MIC Results Research Articles

Antibacterial Activity of Silver Nanoparticles Prepared from Camellia Sinensis Extracts in Multi-Drug Resistant Staphylococcus Aureus.

The nano-method has been used as a technique for creating novel, non-traditional antimicrobial agents. This effective method for treating infectious diseases has many advantages over conventional antibiotics, including increased efficacy against species that have developed drug resistance, and the ability to circumvent the development of resistance that disrupts a number of biological pathways. As a result, the objective of this study was to synthesize and characterize silver nanoparticles using phenolic compounds obtained from Camellia sinensis . The nanoparticles were then used as antibacterial agents on the multidrug resistant Staphylococcus aureus, as well as, biofilm formation mechanism were also investigated. Ten isolates of Staphylococcus aureus were acquired from the labs of the University of Baghdad's Genetic Engineering and Biotechnology Institute. The VITEK-2 system was used to confirm the diagnosis. Aqueous and methanolic extracts of Camellia sinensis leaves were used to create silver nanoparticles and obtain CAgNPs, which were then characterized using Atomic Fluorescence Microscopy (AFM), X-ray diffractometer (XRD), and Zeta potential analyzers. The extracts were put through a series of assays, including High-performance liquid chromatography (HPLC), antibacterial activity assessments, and the microtiter plate method to determine the lowest inhibitory concentration (MIC) and antibiofilm formation. Both aqueous and methanolic extracts containing silver nanoparticles included spherical nanoparticles that may be single or combined. The HPLC results showed the presence of two phenolic compounds (gallic acid and caffeine) by comparing their retention durations to those of the reference compounds. The results of the antibacterial activity of (CAgNPs) showed that the methanolic (CAgNPs) extract was more effective than the aqueous (CAgNPs) extract, producing inhibitory zones of 15.67 ± 0.58 and 20.33 ± 0.58 mm, at 375 and 750 ppm respectively, when compared to the aqueous (CAgNPs) extract, which produced inhibitory zones of 12.33 ± 0.58 and 15.67 ± 0.58 mm, respectively. The MIC result also showed that the CAgNPs methanolic extract was more effective than the CAgNPs aqueous extract. The MIC of the CAgNPs methanolic extract on S. aureus isolates were 11.718 and 23.43 µg/ml, while the MIC of the CAgNPs aqueous extract on all S. aureus isolates were 46.87 µg/ml except isolate No. 3 and 6 which was 11.718 and 93.75 µg/ml respectively. Additionally, The anti-biofilm in S. aureus was increased when CAgNPs methanolic extract were used compared with the CAgNPs aqueous extract, the CAgNPs methanolic extract inhibited 80%, 90% and 100% of the biofilm formation of S. aureus in 23.43, 46.87 and 93.75 µg/ml respectively, while the anti-biofilm activity of the CAgNPs aqueous extract on S. aureus isolates was 80% and 100% of the biofilm formation in 46.87 and 93.75 µg/ml respectively. The methanolic and aqueous leaves extracts from Camellia sinensis are a successful method for producing CAgNPs. The synthesized CAgNPs also have significant antibacterial activity against S. aureus, depending on the concentrations, and inhibit S. aureus biofilm formation.

Construction of BiVO4/g-C3N4/Ag3PO4 ternary heterojunction with double Z-scheme and its photocatalytic and bacteriostatic properties

A novel Bi-composite photocatalyst BiVO4/g-C3N4/Ag3PO4 with a double Z-scheme structure was prepared by hydrothermal method assisted by honeysuckle extract. When the mass fraction of Ag3PO4 in the composite is 10 %, the inactivation rates of E.coli and S. aureus were 6.9 and 4.6 log, respectively. The MIC results were 0.6 and 0.8 mg/ml, respectively. The capture experiment confirmed that h+ and O2− were the main active substances that catalyzed bacteriostasis. Double Z heterojunction reduces the photochemical corrosion of Ag3PO4, changes the transmission path of photogenerated electrons, and then promotes the separation of photogenerated electron holes, thus obtaining significant photocatalytic activity.

Candida auris MIC testing by EUCAST and clinical and laboratory standards institute broth microdilution, and gradient diffusion strips; to be or not to be amphotericin B resistant?

