Aspergillus flavus is a common human pathogen that releases mycotoxin into the host and is frequently treated with synthetic fungicides, but the fungicides have serious human health consequences. Natural products derived from higher plant species have long been investigated as a potential means of controlling pathogenic microorganisms. The indigenous vegetables Boesenbergia rotunda and Syzygium aromaticum are widely distributed in the tropical area. These plants have also been reported in traditional uses for the antimicrobial activity. The purpose of the study was to explore the antifungal susceptibility of dichloromethane and ethanol extracts of B. rotunda rhizomes and S. aromaticum flower buds by Soxhlet’s apparatus against A. flavus using the poison food technique. The effective extract was also subjected to preliminary phytochemical screening tests. The experiment used a completely randomized design with triplications. B. rotunda ethanol extract demonstrated significantly higher potential antifungal activity. The values of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of B. rotunda ethanol extract were 6.25 and 50 mg/ml, respectively, when tested using the macro-dilution method. According to phytochemical tests, the ethanol extract also contained alkaloids, flavonoids, cardiac glycosides, and saponins. The study suggests that a basic guideline for using this as an effective antifungal compound should be separated from the B. rotunda ethanol extract in the future for topical anti-pathogenic fungus.

Quorum sensing (QS) system is related to cell to cell communication as a function of population density, which regulates many physiological functions including biofilm formation and virulence gene expression. The interspecies communication is mediated by autoinducer-2 (AI-2) that is catalysed by LuxS from S-ribosylhomocysteine (RHC). QS inhibitors have emerged as a promising strategy for biofilm and virulence attenuation, which improves the potential of antimicrobial treatments by increasing microbial susceptibility. Among a wide variety of phytochemicals, many of them have been described as QS inhibitors. Driven by the promising phytochemicals clues, this study aimed to identify new active plant natural compounds against LuxS through in silico analysis, followed by in vitro validation. A representative strain for the study of LuxS inhibition, Bacillus subtilis, and a reporter strain sensitive to AI-2, Vibrio harveyi BB170, were used. Firstly, the optimization of a virtual screening protocol was conducted by applying a combination of protein-ligand docking, receptor-based virtual screening, and free energy calculations. Then, optimized virtual screening protocols were applied to screen a phytochemical database containing 3479 drug-like compounds. Based on binding energy scores and compounds cost, the most promising and selected phytochemicals were curcumin, pioglitazone hydrochloride, and 10-undecenoic acid. According to the obtained scores, 10-undecenoic acid could strongly interact with the active binding site of LuxS, while curcumin and pioglitazone hydrochloride had a slight probability compared to natural ligand (RHC). In vitro analysis corroborated the QS inhibitory activity of curcumin and 10-undecenoic acid, however, pioglitazone hydrochloride had no relevant effect. Curcumin (1.25-5 µg/mL) triggered 33-77% reduction of AI-2 accumulation and 10-undecenoic acid (12.5-50 µg/mL) reduced 36-64%. In conclusion, in silico analysis allowed the identification of LuxS antagonistic phytochemicals, revealing curcumin and 10-undecenoic acid as active QS inhibitors. Additionally, this study highlighted that although computational screening is a powerful and quick tool to identify lead compounds, in vitro validation is needed to guarantee high levels of accuracy.

The increasing of multiresistant bacteria worldwide is one of the great concerns in both human and veterinary medicine. Bacterial dermatitis is a frequent problem in small animals, with the primary skin pathogens being Staphylococcus species. Treatment has been made more difficult by the emergence of antibiotic resistance in staphylococcal bacteria in the form of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). The study aimed to investigate MRSA and MRSP in pyodermas admitted to the INNO Veterinary Laboratory (Braga, Portugal), in the period 2013-2021. All microbiological cultures from skin infections from dogs and cats submitted to the INNO Veterinary Laboratory between January 2013 to June 2021 were considered. For the study, only samples with S. aureus and S. pseudintermedius growth were selected. In those two agents’ methicillin resistance was phenotypically detected in the laboratory by MIC determination and by disc diffusion to oxacillin. From a total of 730 samples that tested positive for bacterial growth, 101 (13,8%) were S. pseudointermedius and 27 (3,7%) were S. aureus. The isolates tested for oxacillin 9% (n=6) were MRSP and 6% (n=4) MRSA. The results obtained in this study helps to understand the situation at a national level, where studies in this area are almost non-existent. The presence of MRSA or MRSP in small animals indicates that they are part of the animal-human-environment transmission 'triangle', which should lead us to think of this issue as a public health problem.

