assays

Stationary phase cultures of S. aureus were subcultured 1:100 in fresh sterile TSB and mixed thoroughly. Then, 100 μL of the S. aureus sub-culture was spread onto BD BBL™ MHII agar plates (100 mm diameter) for a final cell volume equivalent to 1/1000th of the original culture density, or approximately 5 x 105 CFU per plate. Next, six mm blank sterile paper filter disks (Fisher Scientific) were aseptically transferred to each spread plate, and 20 μL of each PEO was applied to their respective disks. PEOs observed to have high potency (having full or relatively large zones of inhibition) were tested on plates in the absence of other PEOs so that the zones of inhibition (ZOIs) could be appreciated. All plates were incubated as described above (see strains, media, and growth conditions) for approximately 24 hours (“overnight”). After incubation, the ZOIs on the plates were measured with a Fisher brand metric ruler. The student’s T test was used to determine significance, see Table 1.

Table 1

Average growth inhibition ZOIs and one sample T tests for all PEOs tested in disk diffusion (DDA) and pour plate bead assays.

PEO	Scientific Name	DDA ZOI (mm)	SEM (± mm)	DDA ZOI vs. NH#1 (p values)	Activity* Relative to Vancomycin ZOI	Bead PPZOI (mm)	SEM (± mm)	T-test vs. NH#2 (p values)	PPZOI vs. Vancomycin Bead (p values)
Arborvitae	Thuja plicata	28.5	1.1	<0.0001	Higher	17.4	0.8	<0.0001	Lower
Balsam Fir	Abies balsamea	37.3	1.5	<0.0001	Higher	19.2	0.3	<0.0001	Lower
Basil	Ocimum basilicum	33.7	4.7	0.002	Higher	-	N/A	N/A	Lower
Bergamot	Citrus bergamia	17.6	3.6	0.0225	Similar	5.6	1.2	0.7675	Lower
Black Pepper	Citrus bergamia	18.9	1.3	0.0002	Similar	2.1	1.1	0.0044	Lower
Black spruce	Picea mariana	33.0	5.2	0.0036	Similar	14.7	1.0	0.0002	Lower
Blue Tansy	Tanacetum annuum	15.3	1.4	0.0012	Similar	-	N/A	N/A	Lower
Cardamom	Elettaria cardamomum	25.3	1.0	<0.0001	Higher	5.7	3.0	0.9307	Lower
Cassia	Elettaria cardamomum	32.2	1.7	<0.0001	Higher	21.8	0.6	<0.0001	Lower
Cedarwood	Elettaria cardamomum	12.7	0.7	0.0002	Lower	2.6	1.1	0.0103	Lower
Rom. Chamomile	Elettaria cardamomum	18.3	1.6	0.0005	Similar	-	N/A	N/A	Lower
Cilantro	Coridandrum sativum	80.0	0.0	<0.0001**	Higher	7.4	1.2	0.244	Lower
Cinnamon Bark	Cinnamomum zeylanicum	29.8	2.2	0.0001	Higher	20.7	1.5	<0.0001	Lower
Clary Sage	Salvia sclarea	21.0	1.6	0.0003	Higher	8.0	1.9	0.3126	Lower
Clove	Eugenia carophyllata	16.0	0.9	<0.0001	Similar	7.1	1.5	0.5041	Lower
Copaiba	Copaifera	16.6	1.5	0.0008	Similar	0.5	0.5	<0.0001	Lower
Coriander	Coriandrum sativum	21.7	3.9	0.0101	Similar	5.9	2.2	0.9564	Lower
Cypress	Cupressus sempervirens	32.0	5.3	0.0043	Higher	12.0	2.2	0.0218	Lower
Douglas Fir	Pseudotsuga menziessii	29.7	2.2	0.0001	Higher	12.2	0.8	<0.0001	Lower
Eucalyptus	Eucalyptus radiata	22.8	3.8	0.0071	Similar	11.9	2.6	0.0449	Lower
Fennel	Foeniculum vulgare	13.7	1.7	0.0058	Similar	1.8	0.9	0.0007	Lower
