Spread Plate Method Research Articles

ACTINOBACTERIAL DIVERSITY DETERMINATION IN MALAYSIAN BRIS (BEACH RIDGES INTERSPERSED WITH SWALES) SOIL BY CULTURE-DEPENDENT APPROACH

Globally, the rapid emergence of antibiotic-resistant bacteria poses a major threat to public health, threatening the effectiveness of antibiotics. Addressing the crisis of antimicrobial resistance requires urgency in new drug discovery and the effort in finding novel compounds is dramatically evolving. However, one of the main obstacles is the rediscovery of known compounds from natural resources. To overcome this challenge, exploring underexploited location for new resource of actinomycetes, such as Beach Ridges Interspersed with Swales (BRIS) soil in Malaysia, was selected. Soil samples from Jambu Series and Rudua Series in Setiu, Terengganu were collected and analysed. A total of 87 actinomycetes strains were isolated on yeast malt agar (ISP2), Actinomyces agar and Zobell marine agar, with the highest number from the Jambu series using serial dilution and spread plate method. Antimicrobial screening revealed that 31 isolates exhibited activity against ESKAPE pathogens, with 28 and 12 isolates showing activity against Gram-positive and Gram-negative bacteria, respectively. These isolates showed strong inhibiting response towards one or more ESKAPE pathogens. This study highlights the potential of BRIS soil as an underexploited habitat for discovering actinobacterial communities which could lead to the discovery of a new potential antimicrobial producer to serve as novel drug leads.

Antibacterial Activity and Mechanism of Rose Essential Oil against Aeromonas veronii isolated from Northern snakehead (Channa argus).

To investigate and identify the antibacterial action and mechanism of rose essential oil (REO) against Aeromonas veronii isolated from northern snakehead for the first time by the phenotypic and metabolic analysis. The two-fold broth microdilution and spread-plate method identified that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of REO against A. veronii were 1.25 μL mL-1 and REO impaired the growth in a concentration-dependent manner, indicating that REO possessed a significant bacteriostatic activity. Electron microscopy and live-dead cell staining found that REO caused a severe disruption of cellular morphology and increased the membrane permeability. Additionally, REO treatment induced the leakage of intracellular biomolecules such as proteins and nucleic acids from the bacteria. Metabolomics analysis showed that compared with the control, the REO treatment group exhibited a total of 190 differential metabolites (118 down-regulated and 72 up-regulated), which involved in the main metabolic pathways such as biotin metabolism, arginine biosynthesis, glutathione metabolism, lysine degradation, and histidine metabolism and the TCA cycle. These results verified that REO disturbed the metabolic processes of A. veronii to achieve the bacteriostatic effect. The REO exhibited the effective antibacterial activity against A. veronii via breaking the cellular structure, increasing the membrane permeation and disrupting the metabolic processes.

Decomposition of Polythene by Bacteria Isolated from Waste Disposal Sites in Gusau City

The accumulation of polythene can be hazardous and can cause various environmental problems. This work was aimed at studying the biodegradation of polythene by bacteria isolated from waste disposal areas in North West, (Zamfara state) Nigeria. A total of twenty soil samples were collected from four different locations, such as waste disposal areas, hospitals, roadsides, and petrol pumps station. Spread plate method was carried out on Nutrient agar for the growth of bacteria. A total of 13 bacteria were isolated from different areas. The total bacteria count in hospital ranged from 80 × 10-4 - 248 × 10-4 cfu/mL, roadsides ranged from 116 × 10-4 - 208 × 10-4 cfu/mL, petrol pump ranged from 88 × 10-4 - 184 × 10-4 cfu/mL and waste disposal areas ranged from 116 × 10-4 - 264 × 10-4 cfu/mL. The microbial species associated with the polythene materials were identified as Pseudomonas sp, Bacillus sp, Staphylococcus aureus, Providencia stuartii, Clostridium perfringes, Corynebacterium diphtheria, Listeria monocytogen. The bacteria isolates were screened for polyethylene degradation using mineral salt medium containing polyethylene as sole source of carbon and nitrogen for their ability to degrade polyethylene for the period of 2 months. The efficacy of the microbes in the degradation of polythene were analyzed using liquid culture method, among the bacteria Pseudomonas aeruginosa degraded 75% of polythene, Bacillus subtilis 71.4% in a period of 2 months.

