Evaluation of the Effect of Lactobacillus acidophilus ATCC 4356 Bacteriocin against Staphylococcus aureus.

Background: Lactobacillus acidophilus is lactic acid bacteria that produce bacteriocins. Bacteriocins are antimicrobial peptides or proteins that exhibit activity against closely related bacteria. The aim of this study was to determine the effect of L. acidophilus ATCC 4356 bacteriocin against Staphylococcus aureus. Material and Methods. We used four different phenotypic methods for antimicrobial activities against two standard strains: methicillin-resistant S. aureus (MRSA) ATCC 33591 and methicillin-susceptible S. aureus (MSSA) ATCC 25923. The methods were (1) agar well diffusion, (2) overlay soft agar, (3) paper disk, and (4) modification of punch hole. The ammonium sulfate method was used to concentrate crude bacteriocin, and ultrafiltration and dialysis tubes were used to remove ammonium sulfate from the bacteriocins. Each method was repeated in triplicate. Result: L. acidophilus ATCC 4356 showed antimicrobial activity against both MRSA and MSSA standard strains only by the overlay soft agar method and not by the agar well diffusion, punch hole modification, and paper disk methods. No antimicrobial effects were observed in crude bacteriocins concentrated. Conclusion: The growth inhibition of S. aureus in overlay soft agar method may be due to the production of bacteriocin-like substances. The overlay soft agar method is a qualitative test, so there is a need for further study to optimize the conditions for the production of bacteriocin-like substances in the culture supernatant and precise comparison between the inhibitory activity and pheromone secretion of different strains.

Antimicrobial and antibiofilm activity of specialized metabolites isolated from Centaurea hyalolepis.

The discovery of plant-derived compounds that are able to combat antibiotic-resistant pathogens is an urgent demand. Over years, Centaurea hyalolepis attracted considerable attention because of its beneficial medical properties. Phytochemical analyses revealed that Centaurea plant species contain several metabolites, such as sesquiterpene lactones (STLs), essential oils, flavonoids, alkaloids, and lignans.The organic extract of C. hyalolepis plant, collected in Palestine, showed significant antimicrobial properties towards a panel of Gram-negative and Gram-positive bacterial strains when the Minimal Inhibitory Concentration (MIC) values were evaluated by broth microdilution assays. A bio-guided fractionation of the active extract via multiple steps of column and thin layer chromatography allowed us to obtain three main compounds. The isolated metabolites were identified as the STLs cnicin, 11ß,13-dihydrosalonitenolide and salonitenolide by spectroscopic and spectrometric analyses. Cnicin conferred the strongest antimicrobial activity among the identified compounds. Moreover, the evaluation of its antibiofilm activity by biomass assays through crystal violet staining revealed almost 30% inhibition of biofilm formation in the case of A. baumannii ATCC 17878 strain. Furthermore, the quantification of carbohydrates and proteins present in the extracellular polymeric substance (EPS) revealed the ability of cnicin to significantly perturb biofilm structure. Based on these promising results, further investigations might open interesting perspectives to its applicability in biomedical field to counteract multidrug resistant infections.

Comprehensive analysis of the microbiome in Apis cerana honey highlights honey as a potential source for the isolation of beneficial bacterial strains.

Background: Honey is a nutritious food made by bees from nectar and sweet deposits of flowering plants and has been used for centuries as a natural remedy for wound healing and other bacterial infections due to its antibacterial properties. Honey contains a diverse community of bacteria, especially probiotic bacteria, that greatly affect the health of bees and their consumers. Therefore, understanding the microorganisms in honey can help to ensure the quality of honey and lead to the identification of potential probiotic bacteria. Methods: Herein, the bacteria community in honey produced by Apis cerana was investigated by applying the next-generation sequencing (NGS) method for the V3-V4 hypervariable regions of the bacterial 16S rRNA gene. In addition, lactic acid bacteria (LAB) in the honey sample were also isolated and screened for in vitro antimicrobial activity. Results: The results showed that the microbiota of A. cerana honey consisted of two major bacterial phyla, Firmicutes (50%; Clostridia, 48.2%) and Proteobacteria (49%; Gammaproteobacteria, 47.7%). Among the 67 identified bacterial genera, the three most predominant genera were beneficial obligate anaerobic bacteria, Lachnospiraceae (48.14%), followed by Gilliamella (26.80%), and Enterobacter (10.16%). Remarkably, among the identified LAB, Lactobacillus kunkeei was found to be the most abundant species. Interestingly, the isolated L. kunkeei strains exhibited antimicrobial activity against some pathogenic bacteria in honeybees, including Klebsiella spp., Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus. This underscores the potential candidacy of L. kunkeei for developing probiotics for medical use. Taken together, our results provided new insights into the microbiota community in the A. cerana honey in Hanoi, Vietnam, highlighting evidence that honey can be an unexplored source for isolating bacterial strains with potential probiotic applications in honeybees and humans.