Frankincense	Boswellia	31.7	4.9	0.0035	Higher	10.3	1.6	0.0175	Lower
Geranium	Pelargonium graveolens	15.8	2.0	0.0046	Similar	7.0	2.5	0.6917	Lower
Ginger	Zingiber officinale	12.2	0.8	0.0006	Lower	3.7	1.3	0.0981	Lower
Grapefruit	Citrus x paradisi	14.7	3.1	0.0389	Similar	17.6	0.5	<0.0001	Lower
Green Mandarin	Citrus reticulata	11.8	0.4	<0.0001	Lower	-	N/A	N/A	Lower
Helichrysum	Helichrysum italicum	16.0	3.2	0.0253	Similar	10.2	0.6	0.0003	Lower
Jasmine	Jasminum grandiforum	11.2	1.3	0.0106	Lower	-	N/A	N/A	Lower
Juniper Berry	Juniperis communis	14.0	2.3	0.0171	Similar	9.6	1.7	0.057	Lower
Lavender	Lavandula angustifolia	22.2	2.6	0.0016	Similar	7.7	1.2	0.1914	Lower
Lemon	Citrus limon	21.3	2.5	0.0018	Similar	9.9	1.4	0.0249	Lower
Lemongrass	Cymbopogon flexuosus	29.2	2.8	0.0004	Higher	10.1	0.5	<0.0001	Lower
Lime	Citrus aurantifolia	20.7	3.3	0.0069	Similar	7.7	1.7	0.3305	Lower
Marjoram	Origanum majorana	19.7	2.8	0.0044	Similar	6.1	1.9	0.9524	Lower
Melaleuca	Melaleuca alternifolia	23.2	2.5	0.001	Higher	10.3	2.4	0.1045	Lower
Melissa	Melissa officinalis	19.8	0.6	<0.0001	Higher	1.9	1.0	0.002	Lower
Myrrh	Commiphohra myrrha	10.8	0.4	0.0082	Lower	5.8	1.2	0.8436	Lower
Neroli	Citrus x aurantium	17.8	5.1	0.0689	Similar	2.0	0.9	0.0008	Lower
Oregano	Origanum vulgare	28.5	3.0	0.0006	Higher	15.3	0.7	<0.0001	Lower
Patchouli	Pogostemon cablin	12.8	1.0	0.0009	Lower	5.9	1.3	0.9232	Lower
Peppermint	Mentha piperita	20.0	1.4	0.0002	Higher	8.5	0.5	0.0003	Lower
Petitgrain	Citrus aurantium	22.8	3.4	0.0045	Similar	3.0	1.1	0.0165	Lower
Pink Pepper	Schinus terebinthifolia	10.0	0.3	<0.0001	Lower	2.3	1.0	0.0027	Lower
Rose	Rosa damascena	8.1	0.4	0.0026	Lower	-	N/A	N/A	Lower
Rosemary	Rosmarinus officinalis	29.5	7.4	0.0252	Similar	6.8	1.7	0.6639	Lower
Sandalwood	Santalum album	10.0	1.1	0.0147	Lower	2.2	1.1	0.0086	Lower
Haw. Sandalwood	Santalum paniculatum	9.3	1.0	0.0218	Lower	3.7	1.7	0.2339	Lower
Siberian Fir	Abies sibirica	31.2	5.4	0.0054	Higher	13.2	0.2	<0.0001	Lower
Spearmint	Mentha spicata	22.7	4.2	0.0111	Similar	-	N/A	N/A	Lower
Spikenard	Nardostachys jatamansi	12.7	1.1	0.0017	Lower	4.7	2.4	0.6349	Lower
Tangerine	Citrus reticulata	29.0	4.3	0.0031	Higher	7.4	3.6	0.7139	Lower
Thyme	Thymus vulgaris	42.5	3.4	0.0001	Higher	13.1	1.6	0.0029	Lower
Turmeric	Curcuma longa	8.6	0.2	<0.0001	Lower	2.6	1.3	0.0295	Lower
Vetiver	Vetiveria zizanioides	10.4	0.8	0.0027	Lower	-	N/A	N/A	Lower
Wild orange	Citrus sinensis	9.3	0.8	0.0084	Lower	1.1	1.1	0.0034	Lower
Wintergreen	Gaultheria fragantissima	6.7	2.3	0.7827	Lower	1.1	1.1	0.0023	Lower
Ylang Ylang	Cananga odorata	9.5	0.8	0.0056	Lower	-	N/A	N/A	Lower
Vancomycin		16.0	0.3	<0.0001	N/A	24.1	0.3	<0.0001	N/A

ZOIs listed represent the average ZOI diameters calculated from three biological replicates, where n ≥ 6 (up to 12), and variation is represented as the standard error of the means (SEM). P values of < 0.05 were considered significant.