ObjectivesReported amphotericin B resistance rates for Candida auris vary considerably. This may reflect clinically relevant differences in susceptibility, technical issues with testing, or adoption of a clinical breakpoint that bisects the wild-type population. We compared reference methods and two gradient diffusion strips using a shared C. auris strain collection. MethodsForty C. auris strains from nine U.S. states and ≥3 clades were included. Fourteen MIC data sets were generated using EUCAST E.Def 7.4, Clinical and Laboratory Standards Institute (CLSI) M27Ed4, Etest, and MTS (Liofilchem) strip MICs. MICs ≤1 mg/L were classified as susceptible. ResultsEUCAST and CLSI amphotericin B MIC testing were robust across the included method variables. The modal MIC was 1 mg/L, distributions unimodal and narrow with similar GM-MICs (0.745–1.072); however, susceptibility classification varied (0–28% resistance). Gradient diffusion strip testing resulted in wider and bimodal distributions for 8/9 data sets. If adopting, per manufacturer's protocol, double inoculation for the Etest method, the modal MIC increased to 2–4 mg/L and resistance rates to 45–63% versus 25–30% with the single inoculation. The EUCAST, CLSI, Etest, and MTS strip MICs correlated to the optical density of drug-free control EUCAST wells, suggesting that some isolates grew better than others and that this was associated with MIC. Discussion: The EUCAST and CLSI MIC results were in close agreement, whereas the strip test showed wider and bimodal distributions with reader to reader and centre to centre variation. Our study adds to the concern for commercial MIC testing of amphotericin B against C. auris and suggests the current breakpoint leads to random susceptibility classification.

Screening the antimicrobial, cytotoxic and hemolytic effects of skin-parotoid gland secretions of 13 taxa from six species and one hybrid population of Anatolian endemic Lycian salamanders (Genus: Lyciasalamandra)

Background and purpose: The diverse bioactive content in the skin and parotoid glands of amphibians makes them unique and highly potential sources for pharmacological developments. The main aim of the study is to determine the total protein amounts, in vitro cytotoxicities, antimicrobial activities and hemolytic effects of skin-parotoid secretions of these endemic Lycian salamanders (Genus: Lyciasalamandra). Materials and methods: Lycian salamander specimens of 13 taxa from six species and one hybrid population are collected from southwestern Anatolia. Skin-parotoid secretions were obtained, clarified, supernatants snapfrozen then lyophilized. Total protein amounts were determined by BCA assay kit. The cytotoxicity was determined on eight different cell lines (one healthy and seven cancerous) using MTT assay. The antimicrobial activity was assesed using 11 different microorganisms (nine bacteria and two yeast) from six genus by MIC method. Hemolytic effects were measured on rabbit red blood cells. Results: Lycian salamanders’ skin-parotoid gland secretions showed variable cytotoxic effects on all cell lines with IC50 values from 5.41 ± 0.17 μg/ml to ineffective. MIC results of antimicrobial activity tests were also variable and from 3.9 μg/mL to non-effective. Studied skin-parotoid secretions have no hemolytic activities on rabbit red blood cells at concentrations of 0.5, 5 and 50 μg/mL. Conclusions: Skin-parotoid gland secretions of Anatolian endemic Lycian salamanders have sufficient and remarkable therapeutic potential to be candidate sources of natural and biologically active substances for novel drug discoveries.

Exploring the pharmacological significance of Murraya koenigii (L.) Spreng: Insights from North-East India

Murraya koenigii (L.) Spreng., is a renowned medicinal and aromatic plant having antimicrobial and broad biological activities. In the present study, the essential oil chemical profile of the leaf and floral part were analysed using GC-FID and GC-MS, and the antimicrobial activities were determined by disk diffusion assay. The scientific analysis revealed α-pinene (40.41%) was the major compound of leaf essential oil (MKL) followed by sabinene (29.66%), β-pinene (7.92%), caryophyllene (4.24%), limonene (3.39%), terpinen-4-ol (3.25%), γ-terpinene (2.63%), terpinolene (1.58%), linalool (1.32%), along with other trace components. The floral part (MKF) contains the compounds α-pinene (74.02%) as the major compound followed by β-pinene (7.17%), sabinene (5.72%), limonene (4.19%), β-myrcene (1.98%), γ-terpinene (1.47%) and terpinen-4-ol (1.41%) along with other trace compounds. The MIC result revealed essential oil showed effectiveness against Escherichia coli (40 μg/mL), Micrococcus luteus (80 μg/mL), Aspergillus fumigatus (60 μg/mL) and Aspergillus brasiliensis (80 μg/mL) for MKL while MKF did not exhibit potent antimicrobial activities. The essential oils revealed potent antioxidant potential as determined by DPPH method with IC50 values of 14.02 μL/mL (MKL) and 5.45 μL/mL (MKF) and the ABTS method with IC50 values of 5.07 μL/mL and 8.38 μL/mL in MKL and MKF respectively as compared to that of the standards. The essential oils revealed potent antidiabetic activity with 6.02 μL/mL (MKL) and 8.18 μL/mL (MKF) as that of the standard. However, both MKL and MKF were not much effective in protease inhibitory activity and anti-tyrosinase activity. The study results exhibited a potent bioactivity profile and revealed many new utilization aspects of this essential oil.