Viability and antibacterial properties of Ba2-x AgxFeMoO6(x=0.0, 0.05) double perovskite oxides

Double perovskite Ba2FeMoO6 (BFMO) and doped with Ag ions were synthesized via the sol-gel method. The structural and antibacterial properties and viability of the samples were systematically investigated. The results of the Rietveld refinement indicated that both samples exhibited a single phase with cubic structure and space group Fm-3m. The substitution of Ag ions led to an increase in the unit cell and lattice parameters of BFMO, resulting in a change in the sample's morphology. The direct band gap (∼2eV) was measured for samples by Diffuse Reflectance Spectroscopy (DRS). Antibacterial activity of the Ba2FeMoO6 and Ba1.95Ag0.05FeMoO6 samples was assessed on the Gram-positive bacteria (Staphylococcus aureus) and the Gram-negative bacteria (Escherichia coli) using the spread plate method. The results showed that the samples were effective against Staphylococcus aureus but not against Escherichia coli. The sample doped with Ag ions showed a higher antibacterial effect than the pristine sample. An MTT assay of the normal fibroblast cell line treated with an Ag-doped sample showed a higher percentage of viability.

Novel antibacterial pullulan derivatives modified with quaternary phosphonium salts for infected wound treatments

The development of novel antibacterial agents is urgently needed to tackle bacterial infection, the major global issue menacing human health. Among them, polymeric quaternary phosphonium salts are worth noticing owing to their strong antibacterial activities and other merits including low bacterial drug resistance. Herein, pullulan modified with quaternary phosphonium salts (PQP) was synthesized using esterification reactions of pullulan and (5-carboxypentyl)triphenylphosphonium bromide (CPTPPB) mediated by N,N′‑carbonyldiimidazole (CDI), and was evaluated as novel antibacterial agents for treating wound infection for the first time. The chemical structures, chemical bonding, elemental compositions, crystalline properties and thermostability of PQP were systematically investigated. PQP exhibited in vitro antibacterial activities against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), which were unveiled by the spread plate method and possibly resulted from the damage of bacterial cell walls/membranes mediated by electrostatic and hydrophobic interactions according to preliminary mechanism studies. Weak cytotoxicity and excellent hemocompatibility of PQP at the effective bactericidal concentration were observed. Moreover, in the infected wound model of mice, PQP was capable of disinfecting the wound and accelerating the healing. We opine PQP in this work is promising for antibacterial applications and will inspire the synthesis of novel antibacterial agents derived from natural polymers.

Antibacterial properties enhancement tropical fruit waste biopolymer hydrogel loaded Nephelium Lappaceum leaf extract for sanitizing applications.

Current alcohol-based sanitizers present safety concerns and are not suitable for all applications. To address the issue, biopolymer hydrogels offer a safer, sustainable alternative due to biocompatibility, biodegradability, and customizable properties. In present study, carboxymethyl cellulose (CMC) was prepared from Durian fruit rind, a tropical fruit byproduct rich in polysaccharides and combined with the synthetic polymer Carbopol to form a hydrogel with homogenization technique. Rambutan (Nephelium lappaceum) leaf extract (RLE) as an antibacterial agent was analyzed for functional, morphological, antibacterial, and structural properties. Phytochemical analysis of RLE confirmed the presence of antibacterial compounds, while Minimum Inhibitory Concentrations (MIC) were 33.3μg/mL for Escherichia coli and 28.5μg/mL for Staphylococcus aureus. Additionally, Scanning Electron Microscopy showed significant disruptions in bacterial cell walls. Hydrogel incorporated RLE was produced with improved properties confirmed through viscosity, FT-IR, Disc-diffusion assay and spread plate method analysis. In general, Rambutan leaf extract significantly improves the antibacterial properties of biopolymer-based hydrogels, hence offering a promising, eco-friendly alternative to alcohol-based sanitizers.