* Activity is based on p value comparisons.

** Indicates that cilantro had full inhibition in all DDA trials, therefore we artificially introduced ± 0.5 mm of error to the average value of 80 mm for statistical comparison.

NH#1 = null hypothesis of 6 mm for disk diameter.

NH#2 = null hypothesis of 6 mm for bead diameter.

N/A indicates not applicable.

Determining the pour plate ZOI of PEO- and vancomycin-loaded beads

For PEO-bead growth inhibition assays, we first created 1:1 vortex emulsifications of PEOs in sterile dH2O in 50 mL sterile falcon tubes. Emulsifications totaled 4 mL in volume. PEO:water mixtures were emulsified by vortex at full speed for 15 seconds, and then allowed to rest and separate for 5 minutes. Then, a second vortex emulsification was repeated, the PEO:water emulsates were promptly mixed into 5 g of reagent-grade CaSO4 (Sigma-Aldrich, St. Louis, MO, USA) in an alcohol-sanitized plastic weigh boat, and then hand-mixed using a sanitized spatula (Biocomposites, UK). The PEO-loaded CaSO4 paste was molded into six mm sized hemispherical beads using an estastomer bead mold (Biocomposites, UK). The CaSO4 beads were allowed to harden for one hour, and were then expressed from the mold into a sterile petri plate. Vancomycin-loaded CaSO4 paste was prepared according to a clinically relevant formula of 1 g of vancomycin HCL (Fisher Scientific) per 10 cc of CaSO4, and hardened beads were prepared similarly to PEO-beads described above. To test the beads for growth inhibitory activity, fresh sterile molten MHII agar was cooled to 43°C in a water bath. Then, an overnight grown culture of S. aureus was subcultured 1:1000 (approximately 5 x 105 CFU) in the molten media and stirred thoroughly. The seeded MHII agar was quickly poured into empty sterile petri plates containing pre-positioned PEO or vancomycin beads and allowed to solidify. Hardened pour plates were incubated overnight as described above, and the diameter of the resulting pour plate ZOI (PPZOI) was measured, if applicable. The student’s T test was used to determine significance, see Table 1.

Results

Growth inhibitory activity of 57 PEOs against planktonic S. aureus

Initially we wanted to test the anti-staphylococcal activity of our PEO collection via disk diffusion assays. All of the PEOs tested demonstrated significant growth inhibitory activity against planktonic S. aureus (Table 1) when comparing ZOIs to a null hypothesis of 6 mm (disk size and LOD), except for neroli and wintergreen. 19/57 PEOs tested, including arborvitae, balsam fir, basil, cardamom, cassia, cilantro, cinnamon bark, clary sage, cypress, Douglas fir, frankincense, lemongrass, melaleuca, Melissa, oregano, peppermint, Siberian fir, tangerine, and thyme all demonstrated significantly higher ZOIs compared to a vancomycin 30 μg minimum inhibitory concentration control (Fig 1), while an additional 22/57 PEOs tested had ZOIs statistically similar to the vancomycin control (Table 1). Most notably, cilantro PEO exhibited full growth inhibition of S. aureus in all replicates (n = 6, Table 1). Cedarwood, ginger, green Mandarin, jasmine, myrrh, patchouli, pink pepper, rose, sandalwood and Hawaiian sandalwood, spikenard, turmeric, vetiver, wild orange, wintergreen, and ylang ylang exhibited ZOIs significantly less than that of the vancomycin control (Table 1).

Fig 1

PEOs inhibit the growth of S. aureus in disk diffusion assays.

Representative images of cilantro (A), and cinnamon bark, clary sage, and clove (B) versus a 30 μg vancomycin control (C). Images represent n ≥ 6.

Growth inhibitory activity of PEO-beads

Despite a number of the PEOs exhibiting relatively low-or-no activity in disk diffusion assays compared to vancomycin, we opted to screen all 57 PEOs infused in beads against S. aureus to better understand how disk diffusion assays might be predictive of PEO-bead activity. We operated under the general hypothesis that the disk diffusion data would positively correlate with PEO-bead growth inhibition. That is, for instance, we hypothesized that PEOs with high anti-staphylococcal activity, such as cilantro, would elicit high growth inhibitory activity when infused in beads.

