Agar Dilution Research Articles

Sliding motility of Bacillus cereus mediates vancomycin pseudo-resistance during antimicrobial susceptibility testing.

The glycopeptide vancomycin is the antimicrobial agent-of-choice for the treatment of severe non-gastrointestinal infections with members of Bacillus cereus sensu lato (s.l.). Recently, sporadic detection of vancomycin-resistant phenotypes emerged, mostly for agar diffusion testing such as the disc diffusion method or gradient test (e.g. Etest®) method. In this work, we were able to disprove a preliminarily assumed high resistance to vancomycin in an isolate of B. cereus s.l. using broth microdilution and agar dilution. Microscopic imaging during vancomycin susceptibility testing showed spreading towards the inhibition zone, which strongly suggested sliding motility. Furthermore, transcriptomic analysis using RNA-Seq on the nanopore platform revealed several key genes of biofilm formation (e.g. calY, tasA, krsEABC) to be up-regulated in pseudo-resistant cells, substantiating that bacterial sliding is responsible for the observed mobility. Down-regulation of virulence (e.g. hblABCD, nheABC, plcR) and flagellar genes compared with swarming cells also confirmed the non-swarming phenotype of the pseudo-resistant isolate. The results highlight an insufficiency of agar diffusion testing for vancomycin susceptibility in the B. cereus group, and reference methods like broth microdilution are strongly recommended. As currently no guideline mentions interfering phenotypes in antimicrobial susceptibility testing of B. cereus s.l., this knowledge is essential to obtain reliable results on vancomycin susceptibility. In addition, this is the first report of sliding motility undermining accurate antimicrobial susceptibility testing in B. cereus s.l. and may serve as a basis for future studies on bacterial motility in susceptibility testing and its potential impact on treatment efficacy.

Effects of efflux pumps on antifungal activity of chitosan against Candida albicans

ABSTRACT Background Antifungal resistance is a major problem, commonly caused by drug-efflux pump overexpression. To evaluate if chitosan could be effective in drug-resistant Candida infections, we investigated the effects of efflux pumps on antifungal activity of chitosan. Materials and Methods The minimal fungicidal concentration (MFC) of oligomer (7–9 kD) and polymer (900–1,000 kD) chitosan against Saccharomyces cerevisiae and Candida albicans were evaluated by broth and agar dilution methods. The MFCs of S. cerevisiae with single deletion of efflux pump genes, with deletion of seven efflux pumps (AD∆), and AD∆ overexpressing C. albicans efflux pump genes (CDR1, CDR2 and MDR1) were determined. C. albicans with homozygous deletions of CDR1 and of CDR2 were generated using CRISPR-Cas9 system and tested for chitosan susceptibility. Results While deleting any individual efflux pump genes had no effect on chitosan susceptibility, simultaneous deletion of multiple pumps (in AD∆) increased sensitivity to both types of chitosan. Interestingly, the overexpression of CDR1, CDR2 or MDR1 in AD∆ barely affected its sensitivity. Moreover, C. albicans with homozygous deletions of CDR1 and/or CDR2 showed similar sensitivity to wildtype. Conclusion Thus, C. albicans susceptibility to chitosan was not affected by drug-efflux pumps. Chitosan may be a promising antifungal agent against pump-overexpressing azole-resistant C. albicans.

Antagonistic activity of Trichoderma sp. against pathogens in the leaves of Allium ascalonicum L.

Pathogenic fungi pose constraints and reduce shallot production. Trichoderma sp. is an antagonistic fungus capable of controlling pathogen growth in shallots. The aim of this study was to determine the characteristics of Trichoderma sp. and pathogens in shallots and to assess the antagonistic ability of Trichoderma sp. against shallot pathogens (Allium ascalonicum L.). Trichoderma sp. and shallot pathogens were isolated using a serial dilution agar plate method using potato dextrose agar medium with 7-day incubation. The antagonistic activity of Trichoderma sp. against shallot pathogens was evaluated based on the dual culture method. In this study, we successfully isolated Trichoderma harzianum from the shallot leaf and its root systems. Moreover, four morphologically distinctive pathogens from shallot roots and leaves were successfully isolated (l Aspergillus sp., Colletotrichum sp., Phytium sp. and Penicillium sp. T. harzianum was found to have the ability to inhibit 23.45% growth of Aspergillus sp, 26.19% growth of Colletotrichum sp., 75.40% growth of Phytium sp., and 40.38% growth of Penicillium sp. In conclusion, the isolated T. harzianum had a strong antagonistic activity against some pathogens in the shallot, but the activity was weak against some others.

