Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: Implications for the microbiological diagnosis of implant associated infection.

The diagnosis of implant-associated infections is hampered due to microbial adherence and biofilm formation on the implant surface. Sonication of explanted devices was shown to improve the microbiological diagnosis by physical removal of biofilms. Recently, chemical agents have been investigated for biofilm dislodgement such as the chelating agent ethylenediaminetetraacetic acid (EDTA) and the reducing agent dithiothreitol (DTT). We compared the activity of chemical methods for biofilm dislodgement to sonication in an established in vitro model of artificial biofilm. Biofilm-producing laboratory strains of Staphylococcus epidermidis (ATCC 35984), S. aureus (ATCC 43300), E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 53278) were used. After 3 days of biofilm formation, porous glass beads were exposed to control (0.9% NaCl), sonication or chemical agents. Quantitative and qualitative biofilm analyses were performed by colony counting, isothermal microcalorimetry and scanning electron microscopy. Recovered colony counts after treatment with EDTA and DTT were similar to those after exposure to 0.9% NaCl for biofilms of S. epidermidis (6.3 and 6.1 vs. 6.0 log10 CFU/mL, S. aureus (6.4 and 6.3 vs. 6.3 log10 CFU/mL), E. coli (5.2 and 5.1 vs. 5.1 log10 CFU/mL and P. aeruginosa (5.1 and 5.2 vs. 5.0 log10 CFU/mL, respectively). In contrast, with sonication higher CFU counts were detected with all tested microorganisms (7.5, 7.3, 6.2 and 6.5 log10 CFU/mL, respectively) (p <0.05). Concordant results were observed with isothermal microcalorimetry and scanning electron microscopy. In conclusion, sonication is superior to both tested chemical methods (EDTA and DTT) for dislodgement of S. epidermidis, S. aureus, E. coli and P. aeruginosa biofilms. Future studies may evaluate potential additive effect of chemical dislodgement to sonication.

Introduction

Implants are increasingly used to improve the mobility (joint replacement and bone fixation devices) or prolong the survival and assist the performance of physiological functions (cardiac implantable electronic device (CIED) and neurosurgical shunts). Infections represent a significant complication of implant surgery, resulting in major challenges regarding the diagnosis and treatment [1-5]. Most commonly isolated microorganisms in patients with periprosthetic joint infection are coagulase-negative staphylococci (30–45%) and Staphylococcus aureus (12–23%), followed by streptococci (9–10%), enterococci (3–7%), gram-negative bacilli (3–6%) and anaerobes (2–4%) [6]. Similar distribution of pathogens is observed in CIED [2] and neurosurgical shunt-associated infections [4].

The crucial step in the management of implant-associated infections is an accurate diagnosis. However, as these infections are caused by microorganisms embedded in a polymeric matrix attached to the device surface, the diagnosis may be challenging, especially in chronic low-grade infections. In order to detect the infecting microorganism, dislodgement of the biofilm should precede the standard cultivation methods in solid or liquid growth media [7].

Various approaches had been investigated for biofilm removal from implant surface. Sonication is based on mechanical biofilm dislodgement and showed superior detection yields than other methods and was introduced in routine microbiological diagnosis [8-12].

In vitro studies investigated the ability of chemical dislodgement such as metal-chelating agent ethylenediaminetetraacetic acid (EDTA) and the strong reducing agent dithiothreitol (DTT). The ability of EDTA to chelate and potentiate the cell walls of bacteria and destabilize biofilms by sequestering calcium, magnesium, zinc, and iron suggests to be suitable for the biofilm detachment [13]. Recent reports suggested that treatment of explanted prostheses with a solution containing DTT is superior to sonication for dislodgement of biofilm-embedded bacteria [14].

The aim of the study was to compare the ability of mechanical biofilm dislodgement (i.e. sonication) with chemical dislodgement methods (i.e. EDTA and DTT) in vitro and evaluate their potential role in the routine microbiological diagnosis of implant-associated infections.

Materials and methods

Bacterial strains and biofilm growth conditions

As a model to form the bacterial biofilm porous glass beads (diameter 4 mm, pore sizes 60 μm, ROBU®, Hattert, Germany) were used. Due to the high volume-to-surface ratio, glass beads were used for biofilm studies rather than smooth materials, as investigated in numerous previous research works regarding biofilm formation and anti-biofilm activity [15-20]. To form biofilms, beads were placed in 2 ml of brain heart infusion broth (BHIb, Sigma-Aldrich, St. Louis, MO, USA) containing 1x108 CFU/mL inoculum of Staphylococcus epidermidis (ATCC 35984), S. aureus (ATCC 43300) E. coli (ATCC 25922) or Pseudomonas aeruginosa (ATCC 53278) and incubated at 37°C. After 24 h, beads were re-incubated in fresh BHIb and biofilms were statically grown for further 72 h at 37°C, as previously described [14]. After biofilm formation, beads were washed six times with 2 ml 0.9% NaCl to remove planktonic bacteria.

Biofilm dislodgement by chemical methods (EDTA or DTT) or sonication

To define the minimal chemical concentration and treatment duration for biofilm dislodging, washed beads were placed in 1 ml of EDTA at concentrations 12, 25 and 50 mM or DTT at concentrations 0.5, 1 and 5 g/L and exposed for 5, 15 and 30 min. Untreated beads incubated with 0.9% NaCl were used as negative control. The timing of EDTA and DTT exposure and choice of the concentration for biofilm dislodgement were based on previous studies, which indicated the maximal biofilm disruption without bacterial killing [13, 14].