Antimicrobial Sub-MIC induces Staphylococcus aureus biofilm formation without affecting the bacterial count

BackgroundBiofilm formation is an essential virulence factor that creates a highly protected growth mode for Staphylococcus aureus (S. aureus) to survive in any hostile environment. Antibiotic sub-minimal inhibitory concentration (sub-MIC) may modulate the biofilm formation ability of bacterial pathogens, thereby affecting bacterial pathogenesis and infection outcomes. Intense antimicrobial therapy to treat biofilm-associated infections can control the pathogenic infection aggravation but cannot guarantee its complete eradication.ObjectiveThis study aimed to assess the sub-MICs effect of 5 different antimicrobial classes on biofilm-forming capacity among Staphylococcus aureus clinical isolates using three different biofilm quantitation techniques.MethodsIn this study, the effects of 5 different antimicrobial agents, namely, azithromycin, gentamicin, ciprofloxacin, doxycycline, and imipenem, at sub-MICs of 12.5%, 25%, and 50% were tested on 5 different clinical isolates of S. aureus. The biofilms formed in the absence and presence of different antimicrobial sub-MICs were then assessed using the following three different techniques: the crystal violet (CV) staining method, the quantitative PCR (qPCR) method, and the spread plate method (SPM).ResultsBiofilm formation was significantly induced in 64% of the tested conditions using the CV technique. On the other hand, the qPCR quantifying the total bacterial count and the SPM quantifying the viable bacterial count showed significant induction only in 24% and 17.3%, respectively (Fig. 1). The difference between CV and the other techniques indicates an increase in biofilm biomass without an increase in bacterial growth. As expected, sub-MICs did not reduce the viable cell count, as shown by the SPM. The CV staining method revealed that sub-MICs of imipenem and ciprofloxacin had the highest significance rate (80%) showing an inductive effect on the biofilm development. On the other hand, doxycycline, azithromycin, and gentamicin displayed lower significance rates of 73%, 53%, and 47%, respectively.ConclusionExposure to sub-MIC doses of antimicrobial agents induces the biofilm-forming capacity of S. aureus via increasing the total biomass without significantly affecting the bacterial growth of viable count.

A Simple High-Throughput Technology for Microorganism Detection and Quantitative Analysis.

Normal and damaged microorganisms are related to food safety. The colony-forming unit (CFU) assay and viability of microorganisms have broad applications in food. Traditionally, the CFU assay has been the benchmark for assessing microbial viability across various fields. However, the normal and damaged microorganisms cannot be distinguished. Here, we introduce an improved technology for foods that uses a visible absorbance microplate reader platform for high-throughput quantitative analysis of microbial lag time, doubling time, and CFU. This platform utilizes a 96-well plate and a microplate reader to accurately determine the viable cell number from a five-microliter sample. It boasts the capability to measure a dynamic range spanning from five to seven orders of magnitude, significantly reducing the time required by over 20-fold in comparison to traditional spread plate methods. Additionally, it demonstrates a remarkable ability to detect a single cell within a well. A mild temperature treatment for cell viability detection was implemented and was able to reflect the real microbial quality. Consequently, the high-throughput method as an improved technology provides essential technical support for microbial detection.