Community-acquired pneumonia (CAP) is the leading cause of hospitalization in the Brazilian Unified Health System, with a mortality rate of 18% in children under 5 years of age. Therefore, there is a need for an effective treatment, including antibiotic therapy, based on the main causative agents of the infection. However, there is a risk of the development of bacterial resistance, making it necessary to monitor this use in order to reduce the speed of emergence of multidrug-resistant strains. Thus, this study aims to verify the profile of antibiotic use in children and adolescents treated at a brazilian university hospital. The research consists of a cross-sectional retrospective and descriptive study based on data obtained from medical records provided by the institution, after approval by the ethics committee, and organized in Excel spreadsheets, covering the period from September 2017 to December 2020. It was observed that the profile of this group of patients consists of: a female prevalence in 2017 and 2020 (59% and 57% respectively); while in the years 2018 and 2019, males were higher, 52% and 59%. Regarding age, the age group from 3 months to 4 years was predominant (59.64%). Regarding the use of antibiotics by age group, the following data were found: up to 3 months, the most used were ampicillin (44%) and azithromycin (24.25%); from 4 months to 4 years, ampicillin (32.9%), ceftriaxone (31.7%) and azithromycin (25.9%); and over 5 years, ceftriaxone (33.8%), ampicillin (29.95%) and azithromycin (22.22%). Thus, when comparing the profile found with that recommended by the protocol adopted by the hospital, we can conclude, with the data analyzed, that there is a negligence in the prescription of antimicrobials in the treatment of pediatric CAP, which may corroborate the growth of bacterial resistance, longer hospital stay and, as a result, greater expenditure on care and reduced favorable clinical outcome.

Patients, receiving hematopoietic stem-cell transplantation (HSCT) are prone to develop invasive infections due to disease and transplantation-related immunosuppression. The main causative agents often originate from the digestive tract and are multidrug resistant. Our aim was to investigate the in vitro activity of ceftazidime-avibactam (CZA) against extended spectrum beta-lactamase (ESBL) - producing and carbapenem – resistant (CR) Gram – negative bacteria recovered from blood and fecal samples of patients following HSCT, hospitalized in University Hospital “Saint Marina” - Varna during 2019-2021. A total of 48 isolates (E. coli, n=20; Enterobacter cloacae, n=9; Klebsiella pneumoniae, n=6; Serratia marcescens, n=1; Acinetobacter baumannii, n=2; Pseudomonas putida, n=4; Pseudomonas aeruginosa, n=4; Pseudomonas mendocina, n=1; Pseudomonas composti, n=1) were studied. MALDI Biotyper Sirius (Bruker, Germany) and the automated Phoenix system (BD, USA) were used for species identification and susceptibility testing. Twenty four isolates, included in this study, were resistant to third and fourth generation cephalosporins and therefore identified as ESBL producers (E. coli, n=12; E. cloacae, n=7; K. pneumoniae, n=4; S. marcescens, n=1). A multiplex PCR was used for genes detection, associated with carbapenem resistance. In the studied group, eleven isolates (23%) were CR (E. cloacae, n=1; Pseudomonas spp., n=8; A. baumannii, n=2). All 24 ESBL producing isolates were CZA susceptible. In the group of CR isolates, only 1 P. aeruginosa was susceptible to CZA, while 10 CR isolates were resistant. Genes associated with class B and class D carbapenemases were detected by PCR (bla_VIM and bla_OXA-like). In conclusion, in our study all ESBL producers were susceptible to CZA, while 91% of the CR isolates (all class B and class D carbapenemase producers) were resistant. CZA is a drug combination that is highly active against ESBL producers but its spectrum of activity is limited against carbapenemase producers. Therefore other novel antimicrobial agents are urgently needed.

Background
Levofloxacin is used as salvage therapy in gastric pathogen Helicobacter pylori infection due to its resistance towards first-line therapy antibiotics. Worldwide, the overall prevalence of primary levofloxacin-resistant H. pylori was reported at 14%, emerging as a major concern in eradication failure. Mutations in the gyrA and gyrB genes, especially the quinolone resistance-determining region were reported to be associated with levofloxacin resistance. Therefore, this study aims to identify variants in the levofloxacin-resistance associated genes – gyrA and gyrB of H. pylori in Malaysian patients via sequencing.

