Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: Protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques

Vinícius Magno da Rocha; Carla Ormundo Gonçalves Ximenes Lima; Eliane de Oliveira Ferreira; Gabriel Corrêa de Farias; Fábio César Sousa Nogueira; Luis Caetano Martha Antunes; Keila Mara Cassiano; Rossano Kepler Alvim Fiorelli

doi:10.1371/journal.pone.0271773

. 2023 Feb 27;18(2):e0271773. doi: 10.1371/journal.pone.0271773

Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: Protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques

Vinícius Magno da Rocha ¹, Carla Ormundo Gonçalves Ximenes Lima ², Eliane de Oliveira Ferreira ^2,^*, Gabriel Corrêa de Farias ³, Fábio César Sousa Nogueira ⁴, Luis Caetano Martha Antunes ⁵, Keila Mara Cassiano ⁶, Rossano Kepler Alvim Fiorelli ¹

Editor: D William Cameron⁷

PMCID: PMC9970075 PMID: 36848344

Abstract

Lumbar disc degeneration (LDD) and low back pain (LBP) are two conditions that are closely related. Several studies have shown Cutibacterium acnes colonization of degenerated discs, but whether and how these finding correlates with LBP is unknown. A prospective study was planned to identify molecules present in lumbar intervertebral discs (LLIVD) colonized by C. acnes in patients with LDD and LBP and correlate them with their clinical, radiological, and demographic profiles. The clinical manifestations, risk factors, and demographic characteristics of participants undergoing surgical microdiscectomy will be tracked. Samples will be isolated and pathogens found in LLIVD will be characterized phenotypically and genotypically. Whole genome sequencing (WGS) of isolated species will be used to phylotype and detect genes associated with virulence, resistance, and oxidative stress. Multiomic analyses of LLIVD colonized and non-colonized will be carried out to explain not only the pathogen’s role in LDD, but also its involvement in the pathophysiology of LBP. This study was approved by the Institutional Review Board (CAAE 50077521.0.0000.5258). All patients who agree to participate in the study will sign an informed consent form. Regardless of the study’s findings, the results will be published in a peer-reviewed medical journal. Trials registration number NCT05090553; pre-results.

Introduction

LBP is a common complaint in the general population, and its incidence is about five times higher in patients with LDD and Modic endplate changes [1, 2]. The link between these findings and the isolation of low virulence pathogens in LIVD has already been described in several studies, generating a lot of interest in the topic and promoting the bacterial hypothesis of LDD [3–14].

Albert et al. reported 46% of positive cultures for anaerobes in 61 patients undergoing lumbar discectomy, of which 80% had Modic type 1 changes [15]. Other studies corroborate these findings, with high identification rates for Cutibacterium acnes, an anaerobic and aerotolerant pathogen that is bacillus-shaped, stained by the Gram method, pleomorphic, non-spore-forming, and a biofilm producer [11, 16–20]. Rollason et al. isolated strains of this bacterium from the LIVD of 64 patients, revealing the presence of genotypic profiles distinct from those found in the skin, suggesting that specific variants would be related to LBP [21].

Other authors believe that the presence of this pathogen in the LIVD of asymptomatic patients is due to contamination of the specimens during sampling and/or sample treatment in the laboratory environment. Carricajo et al. found positive cultures for C. acnes in only 3.4% of the discs from 54 patients, defending the possibility of contamination [22]. Rigal et al. also analyzed LIVD from 313 patients who underwent video-assisted retroperitoneal discectomy, with positive cultures for only six patients [23].

These marked divergences in microbiological results cast doubt on the bacterial hypothesis of LDD and, consequently, raise questions about the possibility of anaerobic pathogens acting as triggers or amplifiers of LBP. Urquhart et al. conducted a systematic review of the literature on the subject and concluded that there is evidence correlating the C. acnes isolation with Modic type 1 changes and LBP, but there is a lack of studies producing substantial and irrefutable evidence [24].

Despite the fact that considerable effort has already been expended in determining the presence of C. acnes in LIVD, there are still no standard isolation protocols for this pathogen from this clinical specimen. Furthermore, prior to the preparation of this manuscript, studies have shown no consistency in the collection and analysis techniques used, making comparison and reproducibility of results difficult.

Aware of this reality, Astur et al. proposed a protocol for a cohort study to identify C. acnes from intervertebral discs [25]. Although valid, the initiative has two significant limitations that should be highlighted. First, after collecting clinical specimens, their immediate inoculation in a liquid medium is not foreseen in the operating room, which increases the contact time of the samples with the aerobic environment and reduces the chances of microorganism recovery even with culture-specific techniques. Second, the authors also highlight sonication (ultrasound waves to release bacteria from the biofilm of surgical prostheses) of specimens for subsequent inoculation in automated blood culture vials; however, this technique was originally described for pathogen recovery from orthopedic prostheses rather than biological tissue [26] and, because the sonication parameters used are not clear (time, power, and frequency), the reproduction of results by other authors and the validation of this protocol is compromised.

Specific microbiological identification techniques, such as immediate inoculation of specimens in liquid culture medium while still in the operating room, vortex for sessile cell recovery, incubation time extension in an anaerobic atmosphere with replication every 72 h, and the use of Matrix-Assisted Laser Desorption Ionization Time of Flight Mass spectrometry—MALDI-TOF MS (Bruker Coorporation), and multiplex-touchdown PCR are strategies that assure reliable results regarding the presence of bacteria in intervertebral discs [27, 28], but they do not explain the pathways through which these microorganisms would participate in LDD and/or LBP.

Once LIVD colonization by C. acnes is confirmed using microbiological techniques, in the absence of clinical signs of infection, the main challenge is to understand the implications of this finding for homeostasis of that microenvironment. Rajasekaran et al. showed strong evidence of LIVD colonization by C. acnes using rDNA-16s PCR and proteomic analyses [29]. These authors discovered not only host defense proteins, but also proteins associated with bacterial viability and proliferation. Although these findings have demonstrated the active presence of C. acnes in intervertebral discs, definitively ruling out the possibility of contamination, they do not necessarily reflect the activity of the expressed proteins, which may remain inactive and without interference in pain-generating pathways and/or the inflammatory response.

In this context, the combined use of proteomic and metabolomic techniques to analyze LIVD colonized by C. acnes would be more appropriate in order to assess the subclinical consequences of the colonization by this pathogen [30]. These techniques have already been used successfully to define molecular signatures in other clinical settings [31–34], especially in chronic pain [35–40], and can also provide integrative information on cell function at the molecular level about the role of C. acnes in LDD and LBP [41–44].

Hypotheses and objectives

Our hypothesis is that LDD and LBP are related to LIVD colonization by C. acnes.

The primary goal of this study is to determine the incidence of LIVD colonization by low virulent pathogens using specific sampling and culture techniques, as well as phenotypic and genotypic techniques for microbiological characterization.

Other objectives of the study include: 1- assessing the possibility of clinical specimen contamination in positive cultures; 2- establishing a correlation between the presence of the pathogen and the clinical and radiological profile (Modic type 1 changes) of the study participants; 3- to outline a phylotypic profile of the most isolated C. acnes strains by bacterial WGS; and 4- identify a molecular profile for LDD and LBP in patients with positive cultures for C. acnes through proteomic and metabolomic techniques.

Justifications

Colonization of LIVD by C. acnes in patients with LDD supports the bacterial hypothesis of LBP [4, 8, 29, 45–47]. The determination of a molecular profile for these patients will not only contribute to a better understanding of the pathophysiological basis of LBP associated with LDD [41–43], but it will also positively interfere in the clinical management of this common condition, rationalizing its treatment and optimizing its costs.

Previously studies looked at small groups of patients and did not follow standardized sampling and analysis methods [15, 24, 48, 49]. This makes it difficult to value these findings and while they do suggest a correlation between C. acnes and LBP, the evidence is insufficient to explain how this bacterium could be involved in the process of LIVD degeneration and generation/amplification of LBP.

This is the first protocol to investigate the role of C. acnes in LIVD degeneration, combining isolation with culture techniques, phenotypic and genotypic characterization techniques; and genomic, proteomic, and metabolomic techniques. In addition, the protocol proposes the clinical–radiological characterization of the participants, blinding the researchers and controlling confounding variables, and following appropriate selection criteria and statistical planning to obtain significant results.

Methods and analysis

This study protocol is registered with the Research Ethics Committee (REC) [CAAE: 50077521.0.0000.5258] of the Gaffrée and Guinle University Hospital (HUGG) and at Clinicaltrials.gov under NCT05090553 (https://clinicaltrials.gov/ct2/results?term=NCT05090553).

Ethics and dissemination

The protocol will follow the ethical standards of the HUGG REC. All patients whose biological materials will be analyzed will be well-informed of the research objectives and will sign ICF, agreeing with the availability of samples for further laboratory analysis.

Study design

This analytical study will be performed at a single location (Spine Surgery Center of São Matheus Hospital (HSM) in collaboration with the Gaffrée Guinle University Hospital (HUGG) and Anaerobic Biology Laboratory of Paulo de Góes Microbiology Institute of the Universidade Federal do Rio de Janeiro (UFRJ).

The analyses will be performed sequentially, according to the surgical procedures carried out on the selected participants, taking 6 months after for their recruitment and ending 6 months after the surgical treatment of the last patient. Data from participants will be collected using a form designed specifically for this study.

Population

Participants included in the study must meet the selection criteria listed below.

Inclusion criteria

The inclusion criteria for the study will be participants aged between 18 and 65 years; both sexes; complaint of low back pain lasting more than 3 months; magnetic resonance imaging findings of lumbar disc degeneration (LDD) performed less than 6 months before inclusion in the study; indication for open surgical treatment with isolated microdiscectomy or associated with lumbar arthrodesis; failure of conservative treatment for at least 6 weeks and/or progressive neurological deficit; agreement to follow all phases of the clinical investigation, having signed the informed consent form (ICF) for participation in the study.

Exclusion criteria

The exclusion criteria for the study will be a history of open lumbar spine surgery at any stage of life; chemotherapy or pulse therapy with corticoids; immune deficiency; previous intradiscal therapies (nucleotomy or discography); previous endoscopic surgery; history of spinal infection treated with antibiotics in the 6 months prior to inclusion in the study; use of antibiotics in the 2 months prior to the surgical procedure; incomplete research inclusion form; refusal to participate and/or sign the ICF.