Emergence and genomic characterization of Proteus mirabilis harboring blaNDM-1 in Korean companion dogs

Proteus mirabilis is a commensal bacterium dwelling in the gastrointestinal (GI) tract of humans and animals. Although New Delhi metallo-β-lactamase 1 (NDM-1) producing P. mirabilis is emerging as a threat, its epidemiology in our society remains largely unknown. LHPm1, the first P. mirabilis isolate harboring NDM-1, was detected from a companion dog that resides with a human owner. The whole-genome study revealed 20 different antimicrobial resistance (AMR) genes against various classes of antimicrobial agents, which corresponded to the MIC results. Genomic regions, including MDR genes, were identified with multiple variations and visualized in a comparative manner. In the whole-genome epidemiological analysis, multiple phylogroups were identified, revealing the genetic relationship of LHPm1 with other P. mirabilis strains carrying various AMR genes. These genetic findings offer comprehensive insights into NDM-1-producing P. mirabilis, underscoring the need for urgent control measures and surveillance programs using a “one health approach”.

Biocontrol of multidrug resistant pathogens isolated from fish farms using silver nanoparticles combined with hydrogen peroxide insight to its modulatory effect

This study was divided into two parts. The first part involved the isolation, and detection of the prevalence and antimicrobial resistance profile of Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio species from Nile tilapia fish and marine aquatic water. One hundred freshly dead Nile tilapia fish were collected from freshwater aquaculture fish farms located in Al-Abbassah district, Sharkia Governorate, and 100 samples of marine aquatic water were collected from fish farms in Port Said. The second part of the study focused on determining the in vitro inhibitory effect of dual-combination of AgNPs-H2O2 on bacterial growth and its down regulatory effect on crucial virulence factors using RT-PCR. The highest levels of A. hydrophila and P. aeruginosa were detected in 43%, and 34% of Nile tilapia fish samples, respectively. Meanwhile, the highest level of Vibrio species was found in 37% of marine water samples. Additionally, most of the isolated A. hydrophila, P. aeruginosa and Vibrio species exhibited a multi-drug resistance profile. The MIC and MBC results indicated a bactericidal effect of AgNPs-H2O2. Furthermore, a transcriptional modulation effect of AgNPs-H2O2 on the virulence-associated genes resulted in a significant down-regulation of aerA, exoU, and trh genes in A. hydrophila, P. aeruginosa, and Vibrio spp., respectively. The findings of this study suggest the effectiveness of AgNPs-H2O2 against drug resistant pathogens related to aquaculture.

The mechanism of action underlying antibacterial activity of a diterpene quinone derivative against Staphylococcus aureus through the in vitro and in silico assays

Background: The need for new antibacterials to combat resistance is still constrained by the availability of antibacterials that are safe to use. Diterpene quinone-derived compounds are proven to have antibacterial activity. However, their mechanism of action is unknown. Objective: This study aims to determine the mechanism of antibacterial action of a diterpene quinone (6O7AR) on Staphylococcus aureus through in vitro and in silico assays. Method: MIC tests were performed using microdilution, and MBC determinations were carried out on MHA media. In vitro assays were conducted using membrane permeabilizing with Tris, Triton X-100, and ATPase-inhibiting agents with NaN3. Docking was performed on 2XCT proteins in S. aureus bacteria using AutoDock Vina. Result: The MIC and MBC results of 6O7AR were 300 μM and 2400 μM, respectively. The in vitro assay result suggested that the antimicrobial activities of 6O7AR were associated with the inhibition of ATPase function and disrupting membrane function. The docking results showed that the compound possessed good interactions with the 2XCT proteins of S. aureus. Conclusion: 6O7AR exhibited good antibacterial activity. Based on in vitro and in silico assays, the mechanism of action of this compound is related to the disruption of bacterial membrane function, and it has the potential to inhibit the ATPase enzyme and the S. aureus gyrase.

Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

Characterization and investigation of cytotoxicity and antimicrobial properties of coencapsulated limonene and thymol into the Ferula assafoetida gum microparticles

Thymol (Th) and d-limonene (L) exhibit low stability and are prone to oxidation when exposed to air, light, humidity, and high temperatures. This study examined the coencapsulation of Th and L into Ferula assafoetida gum (AFG) microparticles. Scanning electron microscope (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analyzer (TGA) were done to characterize the obtained complexes. Furthermore, the encapsulation efficiency, antibacterial properties, cytotoxicity, and anticancer properties of both the free and encapsulated forms of L and Th were measured. For all samples, by increasing the percentage of bioactive compound (L, Th, and L-Th) from 2.5 to 5 % w/w, the EE was increased. FTIR and XRD analysis results demonstrated that Th and L were successfully incorporated into the AFG. Additionally, thermogravimetric analysis showed that in the thermal graphs of all samples, the first weight loss occurred between 30 °C and 160 °C, which was due to the evaporation of water. In the free L and Th graph, a sharp reduction peak was observed in which 80 % of compounds were lost. These reduction peaks disappeared in the thermal graphs of L: AFG and Th: AFG revealing that the thermal stability of Th and L was significantly increased upon their incorporation into the AFG. The inclusion of Th into the AFG also led to an increase in its antibacterial activity, while L exhibited acceptable antibacterial activity, albeit not as high as Th. Additionally, according to the MIC results, Th: AFG had the best antibacterial activity among all compounds, especially on gram-positive bacteria. According to the result of the MTT assay, there was a significant difference between the IC50 of free Th (123.4 μg/ml) and Th: AFG (2312 μg/ml), and free L (1762 μg/ml) and L: AFG (2480 μg/ml) showing that encapsulated Th and L into the AFG has decreased the cytotoxicity of free compounds against L929 cell line. Also, Th: AFG had the best anticancer activity against Hella and CT26 cell lines among all compounds. Finally, the flow cytometry analysis demonstrated that the encapsulated particles effectively eliminated cancer cells. The outcomes imply that AFG can be employed as a suitable delivery system to enhance the use of Th and L into the food and pharmaceutical industries.

Antioxidant and antimicrobial effect of sodium alginate nanoemulsion coating enriched with oregano essential oil (Origanum vulgare L.) and Trachyspermum ammi oil (Carum cupticum) on food pathogenic bacteria.

Today, microbial contamination in food is one of the major problems of the food industry and public health in general around the world. Foodborne illnesses, such as diarrheal diseases, kill many people around the world each year. The general objective of this study was to evaluate the antioxidant and antibacterial activity of sodium alginate nanoemulsion coating incorporated with oregano essential oil (Origanum vulgare L.) and Trachyspermum ammi oil (Carum cupticum) on Escherichia coli, and Listeria monocytogenes. To achieve this study, fresh chicken meat was used for this experiment. Listeria monocytogenes ATCC 19111 and Escherichia coli ATCC 35218 were obtained from the American Type Culture Collection (Manassas, VA, USA). After the preparation of the essential oil, the chemical composition of this essential oil was determined by using (GC-MS). The physicochemical properties of the nanoemulsion essential oil prepared were characterized and their antimicrobial activity was evaluated. The results showed that the GC-MS analysis of the volatile constituents of the Origanum vulgare essential oil compounds allowed the identification of 19 compounds representing 93.72% of the total oil. The major components detected in Origanum vulgare essential oil were pulegone (49.25%), eucalyptol (18.23%), and menthone (12.37%). About the Carum cupticum essential oil, 21 compounds representing 98.5% of the total oil were identified. The major components detected in Origanum vulgare essential oil were thymol (23.3%), p-cymene (17.5%), and γ-terpinene (16.8%). The best z-average (d.nm) is 483.4 nm (Carum cupticum essential oil + nano) followed by 470.1 nm (nanochitosan). The results of the antimicrobial test showed that the different preparations have a good inhibitory activity for the invitro growth of Escherichia coli and Listeria monocytogenes. According to the MIC and MBC results of this study, the nanoemulsion also presented a good bacteriostatic activity against the two pathogenic bacteria tested in this study.

