Broth Microdilution Assay Research Articles

Antibacterial activity and anxiolytic-like effect of Ziziphus joazeiro Mart. leaves in adult zebrafish (Danio rerio)

Ziziphus joazeiro Mart. is an endemic plant of the Caatinga that presents a great socioeconomic importance for the Northeast and Semiarid Region of Brazil. In view of this, this study aimed to evaluate the antibacterial activity and anxiolytic-like effects of Ziziphus joazeiro Mart leaves in adult zebrafish (Danio rerio). The characterization of the main classes of metabolites was performed through chemical reactions. The antibacterial and antibiotic potentiating activity was evaluated by broth microdilution assays. The 96 h acute toxicity, open field test and anxiety models test was evaluated in vivo on adult zebrafish. The results obtained in the phytochemical prospection evidenced the presence of flobabenic tannins, leucoanthocyanidins, flavonois, flavonones, catechins, alkaloids, steroids, and triterpenoids. EEFZJ did not show antibacterial activity for all microorganism tested (MIC ≥ 1024 µg/mL), but reduced the concentration required for bacterial growth inhibition in combination with gentamicin and norfloxacin against multidrug-resistant strains of S. aureus (SA10) and E. coli (EC06), exhibiting synergistic effect with these antibiotics (p<0.0001). In the tests in vivo, EEFZJ was found to be nontoxic, performing reduced locomotor activity and demonstrated an anxiolytic-like effect in adult zebrafish via GABAergic and Serotoninergic systems (5-HT1, 5-HT2A/2C and 5-HT3A/3B).

Comprehensive Assessment of Transcorneal Permeation, Antimicrobial, and Antifungal Activities of Andrographolide-Loaded Nanosuspension: In vitro and In vivo Studies

This study aimed to comprehensively assess the transcorneal permeation, antimicrobial, and antifungal activities of a nanosuspension loaded with Andrographolide, a promising herbal compound. Through a combination of in vitro and in vivo studies, the efficacy, and potential applications of the nanosuspension in ocular drug delivery were investigated. In the in vitro phase, transcorneal permeation studies were conducted using Franz diffusion cells with excised rabbit corneas. The nanosuspension demonstrated significantly enhanced permeation compared to a control formulation, indicating its ability to effectively deliver Andrographolide through the cornea and into the ocular tissues. Additionally, the nanosuspension exhibited potent antimicrobial and antifungal activities against various ocular pathogens, as determined by agar diffusion and broth microdilution assays. Building upon the promising in vitro results, in vivo studies were performed using a rabbit model. Ocular tolerability of the nanosuspension was assessed through eye observations, with no observed signs of irritation or adverse effects. Furthermore, the nanosuspension's in vivo antimicrobial and antifungal activities were evaluated by instilling the formulation into the rabbits' eyes and monitoring conjunctival congestion and inflammation. The results demonstrated a significant reduction in conjunctival congestion, highlighting the nanosuspension's potential for combating ocular infections and inflammation. The comprehensive assessment presented in this study establishes the transcorneal permeation capability of the Andrographolide-loaded nanosuspension and its remarkable antimicrobial and antifungal activities. These findings underscore the potential of the nanosuspension as an effective ocular drug delivery system for various ocular infections and inflammatory conditions. The study contributes to the development of novel therapeutic approaches in ophthalmology, aiming to improve patient outcomes and provide alternative treatment options for ocular diseases.&#x0D; Keywords: Transcorneal permeation; Nanosuspension; Andrographolide; Antimicrobial activity; Antifungal activity; Ocular drug delivery

Antibacterial effect of essential oils and their components against Xanthomonas arboricola pv. pruni revealed by microdilution and direct bioautographic assays.