To evaluate the sonication effect, biofilms were sonicated as described previously [10]. Briefly, each bead was inoculated in 1 ml 0.9% NaCl, vortexed for 30 sec, sonicated at 40 kHz at intensity 0.1 Watt/cm2 (BactoSonic, BANDELIN electronic, Berlin, Germany) for 1 min and vortexed again for 30 sec. One-hundred microliter of serial dilutions of the resulting sonication fluid or the solution obtained after chemical treatment with DTT or EDTA were plated onto Tryptic Soy Agar (TSA) (Sigma-Aldrich, St. Louis, MO, USA). After 24 h of incubation at 37°C, the CFU/mL number was counted. The serial dilutions allowed to raise the upper limit of detection providing a reportable range from 0 to 100,000,000 CFU/mL.

Additionally, the viability of planktonic bacteria in presence of chemical agents and sonication was evaluated. Planktonic cells of S. epidermidis, S. aureus, E. coli and P. aeruginosa at final concentration of ≈105 CFU/ml were exposed to EDTA (25 mM) and DTT (1 g/L) for different time periods (5, 15 and 30 min) and sonication. All experiments were performed in triplicates S1 Fig.

Isothermal microcalorimetry analysis

To prove the dislodgement effect of previously described methods and reveal the presence of bacterial cells remained attached on the bead surface, treated beads were washed six times in 2 ml 0.9% NaCl to remove the dislodged biofilm and placed in 4 ml-glass ampules containing 3 ml of BHIb. The ampoules were air-tightly sealed and introduced into the microcalorimeter (TAM III, TA Instruments, Newcastle, DE, USA), first in the equilibration position for 15 min to reach 37°C and avoid heat disturbance in the measuring position. Heat flow (μW) was recorded up to 20 h. The calorimetric time to detection (TTD) was defined as the time from insertion of the ampoule into the calorimeter until the exponentially rising heat flow signal exceeded 100 μW to distinguish microbial heat production from the thermal background [21]. Growth media without bacteria served as negative control.

Scanning electron microscopy (SEM)

Beads with biofilm were fixed with 2.5% (v/v) glutaraldehyde in natrium cacodylat buffer and the samples were dehydrated with increasing concentrations of ethanol for 2 min each. The samples were stored in vacuum until use. Prior to analysis by Scanning electron microscope (GeminiSEM 300, Carl Zeiss, OberkochenDSM 982 GEMINI, Zeiss Oberkochen, Germany), the samples were subjected to gold sputtering (Sputter coater MED 020, Balzer, BingenMED 020, BAL-TEC). All experiments were performed in triplicate.

Statistical methods

Statistical analyses were performed using SigmaPlot (version 13.0; Systat Software, Chicago, IL, USA) and graphics using Prism (version 8; GraphPad, La Jolla, CA, USA). Quantitative data were presented as mean ± standard deviation (SD) or median and range, as appropriate. To compare different groups the ANOVA test was performed and in case of significant differences, nonparametric Kruskal-Wallis test and Wilcoxon signed-rank test for independent samples were used, as appropriate. The significance level in hypothesis testing was predetermined at p-value of <0.05.

Results and discussion

CFU counting method

Bacterial biofilm dislodged after treatment with different concentrations of chemical agents at different time points

S. epidermidis biofilm. EDTA at concentration 25 mM showed significant increase in bacterial count at 15 min compared to 5 min (p = 0.023) and compared to 30 min of exposure (p = 0.012). DTT showed no difference in CFU count when different concentrations were applied (Fig 1A and 1B).

Fig 1

Bacterial biofilm dislodged after treatment with different concentrations of chemical agents at different time points.

S. epidermidis biofilm (A) EDTA, (B) DTT. S. aureus biofilm (C) EDTA, (D) DTT; E. coli biofilm (E) EDTA, (F) DTT; P. aeruginosa biofilm (G) EDTA, (H) DTT. Mean values are shown, error bars represent standard deviation. * Statistically significant difference (p < 0.05).

S. aureus biofilm. EDTA at concentration 25 mM and DTT at concentration 1 g/L at different time points (5, 15 and 30 min) showed no significant difference in CFU count. (Fig 1C and 1D).

E. coli biofilm. EDTA at concentration 25 mM and DTT at concentration 1 g/L at diferent time points (5, 15 and 30 min) showed no significant difference in CFU count. (Fig 1E and 1F).

P. aeruginosa biofilm. EDTA at concentration 25 mM showed significant increase in bacterial count at 15 min (p = 0.042) and 30 min (p = 0.020) compared to 5 min of exposure. DTT at concentration 1 g/L showed increase in bacterial count at 15 min (p = 0.018) compared to 30 min, and a significant increase in CFU at concentration 5 g/L at 15 min compared to 5 min (p = 0.028) was observed (Fig 1G and 1H).

Therefore to evaluate the dislodgement effect of chemical methods the concentrations 25 mM EDTA and 1 g/L DTT were chosen as they showed significant increase in CFU count at 15 min compared to other time points when P. aeruginosa and S. epidermidis biofilms were investigated. The mean colony count obtained after treatment of S. epidermidis biofilms with EDTA (25 mM, 15 min) and DTT (1 g/L, 15 min) was similar to those observed after treatment with 0.9% NaCl used as control (6.3, 6.1 and 6.0 log10 CFU/mL, respectively). In contrast, sonication detected significantly higher CFU counts with 7.5 log10 CFU/mL (p <0.05) (Fig 2A). Similar results were observed when S. aureus biofilms were treated with chemicals (EDTA, 25 mM, 15 min) and DTT, 1 g/L, 15 min) or 0.9% NaCl (6.4, 6.3 and 6.3 log10 CFU/mL, respectively). By using sonication, CFU count of 7.3 log10 CFU/mL (p < 0.05) was observed (Fig 2B).

