Minimum Inhibitory Concentration Results Research Articles

MIC and MBC Levels of Combination Camellia Sinensis and Mentha Piperita Extract Mouthwash Against Streptococcus Mutans

Introduction: Awareness of Indonesian people in maintaining dental and oral health is low, proved by an increase in the percentage of dental and oral health problems by 2.7%. Caries is a dental and oral health problem that occurs in many children. The main cause of dental caries is Streptococcus mutans. To solve this problem, it is necessary to use herbal mouthwash made from a combination of Camellia sinensis and Mentha piperita extract as an antibacterial against Streptococcus mutans. Methods: Mouthwash is made through several processes namely plant determination, extraction, and mouthwash making. Minimal Inhibitory Concentration (MIC) is determined by diluted methods, Minimal Bactericidal Concentration (MBC) is determined by the agar streaking method, and colony tests are calculated using colony counters. Results: The result of plant determination showed the plants in this study were Camellia sinensis and Mentha piperita. At a concentration of 6.25%, no growth of bacteria in each repetition with the number of colonies 0 CFU / ml. While at a concentration of 3.125% found the average number of colonies 13 CFU / ml. Conclusions: Based on good MIC and MBC results, mouthwash containing Camellia sinensis and Mentha piperita has been shown to kill and inhibit the growth of Streptococcus mutans bacteria

Ruta graveolens, Peganum harmala, and Citrullus colocynthis methanolic extracts have in vitro protoscolocidal effects and act against bacteria isolated from echinococcal hydatid cyst fluid.

Echinococcosis is a common and endemic disease that affects both humans and animals. In this study, the in vitro activities of methanolic extracts of Ruta graveolens, Peganum harmala aerial parts, and Citrullus colocynthis seeds against protoscolosis and isolated bacterial strains from hydatid cysts were assessed using disc diffusion methods and Minimum Inhibitory Concentration (MIC). The chemical composition of three methanolic extracts was studied using LC-MS. After 3h of exposure to 40mg/mL R. graveolens extract, a tenfold protoscolocidal effect was seen when compared to the convintional medication (ABZ) for the same duration (P < 0.05). The bacteria listed below were isolated from hydatid cyst fluid collected from a variety of sick locations, including the lung and liver. Micrococcus spp., E. coli, Klebsiella oxytoca, Enterobacter aerogenes, Enterobacter amnigenus, Pseudomonas aeruginosa, Staphylococcus xylosus, and Achromobacter xylosoxidans are among the bacteria that have been identified. The most effective extract was R. graveolens, followed by P. harmala and C. colocynthis, according to the results of antibacterial activity using the disc diffusion method. R. graveolens extract had the lowest MIC values (less than 2mg/mL) against all microorganisms tested. This shows that the R. graveolens extract has additional properties, such as the ability to be both scolocidal and bactericidal. Because these bacteria are among the most prevalent pathogenic bacteria that increase the risk of secondary infection during hydatid cysts, the results of inhibitory zones and MICs of the R. graveolens methanol extract are considered highly promising.

Effect of Wastewater Treatment on Bacterial Community, Antibiotic-Resistant Bacteria and Endoparasites

Wastewater and wastewater treatment plants serve as urban reservoirs of pathogenic microorganisms. Wastewaters frequently contain bacteria, antibiotic-resistant bacteria, and developmental stages of parasites with significant zoonotic potential. Five wastewater treatment plants in the central part of Slovakia were investigated to determine the effect of treatment on bacterial community, antibiotic-resistant bacteria, and the occurrence of helminth eggs. Although all monitored chemical factors (chemical oxygen demand, biochemical oxygen demand, N-NH4, total nitrogen, and total phosphorus) in the effluent were in line with the legislative standards for discharge into public waterways, the results of minimal inhibitory concentrations show that reclaimed water harbors E. coli resistant to several commonly used antibiotics (ampicillin, piperacillin, and tazobactam, combine ampicillin and sulbactam, cefotaxime, tetracycline). The presence of endoparasite developmental stages in wastewater and sludge (Ascaris spp., Hymenolepis nana, eggs from the Ancylostomatidae family, Giardia duodenalis) indicates potential health risks for humans and workers at these sites. Treatment such as composting before applying sludge to land is necessary to reduce human pathogens.