Bacterial spot of stone fruits caused by Xanthomonas arboricola pv. pruni (Xap) is one of the most significant diseases of several Prunus species. Disease outbreaks can result in severe economic losses while the control options are limited. Antibacterial efficacy of essential oils (EOs) of thyme, cinnamon, clove, rosemary, tea tree, eucalyptus, lemon grass, citronella grass, and lemon balm was assessed against two Hungarian Xap isolates. The minimal inhibitory concentration (MIC) was determined by broth microdilution assay and for the identification of active EOs' components a newly introduced high-performance thin-layer chromatography (HPTLC)-Xap (direct bioautography) method combined with solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) was applied. All EOs inhibited both bacterium isolates, but cinnamon proved to be the most effective EO with MIC values of 31.25 µg/mL and 62.5 µg/mL, respectively. Compounds in the antibacterial HPTLC zones were identified as thymol in thyme, trans-cinnamaldehyde in cinnamon, eugenol in clove, borneol in rosemary, terpinen-4-ol in tea tree, citral (neral and geranial) in lemon grass and lemon balm, and citronellal and nerol in citronella grass. Regarding active compounds, thymol had the highest efficiency with a MIC value of 50 µg/mL. Antibacterial effects of EOs have already been proven for several Xanthomonas species, but to our knowledge, the studied EOs, except for lemon grass and eucalyptus, were tested for the first time against Xap. Furthermore, in case of Xap, this is the first report demonstrating that direct bioautography is a fast and suitable method for screening anti-Xap components of complex matrices, like EOs.

P35 Heteroresistance to cefiderocol in clinical Gram-negative isolates

Abstract Background Heteroresistance is a phenotype in which a clinically susceptible bacterial isolate contains less susceptible subpopulations. Unexpectedly high levels of treatment failure for cefiderocol, a novel antibiotic designed to treat multidrug-resistant infections, have been attributed to this phenomenon. Objectives To determine prevalence of cefiderocol heteroresistance amongst clinical isolates at a North London tertiary care centre and study the population dynamics upon exposure to the antibiotic. Methods Gram-negative clinical isolates (n = 146) were screened for cefiderocol susceptibility by standard disc diffusion and Etest assays. Isolates were classified based on EUCAST breakpoints for cefiderocol susceptibility. Heteroresistance was confirmed by performing modified-population analysis profile (mPAP) assay in iron-depleted Mueller Hinton II agar. MIC values were determined by broth microdilution (BMD) assay as per EUCAST guidelines. The heteroresistant isolates and their corresponding less susceptible subpopulations were serially passaged in media with sub-inhibitory concentrations of cefiderocol as well as in cefiderocol-free medium. The changes in frequency and/or susceptibility in the heteroresistant subpopulation were detected using mPAP. Results Eleven heteroresistant (7.53%) and twenty-two resistant (15.06%) isolates were identified. Cefiderocol susceptibility of the heteroresistant isolates after five passages in cefiderocol-free medium partially reverted to that of the parent population. However, this phenomenon was not observed in the cohort that was first passaged in cefiderocol-containing medium before passages in cefiderocol-free medium. When heteroresistant populations were passaged in cefiderocol-containing media at 0.25× MIC values, no MIC changes were observed after five serial passages; however, the proportion of less susceptible subpopulations increased by 1.2 log10 in one out of five isolates. Nevertheless, when the antibiotic pressure was removed by passaging in cefiderocol-free media for a further five serial passages, the MIC and/or frequency of the less susceptible colonies within the heteroresistant population further increased in 80% of the passaged isolates. In one isolate, the MIC of the heteroresistant sub-population increased by &amp;gt;32-fold while the frequency of the heteroresistant sub-population increased by 1.0 log10 in another. Conclusions The prevalence of heteroresistance to cefiderocol was around 7% amongst clinical isolates. The heteroresistant phenotypes were unstable unless exposed to cefiderocol. Such exposure may also increase the MIC and/or frequency of less susceptible cells within the heteroresistant population. This amplification can continue even after removal of antibiotic pressure. Developing methods for detecting heteroresistance to cefiderocol in clinical laboratories is essential. Clinicians need to take this phenomenon into account when planning treatment regimens to optimize the effectiveness of this invaluable addition to the antimicrobial armamentarium.

Chemical Composition, Antioxidant Properties, and Antibacterial Activity of Essential Oils of Satureja macrostema (Moc. and Sessé ex Benth.) Briq.