Fig 2

Quantitative analysis and comparison of biofilm dislodging methods.

(A) S. epidermidis biofilm. (B) S. aureus biofilm. (C) E. coli biofilm. (D) P. aeruginosa biofilm. Mean values are shown, error bars represent standard deviation. * Statistically significant difference (p < 0.05). 0.9% NaCl represents an untreated control. EDTA, ethylenediaminetetraacetic acid. DTT, dithiothreitol.

We found similar colony counts when E. coli biofilms were treated with EDTA (25 mM, 15 min) and DTT (1 g/L, 15 min) as well as 0.9% NaCl (5.2, 5.1 and 5.1 log10 CFU/mL, respectively). Sonication detected significantly higher CFU counts with 6.2 log10 CFU/mL (p < 0.05) (Fig 2C). The results were similar when P. aeruginosa biofilms were investigated. Treatment with chemicals (EDTA, 25 mM, 15 min) and DTT, 1 g/L, 15 min) or 0.9% NaCl (5.1, 5.2 and 5.0 log10 CFU/mL, respectively). Sonication showed significantly higher CFU counts with 6.5 log10 CFU/mL (p < 0.05), (Fig 2D).

Isothermal microcalorimetry

Heat produced by samples containing sonicated glass beads with S. epidermidis biofilm was detected after 11 h. In contrast, heat production exceeding the threshold of 100 μW was observed earlier (after 6.5 and 6.4 h) for the samples that were previously treated with EDTA and DTT, confirming the presence of a higher number of residual bacteria on beads treated with chemical methods, in comparison to those after sonication. This time difference was statistically significant (p <0.05). No difference in heat production was observed after treatment with 0.9% NaCl (control) and EDTA or DTT (6.3 vs 6.5 and 6.4 h, respectively) (p = 0.3) (Fig 3A). Similar results were observed with the analysis of S. aureus biofilm beads. The time of heat detection after sonication of beads was significantly higher (12 h) in comparison to EDTA and DTT (6.1 and 5.8 h, respectively) (p <0.05); no difference between both chemical methods and the control (4.6 h) was observed (Fig 3B). Investigation of E. coli and P. aeruginosa biofilms showed the same results. Time of heat detection in sonicated beads was significantly higher compared to beads treated with chemical agents (EDTA, DTT) as well as control: 7.8 h vs. 4.9, 4.5 and 4.5 h, respectively (p <0.05) for E. coli biofilm and 11h vs. 6.5, 6.5 and 4.6 h, respectively (p <0.05) for P. aeruginosa biofilm, (Fig 3C and 3D).

Fig 3

The microcalorimetric time to detection (TTD) of bacterial growth.

(A) S. epidermidis biofilm. (B) S. aureus biofilm. (C) E. coli biofilm. (D) P. aeruginosa biofilm. 0.9% NaCl represents an untreated control. EDTA, ethylenediaminetetraacetic acid. DTT, dithiothreitol.TTD, the calorimetric time to detection of microbial heat production. * Statistically significant difference (p < 0.05).

Scanning electron microscopy

The use of scanning electron microscopy (SEM) allowed to visualize the biofilms of S. epidermidis, S. aureus, E. coli and P. aeruginosa before and after treatments with either chemicals or sonication. For all microorganisms the scanning electron microscope images showed substantial less biofilm biomass remaining on the beads when sonication was applied compared to control as well as both chemical methods (Figs 4–7).

Fig 4

Scanning electron microscopy (SEM) of S. epidermidis biofilm.

(A) beads after 0.9% NaCl treatment (control). (B) beads after EDTA treatment. (C) beads after DTT treatment. (D) beads after sonication treatment. Scale bars: 200 μm (inserts in the images represent 5 μm).

Fig 7

Scanning electron microscopy (SEM) of P. aeruginosa biofilm.

(A) beads after 0.9% NaCl treatment (control). (B) beads after EDTA treatment. (C) beads after DTT treatment. (D) beads after sonication treatment. Scale bars: 200 μm (inserts in the images represent 5 μm).

Scanning electron microscopy (SEM) of S. aureus biofilm.

Scanning electron microscopy (SEM) of E. coli biofilm.

Implant-associated infections represent a major challenge for the microbiological diagnosis due to biofilm formation [22, 23]. We investigated the ability of different in vitro biofilm dislodgement methods, including sonication as the standard procedure in our institution and chemical treatment using EDTA or DTT as investigational procedures.

For biofilm formation we used laboratory strains of S. epidermidis, S. aureus, E. coli and P. aeruginosa known to be good biofilm formers. We did not use clinical strains as they typically show larger variability and are not suitable for investigation of a new diagnostic method. S. epidermidis and S. aureus were chosen as they are the most common pathogens causing implant-associated infections. P. aeruginosa and E. coli were chosen as representative pathogens of gram-negative bacteria causing about 10–15% of periprosthetic joint infections, up to 40% of fracture-fixation device-associated infections and up to 15% of neurosurgical shunt-associated infections [2-5]. The chosen P. aeruginosa strain was shown to be a good biofilm producer in previous biofilm studies [24].

