Extraction and Characterization of Silver Nitrate Modified Biopolymer and its Antimicrobial Mode of Action

The discovery of antibacterial drugs to combat the spread of infectious diseases brought a great relief to the health community which allowed the medical professionals to treat and reduce death rate arising from infections caused by microorganisms. The need to search and formulate more potent and efficient antimicrobial drugs become necessary due to resistance of some microorganisms to available and existing antimicrobial drugs. Chitosan was extracted from crab shell waste through the stages of demineralization, deproteinization, and deacetylation. The chitosan (CHS) was further modified with silver nitrate (AgNO₃) solution using various concentrations (0.5, 1.0, and 1.5 M) in order to enhance its antimicrobial property. The crab shell powder (CSP) and chitosan (CHS) were characterized using X-ray diffraction (XRD), Fourier Transform Infrared (FT-IR), Scanning Electron Microscopy coupled with Energy Dispersive Spectroscopy. (SEM-EDS). The antimicrobial activity (zone of inhibition) was carried out using disk diffusion method. The result showed that <i>E.coli</i> and <i>Pseudopodium </i>showed the highest susceptibility of 10.2 ± 0.2 mm with extract from chitosan treated with 1.5 M silver nitrate (1.5 SNCHE) at 1000 µg/L whereas the least susceptibility was observed to be E. coli (2.0 ± 0.1 mm) with extract from chitosan treated with 1.0 M silver nitrate (1.0 SNCHE) at 250 µg/L. However, untreated chitosan extract (UCHSE) did not exhibit any antimicrobial effect against any of the tested microorganisms. The proximate analysis of CHS and CHN showed % crude protein to be 12.24 ± 0.01 and 20.54 ± 0.03 respectively. The FT-IR spectra of CHS and CHN showed their characteristic absorption peaks and the diffractograms of CSP and CHS revealed CaCO₃ to be the major mineral component in the samples.

The conversion of seafood waste into valuable materials is essential for advancing seafood sustainability. Crab shells, which are a major byproduct, generate a large volume of waste annually and are a key source of chitin and chitosan. This study explores the extraction and characterisation of chitin and chitosan from Irish brown crab (Cancer pagurus) shell waste using chemical methods. Samples of crab shell waste (CSW), extracted chitin (CT) and chitosan (CTS) were analysed and compared to commercial chitosan (CTS-c) in terms of chemical, thermal and structural properties. The extraction process included steps of demineralisation, deproteinisation, decolourisation and deacetylation, yielding 17.60% CT and 50.45% CTS, which aligns with previously published values. FTIR analysis confirmed structural changes from CSW to CT followed by CTS, shown by distinct spectral shifts, with CTS exhibiting a degree of deacetylation (DD) of 78.53%, comparable to CTS-c (79.53%). DSC findings showed increased enthalpy, ΔH from 72.58 J/g (CSW) to 253.28 J/g (CTS), indicating improved thermal stability. SEM images displayed morphological transformations from porous CT to denser CTS, with porosity measured by ImageJ changing from 2.87% (CSW) to 18.60% CT before decreasing to 2.71% (CTS). These outcomes highlight the feasibility of valorising Irish crab shell waste into high-quality chitin and chitosan suitable for use in end-product applications, thus promoting the concepts of a circular economy.

The increase in crab meat exports increased the amount of crab shells discharged into the environment. Improper crab shell waste management can cause environmental concerns, public health issues, and increased oxygen requirements. This research aims to utilize crab shells from crab meat processing businesses in Indonesia into chitosan using demineralization, deproteination and deacetylation processes, with depolymerization as an additional process. Crab shell powder will be deproteinized with 3% NaOH and demineralized using an HCL solution. Furthermore, the deacetylation process was carried out for 8 hours at 140oC with 50% NaOH to produce chitosan. The resulting chitosan was then depolymerized using H2O2 at a concentration of 13%. The chitosan properties obtained by the deacetylation and depolymerization procedures were evaluated using X-ray diffraction (XRD), Fourier Transform Infrared (FTIR), and scanning electron microscope (SEM) tests. The results show that the depolymerization process exerts a beneficial effect on the chitosan product. The depolymerization treatment process involves the fragmentation of extended chitosan chains, which leads to the formation of smaller fragments and subsequently causes a decrease in the crystallinity, and particle size of the chitosan obtained. In additions, the results of this study indicate that the depolymerization process causes an increase in the degree of deacetylation of chitosan. The chitosan produced from the deacetylation and depolymerization processes produced chitosan with a degree of deacetylation of 81% and 91%, respectively. Increased the degree of deacetylation (DD, %) leads to reduced molecular weight, improved antimicrobial properties, higher water solubility, improved mechanical properties, and indicates better chitosan purity.