When comparing PPZOI diameters against a null hypothesis of 6 mm (bead size and LOD), 17/57 PEO-beads including arborvitae, balsam fir, black spruce, cassia, cinnamon bark, cypress, Douglas fir, eucalyptus, geranium, grapefruit, helichrysum, lemon, lemongrass, oregano, peppermint, Siberian fir, and thyme had significant growth inhibitory activity (Table 1 and Fig 2). However, none of the PEOs tested exhibited growth inhibition statistically higher-or-similar-to a vancomycin-bead control (24.1, ± 0.3 mm). Additionally, we observed that a number of PEOs (22/57) which, according to the disk diffusion data, were hypothesized to elicit high growth inhibitory activity when infused in beads failed to do so (Table 1), including basil, bergamot, black pepper, blue tansy, cardamom, cedarwood, cilantro, clary sage, clove, copaiba, coriander, fennel, geranium, juniper berry, lavender, lime, marjoram, melaleuca, Melissa, neroli, petitgrain, rosemary, spearmint, and tangerine.

Fig 2

PEO-loaded beads inhibit S. aureus growth in agar pour plates.

Representative images of Cassia, Cilantro, Oregano, and Thyme (A), Fennel, Eucalyptus, and Cypress (B), versus the vancomycin 30 μg control (C, in duplicate) loaded beads inhibiting S. aureus growth in three dimensions.

To conserve materials, we tested multiple PEOs in per plate in some tests (in replicate), and this imposes a potential methodological limit in that there could be imperceivable antagonistic or synergistic effects occurring between proximal PEOs. Tests for multi-PEO synergism or antagonism were within the scope of this research, and would require follow-up testing. Regardless, and finally, we calculated the hypothetical SOI that would result if the beads were encased in an adequate amount of seeded media (and/or as if placed in a joint space), and statistically compared the SOIs to the vancomycin control. We found that cinnamon bark PEO would elicit a theoretical SOI (5.5 cm3 ±1.1 cm3) statistically similar to vancomycin (7.4 cm3 ± 0.2 cm3).

Discussion

The antibiotic resistance crisis is well known amongst scientists and physicians. Some creative strategies and inventions for combatting infections, which are gaining considerable focus, include the use of antimicrobial nanoparticles, monoclonal antibodies, antimicrobial peptides, conjugated/tandem antibiotics, vaccines, and phage therapy [17]. Here, we corroborate several decades worth of literature reporting the antimicrobial efficacy of various PEOs against major pathogens (or food spoilage organisms) [13, 18], indicating that PEOs or other plant derivatives represent an abundant and affordable source of natural antimicrobials. Furthermore, we demonstrated that PEOs with anti-staphylococcal properties can be infused in CaSO4 beads and inhibit S. aureus growth in vitro. We observed several PEOs that were seemingly active against S. aureus in agar pour plates, but none that performed as well-or-better than a vancomycin bead control. It is worth considering, though, that the vancomycin beads used in this study were prepared to mimic a clinical formula [19] (see Materials and Methods), which yields beads containing roughly 333 times the minimum inhibitory concentration (30 μg, MIC) for vancomycin against S. aureus. With that being said, the average PEO-bead contained roughly 41.4 μL (±2.8 μL) of PEO per bead, which is approximately two times the amount used in disk diffusion assays. Thus, future work remains to be done with regards to optimizing the PEO:water-CaSO4 mixture described here to determine if the volume of PEO can be increase per bead.

