A fundamental challenge in antibiotic discovery is finding new bioactive compound classes. Due to the longer timeframe and higher cost associated with conventional approaches, it has become imperative to adopt alternative antibiotic discovery paradigms. Advances in computational processing capacity enabled expanding chemical space. The expanded space allowed generation of vast, chemically diverse virtual compound libraries containing billions of compounds. In this study, we exploited the machine learning (ML) model’s ability to make predictive models and applied it to predict growth inhibitory activity in chemical scaffolds outside the training dataset. We employed a Directed-Message Passing Neural Network (D-MPNN) approach to train binary classification and regression ML models on a high-throughput screening dataset performed against Burkholderia cenocepacia previously in our laboratory. The D-MPNN belongs to Spatial-based Convolutional Graph Neural Networks (ConvGNNs), an end-to-end neural network that generates the graph representation of a molecule after iterative message passing process through molecular bonds. To avoid over-fitting and enhance the accuracy of the prediction, we additionally fed the model with 200 global molecular descriptors. The model was further optimized using Bayesian hyperparameter optimization and ensembling. The trained model attained a receiver operating characteristic curve-area under the curve (ROC-AUC) of 0.823. As a proof of principle, we employed the trained ML model to predict the bioactivity of 1,615 FDA-approved compounds and tested the bioactivity of the top 100 ranked compounds in vitro. We found 17 growth-inhibitory compounds with a linear correlation between the predicted rank and the activity. This work highlights the application of ML approaches to rapidly explore chemically diverse, ultra-large compound libraries and discern potential compounds in an inexpensive fashion, thus increasing the chance to discover early lead compounds.

Actinomycetes are the most important group of microorganisms as producers of various useful secondary metabolites, such as antibiotics, antifungal, antitumor agents. There are a potentially a large number of actinomycetes of rare genera that can be producers of new antibiotics, but special conditions are required for their isolation. Different selective methods have been developed to facilitate the frequency of isolation of rare Actinobacteria from untapped ecological niches, which may lead to the identification of novel strains with new natural antibiotic compounds. The goal of this study is to establish effective methods for selective isolation of rare acidophilic actinomycetes - producers of new antibiotics.

The selective isolation approach established here is robust for isolating various acidophilic actinomycetes. A total of 82 strains were isolated from soil samples, using an acid supplement (lemon juice at concentrations of 30% and 50%). A taxonomic analysis, based on the morphological, physiological, biochemical, and molecular investigation of selected strains, revealed that 11 isolates were affiliated to Micromonospora genus. All strains were resistant to low pH =2-2,5, and 6 cultures were able to grow on liquid media for 7 days at a pH=2. According to the literature, actinomycetes of the genus Micromonospora are sensitive to low pH, so in our study, acidotolerant representatives of this genus were identified. An assessment of antimicrobial proprieties of the nine strains, showed moderate to strong antimicrobial activities against fungi and bacteria: Staphylococcus aureus INA 00985, Staphylococcus aureus INA 00761 (MRSA), Micrococcus luteus ATCC 9341, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Saccharomyces cerevisiae ИНА 01042, Candida albicans ATCC 14053, Aspergilus niger ATCC 16404. Isolated acidotolerant Micromonospora strains can be considered as potential producers of new antibiotics.

Bacterial antibiotic resistance (AR) is becoming one of the biggest threats to human health, progressively disarming the current arsenal of antimicrobial drugs. Besides efforts to develop new antimicrobial agents, strategies to avoid the onset of novel resistance mechanisms are strongly needed. The bacterial SOS response to DNA damage (a common outcome of antibiotic treatment), mainly orchestrated by LexA and RecA proteins, is one of the crucial pathways involved in AR acquisition. In previous studies, the SOS response suppression has proved to be an efficient strategy to delay the appearance of drug resistance, but currently known inhibitors of the RecA-LexA axis are limited to few compounds.

From a Fluorescence Polarization (FP)-based high-throughput screening of a small molecule library, a novel hit compound (hereafter “A12”) acting as inhibitor of the Pseudomonas aeruginosa SOS system was discovered. In-vitro dose-response characterization of A12 revealed an inhibitory potency in the high micromolar range, while biophysical assays including differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC) assessed RecA as the main target.

We are currently producing and screening a sub-library based on the structure of compound A12 in order to select more potent derivatives to be tested on P. aeruginosa cultures and determine their effectiveness as antibiotic adjuvants to synergize with bactericidal treatment and delay the onset of resistance.

Emerging infectious diseases (EID) are serious problems caused by fungi in humans and plants and are considered a threat to world food security. Plant origin antifungal peptides are biologically active peptides. They play a vital role in the primary host defense against microbial invasion. The Fusarium oxysporum species complex includes phytopathogenic & non-pathogenic strains and is usually found in the soil. F. oxysporum has gained significant attention from plant pathologists for more than a century owing to economic losses.