Satureja macrostema is a plant that is located in various regions of Mexico and is used in a traditional way against illness. Essential oils (EOs) were obtained from leaves Satureja macrostema and the chemical composition was evaluated by gas chromatography-mass spectrometry (GC-MS). The antioxidant effect of the oil was assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and by Trolox Equivalent Antioxidant Capacity (TEAC). In vitro antibacterial activity against Escherichia coli and Staphylococcus aureus was determined using a broth microdilution assay and thin layer chromatography-direct bioautography (TLC-DB) to identify active antibacterial compounds. The EOs analysis showed 21 compounds, 99% terpenes, and 96% oxygenated monoterpenes, with trans-piperitone epoxide (46%), cis-piperitone epoxide (22%), and piperitenone oxide (11%) as more abundant compounds. Likewise, S. macrostema EOs showed an antioxidant activity of DPPH = 82%, with 50% free radical scavenging (IC50) = 7 mg/mL and TEAC = 0.005, an antibacterial effect against E. coli of 73% inhibition, and 81% over S. aureus at dose of 100 µL of undiluted crude oil. The TLC-DB assay showed that the most active compounds were derived from piperitone. The comparison with other studies on S. macrostema shows variability in the compounds and their abundances, which can be attributed to climatic factors and the maturity of plants with similar antioxidant and antibacterial activities.

Antimicrobial Potential of Biosynthesized Zinc Oxide Nanoparticles Using Banana Peel and Date Seeds Extracts

In the present study, zinc oxide nanoparticles (ZnONPs) were eco-friendly synthesized using banana peel (BPE) and date seed (DSE) extracts. Biosynthesis of both ZnONPs_BPE and ZnONPs_DSE was confirmed by using an ultraviolet–visible spectrophotometer (UV–VIS), then followed by their characterization using different analyses: scanning (SEM), transmission electron microscope (TEM), zeta potential analysis, X-ray diffraction (XRD), and Fourier-transform infrared (FTIR) spectroscopy. The antimicrobial potency of ZnONPs_BPE and ZnONPs_DSE was evaluated using a broth microdilution assay against pathogenic strains to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). UV spectra confirm the formation of ZnONPs-BPE (290 nm) and ZnONP_DSE (400 nm). TEM, SEM, and XRD revealed their hexagonal crystalline structures with nanoscale size ZnONPs_BPE (57.4 ± 13.8 nm, −9.62 mV) and ZnONPs_DSE (72.6 ± 17.1 nm, −5.69 mV). FTIR analysis demonstrated the presence of various functional groups on ZnONPs’ surfaces that act as reducing, capping, and stabilizing agents. The biosynthesized ZnONPs demonstrated a good antimicrobial potential against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Salmonella enteritidis) strains. Especially, ZnONPs-BPE has a higher antimicrobial effect against Salmonella enteritidis (MIC = 0.75 mg/mL, MBC = 1.5 mg/mL), while ZnONP_DSE has a higher effect against Staphylococcus aureus (MIC = 0.75 mg/mL, MBC = 3 mg/mL). The present results are consistent with previous studies that reported the antimicrobial potential of green ZnONPs through ROS induction that in turn damages microbial DNA. Consequently, the present results support the use of different biowastes in NPs’ synthesis, which is a simple and sustainable way that helps with waste management and decreases environmental pollution.

Conjugated carbon monoxide-releasing moleculeshave broad-spectrum antimicrobial activity.

Aim: To test the antimicrobial effect of carbon monoxide-releasing molecules (CORMs) conjugated with azoles on different microorganisms. Methods & results: We used broth microdilution, checkerboard and cytotoxicity assays, as well asimaging, fluorescenceand bioluminescence experiments to study [Re(CO)3(2,2'-bipyridyl)(Ctz)]+ (also known asReBpyCtz). ReBpyCtz exhibits alow minimum inhibitory concentrationvalue, increases the intracellular formation of reactive oxygen species and causes significant alterations on Staphylococcus aureus's membrane. ReBpyCtz is active against fungi, having a more prolonged fungicidal effect on Candida glabrata than clotrimazole andis selectively active onblood-stage malaria parasites, at a concentration that is not toxicto kidney epithelial cells. Conclusion: Conjugated CORMs have the potential to be active against different types of pathogens, thus constituting a promising class of broad-spectrum antimicrobials.