Patient and public involvement

Participant eligibility will be evaluated by the study researchers through a clinical interview and imaging exams evaluation, according to the participants’ interest and availability to participate in the study. If the patient agrees to take part in the study, the predetermined selection criteria will be used to evaluate them. The researchers will explain the details of the study and conduct a joint reading of the ICF. Questions about the study objectives, risks and benefits, stages, and research confidentiality will be answered. A patient will only participate upon signing the ICF at this stage of the research. A copy of this document will be given to the participant, another to the responsible researcher, and a third will be attached to the medical record. After signing the consent form, the patient will undergo an evaluation for demographic and clinical data collection and the forms developed for this purpose will be completed. If the patient is unable to read and sign the written consent form, researchers will verbally explain the study details and the patient will orally provide consent in the presence of a witness who will sign the consent form. The recruitment of participants will take place over a 6-month period, until 120 individuals are included, aiming at a minimum number of 96 collections.

Participant allocation

The participants will be operated by the same surgical team, and the surgical technique will be posterior discectomy followed by arthrodesis of the approached segment.

Blinding

The results of microbiological cultures or molecular analysis will not be disclosed neither to the patients nor to the surgeons. The radiologist who will review the imaging tests will also be blinded to patient data and laboratory results. The researcher who will be analyzing the pain and function scores will be blinded as well.

Withdrawal of a study participant

A participant will be removed from the study in cases of ICF withdrawal, death, recruitment failure identification, loss of post-surgical follow-up, or the presentation of clinical symptoms of infection, intense pain, fever without other infectious foci, increased erythrocyte sedimentation rate and/or C-reactive protein, leukocytosis, imaging tests compatible with spondylodiscitis, or any other condition that leads to blinding interruption.

The reason and circumstances of each participant’s withdrawal from the study will be detailed. The data gathered until the patient is withdrawn from the study will be included in the final analysis.

Selection of outcomes

Primary outcome

The main objective of this study will be to determine the incidence of intervertebral disc colonization by C. acnes in patients with LBP and LDD. Positive cultures will be used to confirm colonization, followed by phenotypic and genotypic confirmation of the isolated pathogen by mass spectrometry (Biotyper) and rDNA-16s PCR analysis, respectively.

Secondary outcomes

Low back pain. At the time of patient recruitment the Visual Numeric Scale (VNS) [[50]] will be used to assess LBP intensity and daily activity limitations. A 30% increase in baseline LBP in the first postoperative month will be considered clinically significant. The VNS and the visual analogue scale have a good correlation and are equally sensitive to quantifying postoperative pain [51].

Quality of life. The validated Portuguese version of the EuroQol questionnaire (EQ-5D) will be used to assess the quality of life of colonized and non-colonized groups with and without Modic changes [52]. The EQ-5D is a self-administrated standardized instrument containing five items (mobility, self-care, usual activities, pain/discomfort, and anxiety/depression).

Functionality. The functionality of the participants will be quantified through the validated Portuguese version of the Oswestry Disability Index (ODI) for LBP [53].

Host multiomic characteristics

Proteomic and metabolomic techniques will be used to examine disc and plasma samples. The comparison of the group of molecules found in disc and plasma samples may contribute to the quantitative and qualitative identification of profiles using peripheral blood that are associated with the presence of the pathogen in the intervertebral disc. After processing the multiomic raw data using proper bioinformatics tools, statistical techniques will be applied to analyze them against the clinical–radiological and microbiological profiles of the participants. We expect to provide a molecular signature relating the presence of C. acnes, LDD and LBP.

Adverse effects

Failures related to surgical treatment, such as surgical wound infection, CSF leak, deep vein thrombosis, pain recurrence, or any other adverse events that may arise in the postoperative period, will be reported and considered in data analysis.

Sampling

The Schedule of enrolment, Interventions, and assessments is shown in Fig 1. During anesthetic induction, intravenous antibiotic prophylaxis will be administered after patient admission to the operating room. Cefazolin at a dose of 1 g replicated every 4 h for the duration of the surgery will be used as an antimicrobial drug. The antibiotic prophylaxis regimen will end with skin closure and will not be extended for additional postoperative time (Fig 1).

The clinical specimens collected will be:

Peripheral blood. A 10 mL aliquot will be obtained 30 min after the infusion of surgical antibiotic prophylaxis through upper limb venoclysis. This sample will be placed in a tube containing clot activator for further separation of the serum, which, in turn, will be stored at −80°C for further analysis.
Skin swab. After skin asepsis with 2% chlorhexidine gluconate degerming solution and 0.5% chlorhexidine gluconate alcoholic solution, a swab will be collected from the region where the surgical incision will be performed.
Intervertebral disc. Five fragments will be collected at each level approached during surgery. A set of sterile surgical tweezers will be used exclusively for collection, one for each tissue fragment collected. The forceps with tissue fragment will be given to a member of the laboratory who will monitor the procedure inside the operating room immediately after collection. Each fragment will be placed in a vial containing thioglycolate broth (Merck^®, Brazil) and 20 glass beads. The vials will be labeled with the initials of the study participants and the type of clinical specimen. After collection, the vials containing the clinical specimens will be kept at room temperature (18°C to 22°C) and transported in a specific case to the laboratory within a maximum period of 2 h.
Muscle–ligament tissue. Before obtaining the LIVD fragments, five fragments of muscle–ligament tissue adjacent to the collected disc will be collected. The same collection and initial cooling precautions used for the disc fragments will be followed when collecting the adjacent tissue.

Laboratory analysis

Cultures

The skin swabs will be opened only in a laboratory setting. Samples will be seeded on blood agar plates (5% defibrinated sheep blood; blood agar base, 40 g/L; agar, 5 g/L) and anaerobic blood agar (5% defibrinated sheep blood; blood agar base, 40 g/L; agar, 5 g/L; hemin, 10 mL/L; menadione, 5 drops/L. After sowing in solid media, the swab will be inoculated in thioglycolate medium, where it will remain for 14 days. One of the plates will be incubated in a capnophilic atmosphere (5% to 10% CO₂), while the other will be incubated in strict anaerobiosis (atmosphere containing 10% CO₂,10% H₂, and 80% N₂) using an anaerobic chamber or Glove Box (Coy Labs^®, USA).

The plates will be read after 24 h of incubation and, in case of growth, the colonies will be identified. In the absence of growth, they will be incubated again, and a new reading will be performed after 72 h. The thioglycolate vials containing skin swabs will be kept in a bacteriological incubator (35°C to 37°C) for 14 days. In a laboratory setting, the tubes containing disc fragments and muscle–ligament tissue will undergo vortexing (Even EVX2800-BI^®, Brazil) for 15 s. All tubes will be kept in a bacteriological incubator (35°C to 37°C) and, after 24 h, the first subculture will be carried out using a 100 μL bacteriological loop. An aliquot of the thioglycolate will be taken and plated on blood agar and anaerobic blood agar. The first plate will be incubated in a capnophilic atmosphere and the second in strict anaerobiosis using an anaerobic chamber or Glove Box (Coy Labs^®, USA). The plates will be read after 24 h and the colonies will be identified in case of growth. In the absence of growth, they will be incubated again, and a new reading will be performed after 72 h. The same procedure will be used for all thioglycolate vials (totaling six vials for each level approached). Thioglycolate tubes containing the clinical specimens will be kept in an oven for 14 days. Subcultures will be carried out after 72 h and always every 3 days, with the last subculture being carried out on the 14^th day of incubation in the same solid culture medium and atmospheres used in the first 24 h.

Quality control of culture media

Sterile saline solution at 0.9% will be used to control the sterility of the culture medium and Pseudomonas aeruginosa ATCC 27853 and Bacteroides fragilis ATCC 25285 strains to evaluate the recovery capacity of facultative microorganisms and strict anaerobes. Each new batch of culture medium prepared will undergo this quality control.

Phenotypic characterization

The phenotypic identification of the species will be carried out through mass spectrometry using MALDI-TOF MS equipment (Bruker Biotyper^®, Germany). The score used for reliable identification of pathogens will follow the manufacturer’s instructions.

Genotypic characterization

The microorganisms isolated from cultures will be subjected to PCR analysis performed in two phases, as described by Barnard et al. [28]. The first to confirm the presence of bacteria (target and non-target) by using species-specific primers targeting the rRNA-16s and a second one, Multiplex-PCR, targeting the virulence and oxidative gens.

Multiomic analyses

Whole genome sequencing

For the WGS, the DNA of the isolated C. acnes strains will be obtained with the Qiagen Blood & Tissue DNA extraction kit (Qiagen^®, USA) and subsequent sample purification using RNase.

The extracted and purified DNA will be dosed, and adjusted to a concentration of 0.2 ng/μL. The ends of its two ribbons will be fragmented and tagged (forward and reverse 5’-3’). This will form fragments of different sizes. This data will be used to create a database on the Nextera Flex Kit platform (Ilumina^®, USA). The samples will then be multiplexed into a cell stream and run on the NextSeq sequencer (Ilumina^®, USA) using paired sequencing to generate files in fastq format files, which will then be filtered using the Trimmomatic tool version 0.36, as recommended by Bolger et al. [54]. This step will be critical for controlling the quality of the analysis because it reduces the possibility of errors during sequencing and allows the use of more reliable reading codes.

The obtained fragments will be sequenced, and their quality will be evaluated with the FastQC tool version 0.11.9. The RedDog software version 1.11 will be used to identify single nucleotide polymorphisms (SNPs) using the GCA_000008345.1 C. acnes reference strain deposited in the GenBank NCBI [https://www.ncbi.nlm.nih.gov/nuccore/] as a comparison. Finally, the PATRIC software version 3.5.21 will be used to create a complete genome phylogeny from the concatenation of SNPs.

Proteomics

In this study, proteomics approaches will apply gel-free and in-gel protein digestion by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique [2].