To compare the ability of chemical agents to dislodge bacterial biofilm, the first step was to find the most optimal concentration and time of exposure dislodging the highest amount of the bacteria from the surface. The biofilms of S. epidermidis, S. aureus, E. coli and P. aeruginosa were treated at different concentrations and time points. The concentrations 25 mM EDTA and 1 g/L DTT were chosen as they showed significant increase in CFU count at 15 min compared to other time points when P. aeruginosa and S. epidermidis biofilms were investigated. Concentration of 1 g/L DTT was also proposed by other authors [14]. We did not observe any difference in CFU count when these concentrations were applied at different time points, therefore the time of 15 min was chosen as a most appropriate time for the routine microbiological examination.

Significantly higher CFU counts of S. epidermidis, S. aureus, E. coli and P. aeruginosa biofilm were detected after sonication compared to chemical dislodgement methods. The concentrations of EDTA (25 mM) and DTT (1 g/L) and sonication showed no impact on bacterial growth (S1 Fig).

Interestingly, our findings contradict the previously published results [14]. In their study, the authors investigated in vitro the dislodgement effect of DTT on polyethylene and titanium discs colonized with S. aureus, S. epidermidis, P. aeruginosa and E. coli biofilms. The authors found that DTT at 1 g/L applied for 15 min dislodged P. aeruginosa and E. coli biofilms with similar yield as with sonication, whereas the dislodgement of S. aureus and S epidermidis biofilms was even more efficient than with sonication.

Recently published ex vivo studies showed that treatment of explanted prosthesis with DTT may be superior to sonication for the diagnosis of periprosthetic joint infection [25-28]. The different type of biomaterial used for ex vivo biofilm studying may in part explain the discordance of results.

Similar discrepancy was found with EDTA. In our study, EDTA was unable to dislodge bacterial biofilms and the colony counts were similar to those obtained after treatment with 0.9% NaCl and were significantly lower compared to sonication. In contrast, previous authors demonstrated that EDTA affects P. aeruginosa biofilms [13, 29]. Banin et al. observed that exposure of P. aeruginosa biofilms to 50 mM EDTA dislodged biofilms. Addition of EDTA to the medium reservoir in a flow system increased the number of dislodged bacteria by >2 log10 CFU/mL after 50 min-incubation in the effluent compared to untreated flow system. The authors also showed that the activity of EDTA in biofilm detachment is mediated by chelation of several divalent cations such as magnesium, calcium, and iron that are required to stabilize the biofilm matrix. Our results derived from colony counting of dislodged bacterial cells were confirmed by two additional independent techniques, namely isothermal microcalorimetry and SEM imaging. Isothermal microcalorimetry is a highly sensitive method that enables a real-time monitoring of bacterial viability in terms of metabolism-related heat production. This method was widely used and validated for testing the anti-biofilm activity [20, 21, 30–34]. Here it was used to evaluate bacteria remaining on the glass beads after dislodging treatments. Isothermal microcalorimetry showed a significant delay in the detection of bacterial metabolism-related heat production from the beads with S. epidermidis, S. aureus. E. coli and P aeruginosa, when sonication was applied, as compared to chemical treatments—EDTA and DTT. These findings suggest that significantly less bacteria remained attached to the beads after sonication.

To visualize the bacteria remaining in the biofilms on the glass beads surface after treatment with either chemicals or sonication methods, the SEM was used. SEM micrographs have a large depth of field yielding a three-dimensional appearance, which is useful for understanding the surface structure of the sample. This method has been employed in various other studies providing good information on spatial structure [35, 36]. In our study, all types of bacterial biofilm SEM images showed less biofilm remaining on the beads when sonication was applied compared to the untreated control as well as both chemical methods.

There are several limitations of this study. First, anaerobes (e.g. Cutibacterium spp.) were not tested. Despite chemical methods were inferior to sonication in our study with all tested microorganisms (S. epidermidis, S. aureus E. coli and P. aeruginosa) it is possible that anaerobes may show better results with chemical methods due to their lower susceptibility to sonication. Before any recommendations about the clinical use in the routine microbiology testing, additional pathogens should be investigated. Second, for biofilm formation we used only laboratory strains. Typically clinical strains show larger variability therefore to evaluate a new diagnostic method in vitro the laboratory strains are more suitable. Third, we used only porous glass beads for biofilm formation. The porous glass beads possess a high volume-to-surface ratio therefore this model to form bacterial biofilm is probably more suitable for biofilm investigation than smooth materials. These results derived from this in vitro analysis represent a fundament for further exploration in the clinical setting with clinical strains and real implants. Forth, we incubated glass beads in the bacterial inoculum for 3 days until a visible biofilm was formed as described previously [14]. We assumed that further cultivation of mature biofilm to compare the ability of different methods for biofilm dislodgement is not needed. However it remains unknown, whether the ability of sonication or chemical methods for biofilm dislodgement would be different in more mature biofilms for example in the clinical setting when we deal with chronic implant-associated infections. Fifth, to study biofilm on glass beads surface, two complementary methods were used for detection of the remaining biofilms (microcalorimetry and scanning electron microscopy). Recently, novel methods for quantitative and qualitative evaluation of biofilm formation were evaluated. The BioTimer assay enumerates adherent microorganisms through microbial metabolism [37]. It showed promising results in the diagnosis of implant-associated infections, especially in combination with sonication, representing a simple and accurate way for the identification and enumeration of microorganisms. Other methods include confocal laser scanning microscopy (CLSM) [38], fluorescence microscopy [39] and atomic force microscopy [40] were not performed since both independent methods used in our study (microcalorimetry and scanning electron microscopy) correlated well.