Methods
A full-length sequencing was performed on gyrA (2484bp) and gyrB (2322bp) using DNA extracted from biopsy samples obtained from 50 treatment-naïve patients infected with H. pylori. The identified DNA variants were translated in-silico and the produced protein sequences were used to predict their relative binding affinity towards levofloxacin using the HPEPDOCK webserver. The molecular docking scores between the wild type and mutant were analysed.

Results and Discussion
In the gyrA gene, three reported mutations (G468E, 80%; P484Q, 76%; A594D, 16%) and two novel polymorphisms (V741I, 80%; S492A, 62%) were identified to have decreased docking scores ranging from 16.36% to 21.25%. For the gyrB gene, two commonly reported mutations (R484K, 26%; D481E, 20%) and a novel polymorphism (S240A, 16%) were reported to have decreased in 13.23%, 5.32% and 10.14% docking scores respectively. Decreased docking scores signify a weaker binding affinity between the levofloxacin and the protein binding sites in mutations compared to the wild type, consequently having a potential impact on the efficacy of levofloxacin treatments.

Conclusion
The novel variants identified in gyrA and gyrB might be attributed to levofloxacin resistance in H. pylori, therefore, warrant for further investigation.

Acknowledgement
This study was funded by the University of Malaya Impact-Oriented Interdisciplinary Research Grant (IIRG029A/B/C-2019).

Aim: Serratia marcescens is a bacterial species associated with different types of infections including hospital acquired. The aim of this study was to examine the resistance rates to 3^rd generation cephalosporins and carbapenems in S. marcescens isolates obtained from various clinical samples of patients hospitalized in two Bulgarian University hospitals. Materials and methods: A total of 180 non-duplicate clinically significant isolates of S. marcescens, collected during the period 2017-2021 were examined: blood, n=19; urine, n=64; respiratory tract secretions, n=36; wounds, n=44; others, n=17. Species identification and antimicrobial susceptibility testing were done by Phoenix (BD) and Vitek 2 (bioMerieux) automated systems. Double Disc Synergy Test (DDST) was used as screening test for detection of ESBL (Extended Spectrum Beta-Lactamase) production. Results: A total of 89 isolates (49.4%) were resistant to 3^rd generation cephalosporins. Among these isolates, the DDST was positive in 32.2% (n=58). Isolates, resistant to 3^rd generation cephalosporins were most commonly obtained from patients in Nephrology (n=31), Urology (n=12) аnd ICU (n=12). The highest rate of 3^rd generation cephalosporin resistance was found among the urine isolates (25.6%, n=46), followed by blood (7.2%, n=13) and wound isolates (5%, n=10). In the studied collection of 180 isolates, cefepime resistant were 47.8% (n=86). Three isolates, resistant to 3^rd generation cephalosporins were susceptible to cefepime. Carbapenem resistance in the whole collection was 3.3% (n=6). Conclusion: The high rates of 3^rd generation cephalosporin resistance and ESBL production among clinically significant isolates of S. marcescens and the detection of carbapenem-resistant isolates are worrisome trends, because are associated with infections with very limited treatment alternatives and usually in immunocompromised patients.

Sodiomyces genus is obligate alkaliphilic ascomycetous fungi of the Plectosphaerellaceae family (Grum-Grzhimaylo et al., 2016, DOI: 10.1007/s13225-015-0320-2). They prefer habitats with alkaline conditions (pH≥10) such as soda salterns and the edge of the soda lakes. Such habitats are located all over the world, although, as a rule, they are small in area. In addition to pH stress, organisms, inhabiting in soda soils, have to survive under fluctuating osmotic and temperature conditions, that vary drastically with a change of drought and rain, heat and freezing. Studies of the antimicrobial activity of alkaline fungi are rare. The prime purpose of this study was to isolate and identify antimicrobial compound from an alkaliphilic fungus Sodiomyces alkalinus. According to previously data, high level antimicrobial culture broth was obtained for three strains of S. alkalinus. Analytical separation by reversed-phase HPLC for the ethyl acetate extract was carried out according to the data described earlier (Kuvarina et al., 2021, DOI: 10.1134/S0003683821010142). It has been suggested that the molecule studied is typical to be a polypeptide; there by its initial structure analysis was conducted by automated Edman sequencing. The polypeptide production yield from the culture broth of S. alkalinus on alkaline medium was achieved in a period of fermentation in 14 days about 25.54 ± 1.4 mg/L. Searching for potential homologies amongst NCBI databases (https://www.ncbi.nlm.nih.gov/) using BLASTP algorithm led to complete matching with fungal hydrophobin F11 (GenBank: ROT36721.1/NCBI Reference Sequence: XP_028464527.1). This data was previously obtained based on whole genome sequence which has been provided for S. alkalinus F11 strain that is typical (Grum-Grzhimaylo et al. 2013, DOI: 10.3767/003158513X673080). The antimicrobial activity of the novel antifungal agent hydrophobin from S. alkalinus was studied in vitro. Strong antifungal effect was against pathogenic and opportunistic fungi strains and MIC was determined. The protein showed growth inhibitory activity of filamentous and yeast fungi. The activity of hydrophobin Sa-HB1 against Aspergillus spp. was comparable to reference polyene cyclic antibiotic Amphotericin B. The inhibition zones for all clinical Candida isolates were found to be 12-14 mm, while C. krusei, C. tropicalis were inactive to Amphotericin B. As a result, the antimicrobial compound from S. alkalinus is hydrophobin. It has high antifungal activity comparable to reference antibiotics. The hydrophobin Sa-HB1 responsible for the reported antifungal activity of S. alkalinus, and may serve as a potential source of lead compounds that can be developed as antifungal drug candidate.