Total Plate Count Analysis and Food-Contaminating Bacterial Identification of Smoked Tatihu (Thunnus albacares) Sold in Several Traditional Markets in Ambon, Indonesia

Highlights:1. This research provides important information regarding the food safety of smoked tatihu (yellowfin tuna) sold in several traditional markets in Ambon, Indonesia.2. We find that smoked tatihu fish samples examined at 2 of 3 traditional markets in Ambon city are unsafe for consumption according to the Indonesian National Standard due to bacterial contamination.3. The findings indicate that smoked fish can be contaminated by Gram-positive bacteria, such as Staphylococcus gallinarum, Staphylococcus sciuri, Rothia kristinae, and Staphylococcus pseudintermedius. Abstract The large marine area and abundant fish resources of Maluku Province, Indonesia, are in contrast to the poor hygiene of its traditional markets, which can cause microbial contamination and taint processed products, such as smoked tatihu (yellowfin tuna). In Ambon city, Maluku, Indonesia, no research had been conducted concerning total plate count analysis and food-contaminating bacterial identification that could guarantee the microbiological safety of smoked tatihu. Therefore, this study aimed to assess the microbiological quality of smoked tatihu according to Indonesian National Standards (INS 2725:2013) and to identify any presence of food-contaminating bacteria. This research was a quantitative descriptive study with a true experimental laboratory approach. The samples used were smoked tatihu collected from three traditional markets in Ambon, Indonesia. The spread plate method was used in the isolation process, while the total plate count analysis was performed to estimate the quantity of colonies on each petri dish. Bacterial identification was carried out macroscopically and microscopically. The microscopic identification involved Gram staining to determine the shape and color of the bacteria. Additionally, the bioMérieux VITEK 2 Compact system was utilized for biochemical identification to ascertain the species of bacteria present. The results revealed that the colony counts in smoked tatihu from the Mardika market and Hative Kecil market were 1.1 x 104 CFU/g and 8.2 x 106 CFU/g, respectively. However, smoked tatihu from the Batu Meja market had an excessive number of colonies that were difficult to quantify. The contaminating bacteria were identified as Staphylococcus gallinarum, Staphylococcus sciuri, Rothia kristinae, and Staphylococcus pseudintermedius. In conclusion, smoked tatihu fish from the Mardika market are considered safe for consumption as the microbiological parameters do not exceed the Indonesian National Standards, whereas those obtained from the Hative Kecil and Batu Meja markets are unsafe for consumption due to the excessive presence of food-contaminating bacteria.

Antibacterial and cytotoxicity properties of a polyherbal mouthwash containing Achyranthes aspera and Trachyspermum ammi against selected periodontal pathogens

BackgroundChlorhexidine (CHX) is considered as a gold standard for its antibacterial efficacy and substantivity in chemical plaque control. However, some adverse effects are associated with its prolonged use. Herbal medicines like Achyranthes aspera and Trachyspermum ammi have been used in many clinical conditions, and they appear to be a valuable substitute next to CHX in the management of periodontal diseases. ObjectiveThis in vitro study was designed to assess and compare the antibacterial potential and cytotoxic effects of novel polyherbal mouthwash containing A. aspera and T. ammi with 0.2% CHX mouthwash against Porphyromonas gingivalis, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans. MethodsEthanolic extracts of A. aspera and T. ammi were prepared by the Soxhlet apparatus method and were subjected to preliminary phytochemical screening. The individual plant extracts and the plant extract mixture (PEM) of A. aspera and T. ammi in the ratio of 1:1, 2:1, 1:2 (w/v) were assessed for minimum inhibitory concentration (resazurin microtitre assay) and minimum bactericidal concentration (spread plating method) against selected periodontal pathogens in comparison to CHX. The polyherbal mouthwash was assessed for zone of inhibition (well diffusion method) and cytotoxicity (MTT assay) on adult human gingival fibroblasts. All the experiments were performed in triplicate. ResultsThe antibacterial activity was evident in the PEMs, and polyherbal mouthwash against tested periodontal pathogens and was comparable to CHX. The cytotoxicity assay findings confirmed that polyherbal mouthwash exhibited 82.1% of surviving cells which proved good biocompatibility. ConclusionA. aspera and T. ammi based mouthwash possess comparable antibacterial activity against periodontal pathogens when compared to CHX.