Conclusions

We showed that sonication is superior to the chemical method for dislodgement of bacterial biofilms of S. epidermidis, S. aureus, E. coli and P. aeruginosa from artificial surface. Therefore, sonication remains the primary assay for biofilm detection in the microbiological diagnosis of implant-associated infection. Future studies may investigate a potential additive effect of chemical dislodgement to sonication.

The viability of planktonic bacteria in presence of chemical agents and sonication.

(TIF)

Click here for additional data file.

20 Nov 2019

PONE-D-19-27719

Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: potential implications for the microbiological diagnosis of implant associated infection

PLOS ONE

Dear Prof. Trampuz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jan 04 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Olivier Habimana

Academic Editor

PLOS ONE

Journal Requirements:

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

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

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. 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.

3.  We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Additional Editor Comments (if provided):

The manuscript has now been reviewed by two experts in the field, with one recommended major revisions, and the other rejection. Major concerns pertained to the clinical relevance of the study that also is in need of more experimentation. The authors are, therefore, kindly requested to present a rebuttal addressing these concerns for further deliberation of the manuscript.

[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: The authors analyzed the activity (not efficacy as this is an in-vitro study) of chemical methods to be used for biofilm removal.

The manuscript is well written; the laboratory methods are suitable. The biofilm dispersion was determined by different methods. However, for this is a preliminary study. The purpose of removing biofilms from implants is medical microbiological diagnostics. For that, more microorganisms (incl. clinical strains) should be included. EDTA may interfere with viability of microorganisms. The supplementary use of chemicals to sonication should included in that study.

Minor remarks. Microorganisms associated with implant infections should be introduced.

How do the glass beads for biofilm formation correspond to clinically used material?

Reviewer #2: The authors performed an interesting in vitro study with the aim to evaluate the diagnostic performance of EDTA and DTT agents in comparison with the reference method (sonication) for bacterial dislodgement of S epidermidis and P aeruginosa biofilms.

Although the study has the following limitations:

- only 2 strains were tested (lacking information on S aureus, which is a well known biofilm producer),

- only reference strains were used,

- only glass beads as foreign body materials,

it undoubtely confirmed the higher bacterial detection rate of sonication method compared with other techniques. cThis finding has important consequences in terms of the clinical application of these new techniques, which might be considered as complentary, and not alternative, to sonication.

Detailed comment:

- Why 3 days of biofilm formation? Would the authors believe the results would be different with shorter or longer incubation time? Please comment on it

- The authors should also stress the potential use of sonication in implant infections other than orthopedics (ie intracardiac devices; neurosurgical shunts). Please add some references and a comment on it in the discussion.

- Insert a reference or an explanation on the use of porous glass beads as suitable model for biofilm formation in vitro in the method part.

- Please add a reference or an explanation for the timing of EDTA and DTT exposition for biofilm dislodgement in the method part, not only in the discussion.

- Furthermore, which is the rationale of choosing the EDTA and DTT concentrations? Please explain also in the method part.

- Insert the limit of detection in the method part (expressed as CFU/mL)

- Were the sonicated beads also submitted to vortexing process?

- The viability experiment should be better explained or addressed in an appropriate reference.

- When comparing all the three quantitative different groups, was the ANOVA test performed? In the case the ANOVA test was not used, please indicate the reason.

- Figure1 and Figure2; Figure5, 6. Please write per extenso EDTA and DTT. NS should be explained.

- Figure3 and figure4. Insert the different methods in the legend.

- Figure4. TTD should be explained. Which is the meaning of the arrow? Please explain.

- Figure3. 0,9% or 0.9%?. Please correct.

- Lines 151-153: the authors should describe that these CFUs obtained with these concentrations and timing derive from the above described experiments.

- Line 155: insert CFU/mL

- Lines 156-157: at which concentration and timing of EDTA and DTT for P. aeruginosa? Please describe.

- Lines 158-160: this sentence might be moved in a more appropriate place (ie, at the beginning of the paragraph).

- Lines 173-177: the results of P aeruginosa are similar to those observed for S epidermidis. Please re-phrase the sentence and remove the term “although”, which might be confounding for the reader.

- Lines 225-226. This sentence is not necessary since it has been already stated.

- It would be interesting to have a comment from the authors regarding other non-culture based methods of diagnosis of implant associated infection (ie, the metabolic assay BTA, BioTimer Assay, which has been recently investigated in intracardiac and central venous catheters infections as a complementary method to sonication)

- Please include a sentence highlighting the limitations of the study

**********

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: No

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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

12 Feb 2020

Karbysheva et al. Manuscript PONE-D-19-27719

Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: implications for the microbiological diagnosis of implant associated infection

Dear Editor

Thank you for the opportunity to re-submit our scientific work again to PLOS ONE for a potential review. Please find below our response to the comments of the Reviewers. Based on these comments, we have already revised our manuscript and hope that with this improved quality it may be suitable to send it for external review and potentially of interest for your prestigious Journal.

With best regards,

Andrej Trampuz

Point-by-point rebuttal

Scientific Editor Comments:

Reviewer #1:

1. The authors analyzed the activity (not efficacy as this is an in-vitro study) of chemical methods to be used for biofilm removal.

COMMENT: We corrected in the manuscript.

“We compared the activity efficacy of chemical methods for biofilm dislodgement to the standard sonication procedure in an established in vitro model of artificial biofilm.”