Nevertheless, if we statistically analyze PEO-bead PPZOI diameters using a null hypothesis of six mm (bead size and LOD), we find that arborvitae, balsam fir, black spruce, cassia, cinnamon bark, cypress, Douglas fir, eucalyptus, frankincense, grapefruit, helichrysum, lemon, lemongrass, oregano, peppermint, rosemary, sandalwood, spearmint, and thyme have significant growth inhibitory activity (Table 1); all of which were predictable by disk diffusion assays. However, because 22/57 PEOs did not elicit statistically significant growth inhibition when infused in beads, as we had predicted, we conclude that disk diffusion assays are unreliable at predicting growth inhibitory activity of PEO-beads in agar pour plates. For instance, one major discrepancy we observed was that cilantro bead PPZOIs were almost immeasurable, having an average diameter of 7.4 mm (±1.2 mm), whereas, cilantro PEO completely inhibited S. aureus growth in all disk diffusion replicates (Table 1). The discrepancy for cilantro and other PEOs across assays may be related to a reduction in elution rate specific to cilantro PEO when it is infused in CaSO4, and/or the hydrophobicity of the active compound(s) in PEOs when encased in an aqueous medium. Consequently, comparison of PEOs predicted to have bead activity with those that actually did may help us predict which PEOs have antimicrobial hydrosolic compounds capable of eluting into aqueous environments; antibacterial hydrosolic compounds would be attractive compounds to identify and research because of their water solubility.

Encasing beads in bacteria-seeded agar is an interesting way to measure their activity in three dimensions, and might represent a useful in vitro approach to measuring the efficacy of potential antimicrobial alternatives, especially for novel compounds that are water soluble. One additional pitfall of the bead pour plate assay is that the normal dissolution process of CaSO4 beads (such that occurs in the weeks following implantation) is not emulated, and this is a crucial aspect of the gradual release of antibiotic locally at a body site. The rate of dissolution is largely related to the fluid levels and movement occurring in the joint space, but typically occurs over a period of several weeks [20]. In a scenario in which antibacterial PEO-beads dissolve over time, we would predict that the PEO loaded into beads would also be gradually released. Thus, although our pour plate assays did not indicate any of the oils to be as effective as a vancomycin bead control, a scenario in which the beads dissolved over time might yield more relevant results. Nevertheless, six of the significantly active PEO-beads had relatively/consistently high activity compared to all others, including arborvitae (17.4 mm), balsam fir (19.2 mm), cassia (21.8 mm), cinnamon bark (20.7 mm), grapefruit (17.6 mm), and oregano (15.28 mm), and these may represent the best candidates for additional study and optimization for use in beads. To the best of our knowledge, this is the first time in which a large set of single plant PEOs have been demonstrated to exhibit growth inhibition of S. aureus in vitro when infused in CaSO4 beads.

14 Jun 2021

PONE-D-21-14096

Calcium sulfate beads made with antibacterial essential oil-water emulsions exhibit growth inhibition against Staphylococcus aureus in agar pour plates.

PLOS ONE

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Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a well done study with a clearly defined focus. It shows that anti-microbial oils may have potential for inhibiting S. aureus in calcium sufate beads. The results are also relevant, in addition to the medical aspects, in the food sector.

Minor comment: line 77 and 79 S. aureus in italics.

Reviewer #2: The study is quite impressive and scientifically elaborated. I advise some minor corrections/clarifications:

- Mention the plants under study in the material and methods section.

- Mention scientific names of the plants in Table 1

- Include some representative images of treatments displaying significantly higher ZOIs compared to vancomycin 30 µg control.

-Include representative image of PEO seeded beads

- What was the basis of selecting 30 µg of vancomycin as control?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Per Saris

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

21 Sep 2021

Review Feedback and Comments pasted from decision letter email with our responses in red.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

o Complete

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

o Complete

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

o Complete

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

- N/A

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

- N/A

We look forward to receiving your revised manuscript.

Kind regards,

Arumugam Sundaramanickam, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

Most of the references are old. Include some recent references between 2019 and 2021.

- Thank you. We updated all the references that could be updated. Some of the references, however, are seminal works or otherwise key pieces of information that support the narrative and project justification. If there are any specific references you would still like us to try to update, please let us know.

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

When submitting your revision, we need you to address these additional requirements.

- We reformatted the references to match the formatting requirements of the journal.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

- Thank you for this comment and the links. This was extremely helpful. We have revised the style of the manuscript exactly as instructed in the links. One issue we were unsure of, though, was table 1, which is quite wide. In order for table 1 to fit underneath the paragraph in which it is first mentioned, we had to flip the pages horizontally. We were not sure if this is appropriate or how we should approach this technical issue.