Biological Activities of Ethanol Extracts of Hericium erinaceus Obtained as a Result of Optimization Analysis.

Mushrooms are one of the indispensable elements of human diets. Edible mushrooms stand out with their aroma and nutritional properties. In this study, some biological activities of the wild edible mushroom Hericium erinaceus were determined. In this context, firstly, the most suitable extraction conditions of the fungus in terms of biological activity were determined. First, 64 different experiments were performed with the Soxhlet device under 40-70 °C extraction temperature, 3-9 h extraction time, and 0.5-2 mg/mL extraction conditions. As a result, a total antioxidant status (TAS) analysis was performed, and the extraction conditions were optimized so that the objective function was the maximum TAS value. The data obtained from the experimental study were modeled with artificial neural networks (ANNs), one of the artificial intelligence methods, and optimized with a genetic algorithm (GA). All subsequent tests were performed using the extract obtained under optimum extraction conditions. The antioxidant capacity of the mushroom was assessed using Rel assay kits and the DPPH and FRAP techniques. The agar dilution method was used to measure the antimicrobial activity. The anti-Alzheimer activity was assessed based on the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The antiproliferative activity was assessed against the A549 cancer cell line. The total phenolic content was measured using the Folin-Ciocalteu reagent. The measurement of total flavonoids was conducted using the aluminum chloride test. LC-MS/MS equipment was used to screen for the presence of standard chemicals. The optimum extraction conditions were found to be a 60.667 °C temperature, 7.833 h, and 1.98 mg/mL. It was determined that the mushroom has high antioxidant potential. It was determined that the substance was successful at combating common bacterial and fungal strains when used at dosages ranging from 25 to 200 µg/mL. The high antiproliferative effect of the substance was attributed to its heightened concentration. The anti-AChE value was found to be 13.85 μg/mL, while the anti-BChE value was confirmed to be 28.00 μg/mL. The phenolic analysis of the mushroom revealed the presence of 13 chemicals. This investigation found that H. erinaceus exhibits robust biological activity when extracted under appropriate circumstances.

Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

Characterization of antibiotic and disinfectant susceptibility in biofilm-forming Acinetobacter baumannii: A focus on environmental isolates.

The clinical role of Acinetobacter baumannii has been highlighted in numerous infectious syndromes with a high mortality rate, due to the high prevalence of multidrug-resistant (MDR) isolates. The treatment and eradication of this pathogen is hindered by biofilm-formation, providing protection from noxious environmental factors and antimicrobials. The aim of this study was to assess the antibiotic susceptibility, antiseptic susceptibility and biofilm-forming capacity using phenotypic methods in environmental A.baumannii isolates. One hundred and fourteen (n = 114) isolates were collected, originating from various environmental sources and geographical regions. Antimicrobial susceptibility testing was carried out using the disk diffusion method, while antiseptic susceptibility was performed using the agar dilution method. Determination of biofilm-forming capacity was carried out using a microtiter-plate based method. Resistance in environmental A. baumannii isolates were highest for ciprofloxacin (64.03%, n = 73), levofloxacin (62.18%, n = 71) and trimethoprim-sulfamethoxazole (61.40%, n = 70), while lowest for colistin (1.75%, n= 2). Efflux pump overexpression was seen in 48.25% of isolates (n = 55), 49.12% (n = 56) were classified as MDR. 6.14% (n = 7), 9.65% (n = 11), 24.65% (n = 28) and 59.65% (n = 68) of isolates were non-biofilm producers, weak, medium, and strong biofilm producers, respectively. No significant differences were observed between non-MDR vs. MDR isolates regarding their distribution of biofilm-producers (P = 0.655). The MIC ranges for the tested antiseptics were as follows: benzalkonium chloride 16-128 μg mL-1, chlorhexidine digluconate 4-128 μg mL-1, formaldehyde 64-256 μg mL-1 and triclosan 2-16 μg mL-1, respectively. The conscientious use of antiseptics, together with periodic surveillance, is essential to curb the spread of these bacteria, and to maintain current infection prevention capabilities.