Isolation and Identification of Swan (Cygnus olor) Digestive Tract Cellulothic Bacteria to Support Fiber Degradation

Cellulolytic bacteria are bacteria that produce cellulase enzymes capable of degrading cellulose substrates. This study aimed to isolate and identify as well as measure the cellulase enzyme activity of cellulolytic bacteria isolated from the digestive tract of swans (Cygnus olor) in Mataram. The bacteria were isolated using de Man Rogosa Sharpe (MRS) selective media with the spread plate method. Then, the extracellular enzyme activity test was conducted by growing selected pure isolates on 1% Carboxy Methyl Cellulose (CMC 1%) media, followed by pouring 0.1% congo red solution and rinsing with 1M NaCl solution to determine cellulolytic activity (cellulolytic potential indicated by the appearance of a clear zone around the colony) and measuring the cellulolytic index. The bacterial isolation results obtained 4 isolates with the potential to support fiber degradation in animal feed. The highest cellulolytic index was produced by the isolate coded S1, reaching 34.5 mm, while the lowest cellulolytic index was produced by 2 isolates coded J21 and I2, reaching 18.8 mm.

Photocatalytic Degradation of Paracetamol and Antibacterial Activity of La-Modified TiO2 Obtained by Non-Hydrolytic Sol–Gel Route

The current study aims to synthesize and analyze both pure and La-doped TiO2, and evaluate the photocatalytic and antibacterial activity of as-prepared samples. Doped and undoped samples were prepared by the non-hydrolytic sol–gel method from titanium(IV) chloride, benzyl alcohol, and lanthanum(III) nitrate followed by thermal treatment. Lanthanum content in synthesized samples was 0.4, 1, and 5 mol%. The resulting nanopowders’ structure and morphology were described using XRD, IR, and UV–Vis analysis. The average particle sizes of pure and doped TiO2 were about 6–15 nm and anatase was found to be a dominant crystalline phase in the samples. It was observed that particle sizes decreased on increasing La content. The photocatalytic activity of the pure and La-doped sol–gel powders was estimated in the decomposition of paracetamol in distilled water using ultraviolet light illumination. Doping with lanthanum ions has been shown to increase the photocatalytic properties on the degradation of paracetamol. Furthermore, the annealed catalysts (pure and La3+ doped) showed increased photocatalytic activity and degradation of the analgesic in comparison with non-annealed materials. In both cases, the highest photocatalytic efficiency is observed at the optimal La3+ (1 mol%) concentration. The antimicrobial activity of 1 mol% La/TiO2 was tested against a reference strain E. coli in the presence of ultraviolet light and in dark conditions. The number of viable bacterial cells was determined by a spread plate method, and kill curves were performed. The results showed that photoactivated 1 mol% La/TiO2 exhibited a strong bactericidal effect, and in concentration, 1 mg/mL efficiently killed bacteria at an initial cell density of about 105 colony forming units in 1 mL within 15 min.

In Vitro Assay of Lytic Bacteriophage to Suppress the Growth of Ralstonia syzygii subsp. indonesiensis, the Causal Pathogen of Potato Wilt Disease

Bacterial wilt disease, the one of the major diseases of potatoes, caused by Ralstonia syzygii subsp. indonesiensis (Rsi). Many efforts have been made to control bacterial wilt disease, including physical control, chemicals, and the use of bacteriophages. Previous studies have shown that bacteriophage application in controlling plant diseases is a fast-expanding area and has great potential to replace chemical methods. This study aims to determine the potential of lytic bacteriophage in suppressing the growth of Rsi in vitro. This study used a Non-Factorial, Completely Randomized Design with 3 replications and 6 treatment levels: R1 (Rsi isolate 1 without bacteriophage), R2 (Rsi isolate 1 with bacteriophage), R3 (Rsi isolate 2 without bacteriophage), R4 (Rsi isolate 2 with bacteriophage), R5 (Rsi isolate 3 without bacteriophage), and R6 (Rsi isolate 3 with bacteriophage). The results showed that bacteriophage could reduce the Rsi population at 24 hours by looking at the optical density (OD) value of 600 nm wavelength and growing on NA medium using the spread-plate method. The best treatment was at R6 with a population of 1 x 108cfu/ml Rsi. This research suggests that bacteriophage has the potential to suppress the growth of Rsi, which causes potato bacterial wilt disease, in vitro.