Effect of brachyura shell particles on glass fibre reinforced epoxy polymer composite

Objective: Socket preservation involves inserting graft material into the tooth socket to prevent alveolar bone resorption and sustain bone volume. Crab shells contain high protein, chitin, and calcium carbonate, making them a potential novel material for bone grafts. This study aims to evaluate the efficacy of chitosan and hydroxyapatite (HA) derived from small crab (Portunus pelagicus) shell waste on Bone Morphogenetic Protein (BMP)-2 expression during socket preservation procedures in experimental animals. Methods: Chitosan derived from crab shells is produced through demineralization, deproteinization, and deacetylation processes. HA is derived from diminutive crab shell through a reaction with calcium and phosphate precursors. A total of 36 male cavia cobaya guinea pigs underwent mandibular incisor extraction, divided into four groups: the chitosan powder from blue crab shells group, the chitosan gel and HA from blue crab shells group, the positive control given commercially available HA bone graft, and the negative control group given a placebo gel. On days 7, 14, and 21, sacrifices were performed to collect the mandibular jaw tissue of the cavia cobaya guinea pigs, and immunohistochemical examinations were conducted to determine BMP-2 expression. Data analysis was conducted using the Shapiro-Wilk test, ANOVA, and post hoc LSD test. Results: On days 7, 14, and 21, there was an increase in BMP-2 expression. Conclusion: Chitosan and hydroxyapatite derived from the shells of Portunus pelagicus have been demonstrated to effectively enhance BMP-2 expression.

The alarming rise of multidrug-resistant (MDR) bacteria, particularly Salmonella spp., has prompted an urgent search for alternative and synergistic antimicrobial strategies. In this study, a novel, green, and multicomponent nanocomposite was synthesized by integrating zinc oxide nanoparticles (ZnO NPs), chitosan (CS), the β-lactam antibiotic ceftazidime (CAZ), and the antidiabetic agent metformin (MTF) straightforward and economical manner. Bacillus subtilis strain ATCC 6633 was used to biosynthesize ZnO NPs, acting as a reliable bio-nanofactory. Various characterization techniques such as FTIR, XRD, TEM, and zeta potential analysis verified the successful integration and structural integrity of the ZnO NPs within the CS nanocomposite containing CAZ and MTF (ZnO/CS/CAZ/MTF). The FTIR spectra confirmed the presence of proteins that act as binding and supportive agents during the biosynthesis process. The produced nanomaterials have a significant positive surface charge of +28.61 mV, which enhances their stability. The particle sizes of the NPs ranged from 9.93 to 17.44nm. The nanocomposite exhibited strong antibacterial activity against MDR Salmonella enterica subsp., enterica serovar Typhi ATCC 19214, showing a significantly increased inhibition zone of 42mm and a greatly reduced minimum inhibitory concentration (MIC) value of 8µg/ml, compared to the separate components. The minimum bactericidal concentration (MBC) value was found to be consistent with the MIC result, emphasizing the potent bactericidal action of the prepared nanocomposite. In silico molecular docking further supported these findings by revealing favorable interactions between the nanocomposite constituents and the outer membrane proteins (OMPs) of Salmonella enterica serovar Typhimurium (PDB ID: 4W4M) and S. typhi (PDB ID: 3UU2). Key interactions included hydrogen bonding, ionic forces, and metal coordination with critical residues. Cytotoxicity assessment using WI-38 lung fibroblast cells revealed an IC₅₀ of 84.26µg/ml, indicating acceptable preliminary biocompatibility. The present study demonstrates the novelty of a ZnO-based multicomponent nanocomposite that uniquely integrates CAZ, MTF, and CS. This novel formulation exhibited synergistic antibacterial effects against multidrug-resistant Salmonella enterica alongside acceptable in vitro safety. The findings underscore the potential of microbially synthesized nanocomposites as promising candidates for combating antibiotic-resistant bacterial infections and support further preclinical investigations.