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This work was supported by intramural funds from the FGCU Department of Biological

Sciences and the FGCU Whitaker Center for Stem Education. We would like to thank doTERRA®

International for the donation of essential oils in support of this work, especially Dr. David Hill,

D.C. and Dr. Cody Beaumont, Ph.D. We would also like to thank Sean Aiken of Biocomposites ®

(UK) for his gracious provision of bead making supplies and scientific input."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Additionally, because some of your funding information pertains to commercial funding, we ask you to provide an updated Competing Interests statement, declaring all sources of commercial funding.

In your Competing Interests statement, please confirm that your commercial funding does not alter your adherence to PLOS ONE Editorial policies and criteria by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests. If this statement is not true and your adherence to PLOS policies on sharing data and materials is altered, please explain how.

Please include the updated Competing Interests Statement and Funding Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

- Thank you. We have revised the acknowledgements section to read as follows:

“We would like to thank doTERRA® International for the donation of essential oils in support of this work, especially Dr. David Hill, D.C. and Dr. Cody Beaumont, Ph.D. We would also like to thank Sean Aiken of Biocomposites ® (UK) for his gracious provision of bead making supplies and scientific input.”

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

- Thank you. We have removed the ‘data not shown” phrase from the manuscript. The manuscript is not significantly altered with/without it.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

- Thank you!

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

- Thank you.

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

- Thank you.

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

- Thank you.

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a well done study with a clearly defined focus. It shows that anti-microbial oils may have potential for inhibiting S. aureus in calcium sufate beads. The results are also relevant, in addition to the medical aspects, in the food sector.

- Thank you!

Minor comment: line 77 and 79 S. aureus in italics.

- Thanks. Corrected!

Reviewer #2: The study is quite impressive and scientifically elaborated. I advise some minor corrections/clarifications:

- Mention the plants under study in the material and methods section. DONE

- Mention scientific names of the plants in Table 1 DONE

- Include some representative images of treatments displaying significantly higher ZOIs compared to vancomycin 30 µg control. DONE. Added fig1

-Include representative image of PEO seeded beads. DONE Added fig2

- What was the basis of selecting 30 µg of vancomycin as control? 30 �  g is the minimum inhibitory concentration for S. aureus species and is used to conduct antibiograms in research and in the clinic to determine susceptibility/resistance. Also, the filter disks come pre-loaded with this amount of vancomycin, and a reference page for determining the susceptibility.

- Thank you for your compliment!

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Per Saris

- Thank you!

Reviewer #2: No

- Thank you!

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

End of Responses

Once again, thank you for the review process and helpful comments and feedback. We look forward to publishing the manuscript. Please do not hesitate to reach out if we have missed anything, or if any additional feedback arises. We would be more than happy to address any other issues.

Submitted filename: Response letter to the reviewers.pdf

Click here for additional data file.

14 Feb 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Filippo Giarratana

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: No further requirements.You have answered the requests in a proper manner. The work presents interesting results.

Reviewer #3: General comments:

Overall, I like the idea on which the work is based. The study tests the possible in vitro antimicrobial activity of several essential oils incorporated into calcium sulphate beads against S. aureus that is among the main causative agent of bone infections in orthopaedic medicine. the authors demonstrated a good command of scientific English. However, there are some limitations. Meanwhile, the results are described on the basis of a not well-defined statistical analysis. Or rather, in the tables this information is reported, however, there is no information about it in the materials and methods. After that, my main concern is that the authors tested multiple essential oils in the same plate. How can they be sure that there has not been an influence between the various oils? I understand that the halos of inhibition are well defined in the plates, however the diffusion of the essential oil in the medium certainly affected the growth.

These are important and relevant methodological limits for the reliability of the results.

Please check the punctuation and italic. I have the impression that a few dots are missing through the text (see lines 65 and 75,77,79 etc.).

Introduction

This paragraph is well written and well explains the background on which the study moves. I appreciated how the aims achieved and the limits of experimentation are briefly mentioned in the final part.

However, I suggest checking, and eventually rephrasing, the lines 79-81. Maybe, at the beginning of line 80, “or” was “of”? I understand what the Authors want to say but is a little bit confusing.

Materials and methods

Line 101 “through preliminary testing”. What are you referring to? Where is this data? Explain better.

Line 102 “ZOIs”: is this the abbreviation of what? Do you mean zone of inhibition? It does not open to me is written by anyone else before. To specify. The same for other abbreviation used such as PPZOI in the results section.

