The efficacy of antimicrobial solutions against multispecies bacterial biofilm with or without negative pressure wound therapy in an in vitro wound model.

OBJECTIVES: Biofilm is the major challenge in chronic wound management. Instilling a wound cleansing solution aids in wound bed cleaning and infectious pathogen elimination. Negative pressure wound therapy (NPWT) improves the wound-healing process. This study investigated the efficacy of two antimicrobials (Vashe Wound Cleanser and Prontosan Wound Irrigation Solution) against a multispecies bacterial biofilm with or without NPWT in an in vitro wound model. METHODS: A mixed multispecies biofilm containing Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, and Acinetobacter baumannii was developed and verified by scanning electron microscopy and fluorescent in situ hybridization. The efficacy of Vashe and Prontosan against multispecies biofilm with or without NPWT was evaluated by colony-forming unit (cfu) of each species and total bacterial number, and visually confirmed by live/dead stain and confocal microscopy. RESULTS: Prontosan reduced biofilm cell numbers significantly: 6 instils over 24 h resulting in 3.86 ±â€Š0.14 cfu log10 reduction without NPWT and 4.75 ±â€Š0.13 cfu log10 reduction combined with NPWT (P < 0.01) and 12 instils over 48 h resulting in 5.24 ±â€Š0.11 cfu log10 reduction without NPWT and biofilm eradication with NPWT (P < 0.001). NPWT alone or combined with Vashe failed to reduce multispecies biofilm numbers significantly over 24 or 48 h. CONCLUSIONS: Prontosan significantly reduced biofilm cell numbers, with better efficacy over 48 than 24 h, emphasizing the necessity for persistent and robust treatment. NPWT enhanced the effectiveness of Prontosan instillation. However, NPWT alone or combined with Vashe showed limited efficacy and difficulty when combating the multispecies biofilm in vitro.

The Low-Abundance Plasma Proteome Reveals Differentially Abundant Proteins Associated with Breast Implant Capsular Contracture: A Pilot Study.

Capsular contracture (CC) is one of the most common postoperative complications associated with breast implant-associated infections. The mechanisms that lead to CC remain poorly understood. Plasma is an ideal biospecimen for early proteomics biomarker discovery. However, as high-abundance proteins mask signals from low-abundance proteins, identifying novel or specific proteins as biomarkers for a particular disease has been hampered. Here, we employed depletion of high-abundance plasma proteins followed by Tandem Mass Tag (TMT)-based quantitative proteomics to compare 10 healthy control patients against 10 breast implant CC patients. A total of 450 proteins were identified from these samples. Among them, 16 proteins were significantly differentially expressed in which 5 proteins were upregulated and 11 downregulated in breast implant CC patients compared to healthy controls. Gene Ontology enrichment analysis revealed that proteins related to cell, cellular processes and catalytic activity were highest in the cellular component, biological process, and molecular function categories, respectively. Further, pathway analysis revealed that inflammatory responses, focal adhesion, platelet activation, and complement and coagulation cascades were enriched pathways. The differentially abundant proteins from TMT-based quantitative proteomics have the potential to provide important information for future mechanistic studies and in the development of breast implant CC biomarkers.

Randomized Controlled Trial to Assess Negative Pressure Wound Therapy versus Standard-of-care Dressings in Breast Surgery: A Pilot Study.

Background: Standard breast reduction dressings such as Prineo are used to cover surgical wounds, in combination with a binder or support bra. The Prevena Restor BellaForm is a negative pressure wound therapy dressing that covers the entirety of the breast mound and is purported to provide further support and reduce swelling. The aim of this study was to compare the Restor to standard-of-care dressings. Methods: The study was a randomized control trial of women undergoing bilateral breast reduction with one breast being dressed with the Prevena Restor BellaForm dressing and the other having standard of care (Prineo). Outcomes measured were drain outputs, postoperative length of stay, quality of scarring, patient preference for dressings, and adverse events. Follow-up was at 1, 2-6, and 26 weeks. Results: The results show a reduction in postoperative days 1 and 2 average drain output on the Restor side compared with standard dressings. Patient-reported outcome measures showed less bruising. There was no difference in postoperative length of stay and no difference in appearance of scars at the 26-week follow-up period. One patient required removal of the dressing due to irritation and one patient required assistance with resealing of the vacuum. Conclusions: We have shown benefits to drain output and comfort using close incisional negative pressure therapy in breast reduction mammaplasty. We plan to continue to investigate close incisional negative pressure therapy in larger comparative trials for other breast procedures including implant-based reconstruction, where a reduction in drain output could be of great benefit to both healing and reduction of infection risk.