Thioglycolate culture mediums will be centrifuged, and the sediment obtained will be washed in phosphate buffer and centrifuged again. The sediment will be dissolved in a lysis solution containing 8 M urea, 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, and 40 mM Tris (hydroxymethyl) aminomethane. This lysis will be complemented with the use of glass beads under a Bead Beater agitation (Biospec Product^®, USA) (3 × 20 s with intervals of 20 s) followed by cooling for 5 min on an ice bath, as described by Shah et al. [55]. Protein concentration will be measured and 20 μg will be used for both the gel and solution techniques.

The SDS-PAGE -based technique will be used to evaluate the profile of the proteins present in the extract. Proteins will be removed from the gel with a sterile scalpel, eluted, and trypsinized (proteolic digestion) for further analysis through mass spectrometry. In the solution technique, the obtained extract will be trypsinized for further analysis through mass spectrometer. Regardless of the technique used, a nano-Liquid Chromatography coupled to a high-resolution mass spectrometer (nLC-HRMS) will be used.

After analysis by both techniques, the raw data (.raw) will be converted into a mascot general file (.mgf) using the Mascot Distiller software version 2.8.0, and proteins will be identified using protein banks referring to C. acnes and eukaryotes (humans), both created using the NCBI as a fasta extension. After this identification, the data will be validated using the Scaffold software version 5 for the same databases. Two biological replicas will be made for both the gel and solution technique. The Interactivenn.net [http://www.interactivenn.net/index.html] [56] and STRING version 11 software [57] will be used for comparative analysis and functional interaction of proteins, respectively.

Metabolomics

The metabolomic analysis in this study will use intervertebral disc fragments with and without isolated C. acnes. The tubes containing the disc fragments in thioglycolate liquid culture medium will be centrifuged and the sediment will be transferred to 2 mL flat-bottom polypropylene tubes containing a titanium bead (one per tube). The liquid content of these tubes will be evaporated by centrifugation with the CentriVap SpeedVac apparatus (Labconco Corporation^®, USA) and then weighed to determine the dry weight. The tubes will then have 500 μL of MS Grade (Fluka) acetonitrile added followed by Bead Beater agitation (Biospec Product^®, USA) (4 x 30 s). When all the material has been dissolved, the tubes will be centrifuged, and the supernatant will be stored at −80°C in new sterile tubes. To identify the metabolites, the material will be injected into a high-resolution mass spectrometer (UHPLC Q-TOF MS/MS). The data obtained will be analyzed in the Metaboanalyst 5.0 database [https://www.metaboanalyst.ca/] and its functions and interactions in the STRING software version 11 [57].

Questionnaires

Questionnaires on pain (VNS), functionality (ODI), and quality of life (EQ-5D) will be given at recruitment as well as at 1, 3, and 6 months postoperatively. A professional who is not involved in the study will collect all questionaires. Follow-up clinical visits will be carried out at 1, 3, and 6 months postoperatively, with acceptance deviation of 7, 14, and 21 days, respectively.

Imaging studies

Imaging studies include 1.5 T magnetic resonance imaging. The tests must be dated no later than 6 months prior to the participant’s inclusion in the study. Any of the following changes will be considered disc degeneration: disc protrusion, extrusion, or sequestration; reduced disc intensity; annular ruptures; Schmorl’s nodes (Depressions on the surface of the vertebral bodies identified on MRI scans that result from the pressure exerted by the intervertebral discs on the endplates, causing intervertebral disc tissue herniation and displacement into the adjacent vertebral bodies); Modic changes; and/or loss of disc height.

Modic changes

The presence of Modic type 1 changes will be considered in the evaluation of magnetic resonance images. This change is characterized by the presence of a high signal on T2-weighted sequences, with reduced signal on T1-weighted sequences [1, 2]. The frequency of Modic type 1 changes in the participants will be calculated for each of the research groups (presence or absence of colonization) and for the total number of discs analyzed in the laboratory.

Pfirrmann classification

Degenerative changes identified in the discs will be categorized according to the Pfirrmann classification [58] using T2-weighted magnetic resonance images.

Confounding variables

As some of this information may change during the study, the data obtained at the time of inclusion in the study will be considered. Below are the confounding variables to be analyzed:

Age
Sex
Education
Leave of absence
Alcohol consumption
1. None or sporadically (< 1 dose/day)
2. Mild (1–2 doses/day)
3. Moderate/High (≥ 3 doses/day)
Smoking
1. Non-smoker
2. Smoker
3. Ex-smoker
Body mass index
1. Underweight (< 18.5 Kg/m²)
2. Normal (18.5–25 Kg/m²)
3. Overweight (25–30 Kg/m²)
4. Obese (> 30 Kg/m²)
Physical activity practice
1. Sedentary (not performing physical activity for at least 10 continuous minutes during the week)
2. Active (vigorous activities for three or more days/week and for 20 min or more per session; or moderate activity or walking for five or more days/week and for 30 min or more per session; or any activity added together 5 or more days/week and 150 min or more/week)
3. Very active (vigorous activity for five or more days/week and 30 min or more per session; vigorous activity for three or more days/week and 20 min or more per session, more moderate activity and/or walking for five or more days/week and for 30 min or more per session)
Use of oral steroids up to 3 months before surgery
Diabetes

Statistical planning

Sample size calculation

Considering the main outcome proposed, the identification of disc colonization by C. acnes, the minimum sample size (n) for the population of interest will be 96 participants, according to the calculation described by Medronho et al., [59] using a confidence interval of 95% and a maximum margin of error of 10%.

The ideal sample size for analysis of secondary outcomes will depend on the incidence of the main outcome. If it is too low, an increase in the number of patients included will be performed with the inclusion of more blocks of participants. In order to have a more accurate dimension, the sample size calculation will be revised as soon as we reach half of the initially planned sample size.

Statistical analysis methodology

The database will be analyzed using the SPSS software, version 22.0, and R software, version 4.0.2. Descriptive analysis will be performed by charts and descriptive statistics. The inferential analysis will consider a maximum significance level of 5%. The chi-square test or Fisher’s exact test will be used for inferential analysis of the distributions of categorical variables. The odds ratio (OR) will be the measure used to estimate risk. The OR’s significance will be evaluated by the OR’s asymptotic confidence interval. The hypothesis of normality will be verified by the Kolmogorov–Smirnov and Shapiro–Wilk tests. Student’s t-test or Mann-Whitney test will be used in the comparison of the two independent groups. More than two independent groups will be compared by the ANOVA or by the Kruskal–Wallis test. Two repeated measures in different assessments will be compared by the paired Student’s t-test or by the Wilcoxon’s signed-rank test. More than two repeated measures will be compared by the ANOVA for repeated measures, or by the Friedman’s test.

The identification of an optimal cutoff point for the marker associated with an outcome will be established through the analysis of the receiver operating characteristic (ROC) curve. The performance will be evaluated by the area under the ROC curve (AUC), and the significance of the AUC will be evaluated by the test that judges the null hypothesis that AUC is equal to 0.5. In addition to the significance test, the asymptotic confidence interval for the AUC will be obtained.

Analysis of the association between two quantitative variables will be visualized by scatter plots and quantified by the Pearson correlation coefficient, in the case of normal distributions, or by the Spearman’s correlation coefficient, if normal distribution is not verified in at least one of the variables. The significance of the correlation coefficient will be evaluated by the correlation coefficient test.

Linear or non-linear regression models will be proposed to explain the relationship between the clinical–radiological variables of the patient and the quantitative outcome variables (colonization and multiomic profile). For the variable colonization (binary), logistic regression models will be proposed. Regression model parameters will be estimated by the maximum likelihood method, and the forward Wald method will be used to choose the variables. The goodness of fit of the model will be analyzed by adherence statistics, residual analysis, and verification of theoretical assumptions of the model.

Supporting information

S1 Checklist. SPIRIT 2013 checklist: Recommended items to address in a clinical trial protocol and related documents*.

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Acknowledgments

The authors gratefully acknowledge the assistance of all undergraduate and graduate students involved in this study.

Data Availability

No datasets were generated or analysed during the current study. All relevant data from this study will be made available upon study completion.

Funding Statement

Yes. Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq), grant # 310875/2020-0. Fundação de Amparo à Pesquisado Estado do Rio de Janeiro (FAPERJ) grant # E-26/211.554/2019 (Programa Redede Pesquisa em Saúde) and Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior - Brasil (CAPES) - Finance Code 001.