Hand-powered vacuum-driven microfluidic gradient generator for high-throughput antimicrobial susceptibility testing

The growth of bacterial resistance to antimicrobials is a serious problem attracting much attention nowadays. To prevent the misuse and abuse of antimicrobials, it is important to carry out antibiotic susceptibility testing (AST) before clinical use. However, conventional AST methods are relatively laborious and time-consuming (18–24 h). Here, we present a hand-powered vacuum-driven microfluidic (HVM) device, in which a syringe is used as the only vacuum source for rapid generating concentration gradient of antibiotics in different chambers. The HVM device can be preassembled with various amounts of antibiotics, lyophilized, and stored for ready-to-use. Bacterial samples can be loaded into the HVM device through a simple suction step. With the assistance of Alamar Blue, the AST assay and the minimum inhibitory concentration (MIC) of different antibiotics can be investigated by comparing the growth results of bacteria in different culture chambers. In addition, a parallel HVM device was proposed, in which eight AST assays can be performed simultaneously. The results of MIC of three commonly used antibiotics against E. coli K-12 in our HVM device were consistent with those obtained by traditional method while the detection time was shortened to less than 8 h. We believe that our platform is high-throughput, cost-efficient, easy to use, and suitable for POCT applications.

786. Facility Reported vs. CLSI MIC Breakpoint Comparison of Carbapenem Non-susceptible (Carb-NS) Pseudomonas aeruginosa (PSA) From 2016-2019: A Multicenter Evaluation

BackgroundCLSI lowered Pseudomonas aeruginosa (PSA) Carbapenem (Carb) interpretive breakpoint minimum inhibitory concentrations (MICs) in 2012. It often takes several years for commercial test manufacturers and microbiology labs to incorporate revised breakpoints. We compare facility-reported rates of Carb-NS PSA to the 2012 CLSI MIC breakpoints, using a large nationwide database for isolates tested in 2016-2020 at United States (US) facilities.Table. Imipenem (IPM)/meropenem (MEM)/doripenem (DOR) interpretation (evaluable isolates) results for PSA. MethodsAll adults with a positive non-contaminant PSA culture (first isolate per 30-day period from blood, respiratory, urine, skin/wound, intra-abdominal, or other) in ambulatory and inpatient settings from 298 US hospitals from Q1 2016-Q4 2020 were evaluated (BD Insights Research Database, Becton, Dickinson & Company). Facility-reported Carb-non susceptible (NS) was defined as lab information system feed designations of susceptible (S), intermediate (I) or resistant (R) to imipenem (IPM), meropenem (MEM) and/or doripenem (DOR) per commercial panels. Where available, MICs were interpreted using CLSI 2012 Carb breakpoints (µg/ml) of ≤2 (S), 4 (I), ≥8 (R) for IPM/MEM/DOR. For evaluable PSA isolates we compared susceptibility results as reported by the facility to those using CLSI MIC breakpoints.ResultsOverall, 86.9% (255,844/294,426) of non-duplicate PSA isolates with facility-reported IPM/MEM/DOR susceptibility interpretations also had interpretable MIC results. S rates were 84.9% and 83.3% as reported by facilities and determined by CLSI criteria, respectively (Table). Facilities under-reported Carb-NS by 9.8%, using CLSI criteria as the standard (10.4% and 7.7% of R and I isolates, respectively, were missed by facility reporting).ConclusionSystematic application of CLSI breakpoints in 2016-20 would have had minimal impact on PSA S rates in the US. However, facility reporting failed to identify ~10% of Carb-NS isolates. The clinical implications of this observation are unknown. Facilities should know their local epidemiology, decide if under-reporting might be an issue, and assess if there is any impact on their patients. Disclosures Vikas Gupta, PharmD, BCPS, Becton, Dickinson and Company (Employee, Shareholder) Kalvin Yu, MD, BD (Employee) Jason M Pogue, PharmD, BCPS, BCIDP, Merck (Consultant)QPex (Consultant)Shionogi (Consultant)Utility Therapeutics (Consultant)VenatoRX (Consultant) Janet Weeks, PhD, Becton, Dickinson and Company (Employee) Cornelius J. Clancy, MD, Merck (Grant/Research Support)

Standardization of resazurin use in susceptibility testing of natural products against yeasts in planktonic cells and in biofilms formation