Protein-peptide interactions (PPI) plays important role in living beings and their structural characterization. The current study discusses the molecular interaction study of plant antifungal peptides with the target fungal protein of F. oxysporum. We have already reported 510 plant origin antifungal peptides in the PlantAFP database. Under the current investigation, we have selected 55 amino acids under the criteria of amino acid length size of 4-30 that are strictly natural amino acids. The protein-peptide interaction study yielded peptide models. The top 5 models namely PHYTO5, PHYTO13, PHYTO28, PHYTO1, and PHYTO52 achieved high cluster density with low RMSD values. The peptide docking study was further validated with simulation studies for thermodynamic properties. PHYTO5 provided important H-bond involvement with SER57, GLU59, ARG65, HIS68, GLU74, and GLU 87. This can be used as a probable anti-fungal peptide inhibitor against fungi-based infectious diseases.

Trimethoprim (TMP) is a synthetic, broad-spectrum chemotherapeutic agent, which inhibits the dihydrofolate reductase (DHFR) enzyme in bacterial cells. It is mostly used to treat community-acquired urinary tract infections in medicine, but TMP is also commonly used in veterinary and during livestock production. An intense use of antibiotics in these sectors leads to the rapid selection of resistant strains which, through the environment, pose a health risk. There are multiple mechanisms by which strains become resistant to trimethoprim and one of the most frequently identified mechanism is bypass. This mechanism is associated with the presence of the genes encoding a non-allelic variants of DHFR. Dfr genes are mostly associated with mobile genetic platforms and their expression is responsible for high levels of trimethoprim resistance. The aim of the study was to assess the differentiation of dfrA genes determining resistance to trimethoprim in commensal Escherichia coli strains isolated from pigs from a breeding farm with intensive supply of antibiotics in metaphylaxis program. A total of one hundred and sixty-four E.coli strains were isolated from feces of fifty animals, from two to six non-identical isolates per animal. Trimethoprim resistance was tested by microdilution method and revealed that 92% of tested strains were resistant with high MIC values (>32 mg/L). Resistance genes were tested by PCR and PCR-RFLP methods and six different genes were detected: dfrA1, dfrA5, dfrA7, dfrA12, dfrA14, dfrA21. 37% of the strains carried two to four different dfrA genes, in different combinations. In only three cases, all the strains derived from one individual had the same pattern of resistance genes, among the others, there was significant variability. DfrA genes were also detected in three strains sensitive to TMP. The study shows the great diversity of the trimethoprim resistance genes, both within the tested animal population and in the individual host. In addition, genes sequence analysis revealed nucleotide changes within some genes, which highlight the potential for alterations leading to the emergence of new resistance gene variants.

Hermetia illucens (Hi) larvae fat is a sustainable reservoir of bioactive rich compounds. This study aimed to evaluate the antimicrobial activity of bioactive compounds sequentially extracted from compressed fat of Hi larvae. From our previous studies was determined that the Hi fat considers as a sustainable reservoir of antimicrobial agents with pronounced multidrug-resistant (MDR) antibacterial activities, which can be isolated through the three sequential extractions approach. The third acidic water-methanol extract (AWME3) turned out to be the most potent among the other extracts. Ten antibiotics used to assess the susceptibility assay against human pathogenic bacteria strains Klebsiella pneumoniae and Bacillus subtilis, and demonstrated that they were MDR strains. Antimicrobial activities for AWME3 against K. pneumoniae and B. subtilis were determined using the disk diffusion method. The inhibition zone diameters caused by the AWME3 ranged between 11.55±0.34 mm and 16.52±0.74 mm at concentration 20 mg/ml against K. pneumoniae M9 and B. subtilis, respectively. B. subtilis was the most resistant strain to AWME3 compared to K. pneumoniae strains. The minimum inhibition concentration (MIC) was 250 µg/ mL for all bacteria strains, while the minimum bactericidal concentration (MBC) values were 250 and 500 µg/ mL against K. pneumoniae and B. subtilis, respectively. Half of the inhibition concentration (IC₅₀) values were ranged between 147.9±0.01 µg/mL and 160.1±0.008 µg/mL against B. subtilis and K. pneumoniae M9, respectively. GC-MS analysis showed that AWME3 composed of 33 compounds, where free fatty acids such as cis-oleic, palmitic, lauric, stearic and myristic acid were the most abundant in the content of AWME3. This study proved the sustainability of the larvae fat constituents during the sequential extraction from the same biomass of H. illucens larvae fat. In addition, the AWME3 might be considered as an alternative to antibiotics against MDR bacteria with high potential for many bacterial diseases treatments, and beneficial for novel antimicrobial drugs discovery against human bacterial pathogens.