References

1.Modic MT, Masaryk TJ, Ross JS, Carter JR. Imaging of degenerative disk disease. Radiology [Internet]. 1o de julho de 1988. [citado 25 de julho de 2021]; Disponível em: https://pubs.rsna.org/doi/abs/10.1148/radiology.168.1.3289089. [DOI] [PubMed] [Google Scholar]
2.Modic MT, Steinberg PM, Ross JS, Masaryk TJ, Carter JR. Degenerative disk disease: assessment of changes in vertebral body marrow with MR imaging. Radiology. 1o de janeiro de 1988;166(1):193–9. doi: 10.1148/radiology.166.1.3336678 [DOI] [PubMed] [Google Scholar]
3.Tang G, Chen Y, Chen J, Wang Z, Jiang W. Higher proportion of low-virulence anaerobic bacterial infection in young patients with intervertebral disc herniation. Exp Ther Med. 2019/10/02 ed outubro de 2019;18(4):3085–9. doi: 10.3892/etm.2019.7910 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Capoor MN, Lochman J, McDowell A, Schmitz JE, Solansky M, Zapletalova M, et al. Intervertebral disc penetration by antibiotics used prophylactically in spinal surgery: implications for the current standards and treatment of disc infections. Eur Spine J. 2018/12/07 ed abril de 2019;28(4):783–91. [DOI] [PubMed] [Google Scholar]
5.Capoor MN, Ruzicka F, Machackova T, Jancalek R, Smrcka M, Schmitz JE, et al. Prevalence of Propionibacterium acnes in Intervertebral Discs of Patients Undergoing Lumbar Microdiscectomy: A Prospective Cross-Sectional Study. PLoS One. 2016/08/19 ed 2016;11(8):e0161676. doi: 10.1371/journal.pone.0161676 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Capoor MN, Konieczna A, McDowell A, Ruzicka F, Smrcka M, Jancalek R, et al. Pro-Inflammatory and Neurotrophic Factor Responses of Cells Derived from Degenerative Human Intervertebral Discs to the Opportunistic Pathogen Cutibacterium acnes. Int J Mol Sci [Internet]. 2021/03/04 ed 26 de fevereiro de 2021;22(5). Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/33652921. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Capoor MN, Ruzicka F, Schmitz JE, James GA, Machackova T, Jancalek R, et al. Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy. PLoS One. 2017/04/04 ed 2017;12(4):e0174518. doi: 10.1371/journal.pone.0174518 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Schmid B, Hausmann O, Hitzl W, Achermann Y, Wuertz-Kozak K. The Role of Cutibacterium acnes in Intervertebral Disc Inflammation. Biomedicines [Internet]. 2020/07/08 ed 30 de junho de 2020;8(7). Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/32629986. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Nguyen HP, Zaroff JG, Bayman EO, Gelb AW, Todd MM, Hindman BJ, et al. Perioperative hypothermia (33 degrees C) does not increase the occurrence of cardiovascular events in patients undergoing cerebral aneurysm surgery: findings from the Intraoperative Hypothermia for Aneurysm Surgery Trial. Anesthesiology. agosto de 2010;113(2):327–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Li B, Dong Z, Wu Y, Zeng J, Zheng Q, Xiao B, et al. Association Between Lumbar Disc Degeneration and Propionibacterium acnes Infection: Clinical Research and Preliminary Exploration of Animal Experiment. Spine Phila Pa 1976. 2015/12/15 ed 1o de julho de 2016;41(13):E764–9. doi: 10.1097/BRS.0000000000001383 [DOI] [PubMed] [Google Scholar]
11.Yuan Y, Zhou Z, Jiao Y, Li C, Zheng Y, Lin Y, et al. Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs. Biomed Res Int. 2017/04/13 ed 2017;2017:6192935. doi: 10.1155/2017/6192935 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Zheng Y, Lin Y, Chen Z, Jiao Y, Yuan Y, Li C, et al. Propionibacterium acnes induces intervertebral discs degeneration by increasing MMP-1 and inhibiting TIMP-1 expression via the NF-kappaB pathway. Int J Clin Exp Pathol. 2018/07/01 ed 2018;11(7):3445–53. [PMC free article] [PubMed] [Google Scholar]
13.Zhou Z, Chen Z, Zheng Y, Cao P, Liang Y, Zhang X, et al. Relationship between annular tear and presence of Propionibacterium acnes in lumbar intervertebral disc. Eur Spine J. 2015/08/20 ed novembro de 2015;24(11):2496–502. doi: 10.1007/s00586-015-4180-y [DOI] [PubMed] [Google Scholar]
14.Wang Y, Che M, Xin J, Zheng Z, Li J, Zhang S. The role of IL-1beta and TNF-alpha in intervertebral disc degeneration. Biomed Pharmacother. 2020/08/28 ed novembro de 2020;131:110660. [DOI] [PubMed] [Google Scholar]
15.Albert HB, Sorensen JS, Christensen BS, Manniche C. Antibiotic treatment in patients with chronic low back pain and vertebral bone edema (Modic type 1 changes): a double-blind randomized clinical controlled trial of efficacy. Eur Spine J. abril de 2013;22(4):697–707. doi: 10.1007/s00586-013-2675-y [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Najafi S, Mahmoudi P, Bassampour SA, Shekarchi B, Soleimani M, Mohammadimehr M. Molecular detection of Propionibacterium acnes in biopsy samples of intervertebral disc with modic changes in patients undergoing herniated disc surgery. Iran J Microbiol. dezembro de 2020;12(6):516–21. doi: 10.18502/ijm.v12i6.5025 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Salehpour F, Aghazadeh J, Mirzaei F, Ziaeii E, Alavi SAN. Propionibacterium acnes Infection in Disc Material and Different Antibiotic Susceptibility in Patients With Lumbar Disc Herniation. Int J Spine Surg. 2019/05/28 ed abril de 2019;13(2):146–52. doi: 10.14444/6019 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Arndt J, Charles YP, Koebel C, Bogorin I, Steib JP. Bacteriology of degenerated lumbar intervertebral disks. J Spinal Disord Tech. 2012/07/27 ed outubro de 2012;25(7):E211–6. doi: 10.1097/BSD.0b013e318269851a [DOI] [PubMed] [Google Scholar]
19.Stirling A, Worthington T, Rafiq M, Lambert PA, Elliott TSJ. Association between sciatica and Propionibacterium acnes. The Lancet. 2001;357(9273):2024–5. doi: 10.1016/S0140-6736(00)05109-6 [DOI] [PubMed] [Google Scholar]
20.Jiao Y, Lin Y, Zheng Y, Yuan Y, Chen Z, Cao P. The bacteria-positive proportion in the disc tissue samples from surgery: a systematic review and meta-analysis. Eur Spine J. 2019/07/18 ed dezembro de 2019;28(12):2941–50. doi: 10.1007/s00586-019-06062-6 [DOI] [PubMed] [Google Scholar]
21.Rollason J, McDowell A, Albert HB, Barnard E, Worthington T, Hilton AC, et al. Genotypic and antimicrobial characterisation of Propionibacterium acnes isolates from surgically excised lumbar disc herniations. Biomed Res Int. 2013/09/26 ed 2013;2013:530382. doi: 10.1155/2013/530382 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Carricajo A, Nuti C, Aubert E, Hatem O, Fonsale N, Mallaval FO, et al. Propionibacterium acnes contamination in lumbar disc surgery. J Hosp Infect. julho de 2007;66(3):275–7. doi: 10.1016/j.jhin.2007.04.007 [DOI] [PubMed] [Google Scholar]
23.Rigal J, Thelen T, Byrne F, Cogniet A, Boissiere L, Aunoble S, et al. Prospective study using anterior approach did not show association between Modic 1 changes and low grade infection in lumbar spine. Eur Spine J. 2016/01/29 ed abril de 2016;25(4):1000–5. [DOI] [PubMed] [Google Scholar]
24.Urquhart DM, Zheng Y, Cheng AC, Rosenfeld JV, Chan P, Liew S, et al. Could low grade bacterial infection contribute to low back pain? A systematic review. BMC Med. 2015/01/23 ed 22 de janeiro de 2015;13:13. doi: 10.1186/s12916-015-0267-x [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Astur N, Martins DE, Wajchenberg M, Ferretti M, Menezes FG, Doi AM, et al. Subclinical Propionibacterium acnes infection estimation in the intervertebral disc (SPInE-ID): protocol for a prospective cohort. BMJ Open. 2017/11/21 ed 17 de novembro de 2017;7(11):e017930. doi: 10.1136/bmjopen-2017-017930 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Evangelopoulos DS, Stathopoulos IP, Morassi GP, Koufos S, Albarni A, Karampinas PK, et al. Sonication: A Valuable Technique for Diagnosis and Treatment of Periprosthetic Joint Infections. Sci World J. 10 de outubro de 2013;2013:e375140. doi: 10.1155/2013/375140 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Suarez S, Nassif X, Ferroni A. [Applications of MALDI-TOF technology in clinical microbiology]. Pathol Biol (Paris). fevereiro de 2015;63(1):43–52. [DOI] [PubMed] [Google Scholar]
28.Barnard E, Nagy I, Hunyadkurti J, Patrick S, McDowell A. Multiplex touchdown PCR for rapid typing of the opportunistic pathogen Propionibacterium acnes. J Clin Microbiol. 2015/01/30 ed abril de 2015;53(4):1149–55. doi: 10.1128/JCM.02460-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Rajasekaran S, Tangavel C, Aiyer SN, Nayagam SM, Raveendran M, Demonte NL, et al. ISSLS PRIZE IN CLINICAL SCIENCE 2017: Is infection the possible initiator of disc disease? An insight from proteomic analysis. Eur Spine J. 2017/02/09 ed maio de 2017;26(5):1384–400. doi: 10.1007/s00586-017-4972-3 [DOI] [PubMed] [Google Scholar]
30.Rinschen MM, Ivanisevic J, Giera M, Siuzdak G. Identification of bioactive metabolites using activity metabolomics. Nat Rev Mol Cell Biol. 2019/03/01 ed junho de 2019;20(6):353–67. doi: 10.1038/s41580-019-0108-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Swank KR, Furness JE, Baker EA, Gehrke CK, Biebelhausen SP, Baker KC. Metabolomic Profiling in the Characterization of Degenerative Bone and Joint Diseases. Metabolites [Internet]. 2020/06/04 ed 29 de maio de 2020;10(6). Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/32485832. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Ou J, Xiao M, Huang Y, Tu L, Chen Z, Cao S, et al. Serum Metabolomics Signatures Associated With Ankylosing Spondylitis and TNF Inhibitor Therapy. Front Immunol. 2021/03/09 ed 2021;12:630791. doi: 10.3389/fimmu.2021.630791 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Fernandez-Garcia M, Rojo D, Rey-Stolle F, Garcia A, Barbas C. Metabolomic-Based Methods in Diagnosis and Monitoring Infection Progression. Exp Suppl. 2018/12/12 ed 2018;109:283–315. doi: 10.1007/978-3-319-74932-7_7 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Johnson CH, Ivanisevic J, Siuzdak G. Metabolomics: beyond biomarkers and towards mechanisms. Nat Rev Mol Cell Biol. 2016/03/17 ed julho de 2016;17(7):451–9. doi: 10.1038/nrm.2016.25 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Shan L, Yang J, Meng S, Ruan H, Zhou L, Ye F, et al. Urine Metabolomics Profiling of Lumbar Disc Herniation and its Traditional Chinese Medicine Subtypes in Patients Through Gas Chromatography Coupled With Mass Spectrometry. Front Mol Biosci. 2021/06/29 ed 2021;8:648823. doi: 10.3389/fmolb.2021.648823 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Teckchandani S, Nagana Gowda GA, Raftery D, Curatolo M. Metabolomics in chronic pain research. Eur J Pain. 2020/10/17 ed fevereiro de 2021;25(2):313–26. doi: 10.1002/ejp.1677 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Liu M, Xie Z, Costello CA, Zhang W, Chen L, Qi D, et al. Metabolomic analysis coupled with extreme phenotype sampling identified that lysophosphatidylcholines are associated with multisite musculoskeletal pain. Pain. 2020/08/25 ed 1o de fevereiro de 2021;162(2):600–8. doi: 10.1097/j.pain.0000000000002052 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Fujita N, Ishihara S, Michikawa T, Azuma K, Suzuki S, Tsuji O, et al. Potential association of metabolic and musculoskeletal disorders with lumbar intervertebral disc degeneration: Cross-sectional study using medical checkup data. J Orthop Sci. 2019/06/10 ed maio de 2020;25(3):384–8. doi: 10.1016/j.jos.2019.05.011 [DOI] [PubMed] [Google Scholar]
39.Aroke EN, Powell-Roach KL. The Metabolomics of Chronic Pain Conditions: A Systematic Review. Biol Res Nurs. 2020/07/16 ed outubro de 2020;22(4):458–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Ji RR, Donnelly CR, Nedergaard M. Astrocytes in chronic pain and itch. Nat Rev Neurosci. 2019/09/21 ed novembro de 2019;20(11):667–85. doi: 10.1038/s41583-019-0218-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Wu X, Xiang H, Zhang G, Hu Y, wang yan, Chen B. A metabolic profiling analysis of degenerative intervertebral disc disease in a rabbit model via GC/TOF-MS. 2019; Disponível em: https://www.researchsquare.com/article/rs-7971/v1. [Google Scholar]
42.Radek M, Pacholczyk-Sienicka B, Jankowski S, Albrecht L, Grodzka M, Depta A, et al. Assessing the correlation between the degree of disc degeneration on the Pfirrmann scale and the metabolites identified in HR-MAS NMR spectroscopy. Magn Reson Imaging. 2015/12/29 ed maio de 2016;34(4):376–80. doi: 10.1016/j.mri.2015.12.005 [DOI] [PubMed] [Google Scholar]
43.Teraguchi M, Yoshimura N, Hashizume H, Muraki S, Yamada H, Oka H, et al. Metabolic Syndrome Components Are Associated with Intervertebral Disc Degeneration: The Wakayama Spine Study. PLoS One. 2016/02/04 ed 2016;11(2):e0147565. doi: 10.1371/journal.pone.0147565 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Veras MA, Lim YJ, Kuljanin M, Lajoie GA, Urquhart BL, Seguin CA. Protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc tissues. JOR Spine. 2020/10/06 ed setembro de 2020;3(3):e1099. doi: 10.1002/jsp2.1099 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Tang G, Han X, Lin Z, Qian H, Chen B, Zhou C, et al. Propionibacterium acnes Accelerates Intervertebral Disc Degeneration by Inducing Pyroptosis of Nucleus Pulposus Cells via the ROS-NLRP3 Pathway. Oxid Med Cell Longev. 2021/02/20 ed 2021;2021:4657014. doi: 10.1155/2021/4657014 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Lin Y, Jiao Y, Yuan Y, Zhou Z, Zheng Y, Xiao J, et al. Propionibacterium acnes induces intervertebral disc degeneration by promoting nucleus pulposus cell apoptosis via the TLR2/JNK/mitochondrial-mediated pathway. Emerg Microbes Infect. 2018/01/13 ed 10 de janeiro de 2018;7(1):1. doi: 10.1038/s41426-017-0002-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Capoor MN, Ruzicka F, Sandhu G, Rollason J, Mavrommatis K, Ahmed FS, et al. Importance of Propionibacterium acnes hemolytic activity in human intervertebral discs: A microbiological study. PLoS One. 2018/11/30 ed 2018;13(11):e0208144. doi: 10.1371/journal.pone.0208144 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Albert HB, Kjaer P, Jensen TS, Sorensen JS, Bendix T, Manniche C. Modic changes, possible causes and relation to low back pain. Med Hypotheses. 2008;70(2):361–8. doi: 10.1016/j.mehy.2007.05.014 [DOI] [PubMed] [Google Scholar]
49.Ohtori S, Koshi T, Yamashita M, Yamauchi K, Inoue G, Suzuki M, et al. Existence of pyogenic spondylitis in Modic type 1 change without other signs of infection: 2-year follow-up. Eur Spine J. 2010/03/10 ed julho de 2010;19(7):1200–5. doi: 10.1007/s00586-010-1358-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Ritter PL, González VM, Laurent DD, Lorig KR. Measurement of pain using the visual numeric scale. J Rheumatol. março de 2006;33(3):574–80. [PubMed] [Google Scholar]
51.Breivik H, Borchgrevink PC, Allen SM, Rosseland LA, Romundstad L, Hals EKB, et al. Assessment of pain. Br J Anaesth. julho de 2008;101(1):17–24. doi: 10.1093/bja/aen103 [DOI] [PubMed] [Google Scholar]
52.Bagattini ÂM, Camey SA, Miguel SR, Andrade MV, de Souza Noronha KVM, de C. Teixeira MA, et al. Electronic Version of the EQ-5D Quality-of-Life Questionnaire: Adaptation to a Brazilian Population Sample. Value Health Reg Issues. 1o de dezembro de 2018;17:88–93. doi: 10.1016/j.vhri.2017.11.002 [DOI] [PubMed] [Google Scholar]
53.Vigatto R, Alexandre NM, Correa Filho HR. Development of a Brazilian Portuguese version of the Oswestry Disability Index: cross-cultural adaptation, reliability, and validity. Spine Phila Pa 1976. 2007/02/17 ed 15 de fevereiro de 2007;32(4):481–6. doi: 10.1097/01.brs.0000255075.11496.47 [DOI] [PubMed] [Google Scholar]
54.Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinforma Oxf Engl. 1o de agosto de 2014;30(15):2114–20. doi: 10.1093/bioinformatics/btu170 [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Lohse M, Bolger AM, Nagel A, Fernie AR, Lunn JE, Stitt M, et al. RobiNA: a user-friendly, integrated software solution for RNA-Seq-based transcriptomics. Nucleic Acids Res. julho de 2012;40 (Web Server issue):W622–627. [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Shah H, Encheva V, Schmid O, Culak RA, Nasir P, Ines I, et al. Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS): A Potentially Powerful Tool for Rapid Characterisation of Microorganisms. In 2005. p. 57–95. [Google Scholar]
57.Heberle H, Meirelles GV, da Silva FR, Telles GP, Minghim R. InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams. BMC Bioinformatics. 22 de maio de 2015;16(1):169. doi: 10.1186/s12859-015-0611-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Urrutia J, Besa P, Campos M, Cikutovic P, Cabezon M, Molina M, et al. The Pfirrmann classification of lumbar intervertebral disc degeneration: an independent inter- and intra-observer agreement assessment. Eur Spine J. 2016/02/18 ed setembro de 2016;25(9):2728–33. doi: 10.1007/s00586-016-4438-z [DOI] [PubMed] [Google Scholar]
59.Medronho R, Bloch K, Luiz R, Werneck G. Epidemiologia. Atheneu. São Paulo. 2009. [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0271773.r001