Natural products, such as the ethanolic propolis extract (PE), have been shown to be a safe and effective therapeutic alternative for the treatment of fungal skin and nail diseases. However, the presence of the resin and the physicochemical characteristics of the extract sometimes difficult the reading and determination of breakpoints of the in vitro tests, evidencing the need for alternatives that facilitate the reading. The present study aimed to standardize the use of resazurin in tests of susceptibility of PE with planktonic yeast cells and biofilm forms. The antifungal activity of PE was determined by minimum inhibitory concentration (MIC) and we observed that, for all Candida spp. tested, the most reproducible MIC results were obtained when resazurin was placed after 24 hours of incubation and remained more 24 hours with yeasts plus PE. For encapsulated yeasts, there was no dye reduction and color transition. Resazurin was also used for the evaluation of minimal biofilm inhibitory concentration and minimal biofilm eradication concentration and it was metabolized and reproduced the action of PE on Candida biofilms. In addition, microdilution checkerboard plates were made with the dye, which assisted reading the result of the interaction between PE and nystatin. We observed that the resin, the color and the turbidity of the PE slightly changed the color of the resazurin in high concentrations of the extract and did not impair the reading. Therefore, the resazurin standardization tests were proven to be efficient and grounds that it should be used as an auxiliary methodology for reading and interpretation of the susceptibility tests for non-encapsulated yeasts with natural products, which form turbidity or precipitation, such as propolis.

Preparation, physicochemical, and antibacterial properties of bovine serum albumin microspheres loaded with sodium nitrite

In this study, NaNO2-embedded bovine serum albumin microspheres (BSAMs) were prepared by oil-in-water emulsification method, which was covalently cross-linked with transglutaminase (TGase). Moreover, the physicochemical properties and antibacterial effect of BSAMs on Escherichia coli, Staphylococcus aureus, Pseudomonas, and beef and pork jerky were also analyzed. The particle size of BSAMs was about 0.5–2.5 μm observed by scanning electron microscopy. Almost all NaNO2 was encapsulated inside BSAMs, and there was no NaNO2 attaching on the surface of BSAMs. The encapsulation rate of NaNO2 first increased from 0 to 20 U/g TGase and then decreased from 20 to 40 U/g TGase; likewise, the release of NO first increased with the increase of TGase (0–20 U/g) and it decreased when the TGase concentration was 30 and 40 U/g. Based the results of minimal inhibitory concentration and minimal bactericidal concentration, E. coli and Pseudomonas were more sensitive to BSAMs than S. aureus. In addition, the slow release of NO from BSAMs could significantly inhibit the growth of bacteria on the surface of beef and pork jerky in a weak acidic environment compared with NaNO2 used directly, so as to achieve long-term antibacterial effect and prolong the shelf life of meat products.

Identification and Characterization of a Novel Chromosomal Aminoglycoside 2'-N-Acetyltransferase, AAC(2')-If, From an Isolate of a Novel Providencia Species, Providencia wenzhouensis R33.

Multidrug-resistant bacteria from different sources have been steadily emerging, and an increasing number of resistance mechanisms are being uncovered. In this work, we characterized a novel resistance gene named aac(2′)-If from an isolate of a novel Providencia species, Providencia wenzhouensis R33 (CCTCC AB 2021339). Susceptibility testing and enzyme kinetic parameter analysis were conducted to determine the function of the aminoglycoside 2′-N-acetyltransferase. Whole-genome sequencing and comparative genomic analysis were performed to elucidate the molecular characteristics of the genome and the genetic context of the resistance gene-related sequences. Among the functionally characterized resistance genes, AAC(2′)-If shares the highest amino acid sequence identity of 70.79% with AAC(2′)-Ia. AAC(2′)-If confers resistance to several aminoglycoside antibiotics, showing the highest resistance activity against ribostamycin and neomycin. The recombinant strain harboring aac(2′)-If (pUCP20-aac(2′)-If/DH5α) showed 256- and 128-fold increases in the minimum inhibitory concentration (MIC) levels to ribostamycin and neomycin, respectively, compared with those of the control strains (DH5α and pUCP20/DH5α). The results of the kinetic analysis of AAC(2′)-If were consistent with the MIC results of the cloned aac(2′)-If with the highest catalytic efficiency for ribostamycin (kcat/Km ratio = [3.72 ± 0.52] × 104 M–1⋅s–1). Whole-genome sequencing demonstrated that the aac(2′)-If gene was located on the chromosome with a relatively unique genetic environment. Identification of a novel aminoglycoside resistance gene in a strain of a novel Providencia species will help us find ways to elucidate the complexity of resistance mechanisms in the microbial population.