Exploring the biological application of Penicillium fimorum-derived silver nanoparticles: In vitro physicochemical, antifungal, biofilm inhibitory, antioxidant, anticoagulant, and thrombolytic performance

This study showed the anti-candida, biofilm inhibitory, antioxidant, anticoagulant, and thrombolytic properties of biogenic silver nanoparticles (AgNPs) fabricated by using the supernatant of Penicillium fimorum (GenBank accession number OQ568180) isolated from soil. The biogenic AgNPs were characterized by using different analytical techniques. A sharp surface plasmon resonance (SPR) peak of the colloidal AgNPs at 429.5 nm in the UV–vis spectrum confirmed the fabrication of nanosized silver particles. The broth microdilution assay confirmed the anti-candida properties of AgNPs with a minimum inhibitory concentration (MIC) of 4 μg mL−1. In the next step, the protein and DNA leakage assays as well as reactive oxygen species (ROS) assay were performed to evaluate the possible anti-candida mechanisms of AgNPs representing an increase in the total protein and DNA of supernatant along with a climb-up in ROS levels in AgNPs-treated samples. Flow cytometry also confirmed a dose-dependent cell death in the AgNPs-treated samples. Further studies also confirmed the biofilm inhibitory performance of AgNPs against Candia albicans. The AgNPs at the concentrations of MIC and 4*MIC inhibited 79.68 ± 14.38% and 83.57 ± 3.41% of biofilm formation in C. albicans, respectively. Moreover, this study showed that the intrinsic pathway may play a significant role in the anticoagulant properties of AgNPs. In addition, the AgNPs at the concentration of 500 μg mL−1, represented 49.27%, and 73.96 ± 2.59% thrombolytic and DPPH radical scavenging potential, respectively. Promising biological performance of AgNPs suggests these nanomaterials as a good candidate for biomedical and pharmaceutical applications.

Phytochemical analysis and evaluation of antimicrobial, antioxidant, and antidiabetic activities of essential oils from Moroccan medicinal plants: Mentha suaveolens, Lavandula stoechas, and Ammi visnaga

Mentha suaveolens, Lavandula stoechas, and Ammi visnaga are widely used in Moroccan folk medicine against several pathological disorders, including diabetes and infectious diseases. This work was designed to determine the chemical profile of M. suaveolens (MSEO), L. stoechas (LSEO), and A. visnaga (AVEO) essential oils and assess their antimicrobial, antioxidant, and antidiabetic effects. The volatile components of LSEO, AVEO, and MSEO were analyzed using Gas Chromatography-Mass Spectrometry (GC–MS). The in vitro antidiabetic activity was assessed using α-amylase and α-glucosidase enzymes, while DPPH, FRAP, and β-carotene/linoleic acid methods were used to determine the antioxidant capacity. The antimicrobial activities were investigated using disc diffusion and broth-microdilution assays. GC-MS investigation revealed that the main components were fenchone (29.77 %) and camphor (24.9 %) for LSEO, and linalool (38.29 %) for AVEO, while MSEO was mainly represented by piperitenone oxide (74.55 %). The results of the antimicrobial evaluation showed that all examined essential oils (EOs) had noticeable antimicrobial activity against both bacteria and yeast, especially Micrococcus luteus and Bacillus subtilis. The MIC, MBC, and MFC values were ranged from 0.015 % to 0.5 %. The MBC/MIC and MFC/MIC ratios were less than or equal to 4.0 % (v/v), indicating their noticeable bactericidal and candidacidal efficacy. Moreover, the three EOs showed significant inhibitory effects on α-amylase and α-glucosidase (p < 0.05). It also exerted remarkable activity on FRAP, β-carotene, and DPPH radicals. These findings demonstrated that the tested plants have promising biological activities, validating their ethnomedicinal value and providing potential applications as natural drugs.

In Vitro Activity of Novel Topoisomerase Inhibitors against Francisella tularensis and Burkholderia pseudomallei.

Antimicrobial resistance is a global issue, and the investigation of alternative therapies that are not traditional antibiotics are warranted. Novel bacterial type II topoisomerase inhibitors (NBTIs) have recently emerged as a novel class of antibiotics with reduced potential for cross-resistance to fluoroquinolones due to their novel mechanism of action. This study investigated the in vitro activity of a series of cyclohexyl-oxazolidinone bacterial topoisomerase inhibitors against type strains of Francisella tularensis and Burkholderia pseudomallei. Broth microdilution, time-kill, and cell infection assays were performed to determine activity against these biothreat pathogens. Two candidates were identified that demonstrated in vitro activity in multiple assays that in some instances was equivalent to ciprofloxacin and doxycycline. These data warrant the further evaluation of these novel NBTIs and future iterations in vitro and in vivo.