Objectives In order to better understand hydroxychavicol’s effectiveness as an oral antibacterial, its structural components were analyzed with respect to minimum inhibitory concentrations and minimum bactericidal concentrations against various oral bacteria. These structural components include the free hydroxy groups and allyl chain connected to hydroxychavicol’s benzene core. Methods Six structural analogs of hydroxychavicol were tested against a range of oral bacteria using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. MIC results were obtained using serial microdilution techniques in 96-well plates with resazurin dye as a colorimetric indicator. Aliquots within each MIC concentration range were then placed on appropriate agar medium and the minimum bactericidal concentration was determined as the lowest concentration with no observed colony growth. Key Findings A synergistic interaction was observed between the allyl chain and hydroxy groups on the benzene core of hydroxychavicol, which resulted in lower MICs against the tested oral bacteria. It was also found that a hydroxy group para to the allyl chain on the benzene ring resulted in more effective inhibition, with a MIC of &amp;lt;50 μg/mL against R. dentocariosa. Additionally, analytes possessing free hydroxy groups ortho to one another on the benzene ring resulted in MICs of 200-300 μg/mL or lower, whereas analytes with free hydroxy groups meta to one another on the benzene ring exhibited MICs of &amp;gt;1000 μg/mL. Conclusions This study helps elucidate the structural components responsible for hydroxychavicol’s effectiveness as an oral antibacterial. The findings herein help to understand the mechanism of hydroxychavicol’s antibacterial properties and will be helpful in the design and synthesis of more effective oral antibacterial treatments.

The crab shell (Portunus Pelagicus) wastes are constituted of major calcium carbonate (account for 70 wt. %) and have the potential to reuse as a starting biomaterial for precipitated calcium carbonate (PCC) products. The purpose of the present work was to synthesize PCC powder using crab shells based on the gas-solid-liquid carbonation route. In this experiment, crab shell powder was prepared by washing, drying, and subsequent grinding. The resulting powder was then calcined at 900 ℃ for 5 h before being used for the synthesis of the PCC product. Later, the calcined crab shell powder was dissolved with 2M HNO3 solution in a glass beaker with the pH solution of 12 set up by adding NH4OH. The solution was subsequently stirred magnetically for 30 min. The carbonation process ended when the PCC solid was obtained at a pH of 7. The crab shell powder, crab shell powder after calcination, and the obtained PCC solid were then characterized using XRD, SEM-EDX, and FTIR methods, respectively. The resulting PCC product has been shown to have calcium carbonate with the major vaterite phase. This experimental work demonstrated the potential application of crab shells for a low-cost biomaterial of future medical applications.

Background/Objectives: The increasing prevalence of antimicrobial-resistant bacteria highlights the need for improved methodologies to evaluate antimicrobial activity beyond conventional minimum inhibitory concentration testing. While resazurin-based assays are widely used for minimum inhibitory concentration determination due to their simplicity and sensitivity, minimum bactericidal concentration assessment still relies on labor-intensive colony-forming unit counting. The objective of this study was to develop and validate a resazurin-based microwell assay capable of determining both the minimum inhibitory concentration and the minimum bactericidal concentration without routine plate counting, thereby simplifying bactericidal evaluation. Methods: A two-step resazurin-based fluorescence assay was designed and performed in microplates. After determining the minimum inhibitory concentration using resazurin as a metabolic indicator, well-showing inhibited bacterial growths were subjected to a regrowth phase by transferring aliquots into fresh antimicrobial-free medium containing resazurin. This additional step allowed discrimination between reversible metabolic inhibition and irreversible bacterial death. The method was evaluated using ciprofloxacin and chloramphenicol against four bacterial species: Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Minimum bactericidal concentration values obtained using this assay were compared with those obtained through conventional colony counting on agar plates. Results: Minimum bactericidal concentration values obtained using the two-step fluorescence assay were fully concordant with the conventional colony-forming unit counting method for all tested antibiotics and bacterial species. Conclusions: The proposed two-step resazurin-based microwell assay represents a rapid, reliable, and less labor-intensive alternative for the determination of both the minimum inhibitory concentration and the minimum bactericidal concentration, with potential applications in clinical and industrial microbiology laboratories.