Synthesis, and characterization of some Cu(I) complexes having propionitrile and pyridine moieties: An investigation on their antibacterial properties

The aim of producing new biologically active compounds lead to the synthesis of some Cu(I) complexes of general formula [Cu(C2H5CN)4][A] and [Cu(C5H5N)4][A] (1–4) {where A: counter anion = B(C6F5)4− and B{C6H3(m-CF3)2}4−} from the reaction of CuCl and silver salt of the corresponding counter anion. The complexes were characterized using elemental analysis, 1H NMR, 11B NMR, FT-IR Spectroscopy, UV–visible spectroscopy and thermo-gravimetric analysis (TGA). The antibacterial activities of all complexes are evaluated by the minimal inhibitory concentration (MIC), using the micro-broth dilution method, against eight bacteria (Gram-negative and Gram-positive), each with fresh clinical isolates. The MIC results were compared with those of Oxytetracycline agent as a positive control. In most cases, the compounds show broad-spectrum activities that were either, more active, or equipotent to, the antibiotic agent in the comparison tests. Complex 4 showed the greatest activity against Proteus mirabilis (Gram- negative) with a minimum inhibitory concentration (MIC) of 8 µg/mL, while complexes 2 and 3 showed the lowest activity against Pseudomonas aeruginosa (Gram-negative) and against Staphylococcus aureus (Gram-positive) with a concentration of 512 µg/mL.

Evaluation of isoeugenol in inhibition of Staphylococcus aureus efflux pumps and their toxicity using Drosophila melanogaster model

The Staphylococcus aureus bacteria is a pathogen considered opportunistic and that has been acquiring resistance to several classes of antibiotics, mainly due to the synthesis of efflux pumps, which are proteins that expel these drugs intracellularly, reducing their effectiveness. The objective of this study was to evaluate the ability of isoeugenol to inhibit S. aureus efflux pumps and to determine its toxicity against a eukaryotic model (Drosophila melanogaster). IS-58, K2068 and K4414 S. aureus strains were used in the study. Isoeugenol minimum inhibitory concentration (MIC) and antibiotic modulation were evaluated in efflux pump inhibitory tests as well as in ethidium bromide (EtBr) assays. Toxicity tests against D. melanogaster assessed mortality and negative geotaxis. Isoeugenol obtained a relevant MIC result and a synergism was observed when isoeugenol was associated with the antibiotics, mainly with ciprofloxacin. Isoeugenol was able to affect all three efflux pumps tested, especially in strain K4414. The mortality of D. melanogaster caused by isoeugenol administration started after 12 h of exposure, being volume dependent and having an LC50 of 81.69 μL/L. In the negative geotaxis test, a statistical difference was observed after 24h of exposure compared to the control, demonstrating that damage to the locomotor apparatus had occurred. Based on the results, isoeugenol is a putative efflux pump inhibitor, becoming an alternative in blocking these proteins, and demonstrated acute toxicity against D. melanogaster.

Synthesis and application of indole esters derivatives containing acrylamide group as antifouling agents

Two new indole ester derivatives containing acrylamide group were synthesized by Michael addition reaction and Friedel-Crafts reaction based on 5-bromo-1H-indole. The synthesized compounds were characterized by 1H Nuclearmagnetic Resonance Spectroscopy (1HNMR) and Fourier Transform Infrared spectroscopy (FTIR). In the step of the Friedel-Crafts reaction, the optimum process conditions were determined as that the catalyst was AlCl3, the reaction time was 3 days and the reaction temperature was 25 °C. The results of minimal inhibitory concentration (MIC) of synthesized compounds showed that the two synthesized compounds have better antibacterial activity than reference. Algal inhibiting experiment shows that indole derivatives have better algae inhibition performance. Anti-protein adsorption experiment of antifouling paints with indole derivative and chlorothalonil as antifouling agents proves that indole derivatives exhibit better protein resistance behaviors. The antifouling paints using indole derivatives as antifouling agents have excellent antifouling properties after 24 months marine field test.

