The Role of Cutibacterium Acnes in the Development of Degenerative Lumbar Intervertebral Disc Disease

The Role of Cutibacterium Acnes in the Development of Degenerative Lumbar Intervertebral Disc Disease and the Clinical Effect of Microdiscectomy

The goal of this study is to clarify the role of the bacterium Cutibacterium acnes in the development of degenerative disc disease in patients with lumbar disc herniation who underwent microdiscectomy as part of their regular medical care.

The project is divided into retrospective and prospective parts. The retrospective part will consist of data processing and analysis using our large cohort of patients enrolled during the previous studies. The prospective part will include a) patient enrollment, b) preoperative data collection, c) perioperative tissue collection, d) microbiological examination, e) postoperative follow-up visits, and f) data management and statistical analyses.

Retrospective data processing and analysis For the retrospective part of the project, the investigators plan to use the C. acnes database stored at the Department of Neurosurgery, St. Anne's University Hospital, Brno, Czech Republic. This database contains 1200 anonymized adult patients with symptomatic lumbar disc herniation scheduled for microdiscectomy between 2015 and 2024. These patients were enrolled during the research project entitled "Presence of the low-virulence bacterium Propionibacterium acnes in the intervertebral disc tissue as a cause of chronic back pain". Each patient signed informed consent forms before enrolling in this study, enabling future data analyses. According to our preliminary review, at least 400 patients of the retrospective cohort have completed epidemiologic, MRI, microbiological, and clinical data. The epidemiological data include age, gender, disc herniation relapse, epidural steroid injection before the surgery, body mass index (BMI), the lumbar segment of herniation, type of herniation, and physical workload. The clinical data include microbiological findings from perioperatively collected disc tissue, preoperative spinal MRI, preoperative and postoperative pain intensity according to the visual analog scale (VAS), and functional disability for low back pain quantified by the Oswestry Disability Questionnaire (ODQ). Although the data is available, no hypotheses between C. acnes positivity and clinical outcomes have yet been tested on the dataset. Therefore, in this part of the project, microbiological findings will be studied together with radiological changes in preoperative MRI and clinical status before and after surgery.

Prospective data cohort

Patient enrollment - the investigators plan to enrol at least 400 patients with lumbar disc herniation indicated for microdiscectomy at the Department of Neurosurgery, Faculty of Medicine, Masaryk University and St. Anne's University Hospital, Brno, Czech Republic. Patients indicated for lumbar microdiscectomy will be prospectively screened, and those who pass the inclusion and exclusion criteria will be asked to sign the Informed Consent form to be enrolled in the study.

Preoperative data collection - baseline epidemiological and clinical data such as age, gender, disc herniation relapse, epidural steroid injection before the surgery, body mass index (BMI), the lumbar segment of herniation, type of herniation, and physical workload will be collected from each patient. Degenerative changes in the intervertebral discs and adjacent spinal structures will be evaluated on MRI as part of a standard preoperative examination. Clinical data will be obtained during the preoperative clinical exam and from patients in the form of questionnaires, including a numerical scale for leg and back pain, the Oswestry questionnaire evaluating the impact of low back pain on activities of daily living, and the Short Form quality of life questionnaire - 36 (SF- 36) to examine the quality of life before surgery.

Perioperative tissue collection - all operations for herniated intervertebral discs in patients included in the project will be performed at the Department of Neurosurgery, Faculty of Medicine, Masaryk University, and St. Anne's University Hospital, Brno, Czech Republic. The surgical site will undergo a comprehensive preparation, including three meticulous applications of povidone-iodine or an adequate antiseptic in case of iodine allergy. The site will then be draped using a standard sterile technique. Common prophylactic antibiotics will be given preoperatively in a single intravenous dose in all cases, with the usual prophylactic antibiotic being cefazolin; clindamycin or vancomycin will be administered in case of allergy. The exact location of the skin incision will be guided by intraoperative fluoroscopy. In all cases, a posterior midline approach will be used. The Caspar-type self-retaining retractor will be placed, and the ligamentum flavum will be removed under an operating microscope using Penfield dissectors and Kerrison rongeurs. The nerve root and dural sac will be gently retracted, and the posterior longitudinal ligament will be incised at the site of the herniated disc. The disc herniation will be exposed and removed with the remnant fragments of the nucleus pulposus in the intervertebral space. To minimize contamination of tissue samples, they will be retained in a sterile sample cup, labeled, and transported to the Department of Microbiology at St. Anne's University Hospital, Brno, Czech Republic. Tissue samples ranging from 3x3x5 mm to 10x5x5 mm will be utilized immediately for microbiological processing.

Microbiological examination - based on the preliminary study, the investigators plan to process the samples (semi)quantitatively. The samples will be homogenized by disintegration in a stomacher. Subsequently, the samples will be inoculated onto suitable culture media and cultured aerobically and anaerobically with subsequent semi-quantitative evaluation of the results. The quantity of bacteria in the sample will be expressed as colony-forming units (CFU) in 1 g of the sample. C. acnes burden in the sample exceeding 103 CFU/gram (≥103 CFU/gram) will be considered a clinically significant finding; C. acnes burden below <102 CFU/gram will indicate contamination; ≥102 - <103 CFU/gram will be interpreted as a borderline result.

Taxonomic identification of isolated strains will be performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as well as conventional phenotypic methods (biochemical tests, microscopy, etc.). For the phylotyping of our isolates, the investigators will use the multiplex PCR. To assess the ability to form a biofilm, the investigators will use the microtiter plate-based assay. The assay needs to be optimized, particularly with regard to suitable culture conditions and suitable treatment of the adhesion surfaces (peptide coating). A CAMP factor will be tested using the CAMP test with Staphylococcus aureus (CCM 6188). Hemolytic activity will be assessed using the blood agar with washed sheep erythrocytes. The production of some other virulence factors, e.g., hyaluronidase decapsulation test, chondroitin sulfatase, hyaluronidase, deoxyribonuclease, gelatinase, phosphatase, and lecithinase, will also be monitored. Antimicrobial susceptibility testing will be performed using phenotypic methods, broth microdilution, and/or standardized disk diffusion methods, according to the European Committee on Antimicrobial Susceptibility Testing - EUCAST Antimicrobial Susceptibility Testing (available at https://www.eucast.org/ast\_of\_bacteria).

Postoperative follow-up visits - postoperative clinical condition and the overall outcome will be evaluated by patients using the same standardized exams and questionnaires as before the surgery (numerical pain scale for leg and back, the Oswestry questionnaire, the Short Form quality of life questionnaire - 36 (SF-36). In addition to the mentioned questionnaires, patients will evaluate the success of the surgical treatment using the modified MacNab criteria and their satisfaction with the surgical treatment using the North American Spine Society (NASS) patient satisfaction index. Follow-up on-site visits will be carried out according to the standards of the Department of Neurosurgery, Faculty of Medicine, Masaryk University and St. Anne's University Hospital, Brno, Czech Republic, 6 weeks (± 1week), 6 months (± 2weeks), and 12 months (± 2weeks) after the operation.

Inclusion Criteria:

• minimum age of 18 years
• MRI of the lumbosacral spine performed one month ± 2weeks before surgery showing a disc protrusion, herniation, or free nucleus pulposus sequestration
• matching physical examination findings including positive straight leg raise test, dermatomal sensory deficits, myotome motor deficits, and diminished deep tendon reflexes.

Exclusion Criteria:

• antibiotics or corticosteroid use the month before surgery
• ongoing bacterial or viral infection
• immunocompromised patient
• trauma
• undefined radiological mass
• inflammatory arthritis
• other rheumatologic diseases.