2. The manuscript is well written; the laboratory methods are suitable. The biofilm dispersion was determined by different methods. However, for this is a preliminary study. The purpose of removing biofilms from implants is medical microbiological diagnostics. For that, more microorganisms (incl. clinical strains) should be included.

COMMENT: Additionally we investigated two more microorganisms (S. aureus and E. coli).

3. EDTA may interfere with viability of microorganisms. The supplementary use of chemicals to sonication should included in that study.

COMMENT: We added supplementary material on the viability of planktonic bacteria in presence of chemical agents and sonication.

4. Minor remarks. Microorganisms associated with implant infections should be introduced.

COMMENT: We added in the manuscript additional information about microorganisms associated with implant infections.

“Most commonly isolated microorganisms in patients with periprosthetic joint infection are coagulase-negative staphylococci (30-45%) and Staphylococcus aureus (12-23%), followed by streptococci (9-10%), enterococci (3-7%), gram-negative bacilli (3-6%) and anaerobes (2-4%) [6]. Similar distribution of pathogens is observed in CIED [2] and neurosurgical shunt-associated infections [4].”

5. How do the glass beads for biofilm formation correspond to clinically used material?

COMMENT: We added the explanation and references in the method part.

“As a model to form the bacterial biofilm porous glass beads (diameter 4 mm, pore sizes 60 μm, ROBU®, Hattert, Germany) were used. Due to the high volume-to-surface ratio, glass beads were used for biofilm studies rather than smooth materials, as investigated in numerous previous research works regarding biofilm formation and anti-biofilm activity [15-20].”

Reviewer #2:

The authors performed an interesting in vitro study with the aim to evaluate the diagnostic performance of EDTA and DTT agents in comparison with the reference method (sonication) for bacterial dislodgement of S epidermidis and P aeruginosa biofilms.

Although the study has the following limitations:

1. only 2 strains were tested (lacking information on S aureus, which is a well known biofilm producer).

Reply: Additionally we investigated two more microorganisms (S. aureus and E. coli).

2. only reference strains were used,

COMMENT: Explained in the manuscript.

“We did not use clinical strains as they typically show larger variability and are not suitable for investigation of a new diagnostic method.”

3. only glass beads as foreign body materials,

COMMENT: We added the explanation and references in the method part.

“As a model to form the bacterial biofilm porous glass beads (diameter 4 mm, pore sizes 60 μm, ROBU®, Hattert, Germany) were used. Due to the high volume-to-surface ratio, glass beads were used for biofilm studies rather than smooth materials, as investigated in numerous previous research works regarding biofilm formation and anti-biofilm activity [15-20].”

It undoubtely confirmed the higher bacterial detection rate of sonication method compared with other techniques. This finding has important consequences in terms of the clinical application of these new techniques, which might be considered as complentary, and not alternative, to sonication.

4. Why 3 days of biofilm formation? Would the authors believe the results would be different with shorter or longer incubation time? Please comment on it

COMMENT: We incubated glass beads for 3 days for biofilm formation as described previously by Drago et al. Clin Orthop Relat Res. 2012. The authors grew the biofilm for 3 days until a visible biofilm was formed therefor further cultivation of mature biofilm is not needed.

5. The authors should also stress the potential use of sonication in implant infections other than orthopedics (ie intracardiac devices; neurosurgical shunts). Please add some references and a comment on it in the discussion.

COMMENT: We added the addition information in the manuscript.

“Implants Orthopedic devices are increasingly used to improve the mobility (joint replacement and bone fixation devices) in the treatment of degenerative joint disease (osteoarthritis) and for the fixation of bone fractures or enhance the survival and assist the performance of physiological functions (cardiac implantable electronic device (CIED) and neurosurgical shunts). Infections represent a significant complication of implant surgery, resulting in major challenges regarding the diagnosis and treatment [1-5].”

6. Insert a reference or an explanation on the use of porous glass beads as suitable model for biofilm formation in vitro in the method part.

COMMENT: We added the explanation and references in the method part.

7. Please add a reference or an explanation for the timing of EDTA and DTT exposition for biofilm dislodgement in the method part, not only in the discussion.

COMMENT: We added the explanation and references for the timing of EDTA and DTT exposition in the method part.

8. Furthermore, which is the rationale of choosing the EDTA and DTT concentrations? Please explain also in the method part.

COMMENT: We added the explanation of choosing EDTA and DTT concentration in the method part.

9. Insert the limit of detection in the method part (expressed as CFU/mL)

COMMENT: We corrected in the manuscript as CFU/mL.

10. Were the sonicated beads also submitted to vortexing process?

COMMENT: We did use the vortexing process in sonication protocol. We corrected the protocol in the method part.

“Briefly, each bead was inoculated in 1 ml 0.9% saline, vortexed for 30 sec, and sonicated at 40 kHz at intensity 0.1 Watt/cm2 (BactoSonic, BANDELIN electronic, Berlin, Germany) for 1 min and vortexed again for 30 sec.”

11. The viability experiment should be better explained or addressed in an appropriate reference.

COMMENT: We added supplementary material on the viability of planktonic bacteria in presence of chemical agents and sonication.

12. When comparing all the three quantitative different groups, was the ANOVA test performed? In the case the ANOVA test was not used, please indicate the reason.

COMMENT: We recalculated the result of the study using ANOVA test. New results are added in the manuscript.

13. Figure1 and Figure2; Figure5, 6. Please write per extenso EDTA and DTT. NS should be explained.

COMMENT: We corrected the information in the Figures.