Antibiotic susceptibility and resistance genes profiles of Vagococcus salmoninarum in a rainbow trout (Oncorhyncus mykiss, Walbaum) farm.

Disease outbreaks negatively affect fish production. Antimicrobial agents used in the treatment of diseases become ineffective over time because of antibiotic resistance developed by bacteria distributed in the aquaculture environment. This study was conducted for 4 months (cold period) in a fish farm to detect the fish disease, cold water streptococcosis. In the study, four brood stock showing disease signs were detected. Bacteria isolates were obtained and identified as Vagococcus salmoninarum. Antimicrobial susceptibility of V. salmoninarum was tested and antibiotic resistance gene profiles of V. salmoninarum isolates were screened. The phylogenetic relation of the isolates with the previously reported strains was evaluated. Antibiotic resistance developed by pathogenic bacteria is distributed in the aquaculture environment. The transfer of resistance genes from one bacterium to another is very common. This situation causes the antimicrobial agents used in the treatment of diseases to become ineffective over time. The disc diffusion test showed that all four isolates developed resistance to 13 (FFC30, AX25, C30, E15, CF30, L2, OX1, S10, T30, CRO30, CC2, PT15 and TY15) of the evaluated antibiotics and were about to develop resistance to six others (AM 10, FM 300, CFP75, SXT25, APR15 and TE30). Furthermore, antibiotic resistance genes tetA, sul1, sul2, sul3, dhfr1, ereB and floR were detected in the isolated strain. Moreover, the phylogenetic analysis showed that isolated V. salmoninarum strain (ESN1) was closely related to the bacterial strains isolated from USA and Jura.

[Fulminant Clostridioides difficile infection during treatment with FLT3 inhibitor for acute myeloid leukemia].

An 80-year-old man with FLT3-TKD mutation-positive acute myeloid leukemia (AML) relapsed during consolidation therapy with venetoclax/azacitidine and was started on gilteritinib as salvage therapy. On the day after treatment initiation, febrile neutropenia was observed, but the fever resolved promptly after initiation of antimicrobial therapy. On the fifth day after completion of antimicrobial therapy, the patient experienced fever and watery diarrhea over 10 times a day, and a diagnosis of Clostridioides difficile infection (CDI) was made based on stool examination. The patient was treated with intravenous metronidazole, but renal dysfunction, hypotension, and hypoxemia developed, and a CT scan showed pleural and intraperitoneal effusion, significant intestinal wall thickening, and intestinal dilatation. Fidaxomicin was started under general monitoring in the intensive care unit and response was achieved. The patient was discharged from the intensive care unit on the 18th day after the onset of CDI. We report this case not only due to the rarity of fulminant CDI during AML treatment, but also because it is a valuable example of effective treatment of fulminant CDI with fidaxomicin.

Australian Gonococcal Surveillance Programme, 1 January to 31 March 2023.

Abstract: The Australian National Neisseria Network (NNN) comprises reference laboratories in each state and territory that report data on antimicrobial susceptibility testing to an agreed group of antimicrobial agents for the Australian Gonococcal Surveillance Programme (AGSP). The AGSP data are presented quarterly in tabulated form, as well as in the AGSP annual report. This report presents national gonococcal antimicrobial resistance surveillance data from 1 January to 31 March 2023.