Inhibitory Effects of Azadirachta indica (Neem) and Bryophyllum pinnatum (Oda Opue) Leaves Extracts on Staphylococcus aureus Isolated from Infected Wound Samples

Aim: to study the antimicrobial effects of different leaves extracts of Azadirachta indica and Bryophyllum pinnatum on Staphylococcus aureus isolated from infected wound.&#x0D; Study Design: This study was done to determine inhibitory effects of extracts on S. aureus isolated from infected wounds.&#x0D; Place and Duration of Study: General hospital Onitsha, Anambra State, Nigeria, January to March 2020.&#x0D; Methodology: wound swabs were collected from 30 patients with infected wound. S. aureus were isolated and identified morphologically and biochemically. The leaves were collected, identified and grinded. Extraction of the leaves were done using soxhlet method with two solvents (ethanol and water). Phytochemical compositions were analyzed using standard methods. In vitro antimicrobial effect of extracts and their combination were evaluated.&#x0D; Results: Phytochemical analysis of extracts showed presence of saponins, flavonoids, steroids, alkaloids, tannins, cardiac glycosides and phenols except B. pinnatum aqueous extract that showed presence of flavonoids, saponins and alkaloids. Minimum inhibitory concentration (MIC) and Minimal bactericidal concentration (MBC) were determined. Among the extracts analysed, A. indica ethanol has highest zone of inhibition against S. aureus with 20.00±0.00 mm at 1000 mg/ml concentration. B. pinnatum ethanol 10.33±0.58 mm at 1000 mg/ml, A. indica aqueous extract 13.33±2.08 mm but B. pinnatum aqueous extract has no inhibitory effect. The combination of A. indica aqueous and B. pinnatum ethanol extracts, B. pinnatum and A. indica ethanol extracts and A. indica ethanol extract and B. pinnatum aqueous extracts have inhibitory effects on S. aureus except combination of A. indica aqueous and B. pinnatum aqueous which has no inhibitory effect. The result of MIC was 500 mg/ml and the MBC showed 1000 mg/ml.&#x0D; Conclusion: The extracts of B. pinnatum and A. indica possess antimicrobial properties for S. aureus and the plants are available and are planted worldwide. There is also need for research on other plants with antimicrobial effects.

Disc Diffusion Testing of Azithromycin Against Clinical Isolates of Typhoidal Salmonellae: A Diagnostic Conundrum.

Introduction: Typhoid remains a major healthcare problem in low and middle-income countries. The emergence of extremely drug-resistant (XDR) typhoid strains from the Indian subcontinent has led to very limited therapeutic options. Azithromycin being the only oral option for XDR typhoid faces a threat of rapid resistance due to its overuse after the COVID-19 pandemic.Objective: To evaluate the reliability of azithromycin disc diffusion testing against clinical isolates of typhoidal salmonellae in comparison with E-test minimum inhibitory concentrations (MICs).Study design: This is a cross-sectional validation study.Place and duration of the study: The Department of Microbiology, Pakistan Navy Ship Shifa hospital, Karachi from June 1 to December 31, 2020.Methodology: Antimicrobial susceptibility was performed by Kirby Bauer disc diffusion method for 60 isolates including Salmonella enterica ser. Typhi and Paratyphi A using Clinical Laboratory Standard Institute (CLSI) guidelines. MICs by the E-test method were determined for Azithromycin only.Results: A significant proportion of the isolates (55%) had high azithromycin MIC in the wild-type distribution range (8-16 µg/ml). Ten (16.6%) isolates showed false resistance, i.e., zone diameter <13 mm by disc diffusion method when compared to E-test MIC results. Isolates with MICs close to breakpoint, i.e., 16 µg/ml were more likely to show discordant results. The sensitivity, specificity, negative predictive value, positive predictive value, and diagnostic accuracy of the disc diffusion method versus E-test were 100%, 83%, 100%, 9%, and 83%, respectively.Conclusions: Disc diffusion method as recommended by CLSI is not reliable for azithromycin susceptibility testing particularly for isolates with high MICs in the susceptible range. The E-test method may be a better alternative to disc diffusion provided appropriate training is done prior to its application.