14. Figure3 and figure4. Insert the different methods in the legend.

COMMENT: We corrected the information in the Figures.

15. Figure4. TTD should be explained. Which is the meaning of the arrow? Please explain.

COMMENT: We corrected the information in the Figures.

16. Figure3. 0,9% or 0.9%?. Please correct.

COMMENT: We corrected the information in the Figures.

17. Lines 151-153: the authors should describe that these CFUs obtained with these concentrations and timing derive from the above described experiments.

COMMENT: We corrected the information in the method part.

18. Line 155: insert CFU/mL

COMMENT: We corrected in the manuscript as CFU/mL.

18. Lines 156-157: at which concentration and timing of EDTA and DTT for P. aeruginosa? Please describe.

COMMENT: We corrected the information in the results part.

19. Lines 158-160: this sentence might be moved in a more appropriate place (ie, at the beginning of the paragraph).

COMMENT: We corrected the information in the results part.

20. Lines 173-177: the results of P aeruginosa are similar to those observed for S epidermidis. Please re-phrase the sentence and remove the term “although”, which might be confounding for the reader.

COMMENT: We corrected the information in the results part.

21. Lines 225-226. This sentence is not necessary since it has been already stated.

COMMENT: We corrected the information in the results part.

22. It would be interesting to have a comment from the authors regarding other non-culture based methods of diagnosis of implant associated infection (ie, the metabolic assay BTA, BioTimer Assay, which has been recently investigated in intracardiac and central venous catheters infections as a complementary method to sonication)

- Please include a sentence highlighting the limitations of the study

COMMENT: We added the additional information on non-culture based methods of diagnosis of implant associated infection in the limitation of the study.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

17 Mar 2020

PONE-D-19-27719R1

Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: implications for the microbiological diagnosis of implant associated infection

PLOS ONE

Dear Prof. Trampuz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by May 01 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Olivier Habimana

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 #2: 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 #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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 #2: 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 #2: 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 #2: (No Response)

Reviewer #3: Thank you for letting me reviewing the revision of this manuscript. I’m impressed by the extensive and multimodal analysis of the activity of different biofilm-dislodging methods by the authors. The results are certainly of clinical relevance.

The former reviewers comments were addressed appropriately with a few exceptions:

Reviewer #2

2. only reference strains were used,

COMMENT: Explained in the manuscript.

“We did not use clinical strains as they typically show larger variability and are not suitable for

investigation of a new diagnostic method.”

3. only glass beads as foreign body materials,

COMMENT: We added the explanation and references in the method part.

“As a model to form the bacterial biofilm porous glass beads (diameter 4 mm, pore sizes 60 μm,

ROBU®, Hattert, Germany) were used. Due to the high volume-to-surface ratio, glass beads

were used for biofilm studies rather than smooth materials, as investigated in numerous

previous research works regarding biofilm formation and anti-biofilm activity [15-20].”

It undoubtedly confirmed the higher bacterial detection rate of sonication method compared with

other techniques. This finding has important consequences in terms of the clinical application of

these new techniques, which might be considered as complementary, and not alternative, to

sonication.

• These two limitations should be stated in the discussion. This in vitro analysis represents a fundament for further exploration in the clinical setting with clinical strains and real implants.

4. Why 3 days of biofilm formation? Would the authors believe the results would be different

with shorter or longer incubation time? Please comment on it

COMMENT: We incubated glass beads for 3 days for biofilm formation as described previously

by Drago et al. Clin Orthop Relat Res. 2012. The authors grew the biofilm for 3 days until a

visible biofilm was formed therefor further cultivation of mature biofilm is not needed.

• I agree with the reviewer. It remains unknown, whether the results would be different in more mature biofilms (as we are dealing with in the clinical setting). Comment this in the limitations setting.

9. Insert the limit of detection in the method part (expressed as CFU/mL)

COMMENT: We corrected in the manuscript as CFU/mL.

• Could not find the detection limit in the methods part. Please make sure to provide the limit of detection.

14. Figure3 and figure4 . Insert the different methods in the legend.

COMMENT: We corrected the information in the Figures.

• Please mention the different methods tested in the legend.

15. Figure4. TTD should be explained. Which is the meaning of the arrow? Please explain.

COMMENT: We corrected the information in the Figures.

• Please explain the meaning of the arrow in the legend

• Please make sure, that the figures are numbered correctly and mentioned accordingly in the manuscript text. Figure 2 is now Fig 5, Fig 3 is now Fig 6, etc.

18. Line 155: insert CFU/mL

COMMENT: We corrected in the manuscript as CFU/mL.

• Please add „CFU/ml“ in al the comparisons in the entire section (e.g. lines 170, 173, 178, 181)

Additional comments from my side:

• Make sure to use the terms dislodgement (vs. dislodgment) and NaCl (vs. saline) uniformally throughout the manuscript

• L 142: Please provide an introducing sentence before reporting the results. For example: „Figure 1 shows CFU counts of residual biofilms after exposure to chemical agents for variable durations.“

**********

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 #2: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

20 Mar 2020

Karbysheva et al. Manuscript PONE-D-19-27719

Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: implications for the microbiological diagnosis of implant associated infection

Dear Editor

Thank you for the opportunity to re-submit our scientific work again to PLOS ONE for a potential review. Please find below our response to the comments of the Reviewers. Based on these comments, we have already revised our manuscript and hope that with this improved quality it may be suitable to send it for external review and potentially of interest for your prestigious Journal.