Australian Gonococcal Surveillance Programme, 1 April to 30 June 2023.

Abstract: The Australian National Neisseria Network (NNN) comprises reference laboratories in each state and territory that report data on antimicrobial susceptibility testing to an agreed group of antimicrobial agents for the Australian Gonococcal Surveillance Programme (AGSP). The AGSP data are presented quarterly in tabulated form, as well as in the AGSP annual report. This report presents national gonococcal antimicrobial resistance surveillance data from 1 April to 30 June 2023.

Australian Gonococcal Surveillance Programme, 1 July to 30 September 2023.

Abstract: The Australian National Neisseria Network (NNN) comprises reference laboratories in each state and territory that report data on antimicrobial susceptibility testing to an agreed group of antimicrobial agents for the Australian Gonococcal Surveillance Programme (AGSP). The AGSP data are presented quarterly in tabulated form, as well as in the AGSP annual report. This report presents national gonococcal antimicrobial resistance surveillance data from 1 July to 30 September 2023.

A Multifunctional Nanozyme Integrating Antioxidant, Antimicrobial and Pro-Vascularity for Skin Wound Management.

Background: Skin wounds are a prevalent issue that can have severe health consequences if not treated correctly. Nanozymes offer a promising therapeutic approach for the treatment of skin wounds, owing to their advantages in regulating redox homeostasis to reduce oxidative damage and kill bacteria. These properties make them an effective treatment option for skin wounds. However, most of current nanozymes lack the capability to simultaneously address inflammation, oxidative stress, and bacterial infection during the wound healing process. There is still great potential for nanozymes to increase their therapeutic functional diversity and efficacy. Methods: Herein, copper-doped hollow mesopores cerium oxide (Cu-HMCe) nanozymes with multifunctional of antioxidant, antimicrobial and pro-vascularity is successfully prepared. Cu-HMCe can be efficiently prepared through a simple and rapid solution method and displays sound physiological stability. The biocompatibility, pro-angiogenic, antimicrobial, and antioxidant properties of Cu-HMCe were assessed. Moreover, a full-thickness skin defect infection model was utilized to investigate the wound healing capacity, as well as anti-inflammatory and pro-angiogenic properties of nanozymes in vivo. Results: Both in vitro and in vivo experiments have substantiated Cu-HMCe's remarkable biocompatibility. Moreover, Cu-HMCe possesses potent antioxidant enzyme-like catalytic activity, effectively clearing DPPH radicals (with a scavenging rate of 80%), hydroxyl radicals, and reactive oxygen species. Additionally, Cu-HMCe exhibits excellent antimicrobial and pro-angiogenic properties, with over 70% inhibition of both E. coli and S. aureus. These properties collectively promote wound healing, and the wound treated with Cu-HMCe achieved a closure rate of over 90% on the 14th day. Conclusion: The results indicate that multifunctional Cu-HMCe with antioxidant, antimicrobial, and pro-angiogenic properties was successfully prepared and exhibited remarkable efficacy in promoting wound healing. This nanozymes providing a promising strategy for skin repair.

Identification and antimicrobial susceptibility profile of bacteria isolated from primary endodontic infections.

This study aimed to identify and characterize the antimicrobial susceptibility profile of bacteria found in primary endodontic infections in the teeth of patients treated at the Dental Clinic of the University of Ribeirão Preto, São Paulo, Brazil. From September to December 2019, samples were obtained from 21 patients with primary endodontic infections. The collections were carried out in triplicate using paper cones placed close to the total length of the root canal. Bacterial isolation was performed in Brain Heart Infusion agar, Blood agar, and other selective culture media cultured at 37°C for up to 48 h under aerobiosis and microaerophilic conditions. The bacterial species were identified using the Vitek 2 automated system. The disk diffusion method on agar Müeller-Hinton was used to assess antimicrobial susceptibility with the recommended antimicrobials for each identified bacterial species. A total of 49 antibiotics were evaluated. Fifteen of the 21 samples collected showed bacterial growth, and 17 bacterial isolates were found. There were 10 different bacterial species identified: Enterococcus faecalis (four isolates), Streptococcus mitis/oralis (three isolates), Streptococcus anginosus (three isolates) being the most common, followed by Staphylococcus epidermidis, Enterococcus faecium, Streptococcus constellatus, Streptococcus alactolyticus, Enterobacter cloacae, Klebsiella variicola, and Providencia rettgeri (one isolate of each species). The analysis demonstrated significant susceptibility to most of the tested antibiotics. However, some Enterococcus isolates resisted the antibiotic's erythromycin, ciprofloxacin, and tetracycline. A Staphylococcus epidermidis isolate was characterized as multidrug-resistant. Five Streptococcus isolates were non-susceptible to all antibiotics tested.