Detection of Colistin Resistance in Carbapenem Resistant Enterobacteriaceae by Reference Broth Microdilution and Comparative Evaluation of Three Other Methods.

Objective Challenges in susceptibility testing of colistin along with increase in the prevalence of colistin-resistant carbapenemase-producing Enterobacteriaceae (CRE) pathogens needs addressal. Evaluation of user-friendly methods is necessary as an alternative to broth microdilution (BMD), the reference susceptibility testing method, for routine implementation in diagnostic clinical microbiology laboratories. Genotypic detection of the plasmid-mediated colistin resistance is also needed for infection control purposes. Materials and Methods Colistin susceptibility of 200 nonduplicate clinical CRE isolates from December 2017 to June 2019 was determined by BMD, agar dilution (AD), E test, and rapid polymyxin NP test and interpreted as per the European Committee on Antimicrobial Susceptibility Testing. The results of AD, E test, and NP test were compared with that of BMD, considering minimal inhibitory concentration (MIC) ≤ 2 µg/mL as susceptible and > 2 µg/mL as resistant. Presence of any plasmid-mediated colistin resistance (mcr-1 and 2) was evaluated in 27 colistin-resistant CRE isolates by polymerase chain reaction. Statistical Analysis Performance of different phenotypic methods was analyzed by comparing MIC results of AD and E test with that of reference BMD method. Agreement between BMD and the other two methods was expressed in terms of categorical agreement and essential agreement. Errors were expressed as very major error (VME: false-susceptible) and major error (ME: false-resistance) by AD/E test. VME and ME of 3% disagreement were considered unacceptable. Results Colistin resistance was found in 27 (13.5%) isolates by BMD method. The VME rates of both AD (11%) and E test (37%) could not meet the Clinical and Laboratory Standards Institute recommendation (< 3% VME rate is acceptable) as alternative tests to the reference BMD. Colistin NP test showed sensitivity and specificity of 85% and 98%, respectively. The percentage discordant result in NP test was highest in Enterobacter spp. (17%). None of the 27 colistin resistant isolates showed presence of mcr-1 and mcr-2 genes. Conclusion High VME rate in AD and E tests precludes their use as alternatives to BMD for colistin susceptibility testing. NP test with moderate sensitivity but excellent specificity can be a good alternative for testing colistin susceptibility in CRE isolates, except in Enterobacter spp. Absence of mcr-1 and mcr-2 gene necessitates the exploration of other mechanisms of colistin resistance.

Minimal Inhibitory Concentration Values of Oxytetracycline for Bacterial Pathogens Isolated from Warmwater Fishes

AbstractThe in vitro susceptibility to oxytetracycline (OTC) was determined through minimal inhibitory concentration (MIC) testing of 10 field isolates from each of seven fish pathogens: Edwardsiella ictaluri, E. piscicida, E. tarda, Flavobacterium columnare, Aeromonas hydrophila, Streptococcus iniae, and S. agalactiae. Bacteria from each species were cultured, diluted, dispensed into 96‐well microdilution plates, and incubated at 28°C. The MIC results were read at 48 h for all species except A. hydrophila, for which results were read at 24 h. The MIC ranges of OTC (μg/mL) for these bacteria were as follows: 0.5–64.0 for E. ictaluri; 0.5 to over 64.0 for E. piscicida; 0.5 to ≥64.0 for E. tarda; less than 0.12 (all) for F. columnare; 0.25 (all) for A. hydrophila; 0.5–16.0 for S. iniae; and 0.5–2.0 for S. agalactiae. Escherichia coli isolate ATCC 25922 was used as the MIC quality control throughout testing, and MIC values were within limits established by the Clinical and Laboratory Standards Institute (CLSI). Although there are no CLSI‐published or other standardized epidemiological cutoff values for these fish pathogens, the isolates with MIC values less than 1.0 μg/mL were deemed susceptible to OTC based on the locally established susceptibility ranges at the College of Veterinary Medicine, Mississippi State University. In the present study, 100% of A. hydrophila and F. columnare isolates; 80–90% of E. ictaluri, E. piscicida, and E. tarda isolates; 50% of S. iniae isolates; and 80% of S. agalactiae isolates were considered susceptible to OTC (MIC &lt; 1.0 μg/mL).