Flowering phenophases influence the antibacterial and anti-biofilm effects of Thymus vulgaris L. essential oil

BackgroundEssential oils are becoming increasingly popular in medicinal applications because of their antimicrobial effect. Thymus vulgaris L. (Lamiaceae) is a well-known and widely cultivated medicinal plant, which is used as a remedy for cold, cough and gastrointestinal symptoms. Essential oil content of thyme is responsible for its antimicrobial activity, however, it has been reported that the chemical composition of essential oils influences its biological activity. In order to explore flowering phenophases influence on the chemical composition of thyme essential oil and its antibacterial and anti-biofilm activity, plant materials were collected at the beginning of flowering, in full bloom and at the end of flowering periods in 2019.MethodsEssential oils from fresh and dried plant materials were distilled and analyzed with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID). The antibacterial activity was performed by broth microdilution and thin layer chromatography-direct bioautography (TLC-DB) assays and the anti-biofilm effect by crystal violet assay, respectively. Scanning electron microscopy was applied to illustrate the cellular changes of bacterial cells after essential oil treatment.ResultsThymol (52.33–62.46%) was the main component in the thyme essential oils. Thyme oil distilled from fresh plant material and collected at the beginning of flowering period exerted the highest antibacterial and anti-biofilm activity against Haemophilus influenzae, H. parainfluenzae and Pseudomonas aeruginosa.ConclusionThe different flowering periods of Thymus vulgaris influence the antibacterial and anti-biofilm activity of its essential oils, therefore, the collection time has to be taken into consideration and not only the full bloom, but the beginning of flowering period may provide biological active thyme essential oil.

Characterization of blaAFM-1-positive carbapenem-resistant strains isolated in Guangzhou, China

BackgroundCarbapenemase-producing makes a great contribution to carbapenem resistance in Gram-negative bacilli. BlaAFM-1 gene was first discovered by us in Alcaligenes faecalis AN70 strain isolated in Guangzhou of China and, was submitted to NCBI on 16 November 2018.MethodsAntimicrobial susceptibility testing was performed by broth microdilution assay using BD Phoenix 100. The phylogenetic tree of AFM and other B1 metallo-β-lactamases was visualized by MEGA7.0. Whole-genome sequencing technology was used to sequence carbapenem-resistant strains including the blaAFM-1 gene. Cloning and expressing of blaAFM-1 were designed to verify the function of AFM-1 to hydrolyze carbapenems and common β-lactamase substrates. Carba NP and Etest experiments were conducted to evaluate the activity of carbapenemase. Homology modeling was applied to predict the spatial structure of AFM-1. A conjugation assay was performed to test the ability of horizontal transfer of AFM-1 enzyme. The genetic context of blaAFM-1 was performed by Blast alignment.ResultsAlcaligenes faecalis strain AN70, Comamonas testosteroni strain NFYY023, Bordetella trematum strain E202, and Stenotrophomonas maltophilia strain NCTC10498 were identified as carrying the blaAFM-1 gene. All of these four strains were carbapenem-resistant strains. Phylogenetic analysis revealed that AFM-1 shares little nucleotide and amino acid identity with other class B carbapenemases (the highest identity (86%) with NDM-1 at the amino acid sequence level). The spatial structure of the AFM-1 enzyme was predicted to be αβ/βα sandwich structure, with two zinc atoms at its active site structure. Cloning and expressing of blaAFM-1 verified AFM-1 could hydrolyze carbapenems and common β-lactamase substrates. Carba NP test presented that the AFM-1 enzyme possesses carbapenemase activity. The successful transfer of pAN70-1(plasmid of AN70) to E.coli J53 suggested that the blaAFM-1 gene could be disseminated by the plasmid. The genetic context of blaAFM indicated that the downstream of the blaAFM gene was always adjacent to trpF and bleMBL. Comparative genome analysis revealed that blaAFM appeared to have been mobilized by an ISCR27-related mediated event.ConclusionsThe blaAFM-1 gene is derived from chromosome and plasmid, and the blaAFM-1 gene derived from the pAN70-1 plasmid can transfer carbapenem resistance to susceptible strains through horizontal transfer. Several blaAFM-1-positive species have been isolated from feces in Guangzhou, China.