Innovative textiles treated with TiO2-AgNPs with succinic acid as a cross-linking agent for medical uses.

Silver and titanium-silver nanoparticles have unique properties that make the textile industry progress through the high quality of textiles. Preparation of AgNPs and TiO2-Ag core-shell nanoparticles in different concentrations (0.01% and 0.1% OWF) and applying it to cotton fabrics (Giza 88 and Giza 94) by using succinic acid 5%/SHP as a cross-linking agent. Ultra-violet visible spectroscopy (UV-Vis), X-ray diffraction (XRD), dynamic light scattering (DLS), zeta potential, transmission electron microscopy (TEM), scanning electron microscopy/energy-dispersive X-ray (SEM-EDX) are tools for AgNPs and TiO2-AgNPs characterization and the treated cotton. The resulting AgNPs and TiO2-AgNPs were added to cotton fabrics at different concentrations. The antimicrobial activities, UV protection, self-cleaning, and the treated fabrics' mechanical characteristics were investigated. Silver nanoparticles and titanium dioxide-silver nanoparticles core-shell were prepared to be used in the treatment of cotton fabrics to improve their UV protection properties, self-cleaning, elongation and strength, as well as the antimicrobial activities to use the produced textiles for medical and laboratory uses and to increase protection for medical workers taking into account the spread of infection. The results demonstrated that a suitable distribution of prepared AgNPs supported the spherical form. Additionally, AgNPs and TiO2-AgNPs have both achieved stability, with values of (- 20.8 mV and - 30 mV, respectively). The synthesized nanoparticles spread and penetrated textiles' surfaces with efficiency. The findings demonstrated the superior UV protection value (UPF 50+) and self-cleaning capabilities of AgNPs and TiO2-AgNPs. In the treatment with 0.01% AgNPs and TiO2-AgNPs, the tensile strength dropped, but the mechanical characteristics were enhanced by raising the concentration to 0.1%. The results of this investigation demonstrated that the cotton fabric treated with TiO2-AgNPs exhibited superior general characteristics when compared to the sample treated only with AgNPs.

Modified polymeric biomaterials with antimicrobial and immunomodulating properties.

The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside) yielded very stable preparations of modified biomaterials. The main emphasis was placed on analyses of their antimicrobial activity and potential immunomodulatory and non-cytotoxic properties towards the CCD841 CoTr model cell line. Cecropin A did not significantly affect the viability or proliferation of the CCD 841 CoTr cells, regardless of its soluble or immobilized form. In contrast, puromycin did not induce a significant decrease in the cell viability or proliferation in the immobilized form but significantly decreased cell viability and proliferation when administered in the soluble form. The covalent immobilization of these two molecules on the surface of biomaterials resulted in stable preparations that were able to inhibit the multiplication of Staphylococcus aureus and S. epidermidis strains. It was also found that the preparations induced the production of cytokines involved in antibacterial protection mechanisms and stimulated the immune response. The key regulator of this activity may be related to TLR4, a receptor recognizing bacterial LPS. In the present study, these factors were produced not only in the conditions of LPS stimulation but also in the absence of LPS, which indicates that cecropin A- and puromycin-modified biomaterials may upregulate pathways leading to humoral antibacterial immune response.

Endophytes: a uniquely tailored source of potential antibiotic adjuvants.