The antimicrobial activity of dehydroabietic acid (DHA) for its use as an antibiofilm agent was tested in this work. DHA was assayed against a collection of Gram-positive, Gram-negative sensitive and resistant bacteria and yeasts through the minimum inhibitory concentration (MIC), MIC with Bioburden challenge, minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), MBIC with Bioburden challenge and growth curve studies. Toxicological studies (Artemia salina, sulforhodamine B (SRB) assay) were done to assess if the compound had antimicrobial and not cytotoxic properties. Furthermore, microencapsulation and stability studies were carried out to evaluate the chemical behavior and stability of DHA. On MIC results, Gram-positive bacteria Staphylococcus aureus ATCC 1228 and Mycobacterium smegmatis ATCC 607 presented a high efficiency (7.81 µg/mL), while on Gram-negative bacteria the highest MIC value of 125 µg/mL was obtained by all Klebsiella pneumoniae strains and Escherichia coli isolate strain HSM 303. Bioburden challenge showed that MIC, MBIC and percentage biofilm inhibition (BI) values suffered alterations, therefore, having higher concentrations. MBIC values demonstrated that DHA has a higher efficiency against S. aureus ATCC 43866 with a percentage of BI of 75.13 ± 0.82% at 0.49 µg/mL. Growth curve kinetic profiles of DHA against S. aureus ATCC 25923 were observed to be bacteriostatic. DHA-alginate beads had a average size of 2.37 ± 0.20 and 2.31 ± 0.17 × 103 µm2 with an encapsulation efficiency (EE%) around 99.49 ± 0.05%, a protection percentage (PP%) of 60.00 ± 0.05% in the gastric environment and a protection efficiency (PE%) around 88.12 ± 0.05% against UV light. In toxicological studies DHA has shown IC50 of 19.59 ± 7.40 µg/mL and a LC50 of 21.71 ± 2.18%. The obtained results indicate that DHA is a promising antimicrobial candidate against a wide range of bacteria and biofilm formation that must be further explored.

Objective One of the properties of an ideal root canal irrigant is the ability to eradicate Enterococcus faecalis which is one of the most resistant microorganisms encountered in persistent peri-radicular lesions. The aim of this study was to test the in vitro antibacterial effectiveness of a naturally occurring agent called phytic acid (IP6) against E. faecalis and compare it to the antibacterial activities of clinically used irrigants: sodium hypochlorite (NaOCl), ethylenediaminetetraacetic (EDTA), phosphoric acid (PA) and chlorhexidine (CHX). Design The antimicrobial activities of 5% IP6, 5% NaOCl, 18% EDTA, 37% PA and 2% CHX against E. faecalis were determined using disk diffusion test. Minimum inhibitory concentration (MIC) was calculated by broth macrodilution method. The minimal bactericidal concentration (MBC) was determined for the used agents by culturing the clear broth of MIC tests. Results The results of agar diffusion test showed statistically significant differences between the groups. PA showed a larger zone when compared to other tested materials (p&lt; 0.05). There was no statistical significant difference between NaOCl, EDTA and CHX (p=0.098). IP6 showed the smallest zone of inhibition when compared to all groups (p&lt; 0.05).The recorded MIC and MBC values for IP6 were 0.156% and 0.625%; respectively. The MIC and MBC values for PA were 0.578% and 4.6% and for NaOCl 0.093% and 0.375%, respectively. EDTA MIC value was 0.14 % but it showed no bactericidal activity. CHX was excluded from MIC test as immediate precipitation and turbidity occurred after mixing CHX with Mueller Hinton Broth. Conclusions Within the limitation of this study and despite that IP6 showed the smallest zone of inhibition in agar diffusion test, the results of MIC and MBC indicated that IP6 exhibits in vitro antibacterial effect against E. faecalis at low concentrations.

Introduction: The use of antimicrobial properties of medicinal plants can solve common problems in the use of antibiotics. The aim of this study was to evaluate the antibacterial properties of the Mentha longifolia essence on bacteria isolated from hospital infections. Materials & methods: In this experimental study the Mentha longifolia essence was extracted using Clevenger device and its antimicrobial activity agar disk diffusion method for determination of bacterial sensitivity and dilution method for determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was used separately. Findings: The results of bacterial sensitivity test in the case of the standard antibiotics of tetracycline, amikacine, and also disks impregnated with Mentha longifolia essence showed that the diameter of the zone of inhibition caused by the compound being tested on four strains of isolates was far larger and this difference about Escherichia coli and Klebsiella isolates was significant (p less-than 0.05). Also the results of MIC and MBC tests indicated that the inhibition of bacterial growth by Mentha longifolia essence had a direct relationship with the amount of available Mentha longifolia essence in the dilution and increasing of Mentha longifolia essence in each dilution reduced the number of cultured bacterial colonies and no bacterial growth was observed in the dilution equivalent to MBC of Mentha longifolia essence. Discussion & conclusions: The results suggest that Mentha longifolia essence can be used as a source of cheap and accessible replacing chemical drugs to treat some bacterial infections.