Extraction Kinetics and Antifungal Activities of Essential Oil from the Aerial Parts of Gros Baume (Hyptis suaveolens) Against Three Fungal Strains in Côte d'Ivoire

Introduction: Microscopic fungi colonize many food substrates resulting in considerable economic losses. Their development leads to an alteration in the organoleptic quality of the food, and produces mycotoxins. The aim of the present work was to evaluate the extraction kinetics and in vitro antifungal activity of the essential oil of Hyptis suaveolens on the growth of three toxigenic fungal strains. Methodology: The essential oil was extracted by steam distillation using a Clevenger-type device. It was tested for in vitro antifungal activity against three mold strains (Aspergillus flavus, Aspergillus niger and Fusarium sp) using the agar dilution method. Results: The results of the extraction kinetics evaluation show that in fifty minutes of distillation, almost all the essential oil contained in the aerial parts of H. suaveolens is recovered. In vitro antifungal tests showed a slight slowdown in mycelial growth for the three strains studied. However, the antifungal parameters determined show that these strains are sensitive to 50 μL/mL with a CMF of 100 μL/mL. This is just a simple fungistatic effect of this extract on the mycelial growth of the strains tested. Aspergillus strains seem to be slightly more affected than Fusarium strains. Conclusion: The antifungal potential of H. suaveolens essential oil does not offer a novel approach to the control of these three fungal strains.

Evaluation of hemostatic, anti-inflammatory, wound healing, skin irritation and allergy, and antimicrobial properties of active fraction from the ethanol extract of Chromolaena odorata (L.) R.M. King & H. Rob

Ethnopharmacological relevanceChromolaenaodorata (L.) R.M. King & H. Rob, a perennial herb, has been traditionally utilized as a herbal remedy for treating leech bites, soft tissue wounds, burn wounds, skin infections, and dento-alveolitis in tropical and subtropical regions. Aim of the studyThe present study was to analyze the active fraction of C. odorata ethanol extract and investigate its hemostatic, anti-inflammatory, wound healing, and antimicrobial properties. Additionally, the safety of the active fraction as an external preparation was assessed through skin irritation and allergy tests. Materials and methodsThe leaves and stems of C. odorata were initially extracted with ethanol, followed by purification through AB-8 macroporous adsorption resin column chromatography to yield different fractions. These fractions were then screened for hemostatic activity in mice and rabbits to identify the active fraction. Subsequently, the hemostatic effect of the active fraction was assessed through the bleeding time of the rabbit ear artery in vivo and the coagulant time of rabbit blood in vitro. The anti-inflammatory activity of the active fraction was tested on mice ear edema induced by xylene and rat paw edema induced by carrageenin. Furthermore, the active fraction's promotion effect on wound healing was evaluated using a rat skin injury model, and skin safety tests were conducted on rabbits and guinea pigs. Lastly, antimicrobial activities against two Gram-positive bacteria (G+, Staphylococcus aureus and S. epidermidis) and three Gram-negative bacteria (G−, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) were determined using the plate dilution method. ResultsThe ethanol extract of C. odorata leaves and stems was fractionated into 30%, 60%, and 90% ethanol eluate fractions. These fractions demonstrated hemostatic activity, with the 30% ethanol eluate fraction (30% EEF) showing the strongest effect, significantly reducing bleeding time (P < 0.05). A concentration of 1.0 g/mL of the 30% EEF accelerated cutaneous wound healing in rats on the 3rd, 6th, and 9th day post-operation, with the healing effect increasing over time. No irritation or allergy reactions were observed in rabbits and guinea pigs exposed to the 30% EEF. Additionally, the 30% EEF exhibited mild inhibitory effect on mice ear and rat paw edema, as well as antimicrobial activity against tested bacteria, with varying minimal inhibitory concentration (MIC) values. ConclusionsThe 30% EEF demonstrated a clear hemostatic effect on rabbit bleeding time, a slight inhibitory effect on mice ear edema and rat paw edema, significant wound healing activity in rats, and no observed irritation or allergic reactions. Antibacterial activity was observed against certain clinically isolated bacteria, particularly the G− bacteria. This study lays the groundwork for the potential development and application of C. odorata in wound treatment.