A New Nanoplatform Under NIR Released ROS Enhanced Photodynamic Therapy and Low Temperature Photothermal Therapy for Antibacterial and Wound Repair.

Skin injury, often caused by physical or medical mishaps, presents a significant challenge as wound healing is critical to restore skin integrity and tissue function. However, external factors such as infection and inflammation can hinder wound healing, highlighting the importance of developing biomaterials with antibiotic and wound healing properties to treat infections and inflammation. In this study, a novel photothermal nanomaterial (MMPI) was synthesized for infected wound healing by loading indocyanine green (ICG) on magnesium-incorporated mesoporous bioactive glass (Mg-MBG) and coating its surface with polydopamine (PDA). In this study, Mg-MBG and MMPI was synthesized via the sol-gel method and characterized it using various techniques such as scanning electron microscopy (SEM), the energy dispersive X-ray spectrometry (EDS) system and X-ray diffraction (XRD). The cytocompatibility of MMPI was evaluated by confocal laser scanning microscopy (CLSM), CCK8 assay, live/dead staining and F-actin staining of the cytoskeleton. The antibacterial efficiency was assessed using bacterial dead-acting staining, spread plate method (SPM) and TEM. The impact of MMPI on macrophage polarization was initially evaluated through flow cytometry, qPCR and ELISA. Additionally, an in vivo experiment was performed on a mouse model with skin excision infected. Histological analysis and RNA-seq analysis were utilized to analyze the in vivo wound healing and immunomodulation effect. Collectively, the new photothermal and photodynamic nanomaterial (MMPI) can achieve low-temperature antibacterial activity while accelerating wound healing, holds broad application prospects.

Isolasi dan Identifikasi Bakteri Asam Laktat dari Hasil Fermentasi Limbah Organik Kulit Buah (Eco-Enzyme)

The aim of this research is the isolation and identification based on the morphological characteristics of lactic acid bacteria in the fermentation of organic waste from fruit peels. Isolation of Lactic Acid Bacteria was carried out using the spread plate method on de Man, Rogosa, and Sharpe Agar (MRSA) media. The culture was incubated at 37 °C for 48 hours. The growing colonies were observed for morphological characteristics, and bacterial Gram staining was carried out. The results of the research showed that 10 bacterial isolates were obtained from the isolation of BAL from ecoenzymes on de Man, Ragosa, and Sharpe Agar (MRSA) media, namely E-BAL 01, E-BAL 02, E-BAL O3, E-BAL 04, E-BAL 05, E-BAL 06, E-BAL 07, E-BAL 08, E-BAL 09, and E-BAL 10, and 10 isolates were characterized as Gram-positive with a bacillary cell form. In conclusion, 10 isolates were found with characteristics that supported MRSA media, namely isolates of lactic acid bacteria of the genus Lactobacillus. This is confirmed by the results of biochemical tests, namely that it is immobile, does not produce the catalase enzyme, and does not have the ability to produce gas. Keywords: Lactic Acid Bacteria (LAB), Ecoenzymes, Fruit Skin

Formulation of silver-loaded zeolitic imidazole framework-67 and its antibacterial coating on titanium surface for bioimplant applications