Decision Letter 0

D William Cameron

7 Sep 2022

PONE-D-22-14386Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniquesPLOS ONE

Dear Dr. Ferreira,

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.

Please see my comments and the reviewers' suggestions below. Minor revisions to this very good protocol would strengthen the proposed study further. Looking forward to seeing this work proceed.

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We look forward to receiving your revised manuscript.

Kind regards,

D. William Cameron, MD

Academic Editor

PLOS ONE

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The authors gratefully acknowledge the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 for supporting this study.

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Additional Editor Comments:

This is a well-written, well informed and well thought out protocol for a timely, and perhaps overdue study of correlation of C. acnes presence, and lumbago due to disc disease, at the point of first surgery. The reviews are well done and should be paid attention to by the authors, as their suggestions will improve the robustness of their findings.

In reading the MS, the authors should remove one double-negative in phrasing, and replace the word incidence with prevalence.

The inclusion of a SPIRIT checklist is appreciated; as this is an observational study, the authors might have used the STROBE reportage guidelines and checklist.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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

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 #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

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Reviewer #1: The central question/controversy in this area concerns the conclusion that any organisms recovered from excised tissues by culture result from skin contamination during sampling. The approach proposed to counter this argument is that proteomic or metabolomic signatures obtained from the tissues might indicate infection, as has been concluded from cited studies. Since no control tissues samples are involved in the study (for example analogous tissues from other surgical sites) reliance is placed on effective skin disinfection prior to surgery. However, viable organisms can remain deep within the skin following pre-operative surgical preparation and these could result in false positive cultures, particularly using the enrichment culture approach in broth before plating. To address this problem, the study protocol proposes swabbing the skin after preparation and before excision. The swab is plated directly onto enriched blood agar without treatment with a neutralizing solution. This omission could give a false idea of the efficacy of skin preparation since residual, active chlorhexidine will be transferred from the skin to the swab and onto the plates. A suitable neutralizer solution such as Dey-Engley broth should be included in the swab transfer tube. Even with addition of this step, there remains concern that skin surface swabs will not pick up viable organisms residing deeper within the skin after preparation and before excision.

PCR methods will be used only to identify and characterize organisms recovered by enrichment culture, no attempt being made to amplify microbial DNA directly from tissues without culture. Whilst this approach will suffer the same problem of possible skin contamination it should at least be considered since it could give some idea of levels of microbial DNA present in the tissues (not possible using the tissue enrichment culture approach).

Reviewer #2: I have read your protocol with great interest, which has the potential to provide an important insights into low virulence infections in lumbar intervertebral discs, and have following optional suggestions based on my experience:

1. Homogenization: Our previous study based on fluorescence in situ hybridization confocal scanning laser microscopy has shown that P. acnes is present deep within intervertebral disc tissue as a biofilm and, as a consequence, it is important that the biofilm is disrupted prior to culture to maximize detection and reduce the possibility of a false-negative result (Capoor 2017). With regard to biofilm disassembly, homogenization has demonstrated the ability to disrupt biofilm deep within the tissue, while sonication has shown demonstrated ability to disrupt biofilm on the surface of implants.