Some biological activities and element levels of Lycoperdon pratense

Mushrooms are natural products with many biological activities. In this study, antioxidant, antimicrobial, anticholinesterase activities and element levels of Lycoperdon pratense Pers. were determined. In this context, the mushroom was extracted with ethanol in a soxhlet device. Antioxidant potential was determined using Rel assay kits. Antimicrobial activity was determined using the agar dilution method. Antialzheimer activity was determined by acetyl- (AChE) and butyrylcholinesterase (BChE) potentials. The levels of elements accumulated in the mushroom were scanned using the wet digestion method. As a result of the analysis, the TAS value of the mushroom was measured as 2.589 ± 0.118 mmol Trolox equiv./L, the TOS value was 10.360 ± 0.197 µmol H2O2 equiv./L and the OSI value was 0.401±0.013. The mushroom extract was found to have high activity against bacteria. It was determined that the extract had an effect against microorganisms at concentrations between 50-400 μg/mL. It was determined that the anti-AChE IC50 value of the mushroom extract was 14.48 ± 0.80 μg/mL and the anti-BChE IC50 value was 23.10±1.21 μg/mL. It is also thought that it can be used as an indicator in terms of element levels within the mushroom. As a result, it was determined that the mushroom has antioxidant, antimicrobial and antiallergic potential.

Antibacterial activity of silver and gold nanoparticles that have been synthesized by curcumin

The green synthesis of metal nanoparticles with curcumin which is a compound with antibiotic effects, was considered for the synthesis of metal nanoparticles. Gold and silver nanoparticles were synthesized with an average diameter of 19.8 ± 6.2 nm and 29.8 ± 6.4 nm respectively. Both particles represent stability in the physiological environment and after a series of washing procedures to remove unreacted precursors. The antimicrobial efficacy of the synthesized nanoparticles was investigated in different techniques such as disk diffusion, agar well diffusion, minimum inhibitory concentration, and minimum bactericidal concentration tests. The results of both curcumin-coated metal nanoparticles in the disk diffusion test were negative. However, in the agar dilution and MIC tests of antibacterial effect of both nanoparticles on the tested bacteria was observed. The silver nanoparticles had an inhibitory effect on all selected bacteria in the agar dilution (8-256 µg/ml). However, gold nanoparticles had a lower inhibitory effect on the growth of some of the selected bacteria (64-512 µg/ml). In conclusion, silver nanoparticles were more effective than gold nanoparticles in most of the investigated methods.

Antibacterial Activity of Ethanol, Ethyl Acetate, and n-Hexane Extracts of Bidara Laut Lignum (Strychnos lucida R.Br) against Staphylococcus aureus and Escherichia coli

Strychnos lucida is empirically believed to have efficacy in treating various diseases such as fever, malaria, toothache, and stomachache and to treat wounds. The secondary metabolite of Strychnos lucida has various polarity. The purpose of this study was to compare the inhibitory activity of Strychnos lucida extracts against E. coli and S. aureus bacteria. The test was carried out by measuring the Diameter of the Inhibitory Region (DIR) using the disc diffusion method on a concentration of 90%, 75%, 60%, 45%, 30%, and 15%. The Minimum Inhibitory Concentration (MIC) was carried out by agar dilution method on a concentration of 15%, 12.5%, 10%, 7.5%, 5%, and 2.5%. The positive control was 1% ampicillin, and the negative control was 10% DMSO. The ethyl acetate extract showed the highest DIR of 14.30 mm against S. aureus and E. coli on a concentration of 45%, ethanol extract was 8,38 mm against S. aureus and 8.71 mm against E. coli, and n-hexane had no inhibitory activity on a concentration 45%. The result of the MIC test for ethanol and ethyl acetate extract for both bacteria was found at a concentration of 15%, while n-hexane extract against Staphylococcus aureus was found at a concentration of 90% and a concentration of 75% against Escherichia coli.