Antimicrobial performance and instrumental analysis for hexagonal ZnO NPs biosynthesized via Ziziphus leaf extract

Abstract In this study, ZnO (NPs) were successfully biosynthesized using Zn(NO3)2·6H2O as the Zn+2 source and fresh Ziziphus leaf extract as the reductive and stabilizer regent. The pH and temperature of the reaction were controlled, and the NPs were calcinated at 500°C for 2 h to produce ZnO. FESEM, EDX, XRD, UV-visible, and FTIR were used to analyze ZnO NPs. UV–Vis spectroscopy confirmed the interaction of the biomolecule with the Zn precursors (λmax = 362 nm). FESEM revealed hexagonal NPs with a size of 41.7 nm. XRD analysis confirmed a hexagonal structure with an average particle size of 17.4 nm. In addition to this work, we use the Minimum Inhibitory Concentration MIC method using a Biotek 800ST plate reader (Biotek, USA) to examine the antimicrobial efficacy of biosynthesized nanoparticles against standard and clinical strains of Pseudomonas aeruginosa (ATCC PAO1), Streptococcus pneumoniae (ATCC BAA-334), and local isolate Candida albicans. The results of MIC explain that MIC value 125 µg/ml with IC50 = 56.2 µg/ml for Pseudomonas aeruginosa (ATCC PAO1), MIC value 125 µg/ml and IC50 = 38.9 µg/ml for Streptococcus pneumoniae (ATCC BAA-334) and MIC value 250 µg/ml with IC50 = 79.3 µg/ml for the local isolate Candida albicans. This green approach offers a potential strategy for developing eco-friendly antimicrobial agents.

Molecular docking and in vitro biological studies of a Schiff base ligand as anticancer and antibacterial agents

In this study, a bis-Schiff base ligand of N,N'-bis(4-dimethylaminobenzylidene)hexane-1,6-diamine (L) was obtained via a condensation reaction of N, N-dimethylaminobenzaldehyde and hexane-1,6-diamine. The ligand (L) was characterized by various analysis tools such as FT-IR, 1H and 13C NMR, Mass, DRS spectra, XRD, and thermal analyses. The ligand (L) inhibitory action was also assessed against two gram-negative and four gram-positive bacterial pathogens. Disk diffusion, MIC, and MBC test results indicate that the investigated ligand (L) has antibacterial activity against bacteria and can be employed as a therapeutic candidate after further research. The MTT assay was used to study the ligand (L) cytotoxicity on the MCF-7 cell line and calculate the IC50. Cell treatment with ligand (L) in various concentrations showed that the bis Schiff base ligand (IC50 = 6.27 ± 0.07 mgmL−1) is the most significant. Assessment of in silico ADMET ligand (L) properties illustrates that showed agreement with Lipinski’s rule of five. Lastly, the interaction between the ligand with 1STE, 1Z11, and 1AJ6 receptors was examined using docking experiments. The best conformation with the lowest affinity energy (−6.57 kcalmol−1) corresponds to the interaction of the ligand (L) with the active site of the 1Z11 receptor.

2115. Rezafungin Activity against Invasive Candidiasis Isolates Globally: Results from the 2022 Rezafungin Surveillance Program

Abstract Background Rezafungin (RZF) is a new echinocandin (ECH) approved by the US FDA to treat candidemia and invasive candidiasis (IC). Fluconazole (FLC) resistance (R) is a raising concern to treat IC, and ECHs are often used as first-line therapy. We evaluated the in vitro activity of RZF, caspofungin (CSF), micafungin (MCF), anidulafungin (ANF), and azoles against a global collection of 500 Candida isolates causing IC. Methods C. albicans (CA; 233 isolates; 46.6%), C. glabrata (CG; 102; 20.4%), C. parapsilosis (CP; 82; 16.4%), C. tropicalis (CT; 41; 8.2%), C. krusei (CK; 18; 3.6%), C. dubliniensis (CD; 15; 3.0%), and C. auris (CARS; 9; 1.8%) isolates were collected (1/patient) in 2022 from 52 medical centers located in Europe (EU; n=203; 19 centers), North America (NA; n=167; 14 centers), Asia-Pacific (AP; n=63; 8 centers), and Latin America (LA, n=67; 7 centers), identified by MALDI-TOF MS and/or sequencing and tested by CLSI broth microdilution. CLSI breakpoints (BP) were applied where available. CARS CDC tentative BPs were applied. Results RFZ exhibited activity against CA (Table) inhibiting 99.1% of the isolates overall, and 98.6%, 99.0%, 100%, and 100% of isolates from NA, EU, AP, and LA. Only 1 CA isolate (from US) displayed a FLC MIC value within the susceptible-dose dependent (SDD) category. CSP, ANF, and MCF showed S rates of 99.1% against CA. All but 1 CG isolate was susceptible (S) to RZF (99.0%S). CG S rates to CSP, ANF, MCF, and FLC were 98.0%, 98.0%, 97.1%, and 96.1% (SDD), respectively. All CP isolates were S to RZF, CSF, and MCF. However, only 84.1% of CP isolates were S to FLC. RZF and other ECHs inhibited all FLC-R isolates (10 total, 9 EU and 1 US). All CT and CK isolates were S to RZF and other ECHs. All CT were also S to azoles, and 94.4% of CK were S to VRC. Only RZF BPs are available by CLSI against CD (86.7%S). All CARS isolates (9 total; 4 EU, 3 NA, and 2 LA) were FLC-R; but 77.8% and 88.9% were S to RZF and other ECHs, respectively. Conclusion RZF demonstrated potent in vitro activity against invasive candidiasis isolates regardless of the region, and remained active against FLC-R CP and most of the FLC-R CARS isolates. Based on MIC50 and MIC90 results, RZF had similar activity to the other ECHs overall. Disclosures Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Paul Rhomberg, BS, MT(ASCP), bioMerieux: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Abby Klauer, BS, Melinta: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support