2. Sample size: Two meta-analyses of previous studies addressing infection of intervertebral discs reported a pooled prevalence of bacteria at 34% and 36.2%, respectively with P. acnes as the predominant species (Urquhart 2015, Ganko 2015). An appropriate sample size estimation should therefore be calculated based upon these prevalence rates [Sample size = (1.96^2 x PR (1-PR))/0.05^2); note PR = prevalence rate] (Naing 2006). To ensure that the 95% confidence interval estimate of the proportion positive cases is within 5% of the true proportion, a sample size of approximately 350 cases is necessary.

3. CFU/Gram: Disc fragments for culture should be weighed, placed into a Micro Bag (Seward) containing 4 mL of Viande-Levure medium, and homogenized with a Stomacher 80 (Seward) under aseptic conditions. 100 µL of the resultant homogenate should be used to inoculate Wilkins Chalgren Anaerobic Agar with 7% sheep’s blood and vitamin K (Hi Media Laboratories). An Anaerobic Work Station Concept 400 (Ruskinn Technology) should be used for culture; inoculated plates will be incubated for 14 days at 37 °C under an atmosphere of 80% N2, 10% CO2, and 10% H2. The same amount of the homogenate should be cultured aerobically on Columbia Blood Agar (Oxoid) for 7 days at 37°C to detect aerobic bacteria. Following incubation, bacterial colonies should be counted and the quantity of each colonial morphotype will be expressed as CFU/g of tissue using the Miles and Misra method.

4. qPCR verification of the presence of P. acnes. The human β-globin gene should be included as an internal control to allow assessment of the specimen quality and the nucleic acid extraction as well as the inhibition amplification process. Our experience shows that found that significant number of samples are eliminated due to:

a) inadequate DNA concentration (outside of range 2-60 ng/µL)

b) inadequate human DNA control (Ct of human β-globin gene outside of range 21-27)

c) inconclusive P. acnes template Ct values (32-35)

Reviewer #3: Lines 30, 121, 135: correlate refers to a particular type of analysis. Is this intended (only appropriate for 2 continuous variables), or would “link” or similar be a more appropriate word?

Line 470+ will baseline measures for patient reported outcomes be included in models for future time points?

**********

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Reviewer #1: No

Reviewer #2: Yes: Manu Capoor

Reviewer #3: No

**********

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PLoS One. 2023 Feb 27;18(2):e0271773. doi: 10.1371/journal.pone.0271773.r002

Author response to Decision Letter 0

19 Oct 2022

Dear Plos One Academic Editor D. William Cameron,

Concerning the manuscript that was recently submitted to your journal as a protocol and untitled “Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques”, we carefully considered all comments/suggestions/corrections, provided by you and the referee, so the manuscript could fit all reviewers.

Herein, we explain how the manuscript was revised based on those comments and recommendations. We extend our appreciation for taking the time and effort necessary to provide such insightful guidance.

Below are the answers based on the journal requirements:

-Request

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

Answer: We apologize for the inconvenience. We did check again all the manuscript’s style so it can meet Plos One style and all the changes were made in the file named marked up copy.

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2. 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.

Answer: We have changed the Funding information and Financial Disclosure with all the information required. The grant number were also included in the manuscript.

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

Answer: We have removed the funding support details from the manuscript in the acknowledgment section and included our amended statement in the cover letter as it follows: The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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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 and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Answer: We have included our amended statement within our cover letter.

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4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

Answer: We have removed from the manuscript, besides from Methods the ethics statements (Line 145)

- Request

5. Please include a caption for figure 1.

Answer: We have included a caption for the figure (Line 256)

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Answer: We have included caption with the supporting information file.

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8. We note that the original protocol file you uploaded contains a confidentiality notice indicating that the protocol may not be shared publicly or be published. Please note, however, that the PLOS Editorial Policy requires that the original protocol be published alongside your manuscript in the event of acceptance. Please note that should your paper be accepted, all content including the protocol will be published under the Creative Commons Attribution (CC BY) 4.0 license, which means that it will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution.Therefore, we ask that you please seek permission from the study sponsor or body imposing the restriction on sharing this document to publish this protocol under CC BY 4.0 if your work is accepted. We kindly ask that you upload a formal statement signed by an institutional representative clarifying whether you will be able to comply with this policy. Additionally, please upload a clean copy of the protocol with the confidentiality notice (and any copyrighted institutional logos or signatures) removed.

Answer: The file we have provided and uploaded as a supporting file contains all the details concerning the approval of our study in the Brazilian ethics committee. This pdf file is provided by the website where all the documentation was sent and the study approved, named “Plataforma Brasil”. When the document/receipt is downloaded it comes with the logo from the website. This document that we are sending to the Plos One Journal contains some details of the research, such as the approval number, title and researcher who had the research approved, and where it will be conducted. Unfortunately, the logo can not be removed.. We have provided a caption together with this document in the supplement file.

- Request

Answer:

We have reviewed the reference list of the manuscript to ensure that it is complete and correct.

Additional Editor Comments:

- Request

In reading the MS, the authors should remove one double-negative in phrasing, and replace the word incidence with prevalence.

Answer:

The double negative was removed from the phrasing (line 487). The word incidence was replaced in the text by prevalence.

- Request

The inclusion of a SPIRIT checklist is appreciated; as this is an observational study, the authors might have used the STROBE reportage guidelines and checklist.

Answer: During the article submission a SPIRIT checklist was included.

Concerning the reviewer’s team collective input, we considered all suggestions/correction as it follows:

1- Endeavored all comments, suggestions and corrections appointed by the referees;

2- Clarified portions of the text, including introduction, material and methods, results and discussion;

3- Corrected any grammar mistakes found along the text together with miswritten sentences.

# Reviewer 1:

Answer: Thank you very much for your comments raised concerning the specimen collection. In addition to the tissue collected at each surgical level (five fragments) for the bacterial isolation, another five fragments from the circumjacent tissue are also collected and used as negative control. This sentence will be included in the methodology section (lines 370-371). Concerning the skin disinfection prior surgery with chlorhexidine, we wanted to ensure that the disinfection before pre-operative procedure was effective and that the C. acnes isolated in the intervertebral tissue was not from skin contamination. In fact, previous studies used the same procedure for cleaning the patient’s skin and some were positive and while others were negative. That’s the reason why we decided to use the same protocol.

- PCR methods will be used only to identify and characterize organisms recovered by enrichment culture, no attempt being made to amplify microbial DNA directly from tissues without culture. Whilst this approach will suffer the same problem of possible skin contamination it should at least be considered since it could give some idea of levels of microbial DNA present in the tissues (not possible using the tissue enrichment culture approach).

Answer: We appreciate your comment, but we do not have a species-specific PCR that can be used to identify C. acnes directly from the skin. And even if we did, there are other species with genomes similar to C. acnes, such as the C. namnetense.

Answer: Thank you so much for your appreciation. We are totally in agreement. That’s why after the samples are collected, they put in a vial containing thioglycolate broth and with glass beads (approximately 20 beads). When the tubes arrive in the laboratory, they are vortexed vigorously so the tissue can be disrupted (Lines 380-381).

Answer: The number of cases was calculated based on the number of surgeries performed at the public hospital where the study will be conducted. This number, according to our calculations, will be 100. As a result, we would most likely have about 400 samples including the swabs from the skin.

Answer: We appreciate the description of the protocol, but as a public hospital we do not have a MicroBag or the Viandre-Levure medium. Instead, after collecting all five samples, including controls into thioglycolate with glass beads, tubes will be kept under them room temperature, until they arrive in the microbiology laboratory at the Federal University of Rio de Janeiro (UFRJ) for anaerobic culture. Prior to the first inoculation in blood agar plates, all tubes are vortexed. Plates are incubated under capnophilic and anaerobic conditions for 14 days or until culture growth occurs; tubes are also kept incubated for 14 days or until culture growth occurs. The capnophilic plate is used as a control for contaminants (aerobic bacteria). We will not quantify it, despite the fact that we identify all microorganism grown on plates.

a) inadequate DNA concentration (outside of range 2-60 ng/µL)

b) inadequate human DNA control (Ct of human β-globin gene outside of range 21-27)

c) inconclusive P. acnes template Ct values (32-35)

Answer: Thank you so much for your advice. We considered it during the protocol, but unfortunately, we do not have this goal for the verification of the presence of C. acnes at this time. However, in the future, we plan to conduct several analyses with the C. acnes isolates, one of which will include qPCR for some virulence genes.

Answer: Thank you so much for your observation. We think that link or associate would be a better word, because we want to correlate the presence of C. acnes with the patient’s condition and intervertebral discs problems. We have changed the word correlate to associate in lines 29, 122, 133.

Line 470+ will baseline measures for patient reported outcomes be included in models for future time points?

Answer: We appreciate the question. Yes, they probably will.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(28.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0271773.r003

Decision Letter 1

D William Cameron

17 Nov 2022

Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques

PONE-D-22-14386R1

Dear Dr. Ferreira,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

D. William Cameron, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The issue of examining "control" tissues has been explained and dealt with satisfactorily.

The thioglycolate broths used for the skin swabs should neutralize any carry-over of chlorhexidine. Primary swabs onto the blood plates may still give false negatives but I note that this has been standard procedure in previous studies.

Reviewer #2: I read reviewer comments and your corrections and believe you did a reasonable and thoughtful job addressing these comments.

Reviewer #3: It would be useful to mention in this text that the baseline measures in the stats analysis section, or at least state a statistician will specify the analyses in a statistical analysis plan.

**********

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 #2: No

Reviewer #3: No

**********

PLoS One. doi: 10.1371/journal.pone.0271773.r004

Acceptance letter

D William Cameron

13 Dec 2022

PONE-D-22-14386R1

Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques

Dear Dr. Ferreira:

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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor D. William Cameron

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Checklist. SPIRIT 2013 checklist: Recommended items to address in a clinical trial protocol and related documents*.

(PDF)

Click here for additional data file.^{(221.2KB, pdf)}

S1 File

(PDF)

Click here for additional data file.^{(77.4KB, pdf)}

S2 File

(PDF)

Click here for additional data file.^{(1.3MB, pdf)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(28.9KB, docx)}

Data Availability Statement

No datasets were generated or analysed during the current study. All relevant data from this study will be made available upon study completion.