Evaluation of phytochemical, in vitro antibacterial and rate of kill assay of &lt;i&gt;Terminalia avicennioides&lt;/i&gt; leaf against some bacteria associated with diarrhoea

The study was aimed to evaluate the phytochemical constituents, in vitro antibacterial and rate of kill assay of Terminalia avicennioides leaf extract against some bacteria associated with diarrhoea. The phytochemical constituents of the ethanol of Terminalia avicennioides leaf, aqueous, n- butanol, and ethyl acetate fractions of the leaf extract were determined using standard analytical methods. The antibacterial activities of the leaf extract and extract fractions against clinical isolates of Salmonella Typhimurium and Escherichia coli isolated from diarrhoeaic patients were determined in vitro by agar diffusion, dilution and time - kill methods. The result of phytochemical screening revealed the presence of carbohydrates, cardiac glycosides, saponins, flavonoides, tannins, alkaloids, phenols and triterpenes. The crude extract and extract fractions of T. avicennioides leaf were effective against the test bacterial isolates at varied concentration of extracts but the n-butanol fraction was more effective with Minimum inhibitory and Minimum Bactericidal Concentration of 6.25 and 12.5mg/ml. The clinical isolates of E. coli and S. Typhimurium were completely killed within 180 minutes of exposure to ethanol leaf extract and extract fractions at varied MBCs of 12.5 mg/ml and 25.0 mg/ml. The clinical isolates of E. coli and S. Typhimurium were more susceptible to n-butanol fraction within 120 minutes of exposure to the extract fraction.

In vitro susceptibility testing of fosfomycin against gram-negative bacteria using agar dilution, gradient diffusion, disc diffusion, and phoenixtm

In vitro susceptibility testing of fosfomycin against gram-negative bacteria using agar dilution, gradient diffusion, disc diffusion, and phoenixtm

In vitro susceptibility testing of fosfomycin against gram-negative bacteria using agar dilution, gradient diffusion, disc diffusion, and phoenixtm

In vitro susceptibility testing of fosfomycin against gram-negative bacteria using agar dilution, gradient diffusion, disc diffusion, and phoenixtm

Antibiotic susceptibility pattern of Helicobacter pylori against eight antibiotics: A study from North India.

The purpose of this analysis is to evaluate the antimicrobial susceptibility of eight drugs effective against Helicobacter pylori (H. pylori) strains and the genetic diversity of H. pylori virulence genes to foresee clinical outcomes in North India. Fifty-eight H. pylori strains isolated from patients suffering from various gastrointestinal (GI) diseases were included in the study. MICs of various antibiotics were determined by the agar dilution method. The chi-squared test and Fisher exact test were used to determine the p-value, which was considered significant at p-value ≤ 0.05. RStudio 4.0 was used to for the data visualization. The prevalence of drug resistance was found to be: cefixime (CFM) (41.3%), furazolidone (FZD) (34.4%), amoxicillin (AMX) (20.7%), levofloxacin (LVFX) (70.7%), metronidazole (MTZ) (39.6%), tetracycline (TET) (20.7%), clarithromycin (CLA) (17.2%), and rifabutin (RIF) (17.2%). Out of 58 H. pylori strains, 3 were pan susceptible. There were H. pylori strains with single-drug resistance (21.8%, 12/55), dual resistance (30.9%, 17/55), triple resistance (20%, 11/55), and multidrug resistance (27.3%, 15/55). The resistance rate in MTZ, CLA and RIF were found to be significantly higher in females as compared to males (p = 0.005, p = 0.002, and p = 0.02), respectively. The resistance to TET exhibited significantly higher levels in gastritis compared to GERD, DU, and other disease groups (p = 0.04) respectively. TET, AMX, CLA, and RIF were found to be more effective antibiotics against H. pylori infections, whereas more studies are required to provide evidence on increasing resistance rate of LVFX.