Lines 101-102 “Some PEOs known to have high potency (through preliminary testing) were

102 tested on plates in the absence” Were the other PEOs tested in common plates? If so, how can you be sure the effect of each PEO does not affect that of the other? Please, clarify.

I don't understand what kind of statistical analysis was done. Although there is some clarification in the tables, the type of statistical analysis that was performed is not specified in this section. Please, improve.

Results

Overall, the data relating to the results are presented, however more than one sentence is not about the results but more about the materials and methods. In addition, there are also repetitions of the materials and methods. Results certainly need to be improved.

Lines 135-139: These are not results; furthermore, these are repetitions of materials and methods. See also lines 189-191.

Line 140: It is not clear how these statistically significant differences were calculated. What results were compared with each other? Are there any replicas of the samples? How much they were? In addition to reporting this information in the tables, it is necessary to report it in the text to understand not only the type of analysis used but whether it is actually possible to use this type of analysis for the type of data available. Specify and improve.

Discussions

The discussions are well written, and the data reported in the results are properly argued. Furthermore, both the limitations and the innovations brought about by this study are highlighted.

Figures

The quality of the figures is poor. Improve.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

2 Jun 2022

Dear (new) Editor and Reviewers,

Thank you for the opportunity to revise our manuscript “Calcium sulfate beads made with antibacterial essential oil-water emulsions exhibit growth inhibition against Staphylococcus aureus in agar pour plates.” We appreciate your feedback and the feedback of the reviewers, we are also grateful for you and the reviewers taking the time to conduct a review process for this manuscript. We have considered the helpful comments and addressed them to the best of our ability. As requested in the decision letter email, we are providing responses to the items highlighted in the feedback about the manuscript.

Update 26Apr2022 - Request from Editor with Response in Red

We've checked your submission and before we can proceed, we need you to address the following issues:

We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

This work was funded with departmental funds from the Department of Biological Sciences at Florida Gulf Coast University, and for which there is no specific grant award number. Therefore, I am selecting “no specific funding for this work.

31Mar2022 - Review Feedback and Comments pasted from decision letter email with our responses in red.

Please include the following items when submitting your revised manuscript:

• Several monor error are presence (S. aureus not in italic line 74: "orthopedic pathogens, including S. aureus (10,14–16)."; ect...)

o Fixed! Thank you!

• Is not reported the diameter of the plat used (90 mm or 120 mm).

o Fixed line 105

• Introduction_Line 77-80: this are "result"...and cannot be insert in the introduction.

o Fixed

• In the results there are repetition of "materials and methods"

o Fixed lines 138-143 and 175-177

Please submit your revised manuscript by 31 March 2022. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

o complete

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

o complete

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

o complete

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Filippo Giarratana

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Partly

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: No

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

________________________________________

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: No further requirements.You have answered the requests in a proper manner. The work presents interesting results.

• Thank you

Reviewer #3: General comments:

Overall, I like the idea on which the work is based. The study tests the possible in vitro antimicrobial activity of several essential oils incorporated into calcium sulphate beads against S. aureus that is among the main causative agent of bone infections in orthopaedic medicine. the authors demonstrated a good command of scientific English. However, there are some limitations. Meanwhile, the results are described on the basis of a not well-defined statistical analysis. Or rather, in the tables this information is reported, however, there is no information about it in the materials and methods. After that, my main concern is that the authors tested multiple essential oils in the same plate. How can they be sure that there has not been an influence between the various oils? I understand that the halos of inhibition are well defined in the plates, however the diffusion of the essential oil in the medium certainly affected the growth.

These are important and relevant methodological limits for the reliability of the results.

Summary of Responses (followed by individual section responses)

• The student’s T test was used to determine statistical significance and this was previously written in the table 1 title in bold. The specific null hypotheses or otherwise were also described underneath the table.

• I added a note about using student’s T test in each methods subsection lines 115 and 135.

• Statistics were also previously written throughout the body of the manuscript, for example, lines 144 – 145

• Lines 188-192 address possible synergism/antagonism in multi-PEO plates. Although not within the scope of this study, we thank you for the call out and agree that acknowledging this limitation strengthens the discussion.

Please check the punctuation and italic. I have the impression that a few dots are missing through the text (see lines 65 and 75,77,79 etc.).