Evaluation of different methods for in vitro susceptibility testing of colistin in carbapenem resistant Gram-negative bacilli.

The increasing antibiotic resistance like the advent of carbapenem resistant Enterobactarales (CRE), Carbapenem Resistant Acinetobacter baumanii (CRAB), and Carbapenem Resistant Pseudomonas aeruginosa (CRPA) has led to to the use of toxic and older drugs like colistin for these organisms. But worldwide there is an increase in resistance even to colistin mediated both by chromosomes and plasmids. This necessitates accurate detection of resistance. This is impeded by the unavailability of a user-friendly phenotypic methods for use in routine clinical microbiology practice. The present study attempts to evaluate two different methods - colistin broth disc elution and MIC detection by Vitek two in comparison to CLSI approved broth microdilution (BMD) for colistin for Enterobactarales, Pseudomonas aeruginosa , and Acinetobacter baumanii clinical isolates. Colistin susceptibility of 6013 carbapenem resistant isolates was determined by BMD, Colistin Broth Disc Elution (CBDE), and Vitek two methods and was interpreted as per CLSI guidelines. The MIC results of CBDE, Vitek two were compared with that of BMD and essential agreement (EA), categorical agreement (CA), sensitivity, specificity, very major error (VME), major error (ME) and Cohen's kappa (CK) was calculated. The presence of any plasmid-mediated colistin resistance (mcr-1, 2, 3, 4 and 5) was evaluated in all colistin-resistant isolates by conventional polymerase chain reaction. Colistin resistance was found in 778 (12.9 %) strains among the carbapenem resistant isolates. Klebsiella pneumoniae had the highest (18.9 %) colistin resistance by the BMD method. MIC of Vitek two had sensitivity ranging from 78.2-84.8% and specificity of >92 %. There were 171 VMEs and 323 MEs by Vitek two method, much more than CLSI acceptable range. The highest percentage of errors was committed for Acinetobacter baumanii (27.8 % of VME and 7.9 % ME). On the other hand, the CBDE method performed well with EA, CA, VME and ME within acceptable range for all the organisms. The sensitivity of the CBDE method compared to gold standard BMD varied from 97.5-98.8 % for different strains with a specificity of more than 97.6 %. None of the isolated colistin resistant organisms harboured mcr plasmids. As BMD has many technical complexities, CBDE is the best viable alternative available for countries like India. A sensitive MIC reported by Vitek two needs to be carefully considered due high propensity for VMEs particularly for Klebsiella spp.

A-273 Multicenter Assessment of the Accuracy of MIC Results for Piperacillin/Tazobactam with MicroScan Dried Gram-Negative MIC Panels using CLSI Breakpoints