IL-9 is a Biomarker of BIA-ALCL Detected Rapidly By Lateral Flow Assay.

BACKGROUND: A delayed seroma around breast implants is the most common clinical presentation of BIA-ALCL. However, most seromas are due to benign causes. Therefore, it is essential to distinguish benign seromas from seromas due to BIA-ALCL. In a prior study mean concentrations of IL-9, IL-10 and IL-13 were found to be significantly higher in BIA-ALCL than in benign seromas. OBJECTIVES: The aim of this research was to test the ability to detect high concentrations of IL-9 rapidly with a lateral flow assay (LFA). Because we previously reported that a LFA for CD30 detected BIA-ALCL in seromas we compared CD30 and IL-9 LFAs in distinguishing BIA-ALCL from benign seromas. METHODS: Thirty microliter samples of 26 seromas (15 benign, 11 malignant) were tested on in-house prepared strips for IL-9 and CD30. Nanoparticle-conjugated antibodies specific to IL-9 and CD30 were used for detection. IL-9 was analyzed in undiluted samples and CD30 samples were optimized at 1:3 dilution. The dynamic range of detection was determined by spiking recombinant IL-9 into a benign seroma. Image analysis measured intensity of both test line (TL) and control line (CL) and a TL/CL ratio was calculated. IL-9 protein and IL-9 transcription factor PU.1 were stained in BIA-ALCL lines and clinical samples. RESULTS: The IL-9 LFA was reliable in distinguishing BIA-ALCL from benign seromas when the concentration of IL-9 was greater than 10 ng/ml. The CD30 LFA was positive in all 11 malignant cases. In one case with only faint CD30 and IL-10 test lines, the IL-9 LFA was clearly positive. Immunohistochemistry showed IL-9 and its essential transcription factor PU.1 were present in tumor cells in BIA-ALCL lines and clinical samples. CONCLUSIONS: IL-9 is a tumor cell biomarker of BIA-ALCL that can be detected by lateral flow assay and immunohistochemistry. Concentrations of IL-9 greater than 10 ng/ml reliably distinguished BIA-ALCL from benign seromas. Moreover, IL-9 LFA could detect BIA-ALCL when CD30 LFA was not definitive and IL-10 was of low concentration with a faint IL-10 TL, suggesting a multiplex LFA including IL-9, CD30 and IL-10 might be more effective in detecting BIA-ALCL in selected cases.

C9orf72-Associated Dipeptide Repeat Expansions Perturb ER-Golgi Vesicular Trafficking, Inducing Golgi Fragmentation and ER Stress, in ALS/FTD.

Hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are debilitating neurodegenerative conditions affecting either motor neurons (ALS) in the brain and spinal cord or neurons in the frontal and/or temporal cortical lobes (FTD). HREs undergo repeat-associated non-ATG (RAN) translation on both sense and anti-sense strands, generating five distinct dipeptide repeat proteins (DPRs), poly-GA, -GR, -GP, -PA and -PR. Perturbed proteostasis is well-recognised in ALS pathogenesis, including processes affecting the endoplasmic reticulum (ER) and Golgi compartments. However, these mechanisms have not been well characterised for C9orf72-mediated ALS/FTD. In this study we demonstrate that C9orf72 DPRs polyGA, polyGR and polyGP (× 40 repeats) disrupt secretory protein transport from the ER to the Golgi apparatus in neuronal cells. Consistent with this finding, these DPRs also induce fragmentation of the Golgi apparatus, activate ER stress, and inhibit the formation of the omegasome, the precursor of the autophagosome that originates from ER membranes. We also demonstrate Golgi fragmentation in cells undergoing RAN translation that express polyGP. Furthermore, dysregulated ER-Golgi transport was confirmed in C9orf72 patient dermal fibroblasts. Evidence of aberrant ER-derived vesicles in spinal cord motor neurons from C9orf72 ALS patients compared to controls was also obtained. These data thus confirm that ER proteostasis and ER-Golgi transport is perturbed in C9orf72-ALS in the absence of protein over-expression. Hence this study identifies novel molecular mechanisms associated with the ER and Golgi compartments induced by the C9orf72 HRE.