Evaluation of Antibiotics by Disk Diffusion and Minimum Inhibitory Concentration Breakpoints in Urinary Tract Infections

Antibiotic resistance (ABR) has made it more challenging to treat uropathogenic organisms. It is impossible to compromise antimicrobial susceptibility testing (AST), which is essential and has a significant impact on infection treatment strategies. Although labor-intensive and technically challenging for everyday laboratory use, the agar dilution technique is appropriate for monitoring and assessing novel antimicrobials. Objective: To determine the minimum inhibitory concentration (MIC), Agar dilution technique and disk diffusion as susceptibility test methodologies. Methods: This study was carried out at Khyber Girls Medical College (KGMC) Peshawar. Keeping in view the Clinical and Laboratory Standards Institute (CLSI) guidelines AST was executed. BIOMÉRIEUX® API® kits and gram staining were utilized for identification of bacteria. The disk diffusion was performed using Thermo ScientificTM OxoidTM antibiotic discs of Co-trimoxazole, Levofloxacin, Nitrofurantoin and Fosfomycin. The MIC and zone of inhibitions for disk diffusion were noted according to the CLSI protocol. Results: 158 culture positive samples were isolated out of 680 total received. Esherechia Coli (E. coli) (74.1%) being the most isolated organism. In comparison of disk diffusion and agar dilution, categorical agreement for Levofloxacin, Cotrimoxazole, Nitrofurantoin and Fosfomycin were (82.28%, 72.15 %, 87.97% and 82.28%) respectively. Kappa coefficients of (0.64, 0.43, 0.57 and 0.37) (p &lt; 0.0001) were calculated for Levofloxacin, Co-trimoxazole, Nitrofurantoin, and Fosfomycin respectively, revealing considerable level of agreement for these antibiotics. Conclusions: It was concluded that Agar dilution is more precise than disk diffusion but being more labor intensive and technical. Disk diffusion can still produce significantly accurate results with less resource consumption.

GC-MS analysis of hospital waste soil and isolation of antimicrobial agent producing bacteria

The current study, deals with the GC-Mass analysis of soil and antibiosis activity of soil bacteria, isolated from district hospital at Kabir Chaura, Varanasi, India. Standard serial dilution plate procedures were used to isolate the bacteria. Gram staining was used to identify the isolates morphologically. Strain KC14 was Gram-negative and all other isolates were Gram-positive. Isolated bacteria were tested against six human pathogens viz., Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, Shigella flexneri and Bacillus subtilis. Primary and secondary screening were done by perpendicular streaking and seed overlay techniques. Based on the results most potential isolates were selected for tertiary screening. The GC-MS of soil samples revealed some major medical pollutants such as para-hydroxy acetanilide, 2-methyl-5-nitro-imidazole and 2-acetoxybenzoic acid. The KC14 isolate was chosen for further studies after demonstrating positive results against S. aureus with a maximum antagonistic activity of 17.3 ± 0.5 zone of inhibition. The molecular identification of KC14 isolate was determined by using 16S rRNA sequencing. The obtained nucleotide sequences were subjected to BLASTN in NCBI, and found similar to the available sequence of Acinetobacter junii. The nucleotide sequences have been deposited in GenBank with accession no. OP265396. The active compound was partially purified by ethyl acetate and tested its activity against S. aureus. This study defines the composition of hospital waste soil and identified a Gram-negative bacterium producing antibacterial activity against important human pathogens.