With best regards,

Andrej Trampuz

Point-by-point rebuttal

Scientific Editor Comments:

Reviewer #2

First revision:

2. only reference strains were used,

COMMENT: Explained in the manuscript.

“We did not use clinical strains as they typically show larger variability and are not suitable for

investigation of a new diagnostic method.”;

3. only glass beads as foreign body materials,

COMMENT: We added the explanation and references in the method part.

“As a model to form the bacterial biofilm porous glass beads (diameter 4 mm, pore sizes 60 μm,

ROBU®, Hattert, Germany) were used. Due to the high volume-to-surface ratio, glass beads

were used for biofilm studies rather than smooth materials, as investigated in numerous

previous research works regarding biofilm formation and anti-biofilm activity [15-20].”

Second revision, Reviewer #2: It undoubtedly confirmed the higher bacterial detection rate of sonication method compared with other techniques. This finding has important consequences in terms of the clinical application of these new techniques, which might be considered as complementary, and not alternative, to sonication.

These two limitations should be stated in the discussion. This in vitro analysis represents a fundament for further exploration in the clinical setting with clinical strains and real implants.

COMMENT: We stated these two limitations in the discussion.

“.., for biofilm formation we used only laboratory strains. Typically clinical strains show larger variability therefore to evaluate a new diagnostic method in vitro the laboratory strains are more suitable. Third, we used only porous glass beads for biofilm formation. The porous glass beads possess a high volume-to-surface ratio therefore this model to form bacterial biofilm is probably more suitable for in vitro biofilm investigation than smooth materials. The results derived from this in vitro analysis represent a fundament for further exploration in the clinical setting with clinical strains and real implants.”

First revision:

4. Why 3 days of biofilm formation? Would the authors believe the results would be different

with shorter or longer incubation time? Please comment on it

COMMENT: We incubated glass beads for 3 days for biofilm formation as described previously

by Drago et al. Clin Orthop Relat Res. 2012. The authors grew the biofilm for 3 days until a

visible biofilm was formed therefor further cultivation of mature biofilm is not needed.

Second revision, Reviewer #2: I agree with the reviewer. It remains unknown, whether the results would be different in more mature biofilms (as we are dealing with in the clinical setting). Comment this in the limitations setting.

COMMENT: We stated this limitation in the discussion.

“Forth, we incubated glass beads in the bacterial inoculum for 3 days until a visible biofilm was formed as described previously [14]. We assumed that further cultivation of mature biofilm to compare the ability of different methods for biofilm dislodgement is not needed. However it remains unknown, whether the ability of sonication or chemical methods for biofilm dislodgement would be different in more mature biofilms for example in the clinical setting when we deal with chronic implant-associated infections.”

First revision:

9. Insert the limit of detection in the method part (expressed as CFU/mL)

COMMENT: We corrected in the manuscript as CFU/mL.

Second revision, Reviewer #2: Could not find the detection limit in the methods part. Please make sure to provide the limit of detection.

COMMENT: We insert the limit of detection in the method part.

“The serial dilutions allowed to raise the upper limit of detection providing a reportable range from 0 to 100,000,000 CFU/mL.”

First revision:

14. Figure3 and figure4 . Insert the different methods in the legend.

COMMENT: We corrected the information in the Figures.

Second revision, Reviewer #2: Please mention the different methods tested in the legend.

COMMENT: Figure 3 is now Figure 2. We insert the methods in the legend.

First revision:

15. Figure4. TTD should be explained. Which is the meaning of the arrow? Please explain.

COMMENT: We corrected the information in the Figures.

Second revision, Reviewer #2: Please explain the meaning of the arrow in the legend

COMMENT: Figure 4 is now Figure 3. We explained the meaning of TTD in the legend.

Second revision, Reviewer #2: Please make sure, that the figures are numbered correctly and mentioned accordingly in the manuscript text. Figure 2 is now Fig 5, Fig 3 is now Fig 6, etc.

COMMENT: We revised the numbers of the figures and mentioned them correctly in the manuscript. We combined Figure 1 and Figure 2 in Figure 1. Figure 3 is now Figure 2. Figure 4 is now Figure 3. Figure 5 is now Figure 4. Figure 6 is now Figure 7. We added two additional Figures (5 and 6).

First revision: 18. Line 155: insert CFU/mL

COMMENT: We corrected in the manuscript as CFU/mL.

Second revision, Reviewer #2: Please add „CFU/ml“ in al the comparisons in the entire section (e.g. lines 170, 173, 178, 181)

COMMENT: We added “CFU/mL” in the manuscript.

Additional comments from Reviewer #2:

• Make sure to use the terms dislodgement (vs. dislodgment) and NaCl (vs. saline) uniformally throughout the manuscript

COMMENT: We corrected the terms in the manuscript.

• L 142: Please provide an introducing sentence before reporting the results. For example: „Figure 1 shows CFU counts of residual biofilms after exposure to chemical agents for variable durations.“

COMMENT: We added the sentence in the results part.

Submitted filename: Response to Reviewers 2.docx

Click here for additional data file.

24 Mar 2020

Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: implications for the microbiological diagnosis of implant associated infection

PONE-D-19-27719R2

Dear Dr. Trampuz,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Olivier Habimana

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

26 Mar 2020

PONE-D-19-27719R2

Comparison of sonication with chemical biofilm dislodgement methods using chelating and reducing agents: implications for the microbiological diagnosis of implant associated infection

Dear Dr. Trampuz:

I am 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.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Olivier Habimana

Academic Editor

PLOS ONE