Bacillus cereus in the Artisanal Cheese Production Chain in Southwestern Mexico.

Bacillus cereus is associated with milk, dairy product, and dairy farm contamination. The aim of this study was to characterize strains of B. cereus in the small-scale artisanal cheese production chain in southwestern Mexico. 130 samples were collected. B. cereus isolation was performed on Mannitol Egg Yolk Polymyxin (MYP) agar. Genotyping, enterotoxigenic profile, and determination of genes involved in the formation of B. cereus biofilm were performed by PCR. An antimicrobial susceptibility test was made by broth microdilution assay. The phylogenetic analysis was performed by amplification and sequencing of 16s rRNA. B. cereus sensu lato was isolated and molecularly identified in 16 samples and B. cereus sensu stricto (B. cereus) was the most frequently isolated and identified species (81.25%). Of all the isolated B. cereus sensu lato strains, 93.75% presented at least one gene for some diarrheagenic toxins, 87.5% formed biofilms, and 18.75% were amylolytic. All B. cereus sensu lato strains were resistant to beta-lactams and folate inhibitors. A close phylogenetic relationship between isolates was found between the cheese isolates and the air isolates. Strains of B. cereus sensu lato were found in small-scale artisanal cheeses on a farm in southwestern Mexico.

Essential oil composition, antimicrobial and antioxidant properties of Pluchea eupatorioides Kurz collected from Vietnam

Pluchea eupatorioides Kurz, which belongs to the family Asteraceae, is known for its medicinal properties and has been traditionally used to treat various diseases. This study aimed to investigate the chemical composition of the essential oil extracted from the aerial parts of P. eupatorioides and evaluate its antimicrobial and antioxidant activities. The essential oil was isolated by hydrodistillation and analysed by gas chromatography/ mass spectrometry (GC/MS). Thirty-three volatile compounds were identified in P. eupatorioides essential oil, including β-caryophyllene (29.36%), 2,5-dimethoxy-p-cymene (18.41%), α-isocomene (15.74%), and caryophyllene oxide (9.77%) as the major compounds. The antimicrobial activity of P. eupatorioides essential oil was investigated using a broth microdilution assay to determine the minimum inhibitory concentration (MIC). The results indicated that P. eupatorioides essential oil demonstrated effectiveness against Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 10231 with a MIC of 100 µg/mL. Moreover, the essential oil was also found to be effective against Bacillus cereus ATCC 14579 and Escherichia coli ATCC 25922, but with a slightly higher MIC of 200 µg/mL. Furthermore, the antioxidant activity of P. eupatorioides essential oil was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging assays. The IC50 values calculated using the DPPH and ABTS assays were 164.2 and 136.1 µg/ mL, respectively. These findings demonstrate that the P. eupatorioides essential oil could be used as a potential source of antimicrobial and antioxidant agents for the food and pharmaceutical industries. This is the first report describing the chemical compositions, antimicrobial and antioxidant activities of P. eupatorioides essential oil.

Bovine Staphylococcus aureus: a European study of contagiousness and antimicrobial resistance.

In dairy herds managements, mastitis is the leading cause of economic losses. One of the most important pathogens responsible for intra-mammary infections is Staphylococcus aureus. The genetic properties of S. aureus have a strong influence on its pathogenicity and contagiousness. In this study, we aimed to obtain a comprehensive overview of the key bovine S. aureus clinical properties, such as contagiousness and antimicrobial resistance, present in European strains. For this, 211 bovine S. aureus strains from ten European countries that were used in a previous study were used in this study. Contagiousness was assessed using qPCR for the detection of the marker gene adlb. Antimicrobial resistance was evaluated using a broth microdilution assay and mPCR for the detection of genes involved in penicillin resistance (blaI, blaR1, and blaZ). It was found that adlb was present in CC8/CLB strains; however, in Germany, it was found in CC97/CLI and in an unknown CC/CLR strains. CC705/CLC strains from all countries were found to be susceptible to all tested antibiotics. Major resistance to penicillin/ampicillin, chloramphenicol, clindamycin and tetracycline was detected. Resistance to oxacillin, trimethoprim/sulfamethoxazole and cephalosporins was rarely observed. In addition, contagiousness and antibiotic resistance seem to correlate with different CCs and genotypic clusters. Hence, it is recommended that multilocus sequence typing or genotyping be utilized as a clinical instrument to identify the most appropriate antibiotic to use in mastitis treatment. Actualization of the breakpoints of veterinary strains is necessary to address the existing antibiotic resistance of the bacteria involved in veterinary mastitis.