Artificial bioimplant infections caused by bacteria have been a persistent problem that requires re-surgery and is harmful to human tissue. A facile, inexpensive and stable antibacterial agent coating can be a significant alternative. The present study attempted to construct titanium (Ti) implant coated with silver loaded zeolitic imidazole framework (ZIF)-67 (Ag@ZIF-67)-chitosan composite through a casting technique. The as-prepared nanocomposite and its coating were subjected to various characterization techniques, including XPS, XRD, SEM, and TEM to understand the material properties. Antibacterial activity was assessed through a standard spread-plate method, and the cytocompatibility of the fabricated materials was examined upon the skin fibroblast (L929) cell line. Corrosion studies were performed by using anodic polarization and impedance methods. The bactericidal efficacy of the coating was validated in vivo by utilizing a subcutaneous mouse model. The nanocomposite and coating exhibited a significant antibacterial efficiency of above 95 % against S. aureus and E. coli as the combined release of Co2+ and Ag+ ions entailed cellular membrane disintegration. The Ag@ZIF-67 composite revealed negligible cytotoxicity and its coating exhibited strong bone cell attachment. In contrast to pure Ti surface, the Ag@ZIF-67-chitosan coated Ti exhibited a minor reduction in corrosion resistance and modifications to its passivation properties as a result of the formation of a galvanic cell. A remarkable antibacterial effect was evinced through the in vivo experiments demonstrating a complete disinfection with 99 % of S. aureus lysis with no bacterial growth in the nearby healthy tissues. In conclusion, the current results demonstrated that the fabricated Ag@ZIF-67 could be applied as an antibacterial coating material of Ti for bioimplant applications.

Effect of Specific Spoilage Organisms on the Degradation of ATP-Related Compounds in Vacuum-Packed Refrigerated Large Yellow Croaker (Larimichthys crocea).

This study examined the spoilage potential of specific spoilage organisms on the degradation of adenosine triphosphate (ATP)-related compounds in vacuum-packed refrigerated large yellow croaker. The total viable count (TVC), ATP-related compounds and related enzymes of vacuum-packed refrigerated large yellow croaker inoculated with different bacteria (Pseudomonas fluorescens and Shewanella putrefaciens) were characterized using the spread plate method, high-performance liquid chromatography and assay kits, respectively. Results indicated that the TVC for both control and Shewanella putrefaciens groups reached spoilage levels at days 9 and 15, respectively. The changes of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine deaminase activity across all groups showed no significant difference attributable to microbial growth. The results suggested that ATP to inosine monophosphate (IMP) degradation primarily occurs via fish's endogenous enzymes, with minimal microbial involvement. On day 12, the IMP content in fillets inoculated with Pseudomonas fluorescens (0.93 μmol/g) was half higher than in those inoculated with Shewanella putrefaciens (0.57 μmol/g). Both spoilage organisms facilitated IMP degradation, with Shewanella putrefaciens making a more substantial contribution. Analysis of K values and correlation coefficients revealed that Shewanella putrefaciens was the primary factor in the freshness loss of refrigerated vacuum-packed large yellow croaker. These findings offer a reference for understanding quality changes in refrigerated large yellow croaker, especially regarding umami degradation at the microbial level.

Comparing the population density, colony diversity, and phosphate solubilization ability between rice root endophytic and rice rhizospheric soil bacteria from organic wetland

Abstract Phosphate-solubilizing bacteria (PSB) play important roles in the agricultural systems. The present study aims to compare the population density, colony diversity, and phosphate solubilization ability between rice root endophytic bacteria (RRoEB) and rice rhizospheric soil bacteria (RRzSB) from organic wetland. By spread plate method, bacteria were isolated directly using Pikovskaya’s agar and indirectly through other media, namely Nutrient Agar (NA), Starch Casein Agar (SCA), Jensen, and Yeast Extract Mannitol Agar (YEMA). Each distinct colony based on the morphology on the respective media was screened for phosphate solubilization ability by subculturing on Pikovskaya’s agar. The phosphate solubilizing index (PSI) was measured from the halo zone formation. The results showed that by direct isolation, 6 RRoEB and 5 RRzSB were able to phosphate solubilization. By indirect isolation from NA, SCA, Jensen, and YEMA, 2, 3, 1, and 3 isolates of RRoEB and 4, 3, 1, and 3 isolates of RRzSB showed the capability as phosphate solubilizer. Although the highest colony diversity of PSB was obtained from direct isolation, however the highest PSI was yielded from indirect isolation, namely isolates J1R1 (3.53) and Y1S1 (2.48). The highest PSI from direct isolation was obtained by P1R5 (1.90) and P1S1 (1.51).