• This was noted by the editor, and was completed. Thank you!

Introduction

This paragraph is well written and well explains the background on which the study moves. I appreciated how the aims achieved and the limits of experimentation are briefly mentioned in the final part.

• Thank you!

However, I suggest checking, and eventually rephrasing, the lines 79-81. Maybe, at the beginning of line 80, “or” was “of”? I understand what the Authors want to say but is a little bit confusing.

• Fixed. Thank you for the call-out.

Materials and methods

Line 101 “through preliminary testing”. What are you referring to? Where is this data? Explain better.

• Fixed the wording to not be less vague or misleading.

Line 102 “ZOIs”: is this the abbreviation of what? Do you mean zone of inhibition? It does not open to me is written by anyone else before. To specify. The same for other abbreviation used such as PPZOI in the results section.

• Defined ZOI line 111

• Defined PPZOI 133

Lines 101-102 “Some PEOs known to have high potency (through preliminary testing) were

• Fixed the wording, also addressed above

102 tested on plates in the absence” Were the other PEOs tested in common plates? If so, how can you be sure the effect of each PEO does not affect that of the other? Please, clarify.

• Lines 188-192 address possible synergism/antagonism in multi-PEO plates. Although not within the scope of this study, we thank you for the call out and agree that acknowledging this limitation strengthens the discussion.

I don't understand what kind of statistical analysis was done. Although there is some clarification in the tables, the type of statistical analysis that was performed is not specified in this section. Please, improve.

• I added a note about using student’s T test in each methods subsection lines 115 and 135.

Results

Overall, the data relating to the results are presented, however more than one sentence is not about the results but more about the materials and methods. In addition, there are also repetitions of the materials and methods. Results certainly need to be improved.

• Fixed

Lines 135-139: These are not results; furthermore, these are repetitions of materials and methods. See also lines 189-191.

• Fixed

Line 140: It is not clear how these statistically significant differences were calculated. What results were compared with each other? Are there any replicas of the samples? How much they were? In addition to reporting this information in the tables, it is necessary to report it in the text to understand not only the type of analysis used but whether it is actually possible to use this type of analysis for the type of data available. Specify and improve.

• Again, we used student’s t test and this is stated several times in the manuscript, and we now added lines to both methods subsections explicitly stating this. Table 1 was designed to comprehensively show the ZOI data and how comparisons used for p values.

o For instance, a T test was used to determine that [for arborvitae] the disk diffusion assay ZOI was significantly higher than a vancomycin control.

Discussion

The discussions are well written, and the data reported in the results are properly argued. Furthermore, both the limitations and the innovations brought about by this study are highlighted.

Figures

The quality of the figures is poor. Improve.

o This comment is subjective and/or too vague to enact specific change. The images were taken with a high resolution camera, and are at the maximum resolution of 300 dpi. Some oils do not elicit a perfect margin for ZOIs or especially PPZOIs which are 3 dimensional, and can create and impression of blurriness. At this point, I can’t retroactively improve image quality, nor is it acceptable to “doctor” images.

________________________________________

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Reviewer #1: No

Reviewer #3: No

Once again, thank you for the review process and helpful comments and feedback. We look forward to publishing the manuscript. Please do not hesitate to reach out if we have missed anything, or if any additional feedback arises. We would be more than happy to address any other issues.

Most Sincerely,

Matthew C. Swearingen, PhD

p.s. Editor, this work was originally completed at Florida Gulf Coast University in Fort Myers, Florida. I have since left that institution and now work in private industry as a Senior Microbiologist. Hence, I am no longer using the FGCU contact information, but instead my personal information for correspondences.

Submitted filename: Response letter to the reviewers part 2.docx

Click here for additional data file.

27 Jun 2022

Calcium sulfate beads made with antibacterial essential oil-water emulsions exhibit growth inhibition against Staphylococcus aureus in agar pour plates.

PONE-D-21-14096R2

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Additional Editor Comments (optional):

The authors have improved the paper with all the required suggestion/revision.

The paper now can be accepted.

30 Jun 2022

PONE-D-21-14096R2

Calcium sulfate beads made with antibacterial essential oil-water emulsions exhibit growth inhibition against Staphylococcus aureus in agar pour plates.

Dear Dr. Swearingen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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