Development of a microencapsulated probiotic containing Pediococcus acidilactici WU222001 against avian pathogenic Escherichia coli.

Probiotics are beneficial microorganisms for humans and animals. In this study, we developed a microencapsulated probiotic with antibacterial activity against avian pathogenic Escherichia coli (APEC). Alignment of the 16S rRNA sequences of the isolate WU222001 with those deposited in GenBank revealed that the isolate was Pediococcus acidilactici with 99.6% homology. This bacterium was characterized as a probiotic based on its tolerance toward in vitro gastrointestinal tract (GIT) conditions, hydrophobicity, and auto-aggregation. The antibacterial activity of the probiotic's culture supernatant against APEC was investigated using a broth microdilution assay. Pediococcus acidilactici was microencapsulated using sodium alginate and agar with diameters ranging from 47 to 61 μm. Then, physicochemical characteristics and stability of the microcapsules were determined. The isolate was characterized as a probiotic based on its resistance to low pH, bile salts, and pancreatin, with relative values of 79.2%, 70.95%, and 90.64%, respectively. Furthermore, the bacterium exhibited 79.56% auto-aggregation and 55.25% hydrophobicity at 24 h. The probiotic's culture supernatant exhibited strong antibacterial activity against clinical APEC isolates with minimum inhibitory concentration and minimum bactericidal concentration of 12.5% and 25% v/v, respectively. Microencapsulation-enhanced bacterial viability in GIT compared to free cells. Moreover, 89.65% of the encapsulated cells were released into the simulated intestinal fluid within 4 h. The viable count in microcapsules was 63.19% after 3 months of storage at 4°C. The results indicated that the culture supernatant of P. acidilactici inhibited the growth of APEC. In addition, microencapsulation extends the viability of P. acidilactici under harsh conditions, indicating its potential application in the feed production.

Structure-based drug design of multi-targeting inhibitors of human pathogen fungi

The development of multi-targeting inhibitors is proposed to improve the antifungal effect. In a single chemical entity it is possible to inhibit two different targets. Thioredoxin reductase and glutathione reductase belong to the two main systems responsible for the maintenance of cell homeostasis. Herein, we describe the discovery and selection of dual-inhibitors targeting thioredoxin reductase and glutathione reductase proteins of Aspergillus fumigatus. Both proteins were modeled and molecular dynamics were performed to obtain different conformational frames to ensemble docking. After the independent virtual screening of each protein, a ranking containing molecules selected for both proteins were generated. Fourteen molecules were tested in vitro against A. fumigatus, Fusarium oxysporum, Paracoccidioides brasiliensis, Candida albicans and Cryptococcus neoformans by broth microdilution assay. Octenidine (OCT) was the most promising molecule which exhibited MIC < 0.5 µg/mL for all fungi tested. The literature describes the action of OCT in the cell membrane of gram-negative bacteria. Thus, we assume that the mechanism in which OCT acts on fungal cells is an association of disrupting the cell membrane and also the dual-inhibition of thioredoxin reductase and glutathione reductase which implies the increase of ROS and further fungal cell death.

Epidemiological and Antimicrobial Resistant Patterns, and Molecular Mechanisms of Carbapenem-Resistant Klebsiella pneumoniae Infections in ICU Patients.