Evaluating the efficiency of indigenous soil bacterial strains for heavy metal removal

Significant quantities of toxic heavy metals are regularly deposited into soil environments every day as a result of industrial and human activities, as well as natural emissions. Today, biological methods for reducing heavy metal pollution are becoming increasingly common as an innovative approach to combating heavy metal pollution. The purpose of this study was to identify soil bacteria capable of removing heavy metals and to explore the potential of native bacteria in the bioremediation process. During this experiment, various soil samples were collected from several sites (river bank Rvs1, bus station Bt2, agricultural site Ag3, landfill Gr4 and construction site Cn5). Five bacterial strains were isolated from soil samples using a conventional serial plate dilution method. Morphologically distinct colonies were selected and purified during incubation. Pure cultures of bacterial colonies were established and maintained as slant cultures. Depending on preliminary physical, chemical and morphological characteristics, isolates RvsS1, Bt2, Ag3, Gr4 and Cn5 were compared with the bacterial species: “Proteus”, “Staphylococcus”, “Pseudomonas”, “Bacilli” and “Klebsiella” respectively. Bacterial isolates were examined for their uptake of Cu, Cd and Pb in flask bioreactors. Strains RV1, Bt2, Ag3 and Gr4 removed 48.7%, 25.6%, 59.8% and 23.5% of lead, respectively. The removal efficiency of isolate Cn5 was 35.68% for lead and 48.53% for copper. Thus, the data demonstrate that the use of indigenous bacteria is potentially effective in the bioremediation of heavy metals. Keywords: BIOREMEDIATION, MICROORGANISMS, HEAVY METALS, LOCAL BACTERIA, ISOLATES, STRAINS, ABSORPTION

Chemical composition and antimicrobial activity of an apolar extract from Lactuca serriola L. leaves

Lactuca serriola L. is an annual herbaceous plant species belonging to the Asteraceae family, called Prickly lettuce, Wild lettuce, and Jagged lettuce. L. serriola grows on grassy and rocky slopes in many regions in Europe, Asia, Australia, Africa, and North, Central and South America. The plant is used as a natural pharmaceutical agent in primary health care due to its analgesic, anti-inflammatory, antibacterial, and antioxidant properties, which are attributed to the compounds it contains. However, this is the first study demonstrating the antimicrobial activity of L. serriola essential oil. In this study, L. serriola essential oil was obtained by hydrodistillation of plant leaves and analyzed by Gas Chromatography-Mass Spectrometry (GS-MS); subsequently, antimicrobial properties of the essential oil were evaluated using agar dilution and broth microdilution methods. As a result of the GS-MS analysis, hexadecanoic acid, oleic acid, myristic acid, hexahydrofarnesyl acetone, β-ionone, and n-tetradecyl butanoate were found as dominant components in the plant essential oil with the rate of 6.61%, 4.84%, 2.05, 1.55%, 1.3%, and 1.09% (v/v), respectively. The MIC value of L. serriola essential oil against Enterobacter aerogenes,Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Candida tropicalis was found to be 0.94 μLmL−1, and MIC values were determined as 1.87 μLmL−1 and 0.47 μLmL−1 against Klebsiella pneumonia and Candidaparapsilosis, respectively. These results have opened a new horizon regarding the usability of L. serriola essential oil as a pharmacological therapeutic antibacterial and antifungal agent.

Exploring the Molecular Mechanisms of Macrolide Resistance in Laboratory Mutant Helicobacter pylori.

Resistance to clarithromycin, a macrolide antibiotic used in the first-line treatment of Helicobacter pylori infection, is the most important cause of treatment failure. Although most cases of clarithromycin resistance in H. pylori are associated with point mutations in 23S ribosomal RNA (rRNA), the relationships of other mutations with resistance remain unclear. We examined possible new macrolide resistance mechanisms in resistant strains using next-generation sequencing. Two resistant strains were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations using the agar dilution method. Sanger sequencing and whole-genome sequencing were performed to detect resistance-related mutations. Both strains carried the A2142G mutation in 23S rRNA. Candidate mutations (T1495A, T1494A, T1490A, T1476A, and G1472T) for clarithromycin resistance were detected in the Mutant-1 strain. Furthermore, a novel mutation in the gene encoding for the sulfite exporter TauE/SafE family protein was considered to be linked to clarithromycin resistance or cross-resistance, being identified as a target for further investigations. In the Mutant-2 strain, a novel mutation in the gene that encodes DUF874 family protein that can be considered as relevant with antibiotic resistance was detected. These mutations were revealed in the H. pylori genome for the first time, emphasizing their potential as targets for advanced studies.