Preventing Bacterial Contamination of Breast Implants Using Infection Mitigation Techniques: An In Vitro Study.

BACKGROUND: Bacterial contamination of implants has been linked to biofilm formation and subsequent infection, capsular contracture, and breast implant-associated anaplastic large cell lymphoma. Reducing contamination during implant insertion should therefore reduce biofilm formation disease sequelae. OBJECTIVES: The aim of this study was to compare levels of contamination between preventative techniques. METHODS: A model to simulate the passage of implants through a skin incision was designed that utilized a sterile textured polyvinyl plastic sheet contaminated with Staphylococcus epidermidis. In the first stage of the polyvinyl contamination model, implants were subject to infection-mitigation techniques and passed through the incision, then placed onto horse blood agar plates and incubated for 24 hours. In the second stage of the study the same contamination was applied to human abdominal wall specimens. A 5 cm incision was made through skin and fat, then implants were passed through and levels of contamination were measured as described. RESULTS: Smooth implants grew a mean of 95 colony-forming units (CFUs; approximately 1 CFU/cm2) and textured implants grew 86 CFUs (also approximately 1 CFU/cm2). CFU counts were analyzed by the Mann-Whitney U-test which showed no significant difference between implant types (P < .05); independent-sample t-tests showed a significant difference. The dependent-variable techniques were then compared as groups by one-way analysis of variance, which also showed a significant reduction compared with the control group (P < .01). CONCLUSIONS: This in vitro study has shown the effectiveness of antiseptic rinse and skin/implant barrier techniques for reducing bacterial contamination of breast implants at the time of insertion.

CD30 Lateral Flow and Enzyme-Linked Immunosorbent Assays for Detection of BIA-ALCL: A Pilot Study.

INTRODUCTION: Breast Implant-Associated Anaplastic Large Cell Lymphoma (BIA-ALCL) commonly presents as a peri-implant effusion (seroma). CD30 (TNFRSF8) is a consistent marker of tumor cells but also can be expressed by activated lymphocytes in benign seromas. Diagnosis of BIA-ALCL currently includes cytology and detection of CD30 by immunohistochemistry or flow cytometry, but these studies require specialized equipment and pathologists' interpretation. We hypothesized that a CD30 lateral flow assay (LFA) could provide a less costly rapid test for soluble CD30 that eventually could be used by non-specialized personnel for point-of-care diagnosis of BIA-ALCL. METHODS: We performed LFA for CD30 and enzyme-linked immunosorbent assay (ELISA) for 15 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas. To determine the dynamic range of CD30 detection by LFA, we added recombinant CD30 protein to universal buffer at seven different concentrations ranging from 125 pg/mL to 10,000 pg/mL. We then performed LFA for CD30 on cryopreserved seromas of 10 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas. RESULTS: Recombinant CD30 protein added to universal buffer produced a distinct test line at concentrations higher than 1000 pg/mL and faint test lines at 250-500 pg/mL. LFA produced a positive test line for all BIA-ALCL seromas undiluted and for 8 of 10 malignant seromas at 1:10 dilution, whereas 3 of 10 benign seromas were positive undiluted but all were negative at 1:10 dilution. Undiluted CD30 LFA had a sensitivity of 100.00%, specificity of 70.00%, positive predictive value of 76.92%, and negative predictive value of 100.00% for BIA-ALCL. When specimens were diluted 1:10, sensitivity was reduced to 80.00% but specificity and positive predictive values increased to 100.00%, while negative predictive value was reduced to 88.33%. When measured by ELISA, CD30 was below 1200 pg/mL in each of six benign seromas, whereas seven BIA-ALCL seromas contained CD30 levels > 2300 pg/mL, in all but one case calculated from dilutions of 1:10 or 1:50. CONCLUSIONS: BIA-ALCL seromas can be distinguished from benign seromas by CD30 ELISA and LFA, but LFA requires less time (<20 min) and can be performed without special equipment by non-specialized personnel, suggesting future point-of-care testing for BIA-ALCL may be feasible.

Developing an International Framework for Informed Consent in Plastic Surgery: A Focus on Cosmetic Breast Augmentation.