The vicious spread of undesired antibiotic resistance among all the possible horizons of the living world is a cause of great concern and requires immediate attention. As we are concerned about the prevalence of these antibiotic resistance genes (ARGs), studies have suggested the presence of antibiotic resistant determinants in highly controlled environments such as neonatal-intensive care units (ICU). The presence of ARGs in the typical neonatal facilities are a kind of modern nightmare. In the present work, neonatal gut resistome from infants under ICU care was analyzed by metagenomic approach, to examine the possibility of spread of ARGs in neonatal care units. All samples were found to be rich in ARGs and were containing 176 to 291 ARGs per sample and the abundance ranging from 7.68 to 12.86 copies of ARGs per copy of the 16S rRNA gene. Among the all ARGs, Aminocoumarins (mdtA, mdtC), Aminoglycoside (cpxA, APH(3'')-Ib) and Bacitracin (BacA) were the most abundant. The analysis also found that chromosomal ARGs were having significantly higher abundance compared to plasmid ARGs (p < 0.05). We also found that ARGS were highly correlated to metal resistant genes, as compares to mobile genetic elements. Moreover, Efflux pump-based virulence factor genes were also highly associated with ARGs. While, the taxonomy of ARGs carrying contigs showed the majority of genera Klebsiella and Enterobacter. The present study showed, that the higher gut resistome in neonatal ICUs could be due to the compromised sterile conditions in the neonatal units, which present a greater risk to the neonates even in the controlled environment.

Although antimicrobial resistance is an anticipated threat to the globe, measures taken by developing countries to combat this problem are insufficient. If neglected, many surgical procedures and treatments that exist today could come to a standstill, because all of those carry a sizeable risk of infection. An enormous majority of the global population could die from incurable diseases. This review emphasizes the significance of an antimicrobial stewardship team in the hospitals of developing countries as well as the steps to be taken to drive it forward. The pace at which antimicrobial resistance develops will multiply if hospitals fail to impose restrictions on antimicrobial prescribing. The nonchalant attitude of hospital administrations, infection control committees and pharmaceutical companies have a significant impact on this situation. To delay the accelerated progression of AR there should be further developments made in the field of diagnostic testing and alternatives to antimicrobial treatments. There should also be more trials conducted, to determine the effectiveness of antimicrobial stewardship, and the same should be implemented if possible, in every clinical setting. Clinical Pharmacists are undoubtedly the key to propel this movement towards Antimicrobial Stewardship.

Abstract

Antimicrobial peptides (AMPs) are short, amphipathic cationic molecules. Tryptophan amino acid has been found to play an important role in protein folding and in altering the biological activities of peptides. Unlike conventional antibiotics, most AMPs act by lysing or disrupting the bacterial membrane. However, translation of AMPs to therapeutics has some limitations such as sensitivity to enzymatic degradation, low bioavailability, and high production cost. Oligo-N-substituted peptides (e.g. peptoids) are good alternatives to overcome the AMPs limitations. In this work, we designed and synthesized short peptoids containing the aromatic residues with cationic ammonium, guanidinium and quaternary ammonium groups. The effect of amino groups with different alkyl chains on antibacterial activity was investigated. Additionally, we examined the effect of an indole ring in the peptoids on their antibacterial and membrane-disruptive activities by comparing them with simple aromatic substituents. The antibacterial and hemolysis results of the synthesized compounds will also be presented.

Introduction

Antimicrobial resistance became a major worldwide threat. Research have been focused recently on the reassessment of natural products as molecules with high biological and chemical potential. We aimed to study the efficacy of several extracts and essential oils with less known antimicrobial potential.

Method

Antimicrobial and antibiofilm activities of Ocimum basilicum and Eugenia caryophyllus essential oils dissolved in DMSO 1% on two multidrug-resistant bacterial strains of S. aureus ES5168 and E. coli ES5649 and antimicrobial activity of Ocimum basilicum, Robinia pseudocacia, Allium arsinum, Artemisia absinthium, Equisetum arvense alchoolic extracts on four multidrug-resistant bacterial strains of S. aureus ES5168, RC0831 and E. coli ES5649, CA0422 were tested. All bacterial strains exhibited resistance phenotype by a series of cumulative mechanisms. The essential oils and the plant extracts were analyzed by GC-MS. Antibacterial activity was evaluated using the microdilutions technique and anti-biofilm activity by crystal violet-based microtiter plate assay.

Results

Ocimum basilicum essential oil had no inhibitory effect on the S. aureus culture and biofilm. Both essential oils elicited values of 8 mg/ml on the E. coli and this concentration also had the higher inhibiting action on biofilm formation. All the tested extracts inhibited bacterial growth, although in the case of E. Coli no extract caused inhibition rates higher than 50%, while the Equisetum arvense led to a high inhibition rate of S. aureus, 79.06% and 80.32% respectively.

Conclusions

Even though the antimicrobial effect of plant extracts on the multidrug-resistant bacteria is not very high in all cases, the Equisetum arvense extract, a plant less used for its antibacterial properties, strongly inhibits the S. aureus strains both in culture and biofilms.