To study the epidemiological and antimicrobial resistant patterns, clinical characteristics and risk factors of critically ill patients infected with carbapenem-resistant Klebsiella pneumoniae (CRKP) from intensive care units (ICUs). The potential molecular mechanisms of antimicrobial resistance and virulence of CRKP were investigated through evaluation of associated genes. Totally, 201 ICU patients infected with K. pneumoniae were recruited from January 2020 through January 2021. K. pneumoniae strains were collected from diverse clinical specimens and identified by microbial cultures and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Antimicrobial resistance was measured through broth micro-dilution or Kirby-Bauer assays. The carbapenemase-, virulence-, and capsular serotype-associated genes of CRKP were individually detected by PCR and sequencing. Demographic and clinical profiles were acquired from hospital databases to evaluate the correlation of CRKP infection incidence with clinical risk factors. Of the 201 K. pneumoniae strains, CRKP accounted for 41.29%. Seasonal bias existed in local prevalence of CRKP infections. CRKP strains mounted significantly strong resistance against major antimicrobial agents except ceftazidime-avibactam, tigecycline and minocycline. Recent exposure to certain antibiotics and prior treatment with invasive interventions were prone to increase CRKP infection risks with worsened infectious outcomes. The local top carbapenemase-encoding and virulence-associated genes of CRKP were blaKPC and irp2, respectively. Nearly half of CRKP isolates harbored a capsular polysaccharide serotype of K14.K64 (wzi-64) which preferentially emerged in the cohort with worse outcomes of infection. Featured epidemiology and typical clinical characteristics existed extensively in K. pneumoniae infections among ICU patients. The CRKP cohort exhibited substantially high antimicrobial resistance. Distinctive carbapenemase-, virulence-, and serotype-associated genes were intensively involved in the spread and pathogenesis of CRKP. These findings supported careful management of critically ill patients potentially infected with virulent CRKP in the ICUs.

Minimum inhibitory concentrations and sequencing data have to be analysed in more detail to set provisional epidemiological cut-off values forMycobacterium tuberculosiscomplex

There is a clear need for a high-quality broth microdilution (BMD) assay for quantitative antimicrobial susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC) [1]. The BMD plate developed by the CRyPTIC Consortium, therefore, represents an important advance [2]. However, the analysis of the resulting data has several limitations. Provisional epidemiological cut-off values for M. tuberculosis complex need to be set carefully <http://bit.ly/3XjJ8px> C.U. Köser is a visiting scientist at the Department of Genetics, University of Cambridge, and research associate at Wolfson College, University of Cambridge. Feedback from G. Kahlmeter, T. Schön and J. Turnidge was gratefully received. We thank L. Sonnenkalb for clarifications regarding her work.

Comparative Study of the Antibacterial Activity of Algerian Honeys and Manuka Honey Toward Pathogenic Bacteria from Burn Wound Infections

Objective. Honey is an extremely promising agent in the treatment of infected wounds of burned patients. This study aims to evaluate the antibacterial activity of 14 Algerian honey samples in comparison to Manuka honey towards pathogenic bacteria isolated from burn wound infections. Methods. The antibacterial effect of 14 Algerian honey samples and the Manuka honey was assessed against six multidrug-resistant bacteria: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus saprophyticus, and Enterococcus faecalis. Well agar diffusion, microdilution broth assay, and time-kill assay were used to evaluate the effects of honey samples on the growth of pathogenic bacteria. Results. The results obtained show that all tested honey samples have good antibacterial effects and there is no significant difference between Algerian honey samples and Manuka honey, except honey samples H12 and H13. The Gram-positive bacteria were more susceptible to honey samples than Gram-negative bacteria. The inhibitory diameters were between 14 to 38 mm for Gram-positive bacteria and from 8 to 28 mm for Gram-negative bacteria. The minimal inhibitory concentration of Algerian honey was between 5 and 80% (v/v) and minimal bactericidal concentration was between 10 and 80 % (v/v). However, the minimal inhibitory concentration of Manuka honey was between 5 and 40% (v/v) and minimal bactericidal concentration was between 10 and 80% (v/v). The MBC/MIC ratio was from 1 to 2, which proves that both Algeria honeys and Manuka honey have a bactericidal effect rather than a bacteriostatic effect. A time-kill assay showed that the inhibition effect of honey samples started after the first 3 hours of incubation. Honey samples 3 and 7 inhibited the growth of S. aureus and S. saprophyticus in 15 hours; however, they inhibited the growth of the other pathogenic bacteria in 18 hours. Conclusions. This study proposes honey as an extremely promising treatment against multidrug-resistant bacteria from burn infections.