Background: Informed consent is a fundamental pillar of patient rights and is an essential part of good clinical practice. In 2019, the International Confederation of Plastic Surgery Societies launched a survey to collect feedback on informed consent practices, with an aim to develop an international guideline for cosmetic surgery. Methods: A 15-question survey was sent to delegates of the International Confederation of Plastic Surgery Societies for dissemination to their national society members. The survey comprised a range of quantitative and qualitative questions. Descriptive and thematic analysis was performed. Results: There were 364 respondents. Over half of the respondents reported no local informed consent policy, whereas others noted national society, specialist college, or government policies. The majority of respondents believed that the performing surgeon should be responsible for obtaining informed consent with at least two face-to-face consultations. Most respondents agreed with a cooling-off period (duration based on procedure type and use of high-risk devices). Regarding cosmetic breast augmentation, the majority of respondents felt that the performing surgeon should be responsible for postoperative management, including cases that occur as part of surgical tourism. Some respondents incorporate financial consent as part of their informed consent practice. Most supported the development of an international informed consent guideline. Conclusions: Informed consent should result from face-to-face consultations with the performing surgeon. There should be a minimum cooling-off period. Postoperative surveillance should be available in all settings. The findings of this survey will help inform an international standardized informed consent guideline for cosmetic surgery.

Diagnosis of breast implant associated anaplastic large cell lymphoma by analysis of cytokines in peri-implant seromas.

More than 1300 women with breast implants have developed an anaplastic large cell lymphoma (ALCL) in fluid (seroma) around their implant. More often, seromas are due to benign causes, for example, capsule contracture, leakage, or trauma. Our report in American Journal of Hematology identified several cytokines (IL-9, IL-10, IL-13) as significantly elevated only in seromas due to ALCL. We further showed that the most robust biomarker, IL-10, could be detected by a lateral flow assay (similar to COVID detection) within minutes allowing physicians to quickly plan management, eliminate or reduce costly testing and patient time away from family. Early detection of ALCL in seromas before infiltration may avoid need for cytotoxic or immunotherapy and is possibly life-saving.

Topical Tranexamic Acid in Primary Breast Augmentation Surgery: Short- and Long-term Outcomes.

BACKGROUND: Breast augmentation mammaplasty (BAM) remains the most popular cosmetic procedure done worldwide. Bleeding in this procedure increases the chance of capsular contracture. Tranexamic acid (TXA), an antifibrinolytic, has been widely used by other surgical specialties to reduce bleeding. OBJECTIVES: We aimed to evaluate the use of TXA in BAM surgery. METHODS: This was a single-surgeon case series of all patients who underwent primary BAM from March 2017 to March 2018 and received topical TXA spray to the implant pocket before implant insertion. Early postoperative complications and long-term outcomes, such as capsular contracture and revisional surgery, were recorded and described. RESULTS: Two hundred and eighty-eight patients were included in the study with an overall complication rate of 2.8% over 5 years. No patients had postoperative bleeding or hematoma formation. One patient had a seroma, managed with ultrasound drainage. Complications requiring reoperation included rippling (3, 1.0%), pocket revision (2, 0.7%), capsule contracture (1, 0.3%) and rupture (1, 0.3%). CONCLUSIONS: This study highlights the safety and potential benefits of the use of topical TXA in breast augmentation, with low bleeding and capsular contracture rates.

Disease Focused Integrated Care - a New Model of Healthcare Delivery for the Treatment of Skin Cancer.

Introduction: As the most common cancer in Australia, skin cancer generates a considerable health burden. This study outlines the establishment of a new model of integrated care for the diagnosis and management of skin cancer. Methods: A new model of integrated care was established to provide access to all aspects of skin cancer management. General practitioners (GPs) were upskilled through hands-on training and a 6-month skin cancer education program and partnered with specialist Dermatologists and Plastic Surgeons co-located in the same clinic. Data including median wait times between the initial consultation and treatment were prospectively collected and compared patients seen through the integrated pathway to patients referred from their primary GP to specialist Dermatologists and Plastic Surgeons directly (non-integrated pathway). The percentage of patients needing co-consultation with a specialist in the integrated pathway was also measured over time. Results: A total of 25341 patients were seen from the commencement of the clinic in August 2015 to June 2021. In 2017 and 2018 the median wait time to be treated was 7 days for the integrated model compared to 54 days (2017) and 46 days (2018) for non-integrated care (p < 0.0001). The percentage of GPs requesting specialist co-consultations for assessment of skin cancer fell from 98% in 2015, to 5.6% in 2021. Histopathology shows that 66% of lesions excised by GPs in this model were malignant or pre-malignant. Conclusions: This study firstly shows a significant reduction in time to treatment in an integrated skin cancer model over traditional models of health. Secondly it demonstrates GP upskilling over time in the integrated program. Integrating GP and specialist medical practitioners in the treatment of skin cancer offers potential for more efficient, accessible, and affordable care. This cooperative, co-located model may provide a template for the integrating the management of other conditions.