[pone.0271773.ref001] 1.Modic MT, Masaryk TJ, Ross JS, Carter JR. Imaging of degenerative disk disease. Radiology [Internet]. 1o de julho de 1988. [citado 25 de julho de 2021]; Disponível em: https://pubs.rsna.org/doi/abs/10.1148/radiology.168.1.3289089. [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref002] 2.Modic MT, Steinberg PM, Ross JS, Masaryk TJ, Carter JR. Degenerative disk disease: assessment of changes in vertebral body marrow with MR imaging. Radiology. 1o de janeiro de 1988;166(1):193–9. doi: 10.1148/radiology.166.1.3336678 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref003] 3.Tang G, Chen Y, Chen J, Wang Z, Jiang W. Higher proportion of low-virulence anaerobic bacterial infection in young patients with intervertebral disc herniation. Exp Ther Med. 2019/10/02 ed outubro de 2019;18(4):3085–9. doi: 10.3892/etm.2019.7910 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref004] 4.Capoor MN, Lochman J, McDowell A, Schmitz JE, Solansky M, Zapletalova M, et al. Intervertebral disc penetration by antibiotics used prophylactically in spinal surgery: implications for the current standards and treatment of disc infections. Eur Spine J. 2018/12/07 ed abril de 2019;28(4):783–91. [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref005] 5.Capoor MN, Ruzicka F, Machackova T, Jancalek R, Smrcka M, Schmitz JE, et al. Prevalence of Propionibacterium acnes in Intervertebral Discs of Patients Undergoing Lumbar Microdiscectomy: A Prospective Cross-Sectional Study. PLoS One. 2016/08/19 ed 2016;11(8):e0161676. doi: 10.1371/journal.pone.0161676 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref006] 6.Capoor MN, Konieczna A, McDowell A, Ruzicka F, Smrcka M, Jancalek R, et al. Pro-Inflammatory and Neurotrophic Factor Responses of Cells Derived from Degenerative Human Intervertebral Discs to the Opportunistic Pathogen Cutibacterium acnes. Int J Mol Sci [Internet]. 2021/03/04 ed 26 de fevereiro de 2021;22(5). Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/33652921. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref007] 7.Capoor MN, Ruzicka F, Schmitz JE, James GA, Machackova T, Jancalek R, et al. Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy. PLoS One. 2017/04/04 ed 2017;12(4):e0174518. doi: 10.1371/journal.pone.0174518 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref008] 8.Schmid B, Hausmann O, Hitzl W, Achermann Y, Wuertz-Kozak K. The Role of Cutibacterium acnes in Intervertebral Disc Inflammation. Biomedicines [Internet]. 2020/07/08 ed 30 de junho de 2020;8(7). Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/32629986. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref009] 9.Nguyen HP, Zaroff JG, Bayman EO, Gelb AW, Todd MM, Hindman BJ, et al. Perioperative hypothermia (33 degrees C) does not increase the occurrence of cardiovascular events in patients undergoing cerebral aneurysm surgery: findings from the Intraoperative Hypothermia for Aneurysm Surgery Trial. Anesthesiology. agosto de 2010;113(2):327–42. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref010] 10.Li B, Dong Z, Wu Y, Zeng J, Zheng Q, Xiao B, et al. Association Between Lumbar Disc Degeneration and Propionibacterium acnes Infection: Clinical Research and Preliminary Exploration of Animal Experiment. Spine Phila Pa 1976. 2015/12/15 ed 1o de julho de 2016;41(13):E764–9. doi: 10.1097/BRS.0000000000001383 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref011] 11.Yuan Y, Zhou Z, Jiao Y, Li C, Zheng Y, Lin Y, et al. Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs. Biomed Res Int. 2017/04/13 ed 2017;2017:6192935. doi: 10.1155/2017/6192935 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref012] 12.Zheng Y, Lin Y, Chen Z, Jiao Y, Yuan Y, Li C, et al. Propionibacterium acnes induces intervertebral discs degeneration by increasing MMP-1 and inhibiting TIMP-1 expression via the NF-kappaB pathway. Int J Clin Exp Pathol. 2018/07/01 ed 2018;11(7):3445–53. [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref013] 13.Zhou Z, Chen Z, Zheng Y, Cao P, Liang Y, Zhang X, et al. Relationship between annular tear and presence of Propionibacterium acnes in lumbar intervertebral disc. Eur Spine J. 2015/08/20 ed novembro de 2015;24(11):2496–502. doi: 10.1007/s00586-015-4180-y [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref014] 14.Wang Y, Che M, Xin J, Zheng Z, Li J, Zhang S. The role of IL-1beta and TNF-alpha in intervertebral disc degeneration. Biomed Pharmacother. 2020/08/28 ed novembro de 2020;131:110660. [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref015] 15.Albert HB, Sorensen JS, Christensen BS, Manniche C. Antibiotic treatment in patients with chronic low back pain and vertebral bone edema (Modic type 1 changes): a double-blind randomized clinical controlled trial of efficacy. Eur Spine J. abril de 2013;22(4):697–707. doi: 10.1007/s00586-013-2675-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref016] 16.Najafi S, Mahmoudi P, Bassampour SA, Shekarchi B, Soleimani M, Mohammadimehr M. Molecular detection of Propionibacterium acnes in biopsy samples of intervertebral disc with modic changes in patients undergoing herniated disc surgery. Iran J Microbiol. dezembro de 2020;12(6):516–21. doi: 10.18502/ijm.v12i6.5025 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref017] 17.Salehpour F, Aghazadeh J, Mirzaei F, Ziaeii E, Alavi SAN. Propionibacterium acnes Infection in Disc Material and Different Antibiotic Susceptibility in Patients With Lumbar Disc Herniation. Int J Spine Surg. 2019/05/28 ed abril de 2019;13(2):146–52. doi: 10.14444/6019 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref018] 18.Arndt J, Charles YP, Koebel C, Bogorin I, Steib JP. Bacteriology of degenerated lumbar intervertebral disks. J Spinal Disord Tech. 2012/07/27 ed outubro de 2012;25(7):E211–6. doi: 10.1097/BSD.0b013e318269851a [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref019] 19.Stirling A, Worthington T, Rafiq M, Lambert PA, Elliott TSJ. Association between sciatica and Propionibacterium acnes. The Lancet. 2001;357(9273):2024–5. doi: 10.1016/S0140-6736(00)05109-6 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref020] 20.Jiao Y, Lin Y, Zheng Y, Yuan Y, Chen Z, Cao P. The bacteria-positive proportion in the disc tissue samples from surgery: a systematic review and meta-analysis. Eur Spine J. 2019/07/18 ed dezembro de 2019;28(12):2941–50. doi: 10.1007/s00586-019-06062-6 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref021] 21.Rollason J, McDowell A, Albert HB, Barnard E, Worthington T, Hilton AC, et al. Genotypic and antimicrobial characterisation of Propionibacterium acnes isolates from surgically excised lumbar disc herniations. Biomed Res Int. 2013/09/26 ed 2013;2013:530382. doi: 10.1155/2013/530382 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref022] 22.Carricajo A, Nuti C, Aubert E, Hatem O, Fonsale N, Mallaval FO, et al. Propionibacterium acnes contamination in lumbar disc surgery. J Hosp Infect. julho de 2007;66(3):275–7. doi: 10.1016/j.jhin.2007.04.007 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref023] 23.Rigal J, Thelen T, Byrne F, Cogniet A, Boissiere L, Aunoble S, et al. Prospective study using anterior approach did not show association between Modic 1 changes and low grade infection in lumbar spine. Eur Spine J. 2016/01/29 ed abril de 2016;25(4):1000–5. [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref024] 24.Urquhart DM, Zheng Y, Cheng AC, Rosenfeld JV, Chan P, Liew S, et al. Could low grade bacterial infection contribute to low back pain? A systematic review. BMC Med. 2015/01/23 ed 22 de janeiro de 2015;13:13. doi: 10.1186/s12916-015-0267-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref025] 25.Astur N, Martins DE, Wajchenberg M, Ferretti M, Menezes FG, Doi AM, et al. Subclinical Propionibacterium acnes infection estimation in the intervertebral disc (SPInE-ID): protocol for a prospective cohort. BMJ Open. 2017/11/21 ed 17 de novembro de 2017;7(11):e017930. doi: 10.1136/bmjopen-2017-017930 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref026] 26.Evangelopoulos DS, Stathopoulos IP, Morassi GP, Koufos S, Albarni A, Karampinas PK, et al. Sonication: A Valuable Technique for Diagnosis and Treatment of Periprosthetic Joint Infections. Sci World J. 10 de outubro de 2013;2013:e375140. doi: 10.1155/2013/375140 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref027] 27.Suarez S, Nassif X, Ferroni A. [Applications of MALDI-TOF technology in clinical microbiology]. Pathol Biol (Paris). fevereiro de 2015;63(1):43–52. [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref028] 28.Barnard E, Nagy I, Hunyadkurti J, Patrick S, McDowell A. Multiplex touchdown PCR for rapid typing of the opportunistic pathogen Propionibacterium acnes. J Clin Microbiol. 2015/01/30 ed abril de 2015;53(4):1149–55. doi: 10.1128/JCM.02460-14 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref029] 29.Rajasekaran S, Tangavel C, Aiyer SN, Nayagam SM, Raveendran M, Demonte NL, et al. ISSLS PRIZE IN CLINICAL SCIENCE 2017: Is infection the possible initiator of disc disease? An insight from proteomic analysis. Eur Spine J. 2017/02/09 ed maio de 2017;26(5):1384–400. doi: 10.1007/s00586-017-4972-3 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref030] 30.Rinschen MM, Ivanisevic J, Giera M, Siuzdak G. Identification of bioactive metabolites using activity metabolomics. Nat Rev Mol Cell Biol. 2019/03/01 ed junho de 2019;20(6):353–67. doi: 10.1038/s41580-019-0108-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref031] 31.Swank KR, Furness JE, Baker EA, Gehrke CK, Biebelhausen SP, Baker KC. Metabolomic Profiling in the Characterization of Degenerative Bone and Joint Diseases. Metabolites [Internet]. 2020/06/04 ed 29 de maio de 2020;10(6). Disponível em: https://www.ncbi.nlm.nih.gov/pubmed/32485832. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref032] 32.Ou J, Xiao M, Huang Y, Tu L, Chen Z, Cao S, et al. Serum Metabolomics Signatures Associated With Ankylosing Spondylitis and TNF Inhibitor Therapy. Front Immunol. 2021/03/09 ed 2021;12:630791. doi: 10.3389/fimmu.2021.630791 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref033] 33.Fernandez-Garcia M, Rojo D, Rey-Stolle F, Garcia A, Barbas C. Metabolomic-Based Methods in Diagnosis and Monitoring Infection Progression. Exp Suppl. 2018/12/12 ed 2018;109:283–315. doi: 10.1007/978-3-319-74932-7_7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref034] 34.Johnson CH, Ivanisevic J, Siuzdak G. Metabolomics: beyond biomarkers and towards mechanisms. Nat Rev Mol Cell Biol. 2016/03/17 ed julho de 2016;17(7):451–9. doi: 10.1038/nrm.2016.25 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref035] 35.Shan L, Yang J, Meng S, Ruan H, Zhou L, Ye F, et al. Urine Metabolomics Profiling of Lumbar Disc Herniation and its Traditional Chinese Medicine Subtypes in Patients Through Gas Chromatography Coupled With Mass Spectrometry. Front Mol Biosci. 2021/06/29 ed 2021;8:648823. doi: 10.3389/fmolb.2021.648823 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref036] 36.Teckchandani S, Nagana Gowda GA, Raftery D, Curatolo M. Metabolomics in chronic pain research. Eur J Pain. 2020/10/17 ed fevereiro de 2021;25(2):313–26. doi: 10.1002/ejp.1677 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref037] 37.Liu M, Xie Z, Costello CA, Zhang W, Chen L, Qi D, et al. Metabolomic analysis coupled with extreme phenotype sampling identified that lysophosphatidylcholines are associated with multisite musculoskeletal pain. Pain. 2020/08/25 ed 1o de fevereiro de 2021;162(2):600–8. doi: 10.1097/j.pain.0000000000002052 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref038] 38.Fujita N, Ishihara S, Michikawa T, Azuma K, Suzuki S, Tsuji O, et al. Potential association of metabolic and musculoskeletal disorders with lumbar intervertebral disc degeneration: Cross-sectional study using medical checkup data. J Orthop Sci. 2019/06/10 ed maio de 2020;25(3):384–8. doi: 10.1016/j.jos.2019.05.011 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref039] 39.Aroke EN, Powell-Roach KL. The Metabolomics of Chronic Pain Conditions: A Systematic Review. Biol Res Nurs. 2020/07/16 ed outubro de 2020;22(4):458–71. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref040] 40.Ji RR, Donnelly CR, Nedergaard M. Astrocytes in chronic pain and itch. Nat Rev Neurosci. 2019/09/21 ed novembro de 2019;20(11):667–85. doi: 10.1038/s41583-019-0218-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref041] 41.Wu X, Xiang H, Zhang G, Hu Y, wang yan, Chen B. A metabolic profiling analysis of degenerative intervertebral disc disease in a rabbit model via GC/TOF-MS. 2019; Disponível em: https://www.researchsquare.com/article/rs-7971/v1. [Google Scholar]