Ceftriaxone-based Schiff base transition metal(II) complexes. Synthesis, characterization, bacterial toxicity, and DFT calculations. Enhanced antibacterial activity of a novel Zn(II) complex against S. aureus and E. coli

From the reaction of ceftriaxone 1 antibiotic with 2,6-diaminopyridine 2 a ceftriaxone-based Schiff base (H2L,3) was obtained and its transition metal complexes were synthesized. Spectroscopic and physicochemical techniques, namely, UV–visible, FT-IR, 1H NMR, EPR, mass spectrometry, molar conductance, magnetic susceptibility and density functional theory (DFT) calculations, together with elemental and thermal analyses, were used to find out the binding mode and composition of these complexes. The ceftriaxone-based Schiff base 3 behaves as a monoanionic tridentate N,N,O ligand. Spectral and magnetic data suggest an octahedral geometry for all complexes and the general formulae [M(HL)(OAc)(H2O)2] (M(II) = Mn2+4, Co2+5, Ni2+6, Cu2+7, Zn2+8), are proposed for them. All compounds were screened for antibacterial activity using both the agar disc diffusion method and the minimal inhibitory concentration (MIC). It was found that complex 8 exhibited the most promising bactericidal activity against S. aureus (MIC = 0.0048 μmol/ml) and E. coli (MIC = 0.0024 μmol/ml). It is more active than the free ligand 1 (MIC = 0.0560 μmol/ml for S. aureus and 0.0140 μmol/ml for E. coli). These MIC results were compared with those obtained using similar zinc(II) Schiff base complexes, and with the values obtained using ceftriaxone conjugated with silver and gold nanoparticles (NPs), using earlier published data. Synthesized metal complexes exhibited LC50 values >1000 ppm indicating their nontoxicity against brine shrimp nauplii (Artemia Salina).

Investigation of Mutations in the Rifampin-Resistance-Determining Region of the rpoB Gene of Brucella melitensis by Gene Analysis

Background: RNA polymerase beta subunit (rpoB) gene analysis in bacterial communities is known as a method for determining rifampin sensitivity and genetic diversity among Brucella spp. Detection of antibiotic resistance among Brucella isolates can be a critical approach to control brucellosis. However, rpoB gene analysis of Brucella melitensis for assessing rifampicin resistance has not yet been performed in Iran, which is considered an endemic area for brucellosis. Objectives: The aim of this study was to analyze the whole sequence of rpoB genes of different B. melitensis isolates from humans to identify the single-nucleotide polymorphisms (SNPs) and mutations related to rifampin resistance and to analyze the genetic diversity of these bacteria in Iran. Methods: Between 2017 and 2019, a total of 156 blood samples along with 12 synovial fluid specimens were collected from brucellosis patients in different Iranian provinces and subjected to bacterial culture in Brucella selective media. Brucella identification was carried out using classical biotyping and molecular examinations. Polymerase chain reaction (PCR)-based amplification of the rpoB gene was performed by specific rpoB primers for whole gene sequencing. The antimicrobial susceptibility of Brucella isolates was assessed using disk diffusion susceptibility tests and minimal inhibitory concentration (MIC) methods. The presence of rifampin-binding sites and SNPs were investigated through rpoB whole gene sequencing. Results: Clinical B. melitensis isolates were obtained from blood (13) and synovial fluid (1) samples of patients from different regions of Iran. The results of MIC and disk diffusion susceptibility tests showed that all the isolates were sensitive to rifampin except for one isolate showing intermediate rifampin resistance based on the standards defined for slow-growing bacteria by the Clinical and Laboratory Standards Institute (CLSI). Gene analysis for identifying the mutations related to rifampin resistance and investigating genetic diversity showed that none of the B. melitensis isolates had missense mutations, confirming the susceptibility of all the studied isolates to rifampin. Conclusions: The present study revealed that rpoB gene analysis could be used for the efficient and precise identifying of the mutations related to rifampin resistance, investigating rifampin binding sites, and genotyping Brucella species. Furthermore, the identification of B. melitensis isolates with intermediate resistance to rifampicin highlighted the importance of periodically carrying out antibiotic susceptibility testing. The molecular detection of rpoB mutations in different Brucella isolates may help to prevent the spread of rifampin-resistant Brucella spp. among humans and livestock.