Enhanced Skin Penetration of Cannabidiol Using Organosilane Particles as Transdermal Delivery Vehicles.

There is potential for cannabidiol to act as an analgesic, anxiolytic and antipsychotic active ingredient; however, there is a need to find alternate administration routes to overcome its low oral bioavailability. In this work, we propose a new delivery vehicle based on encapsulation of cannabidiol within organosilica particles as drug delivery vehicles, which are subsequently incorporated within polyvinyl alcohol films. We investigated the long-term stability of the encapsulated cannabidiol, as well as its release rate, in a range of simulated fluids with different characterization techniques, including Fourier Transform Infrared (FT-IR) and High-performance Liquid Chromatography (HPLC). Finally, we determined the transdermal penetration in an ex vivo skin model. Our results show that cannabidiol is stable for up to 14 weeks within polyvinyl alcohol films at a range of temperatures and humidity. Release profiles are first-order, consistent with a mechanism involving diffusion of the cannabidiol (CBD) out of the silica matrix. The silica particles do not penetrate beyond the stratum corneum in the skin. However, cannabidiol penetration is enhanced and is detected in the lower epidermis, which was 0.41% of the total CBD in a PVA formulation compared with 0.27% for pure CBD. This is partly due to an improvement of its solubility profile as it is released from the silica particles, but we cannot rule out effects of the polyvinyl alcohol. Our design opens a route for new membrane technologies for cannabidiol and other cannabinoid products, where administration via non-oral or pulmonary routes can lead to better outcomes for patient cohorts in a range of therapeutics.

Staphylococcus aureus Cell Wall Phenotypic Changes Associated with Biofilm Maturation and Water Availability: A Key Contributing Factor for Chlorine Resistance.

Staphylococcus aureus biofilms are resistant to both antibiotics and disinfectants. As Staphylococci cell walls are an important defence mechanism, we sought to examine changes to the bacterial cell wall under different growth conditions. Cell walls of S. aureus grown as 3-day hydrated biofilm, 12-day hydrated biofilm, and 12-day dry surface biofilm (DSB) were compared to cell walls of planktonic organisms. Additionally, proteomic analysis using high-throughput tandem mass tag-based mass spectrometry was performed. Proteins involved in cell wall synthesis in biofilms were upregulated in comparison to planktonic growth. Bacterial cell wall width (measured by transmission electron microscopy) and peptidoglycan production (detected using a silkworm larva plasma system) increased with biofilm culture duration (p < 0.001) and dehydration (p = 0.002). Similarly, disinfectant tolerance was greatest in DSB, followed by 12-day hydrated biofilm and then 3-day biofilm, and it was least in the planktonic bacteria--suggesting that changes to the cell wall may be a key factor for S. aureus biofilm biocide resistance. Our findings shed light on possible new targets to combat biofilm-related infections and hospital dry surface biofilms.

Making Sense of Late Tissue Nodules Associated With Hyaluronic Acid Injections.

BACKGROUND: The pathogenesis of delayed-onset tissue nodules (DTNs) due to hyaluronic acid (HA) injections is uncertain. OBJECTIVES: To formulate a rational theory for DTN development and their avoidance and treatment. METHODS: A multidisciplinary and multicountry DTN consensus panel was established, with 20 questions posed and consensus sought. Consensus was set at 75% agreement. RESULTS: Consensus was reached in 16 of 20 questions regarding the pathogenesis of DTNs, forming the basis for a classification and treatment guide. CONCLUSIONS: The group believes that filler, pathogens, and inflammation are all involved in DTNs and that DTNs most likely are infection initiated with a variable immune response. Injected filler may incorporate surface bacteria, either a commensal or a true pathogen, if the skin barrier is altered. The initially high molecular weight HA filler is degraded to low molecular weight HA (LMWHA) at the edge of the filler. Commensals positioned within the filler bolus may be well tolerated until the filler is degraded and the commensal becomes visible to the immune system. LMWHA is particularly inflammatory in the presence of any local bacteria. Commensals may still be tolerated unless the immune system is generally heightened by viremia or vaccination. Systemic pathogenic bacteremia may also interact with the filler peripheral LMWHA, activating Toll-like receptors that induce DTN formation. Given this scenario, attention to practitioner and patient hygiene and early systemic infection treatment deserve attention. Classification and treatment systems were devised by considering each of the 3 factors-filler, inflammation, and infection-separately.