Abstract Background MIC data from MicroScan Dried Gram-negative MIC (MSDGN) Panels with piperacillin/tazobactam were evaluated with CLSI-M100-ED32 breakpoints for Acinetobacter, Enterobacterales, Other-Non-Enterobacterales, and proposed CLSI breakpoints for Pseudomonas aeruginosa from a multicenter clinical study. MIC results were compared to results obtained with CLSI frozen broth microdilution panels. Materials/Methods The study included a total of 683 clinical isolates tested using Prompt and turbidity during efficacy and challenge phases. MSDGN panels were evaluated at three clinical sites, comparing MIC values obtained using MSDGN panels to MICs utilizing CLSI reference panel. MSDGN panels were incubated at 35 ± 1°C and read on WalkAway System, autoSCAN-4 instrument, and read visually at 16–20 h. Frozen reference panels were prepared according to CLSI methodology, incubated for 16–20 h for Enterobacterales, Other Non-Enterobacterales, and Pseudomonas aeruginosa and for 20–24 h for Acinetobacter and read visually. CLSI breakpoints(mg/L) used for interpretation of MIC results were: ≤16/4 S, 32/4–64/4 I, ≥128/4 R for Acinetobacter and Other Non Enterobacterales, ≤8/4 S, 16/4 SDD, ≥32/4 R for Enterobacterales, and ≤16/4 S, 32/4 I, >64/4 R for Pseudomonas aeruginosa. Results When compared to frozen reference panel results, essential and categorical agreements for all isolates tested are as follows with these recommendations: Due to elevated major error rates for S. marcescens and WalkAway reads with Prompt, results should be confirmed visually prior to reporting. Due to performance with P. rettgeri and S. liquefaciens complex, do not report drug, therapy, or MIC. Conclusion Piperacillin/tazobactam MIC results for gram-negative clinical isolates obtained with the MSDGN panel correlate with MICs obtained using frozen reference panels with updated CLSI interpretive criteria.

A-274 Validation of Cefepime-Taniborbactam MIC Antimicrobial Susceptibility Test for MicroScan Dried Gram-Negative MIC Panels from a Multicenter Assessment of Enterobacterales and Pseudomonas aeruginosa

Abstract Background Cefepime-taniborbactam is an investigational agent with activity against carbapenem and multidrug-resistant Enterobacterales and Pseudomonas aeruginosa.1 Given the need for accurate antimicrobial susceptibility testing results to support patient care decisions, a multicenter study was conducted with Enterobacterales and Pseudomonas aeruginosa clinical isolates on investigational MicroScan Dried Gram-negative MIC (MSDGN) panels with cefepime-taniborbactam.2 Materials/Methods MSDGN panels were evaluated at three clinical sites by comparing MIC values obtained using MSDGN panels to MICs utilizing CLSI broth microdilution reference panels. The study included 490/491 Enterobacterales and 65 Pseudomonas aeruginosa clinical isolates tested using Prompt® and turbidity methods of inoculation during the combined efficacy and challenge phases. MSDGN panels were incubated at 35 ± 1°C and read on the WalkAway System, the autoSCAN-4 instrument, and visually at 16–20 h. Frozen reference panels were prepared according to CLSI/ISO methodology, incubated for 16–20 h and read visually. Results Essential agreement was calculated compared to MIC results from frozen reference panels for isolates tested in efficacy and challenge and found in the following table. Conclusion Cefepime-taniborbactam MIC results for Enterobacterales and Pseudomonas aeruginosa obtained with the MSDGN panel correlate well with MICs obtained using frozen reference panels in this multicenter study.Pending approval by the US Food and Drug Administration; not available for in vitro diagnostic use in the US. Not for Distribution in the US.Pending submission and clearance by the US Food and Drug Administration; not yet available for in vitro diagnostic use in the US.For Investigational Use Only. Performance characteristics of this product have not been established.Read methodEnterobacterales Essential agreement (EA) % (n/N)Pseudomonas aeruginosaEssential agreement (EA) % (n/N)PromptTurbidityPromptTurbidityWalkAway90.0 (441/490)99.2 (487/491)100.0 (65/65)100.0 (65/65)autoSCAN-494.3 (462/490)99.0 (486/491)98.5 (64/65)98.5 (64/65)Visually92.7 (454/490)99.2 (487/491)100.0 (65/65)100.0 (65/65)Abbreviations: Prompt, Prompt inoculation method, Turbidity, Turbidity inoculation method.

Identification and characterization of SCCmec typing with psm-mec positivity in staphylococci from patients with coagulase-negative staphylococci peritoneal dialysis-related peritonitis