Dehydroabietic Acid Microencapsulation Potential as Biofilm-Mediated Infections Treatment.

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.

Antibacterial effects assessment on some livestock pathogens, thermal stability and proposing a probable reason for different levels of activity of thanatin

There is a continuing need to prevent the increasing use of common antibiotic and find the replacement to combat the drug/antibiotic resistant bacteria such as antimicrobial peptides (AMPs) such as thanatin peptide. In this study, recombinant thanatin peptide was expressed in the HEK293 cell line. Then the antimicrobial properties of this peptide on some poultry and farm animal’s pathogen strains were assessed. The thermal-stability of thanatin was predicted in various temperatures through in silico analysis. Afterwards, according to Minimum Inhibitory Concentration (MIC) results, Escherichia coli and Pseudomonas aeruginosa were chosen to test the hypothesis of LptA/LptD–thanatin interaction, computationally. Relative amino acid sequences and crystallography structures were retrieved and missed tertiary structures were predicted. The interaction of thanatin with LptA and LptD of Escherichia coli and Pseudomonas aeruginosa were analyzed subsequently. The antibacterial activity of thanatin peptide was evaluated between 6.25 and 100 μg/mL using minimum inhibitory concentration. Also, the amounts of minimum bactericidal concentrations (MBC) were between 12.5 and 200 μg/mL. The bioinformatics analysis followed by the in vitro assessment, demonstrated that thanatin would be thermally stable in the body temperature of poultry and farm animals. Thanatin could penetrate to the outer membrane domain of LptD in Escherichia coli and it could block the transition path of this protein while the entrance of LptD in Pseudomonas aeruginosa was blocked for thanatin by extra residues in comparison with Escherichia coli LptD. In addition, the quality of interaction, with regard to the number and distance of interactions which leads to higher binding energy for thanatin and LptD of Escherichia coli was much better than Pseudomonas aeruginosa. But the site and quality of interaction for thanatin and LptA was almost the same for Escherichia coli and Pseudomonas aeruginosa. Accordingly, thanatin can prevent the assembly of LptA periplasmic bridge in both pathogens. The antibacterial and thermal stability of the thanatin peptide suggested that thanatin peptide might serve as a natural alternative instead of common antibiotics in the veterinary medicine. The outcome of this in silico study supports the MIC results. Therefore, a probable reason for different level of activity of thanatin against Escherichia coli and Pseudomonas aeruginosa might be the quality of LptA/LptD–thanatin interaction.

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

Biopolymer (chitosan) was isolated from crab shell waste through the processes of demineralization, deproteinization, decolourization and deacetylation. The resulting chitosan (CHS) was further treated with silver nitrate (AgNO3) solution at various concentrations (0.5, 1.0 and 1.5 M) in order to enhance the antimicrobial activity of chitosan. The crab shell powder (CSP) and (CHS) were characterized using X-ray Diffractometer (XRD), Fourier Transform Infrared (FT-IR), and Scanning Electron Microscopy coupled with Energy Dispersive Spectroscopy (SEM-EDS). The antimicrobial mode of action of AgNO3 treated chitosan was performed using serial dilution (1:2) technique for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) and tested against four microorganisms (Escherichia coli, Klebsiella pneumonia, Staphlococcusaureus and Pseudopodium). The result of proximate analysis of CHS and chitin (CHN) showed % crude protein to be 12.24±001 and 20.54±0.03 respectively, % ash was found to be 4.1±0.01 for CHS and 3.80±0.01 for CHN. The FTIR spectra of CHS and CHN showed their characteristic absorption peaks and the diffractograms of CSP and CHS showed CaCO3 to be the major mineral present in the samples. The antimicrobial evaluation revealed that untreated chitosan extract (UCHSE) showed no antimicrobial activity against the four tested microorganisms. The results of MIC and MBC showed that the organisms responded to the antimicrobial agent at different dilution concentration. It was observed that CHS treated with 0.5 M AgNO3 (0.5 SNCHSE) inhibited the growth of E. coli at 1000 µg/mL, S. aureus at 500 µg/mL while it exhibited bactericidal (MBC) activity against all the organisms at 1000 µg/mL.