Prospective Study of Clinical Outcomes From a Breast Implant Assessment Service.

BACKGROUND: Breast augmentation remains the commonest cosmetic surgical procedure worldwide, in spite of recent regulatory action. OBJECTIVES: The aim of this study was to evaluate women with breast implants attending a breast implant assessment clinic and to capture clinical and implant data in women presenting to the service. METHODS: Patients were enrolled prospectively between January 2018 and December 2021. Clinical, implant, and practitioner data were recorded. Patients reported satisfaction on size, shape, and overall outcome as well as the presence or pain. Radiological evaluation, where indicated, was performed and data were included on these findings. RESULTS: A total of 603 patients were assessed. Their mean age was 42.7 years and mean age at implantation was 29.1 years. The most common complications were capsular contracture followed by pain, waterfall deformity, and double bubble, with rupture/contracture rates increasing after the 10-year mark. The risk of double bubble was significantly lower if patients were operated on by certified practitioners (odds ratio = 0.49, P = 0.011). There was almost universally poor awareness of the risks of breast implants in patients presenting for evaluation. CONCLUSIONS: This study has shown benefit in a breast implant assessment clinic to gather information on adverse events and patient-reported outcomes following breast implant surgery. Having appropriately trained and certified practitioners perform cosmetic augmentation significantly lowers the risk of implant malposition and deformity. Any adverse event occurring within 5 years of initial surgery should be flagged as a mandatory reportable clinical indicator and trigger further investigation.

One Step Forward with Dry Surface Biofilm (DSB) of Staphylococcus aureus: TMT-Based Quantitative Proteomic Analysis Reveals Proteomic Shifts between DSB and Hydrated Biofilm.

The Gram-positive bacterium Staphylococcus aureus is responsible for serious acute and chronic infections worldwide and is well-known for its biofilm formation ability. Recent findings of biofilms on dry hospital surfaces emphasise the failures in current cleaning practices and disinfection and the difficulty in removing these dry surface biofilms (DSBs). Many aspects of the formation of complex DSB biology on environmental surfaces in healthcare settings remains limited. In the present study, we aimed to determine how the protein component varied between DSBs and traditional hydrated biofilm. To do this, biofilms were grown in tryptic soy broth (TSB) on removable polycarbonate coupons in the CDC biofilm reactor over 12 days. Hydrated biofilm (50% TSB for 48 h, the media was then changed every 48 h with 20% TSB, at 37 °C with 130 rpm). DSB biofilm was produced in 5% TSB for 48 h at 35 °C followed by extended periods of dehydration (48, 66, 42 and 66 h at room temperature) interspersed with 6 h of 5% TSB at 35 °C. Then, we constructed a comprehensive reference map of 12-day DSB and 12-day hydrated biofilm associated proteins of S. aureus using a high-throughput tandem mass tag (TMT)-based mass spectrometry. Further pathway analysis of significantly differentially expressed identified proteins revealed that proteins significantly upregulated in 12-day DSB include PTS glucose transporter subunit IIBC (PtaA), UDP-N-acetylmuramate-L-alanine ligase (MurC) and UDP-N-acetylenolpyruvoylglucosamine (MurB) compared to 12-day hydrated biofilm. These three proteins are all linked with peptidoglycan biosynthesis pathway and are responsible for cell-wall formation and thicker EPS matrix deposition. Increased cell-wall formation may contribute to the persistence of DSB on dry surfaces. In contrast, proteins associated with energy metabolisms such as phosphoribosyl transferase (PyrR), glucosamine--fructose-6-phosphate aminotransferase (GlmS), galactose-6-phosphate isomerase (LacA), and argininosuccinate synthase (ArgG) were significantly upregulated whereas ribosomal and ABC transporters were significantly downregulated in the 12-day hydrated biofilm compared to DSB. However, validation by qPCR analysis showed that the levels of gene expression identified were only partially in line with our TMT-MS quantitation analysis. For the first time, a TMT-based proteomics study with DSB has shed novel insights and provided a basis for the identification and study of significant pathways vital for biofilm biology in this reference microorganism.