Multidrug microbial resistance is risking an annual loss of more than 10 million people' lives by 2050. Solutions include the rational use of antibiotics and the use of drugs that reduce resistance or completely obliterate them. Here endophytes come to play due to their high-yield production and inherent nature to produce antimicrobial molecules. Around 40%, 45% and 17% of antibacterial agents were obtained from fungi, actinomycetes, and bacteria, respectively, whose secondary metabolites revealed effectiveness against resistant microbes such as MRSA, MRSE, and Shigella flexneri. Endophyte's role was not confined to bactericidal effect but extended to other mechanisms against MDR microbes, among which was the adjuvant role or the "magic bullets". Scarce focus was given to antibiotic adjuvants, and many laboratories today just screen for the antimicrobial activity without considering combinations with traditional antibiotics, which means real loss of promising resistance combating molecules. While some examples of synthetic adjuvants were introduced in the last decade, the number is still far from covering the disused antibiotics and restoring them back to clinical use. The data compiled in this article demonstrated the significance of quorum sensing as a foreseen mechanism for adjuvants from endophytes secondary metabolites, which call for urgent in-depth studies of their molecular mechanisms. This review, comprehensively and for the first time, sheds light on the significance of endophytes secondary metabolites in solving AMR problem as AB adjuvants.

Plackett-Burman screening of physico-chemical variables affecting Citrus peel-mediated synthesis of silver nanoparticles and their antimicrobial activity.

With the growing resistance of pathogenic microbes to traditional drugs, biogenic silver nanoparticles (SNPs) have recently drawn attention as potent antimicrobial agents. In the present study, SNPs synthesized with the aid of orange (Citrus sinensis) peel were engineered by screening variables affecting their properties via Plackett-Burman design. Among the variables screened (temperature, pH, shaking speed, incubation time, peel extract concentration, AgNO3 concentration and extract/AgNO3 volume ratio), pH was the only variable with significant effect on SNPs synthesis. Therefore, SNPs properties could be enhanced to possess highly regular shape with zeta size of 11.44 nm and zeta potential of - 23.7 mV. SNPs purified, capped and stabilized by cloud point extraction technique were then checked for their antimicrobial activity against Bacillus cereus, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium and Candida albicans. The maximum antimicrobial activity of SNPs was recorded against E. coli, L. monocytogenes and C. albicans with clear zone diameter of 33.2, 31.8 and 31.7 mm, respectively. Based on minimum inhibition concentration and minimum bactericidal concentration of SNPs (300 mg/l) as well as their effect on respiratory chain dehydrogenases, cellular sugar leakage, protein leakage and lipid peroxidation of microbial cells, E. coli was the most affected. Scanning electron microscopy, protein banding and DNA fragmentation proved obvious ultrastructural and molecular alterations of E. coli treated with SNPs. Thus, biogenic SNPs with enhanced properties can be synthesized with the aid of Citrus peel; and such engineered nanoparticles can be used as potent antimicrobial drug against E. coli.

Interventions to address antimicrobial resistance: an ethical analysis of key tensions and how they apply in low- income and middle-income countries.

Antimicrobial resistance (AMR) is a global health and one health problem. Efforts to mitigate the problem of AMR are challenging to implement due to unresolved ethical tensions. We present an in-depth ethical analysis of tensions that might hinder efforts to address AMR. First, there is a tension between access and excess in the current population: addressing lack of access requires facilitating use of antimicrobials for some populations, while addressing excessive use for other populations. Second, there is a tension between personal interests and a wider, shared interest in curbing AMR. These personal interests can be viewed from the perspective of individuals seeking care and healthcare providers whose livelihoods depend on using or selling antimicrobials and who profit from the sales and use of antimicrobials. Third, there is a tension between the interests of current populations and the interests of future generations. Last, there is a tension between addressing immediate health threats such as pandemics, and AMR as a 'silent', chronic threat. For each of these tensions, we apply 'descriptive ethics' methods that draw from existing evidence and our experiences living and working in low-income and middle-income countries to highlight how these ethical tensions apply in such settings.

Contribution of the patient microbiome to surgical site infection and antibiotic prophylaxis failure in spine surgery.