This study was supported by the Russian Science Foundation (grant no. 22-25-00353)

The aromatic polyketide tetracenomycin X (TcmX) was recently found to be a potent inhibitor of protein synthesis; its binding site is located in a unique locus within the tunnel of the large ribosomal subunit. The distinct mode of action makes this relatively narrow class of aromatic polyketides promising for drug development in the quest to prevent the spread of drug-resistant pathogens. We isolated two novel tetracenomycin congeners – 6-hydroxytetracenomycin X (6-OH-TcmX) and O⁴-Me-Tcm C (TcmX isomer). Spectral properties of the compounds were studied. 6-OH-TcmX exhibited lower antimicrobial and cytotoxic activity, whereas TcmX isomer was found to be completely inactive. Interestigly, in vitro protein synthesis inhibition ability of TcmX and 6-OH-TcmX were found to be comparable, suggesting a significant influence of 6-hydroxylation on the tetracenomycin X cell penetration ability. Complete absence of both antimicrobial activity and in vitro protein synthesis inhibition ability of TcmX isomer corroborates crucial role of 4-OH group in ribosome binding. This work was supported in part by Ministry of Science and Higher Education of the Russian Federation (Agreement No 075–15-2021-1085). V.A. Alferova was supported by a RF President’s Scholarship (project SP-4094.2022.4).

Natural extracts are emerging as potential alternatives to the use of antibiotics to fight infections, by exhibiting excellent antimicrobial profiles. In particular, the biomolecules thymol and eugenol, have been explored in the prevention/treatment of skin infections with effective outcomes. Different fiber-based or hydrogel-based scaffolds have been employed in the treatment of skin infections because of their similarities to the skin structure, specifically the matrix and fibrillar elements, respectively. In this study, we explore these hybrid fiber-hydrogel architectures further, by examining the antimicrobial potentialities of polycaprolactone (PCL) wet-spun fibers functionalized with thymol encased within a poly(vinyl alcohol) (PVA) hydrogel modified with eugenol, against Staphylococcus aureus and Staphylococcus epidermidis. Thymol needed only 0.313 and 0.627 mg/mL to inhibit the growth of these bacteria, respectively. While eugenol, required 5 mg/mL to induce the same effect. PCL fibers (9wt.% in dimethylformamide) were processed by wet-spun at an extrusion rate of 0.1 mL/min directly into a distilled water (dH₂O) coagulation bath. Thymol dissolved in dimethyl sulfoxide was combined with the PCL solution at 1,254 mg/mL (2×0.627 mg/mL) to generate modified fibers. All PCL-based fibers presented a uniform and homogeneous appearance, even though thymol’s presence reduced the diameter of the fibers (247.49 to 146.99 µm) and their elongation at break (159.32 to 93.26%). Eugenol-loaded and unloaded PVA hydrogels were prepared at 10wt.% in dH₂O) in a coagulation bath of 8% NaOH and 4% Na₂SO₄. PVA-based hydrogels with a soft, flexible, and malleable structure were generated. The incorporation of eugenol appeared to increase the flexibility of the hydrogel while maintaining its integrity. Incorporation of PCL fibers in the PVA hydrogel, both functionalized with the respective biomolecules, was successful. In general, the potential of these biomolecules and both structures to work synergistically for the treatment of skin infections was demonstrated.