[pone.0271773.ref042] 42.Radek M, Pacholczyk-Sienicka B, Jankowski S, Albrecht L, Grodzka M, Depta A, et al. Assessing the correlation between the degree of disc degeneration on the Pfirrmann scale and the metabolites identified in HR-MAS NMR spectroscopy. Magn Reson Imaging. 2015/12/29 ed maio de 2016;34(4):376–80. doi: 10.1016/j.mri.2015.12.005 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref043] 43.Teraguchi M, Yoshimura N, Hashizume H, Muraki S, Yamada H, Oka H, et al. Metabolic Syndrome Components Are Associated with Intervertebral Disc Degeneration: The Wakayama Spine Study. PLoS One. 2016/02/04 ed 2016;11(2):e0147565. doi: 10.1371/journal.pone.0147565 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref044] 44.Veras MA, Lim YJ, Kuljanin M, Lajoie GA, Urquhart BL, Seguin CA. Protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc tissues. JOR Spine. 2020/10/06 ed setembro de 2020;3(3):e1099. doi: 10.1002/jsp2.1099 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref045] 45.Tang G, Han X, Lin Z, Qian H, Chen B, Zhou C, et al. Propionibacterium acnes Accelerates Intervertebral Disc Degeneration by Inducing Pyroptosis of Nucleus Pulposus Cells via the ROS-NLRP3 Pathway. Oxid Med Cell Longev. 2021/02/20 ed 2021;2021:4657014. doi: 10.1155/2021/4657014 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref046] 46.Lin Y, Jiao Y, Yuan Y, Zhou Z, Zheng Y, Xiao J, et al. Propionibacterium acnes induces intervertebral disc degeneration by promoting nucleus pulposus cell apoptosis via the TLR2/JNK/mitochondrial-mediated pathway. Emerg Microbes Infect. 2018/01/13 ed 10 de janeiro de 2018;7(1):1. doi: 10.1038/s41426-017-0002-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref047] 47.Capoor MN, Ruzicka F, Sandhu G, Rollason J, Mavrommatis K, Ahmed FS, et al. Importance of Propionibacterium acnes hemolytic activity in human intervertebral discs: A microbiological study. PLoS One. 2018/11/30 ed 2018;13(11):e0208144. doi: 10.1371/journal.pone.0208144 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref048] 48.Albert HB, Kjaer P, Jensen TS, Sorensen JS, Bendix T, Manniche C. Modic changes, possible causes and relation to low back pain. Med Hypotheses. 2008;70(2):361–8. doi: 10.1016/j.mehy.2007.05.014 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref049] 49.Ohtori S, Koshi T, Yamashita M, Yamauchi K, Inoue G, Suzuki M, et al. Existence of pyogenic spondylitis in Modic type 1 change without other signs of infection: 2-year follow-up. Eur Spine J. 2010/03/10 ed julho de 2010;19(7):1200–5. doi: 10.1007/s00586-010-1358-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref050] 50.Ritter PL, González VM, Laurent DD, Lorig KR. Measurement of pain using the visual numeric scale. J Rheumatol. março de 2006;33(3):574–80. [PubMed] [Google Scholar]

[pone.0271773.ref051] 51.Breivik H, Borchgrevink PC, Allen SM, Rosseland LA, Romundstad L, Hals EKB, et al. Assessment of pain. Br J Anaesth. julho de 2008;101(1):17–24. doi: 10.1093/bja/aen103 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref052] 52.Bagattini ÂM, Camey SA, Miguel SR, Andrade MV, de Souza Noronha KVM, de C. Teixeira MA, et al. Electronic Version of the EQ-5D Quality-of-Life Questionnaire: Adaptation to a Brazilian Population Sample. Value Health Reg Issues. 1o de dezembro de 2018;17:88–93. doi: 10.1016/j.vhri.2017.11.002 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref053] 53.Vigatto R, Alexandre NM, Correa Filho HR. Development of a Brazilian Portuguese version of the Oswestry Disability Index: cross-cultural adaptation, reliability, and validity. Spine Phila Pa 1976. 2007/02/17 ed 15 de fevereiro de 2007;32(4):481–6. doi: 10.1097/01.brs.0000255075.11496.47 [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref054] 54.Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinforma Oxf Engl. 1o de agosto de 2014;30(15):2114–20. doi: 10.1093/bioinformatics/btu170 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref055] 55.Lohse M, Bolger AM, Nagel A, Fernie AR, Lunn JE, Stitt M, et al. RobiNA: a user-friendly, integrated software solution for RNA-Seq-based transcriptomics. Nucleic Acids Res. julho de 2012;40 (Web Server issue):W622–627. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref056] 56.Shah H, Encheva V, Schmid O, Culak RA, Nasir P, Ines I, et al. Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS): A Potentially Powerful Tool for Rapid Characterisation of Microorganisms. In 2005. p. 57–95. [Google Scholar]

[pone.0271773.ref057] 57.Heberle H, Meirelles GV, da Silva FR, Telles GP, Minghim R. InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams. BMC Bioinformatics. 22 de maio de 2015;16(1):169. doi: 10.1186/s12859-015-0611-3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0271773.ref058] 58.Urrutia J, Besa P, Campos M, Cikutovic P, Cabezon M, Molina M, et al. The Pfirrmann classification of lumbar intervertebral disc degeneration: an independent inter- and intra-observer agreement assessment. Eur Spine J. 2016/02/18 ed setembro de 2016;25(9):2728–33. doi: 10.1007/s00586-016-4438-z [DOI] [PubMed] [Google Scholar]

[pone.0271773.ref059] 59.Medronho R, Bloch K, Luiz R, Werneck G. Epidemiologia. Atheneu. São Paulo. 2009. [Google Scholar]

PERMALINK

Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: Protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques

Vinícius Magno da Rocha

Carla Ormundo Gonçalves Ximenes Lima

Eliane de Oliveira Ferreira

Gabriel Corrêa de Farias

Fábio César Sousa Nogueira

Luis Caetano Martha Antunes

Keila Mara Cassiano

Rossano Kepler Alvim Fiorelli

Roles

Abstract

Introduction

Hypotheses and objectives

Justifications

Methods and analysis

Ethics and dissemination

Study design

Population

Inclusion criteria

Exclusion criteria

Patient and public involvement

Participant allocation

Blinding

Withdrawal of a study participant

Selection of outcomes

Primary outcome

Secondary outcomes

Host multiomic characteristics

Adverse effects

Sampling

Fig 1. Flowchart showing all stages of the study.

Laboratory analysis

Cultures

Quality control of culture media

Phenotypic characterization

Genotypic characterization

Multiomic analyses

Whole genome sequencing

Proteomics

Metabolomics

Questionnaires

Imaging studies

Modic changes

Pfirrmann classification

Confounding variables

Statistical planning

Sample size calculation

Statistical analysis methodology

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

D William Cameron

Roles

Author response to Decision Letter 0

Decision Letter 1

D William Cameron

Roles

Acceptance letter

D William Cameron

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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