Optimal Extraction Technique and Antibacterial Properties of Essential Oil Through Magnolia sieboldii K. Koch Leaves

To provide scientific support for the exploration and application of the essential oil derived through leaves of Magnolia sieboldii K. Koch, this paper explores extraction technology optimization, composition analysis, and antibacterial properties for the essential oil derived through Magnolia sieboldii K. Koch leaves. Essential oil derived through Magnolia sieboldii K. Koch leaves was extracted by adopting hydrodistillation. With single-factor test and reaction surface improvement methods, the extraction technology of essential oil derived through Magnolia sieboldii K. Koch leaves was optimized. The structure of chemicals has been explored through the use of gas chromatographmass spectrometer (GC-MS) technology. By conducting Kirby-Bauer test and applying spread plate method, the antibacterial properties for essential oil has been measured. The liquid-solid ratio was 7.15 mL/g, the dilution period was 4.00 h, and the NaCl mass percentage was 2.26% as a consequence of the response surface optimization. The rate about gathered essential oil under such circumstances was 0.31537%. By performing chemical composition analysis, 43 compounds were measured from essential oil. Thel oil was sensitive to Staphylococcus aureus, Bacillus subtilis, and Escherichia coli with inhibitory area diameters of 17.27, 10.85, and 9.56 mm, respectively. The minimum inhibitory concentration (MIC) values for the three bacteria strains were 2.5 × 10–6, 5 × 10–6, and 10 × 10–6 g/mL, respectively. The optimum extraction rate of essential oil derived through leaves of Magnolia sieboldii K. Koch by hydro distillation is 0.35%, with the cancer-fighting substance β-elemene reaching up to a maximum of 42.53%. Simultaneously, the essential oil derived through leaves of Magnolia sieboldii K. Koch exhibits notable antibacterial effect and large development potential, suggesting the comprehensive development and utilization.

Occurrence of Antimicrobial-Resistant Bacteria in Intestinal Contents of Wild Marine Fish in Chile.

Antimicrobial-resistant bacteria (ARB) from the intestinal contents of wild fish may have a relevant ecological significance and could be used as indicators of antimicrobial-resistance dissemination in natural bacterial populations in water bodies impacted by urban contamination. Thus, the occurrence of ARB in the intestinal contents of pelagic and demersal wild fishes captured in anthropogenic-impacted Coquimbo Bay in Chile was studied. Culturable counts of total and antimicrobial-resistant bacteria were determined by a spread plate method using Trypticase soy agar and R2A media, both alone and supplemented with the antimicrobials amoxicillin, streptomycin, florfenicol, oxytetracycline and ciprofloxacin, respectively. Heterotrophic plate counts of pelagic and demersal fishes ranged from 1.72 × 106 CFU g-1 to 3.62 × 109 CFU g-1, showing variable proportions of antimicrobial resistance. Representative antimicrobial-resistant isolates were identified by 16S rRNA gene sequencing, and isolates (74) from pelagic fishes mainly belonged to Pseudomonas (50.0%) and Shewanella (17.6%) genera, whereas isolates (68) from demersal fishes mainly belonged to Vibrio (33.8%) and Pseudomonas (26.5%) genera. Antimicrobial-resistant isolates were tested for susceptibility to 12 antimicrobials by an agar disk diffusion method, showing highest resistance to streptomycin (85.2%) and amoxicillin (64.8%), and lowest resistance to oxytetracycline (23.2%) and ciprofloxacin (0.7%). Only furazolidone and trimethoprim/sulfamethoxazole were statistically different (p < 0.05) in comparisons between isolates from pelagic and demersal wild fishes. Furthermore, an important number of these isolates carried plasmids (53.5%) and produced Extended-Spectrum-β-lactamases (ESBL) (16.9%), whereas the detection of Metallo-β-Lactamases and class 1-integron was rare. This study provides evidence that wild fish are important reservoirs and spreading-vehicles of ARB, carrying plasmids and producing ESBLs in Chilean marine environments.