BackgroundPeritonitis is the most important complication of peritoneal dialysis (PD) and coagulase-negative staphylococci (CNS) are a frequent cause of dialysis-related infections. The association between SCCmec typing with psm-mec positivity in staphylococci and PD-related infections has not been identified. We aim to investigate the molecular epidemiology of CNS isolated from PD-peritonitis in a single Chinese center, focusing on the genetic determinants conferring methicillin resistance.MethodsWe collected 10 genetically unrelated CNS isolates from 10 patients with CNS PD-related peritonitis. The patients were divided into two groups based on the results of MIC to oxacillin: the methicillin-resistant CNS (MRCNS) and methicillin-sensitive CNS (MSCNS) groups. The biofilm formation group (BFG) and the non-biofilm formation group (NBFG) were used as the control groups. Phenotypic and molecular methods were used to analyze SCCmec types I, II and III, associated genes and biofilm formation and the existence of psm-mec. The demographic data and clinical indicators were collected.ResultsTen CNS PD-related peritonitis patients were enrolled for this study. There were 6 MRCNS and 4 MRCNS isolates. SCCmec types were fully determined in 10 isolates. Seven staphylococci (70%) carried SCCmec, of which 4 isolates carried single SCCmec type I (40%) and 3 isolates had multiple SCCmec elements (I + III). Of the 6 MRCNS isolates, 3 carried SCCmec type I (50%) and 2 isolates carried SCCmec type I + III (33.3%). A high diversity of ccr types, mec complexes and ccr-mec complex combinations was identified among the 10 CNS isolates. The psm-mec gene was detected in 2/10 (20%) CNS isolates. There was no mutation in the psm-mec gene.ConclusionsThe majority of isolates were hospital-associated isolates. Furthermore, 2 psm-mec positive isolates were MRCNS in the NBFG. The PD patients frequent exposure to hospital would be the main risk factor. The presence of the psm-mec signal in the spectra of the MRCNS tested here demonstrates the presence of certain SCCmec cassettes that convey methicillin resistance.

Antimicrobial and Phytochemical Activities of Garlic (Allium sativum) on Staphylococcus aureus and Candida albicans Isolated from High Vaginal Swab samples and Female Students with UTI

Studies on antimicrobial and phytochemical activities of garlic on Staphylococcus aureus and Candida albicans isolated from HVS samples and female students with urinary tract infection were investigated. Garlic is used in many homes as condiment or as flavoring agent. The garlic used was purchased from Eke market Agbani in Nkanu West Enugu State Nigeria. Ten Urine and HVS samples were collected from female students using a universal container and swab sticks. Preliminary qualitative phytochemical analysis was done using standard methods. The powder of garlic was macerated in ethanol and chloroform to produce crude extract reconstituted with distilled water to concentrations of 200mg/ml, 100mg/ml, 50mg/ml, 25mg/ml and 12.5mg/ml. The ethanol and chloroform extract of garlic obtained was used to evaluate antimicrobial activity against Staphylococcus aureus and Candida albicans. Also, minimum inhibitory concentration was determined. The garlic extract contained alkaloids, tannins, flavonoids, terpenoids and saponins. The inhibitory effect of ethanol extract started at 200mg/ml with inhibition zones of 2mm against Staphylococcus aureus and Candida albicans. MIC result for garlic extract on Staphylococcus aureus and Candida albicans showed that the extract has inhibitory effect at low concentration of 50mg/ml and inhibition increased at 100mg/ml. Ethanol extract was the most effective extract retarding microbial growth of Staphylococcus aureus and Candida albicans. This study however, justify the use of garlic extract in traditional medicine practice as a therapeutic agent. In cases where possible, the ethanol extracts of garlic should be used at a concentration up to 100mg/ml so as to give a better treatment.

The Antioxidant, Antimicrobial, and Total Phenolic Potential of Clove Extracts for Inhibition of Food Pathogens

Food spoilage pathogens cause food waste and consumption of pathogen-contaminated food threatens human health. New approaches that do not harm the environment are needed for decreasing the enlargement of pathogenic microorganisms without using chemical preservatives. The current work intended to appraise the TPC value, anti-oxidant, anti-bacterial, and anti-fungal properties of clove different extracts. The antimicrobial tests were evaluated by disc diffusion, MIC, and MBC tests. Antioxidant potential was conducted using ABTS• and DPPH• radical, and TPC was tested by the Folin–Ciocalteu reagent assay. As a result, the highest antimicrobial activity was found against E. faecalis by 19,30±0,17 mm zone diameter from methanol extract. The lowest activity was obtained from aqueous extract over S. Typhimurium by 7,17±0,29 mm zone diameter. MIC and MBC results were examined, and it was determined that clove ethanol extract showed the highest MIC value was 2.5-10 mg/ml. MBC test results also revealed that cloves ethanol extract has the highest activity with 5->10 mg/ml. The antioxidant data of cloves were examined, and the highest DPPH• and ABTS• sweep activities were determined in 60.93±1.67% aqueous and 85.81±1.08 ethanol extract, respectively. The TPC results revealed that the highest content was provided from the aqueous extract with 189.84±2.84 mg/g GA. The results gained from the study bring to light that clove has a high potential for antimicrobial, antioxidant, and total phenolic content.