Despite modern antiseptic techniques, surgical site infection (SSI) remains a leading complication of surgery. However, the origins of SSI and the high rates of antimicrobial resistance observed in these infections are poorly understood. Using instrumented spine surgery as a model of clean (class I) skin incision, we prospectively sampled preoperative microbiomes and postoperative SSI isolates in a cohort of 204 patients. Combining multiple forms of genomic analysis, we correlated the identity, anatomic distribution, and antimicrobial resistance profiles of SSI pathogens with those of preoperative strains obtained from the patient skin microbiome. We found that 86% of SSIs, comprising a broad range of bacterial species, originated endogenously from preoperative strains, with no evidence of common source infection among a superset of 1610 patients. Most SSI isolates (59%) were resistant to the prophylactic antibiotic administered during surgery, and their resistance phenotypes correlated with the patient's preoperative resistome (P = 0.0002). These findings indicate the need for SSI prevention strategies tailored to the preoperative microbiome and resistome present in individual patients.

Treatment of peri-implantitis using nonsurgical debridement combined with bioresorbable doxycycline nanospheres: a case report with 3-year follow-up.

Treatment of peri-implant diseases focuses on reducing the bacterial load and consequent infection control. The use of local antimicrobials as an adjunct to mechanical therapy may result in a better outcome. Among antimicrobials, doxycycline stands out because of its local modulation of cytokines, microbial reduction, and clinical parameters in the treatment of periodontal diseases. The objective of this case report was to describe the combined application of mechanical debridement and bioresorbable doxycycline-loaded nanospheres for the treatment of peri-implantitis in a 71-year-old man. At the 3-year evaluation, the peri-implant tissues had improved, showing decreased probing depths, an absence of bleeding on probing, and no suppuration. This case report highlights the importance of supportive therapy, which is essential for the long-term success of peri-implantitis treatment.

The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response.

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation.

Immature neutrophils in cord blood exert increased expression of genes associated with antimicrobial function.

Introduction: The immune systems of both the mother and the newborn face significant challenges during birth. Proper immune regulation after birth is essential for the survival of neonates. Numerous studies have demonstrated that the neonatal immune system is relatively immature, particularly in its adaptive arm, placing the primary responsibility for immune surveillance on innate immunity. Methods: Given the significant role of neutrophils in protecting the neonate after birth, we conducted a study investigating the properties of neutrophils in newborn cord blood using various methodological approaches. Results: Our findings demonstrate the presence of immature low-density neutrophils in the cord blood, which are likely responsible for the observed elevated expression of genes coding for proteins essential to antimicrobial response, including myeloperoxidase, neutrophils elastase, and defensins. Discussion: We propose that these cells function normally and support the protection of newborns early after birth. Furthermore, our results suggest that the mode of delivery might significantly influence the programming of neutrophil function. The presented findings emphasize the importance of distinct neutrophil subpopulations in neonatal immunity and their potential impact on early postnatal health.

Stability Evaluation of Bracketed Diclofenac Sodium USP in VersaPro™ Cream Base, VersaPro™ Gel Base and PLO Gel Mediflo™ 30 Compound Kit.

Diclofenac Sodium is a nonsteroidal anti-inflammatory drug (NSAID) that has analgesic, anti-inflammatory, and antipyretic properties, and has been found to be effective in treating a variety of acute and chronic pain and inflammatory conditions. A stability study was designed to assess the physical, chemical, and antimicrobial stability of three extemporaneously compounded bracketed Diclofenac Sodium formulations over time using a validated, stability indicating HPLC method. Diclofenac Sodium 1% and 15% were compounded in Medisca VersaPro™ Cream Base, VersaPro™ Gel Base and PLO Gel Mediflo™30 Compound Kit and stored at room temperature, in tightly closed, light resistant, plastic containers for 180 days. The organoleptic properties, pH, viscosity, and Diclofenac Sodium concentration of each formulation were evaluated at predetermined time points. Antimicrobial effectiveness testing of the compounded formulation according to USP <51> was also evaluated at the initial time point and after 180 days. The results demonstrated that all formulations remained within the specified stability criteria for the duration of the study. Therefore, an extended beyond-use-date of 180 days may be assigned to these compounded formulations under the studied conditions.