The aim of the preset study was to determine the efficacy of Annona Senegalensis stem and root extracts against Salmonella typhimurium Shigella flexneri and Escherichia coli through the evaluation of bacterial sensitivity and determination of the minimum inhibitory and bactericidal concentration of the extracts against the test isolates. Plant materials were collected and duly authenticated. The methanoic and aqueous extracts prepared from the powdered forms were tested on the bacterial after cultural and biochemical identification of the isolates. The antibiotic sensitivity test was carried out using the Kirby Bauer disk diffusion method while chloramphenicol was used as the standard control. The Minimum Inhibitory Concentration (MIC) of the plant extracts were determined by broth dilution method while the Minimum Bactericidal Concentration (MBC) was determined by a method described using standard protocols. The ratio of MBC:MIC was computed to determine the bactericidal or bacteriostatic effects of the extracts. Data were analyzed using the Minitab 16 statistical package. Descriptive statistics (mean and standard error) and analysis of variance tools were applied while mean separation was done Fischer’s method at 5% level of significance. Antibacterial sensitivity test showed that the control test (Chloramphenicol) had significantly higher antibacterial sensitivity ( P&lt;0.05) than any of the plant extract. Minimum Inhibitory Concentration (MIC) of the plant extract ranged from 6.25 mg/ml to 25.0 mg/ml. The lowest MIC of 6.25mg/ml was observed in Salmonella typhimurium among all extract types. Root and stem had similar effects on the test organisms (P&gt;0.05) but, methanoic root extract had the lowest MBC of 6.25mg/ml against S.typhimurium and S. flexneri. Based on the MBC/MIC ratio, all extract types had bactericidal effects on Salmonella typhimurium and Shigella flexneri except aqueous root extract that showed bacteriostatic effect. Only the methanoic root and stem extracts exhibited bactericidal effects on Escherichia coli. Annona Senegalensis root and stem could possibly be explored commercially as an antibacterial agent against species of Salmonella, Shigellia and Escherichia.

In vitro antibacterial activities of five extracts from dietary medicinal plants were investigated by agar-well diffusion method (AWD), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against 13 foodborne pathogenic bacteria reference strains; four Gram positive bacteria including Staphylococcus aureus (NCINB 50080), Bacillus cereus (NCINB 50014), Citrobacter freundi (ATCC 8090) and Listeria innocua (ATCC 33090) as well as nine Gram negative bacterial reference strains including Escherichia coli (ATCC 11775), E. coli O157 (ATCC 700728), Salmonella typhimurium (ATCC 13311), Shigella boydii (ATCC 9207), Shigella sonnei (ATCC 25931), Shigella flexneri (ATCC&nbsp;12022), Pseudomonas aeruginosa (NCINB 50067), Klebsiella pneumoniae (NCTC 9633) and Proteus mirabilis (ATCC 14153). Four ethanolic extracts underwent acetone wash then analyzed for their principal components using gas chromatography-mass spectrometry (GC-MS), Oleamide was the predominant compound in onion, garlic, wheat germ and Nigella sativa which have great antibacterial effect. The tested acetone extracts exhibit variable antibacterial activity against foodborne pathogens which differ according to the compounds clarified in the GC-MS analysis. Garlic extract showed the best antibacterial activities, GC-MS analysis showed the presence of five compounds including; tetrasulfide, monosilane, oleamide, stearoylamide and vitamin E. &nbsp;Testing for the presence of 91 pesticides in the tested extracts using GC-MS analysis proved complete absence of pesticides which indicate that the antibacterial activities showed was due to the active components in the tested extracts and not due to the pesticides contaminants. Antimicrobial activities of plant extracts revealed that garlic has greatest inhibitory effect against S. aureus NCINB 50080 followed by S. Typhimurium ATCC 13311 with zone of inhibition 28 mm, 30 mm for AWDT and and 2.61 &micro;g/ml for MIC, respectively. The best hindrance abilities was shown with garlic extracts with mean zone of inhibition (23.46 mm) followed by onion (18.15 mm), wheat germ &nbsp;extract (17.38 mm), mint (17.15 mm) then Nigella sativa (15.69 mm). Results of MIC and MBC confirm the antibacterial activities of the tested extracts. &nbsp; Key words: Antibacterial activity, agar well diffusion test (AWDT) minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), seed, bulb, ethanolic extracts.

Antimicrobial effect of different herbal plant extracts against different microbial population.