Drug-resistant Pathogenic Bacteria Research Articles

Cyclodextrin-based ternary supramolecular deep eutectic solvents for efficient extraction and analysis of trace quinolones and sulfonamides in wastewater by adjusting pH

BackgroundAntibiotics residues can accelerate the growth of drug-resistant bacteria and harm the ecological environment. Under the effect of enrichment and biomagnification, the emergence of drug-resistant pathogenic bacteria may eventually lead to humans being ineffective to drugs in the face of bacterial or fungal disease infections in the future. It is urgent to develop an efficient separation medium and analytical method for simultaneous extraction and determination of antibiotics in the water environment. ResultsThis work doped 2,6-Di-O-methyl-β-cyclodextrin, randomly methyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin with thymol:fatty acid respectively to construct non-covalent interaction-dominated pH-responsive ternary supramolecular deep eutectic solvents (SUPRADESs), which can undergo a hydrophilic/hydrophobic transition with aqueous phase to achieve an efficient microextraction. Semi-empirical method illustrated that SUPRADESs have a wide range of hydrogen bond receptor sites. We developed a SUPRADES-based analytical method combined with liquid chromatography-triple quadrupole mass spectrometry for the extraction and determination of trace quinolones and sulfonamides in wastewater. The overall limits of detection of the method were 0.0021-0.0334 ng mL-1 and the limits of quantification were 0.0073-0.1114 ng mL-1. The linearity maintained good in the spiked level of 0.01-100 ng mL-1 (R2>0.99). The overall enrichment factors of the method were 157-201 with lower standard deviations (≤ 8.7). SignificanceThe method gave an extraction recovery of 70.1-115.3% for 28 antibiotics in livestock farming wastewater samples from Zhejiang, China, at trace levels (minimum 0.5 ng mL-1). The results demonstrated that inducing the phase transition between SUPRADES and aqueous phase by adjusting pH for extraction is a novel and efficient pretreatment strategy. To our knowledge, this is the first application of cyclodextrin-based ternary SUPRADESs with pH-responsive reversible hydrophobicity-hydrophilicity transition behavior in wastewater analysis.

Discovery of Bacterial Key Genes from 16S rRNA-Seq Profiles That Are Associated with the Complications of SARS-CoV-2 Infections and Provide Therapeutic Indications.

SARS-CoV-2 infections, commonly referred to as COVID-19, remain a critical risk to both human life and global economies. Particularly, COVID-19 patients with weak immunity may suffer from different complications due to the bacterial co-infections/super-infections/secondary infections. Therefore, different variants of alternative antibacterial therapeutic agents are required to inhibit those infection-causing drug-resistant pathogenic bacteria. This study attempted to explore these bacterial pathogens and their inhibitors by using integrated statistical and bioinformatics approaches. By analyzing bacterial 16S rRNA sequence profiles, at first, we detected five bacterial genera and taxa (Bacteroides, Parabacteroides, Prevotella Clostridium, Atopobium, and Peptostreptococcus) based on differentially abundant bacteria between SARS-CoV-2 infection and control samples that are significantly enriched in 23 metabolic pathways. A total of 183 bacterial genes were found in the enriched pathways. Then, the top-ranked 10 bacterial genes (accB, ftsB, glyQ, hldD, lpxC, lptD, mlaA, ppsA, ppc, and tamB) were selected as the pathogenic bacterial key genes (bKGs) by their protein-protein interaction (PPI) network analysis. Then, we detected bKG-guided top-ranked eight drug molecules (Bemcentinib, Ledipasvir, Velpatasvir, Tirilazad, Acetyldigitoxin, Entreatinib, Digitoxin, and Elbasvir) by molecular docking. Finally, the binding stability of the top-ranked three drug molecules (Bemcentinib, Ledipasvir, and Velpatasvir) against three receptors (hldD, mlaA, and lptD) was investigated by computing their binding free energies with molecular dynamic (MD) simulation-based MM-PBSA techniques, respectively, and was found to be stable. Therefore, the findings of this study could be useful resources for developing a proper treatment plan against bacterial co-/super-/secondary-infection in SARS-CoV-2 infections.

The impact of Lactococcus lactis KUST48 on the transcription profile of Aeromonas hydrophila-infected zebrafish spleen.

Aeromonas hydrophila, an aquatic pathogenic bacterium, has been found to infect many fish species and cause huge aquaculture losses. Antibiotics are the most common drugs used to treat these infections. However, antibiotic abuse can lead to the development of antibiotic resistance. Probiotics have the potential to replace antibiotics for preventing infections. Zebrafish (Danio rerio) is a model organism used to study the innate immune system and host-pathogen interactions. Currently, there is little information on how the fish immune system responds to A. hydrophila and probiotic treatment. To increase the understanding of the molecular mechanisms behind the zebrafish defense against A. hydrophila and provide evidence that antibiotics can be replaced by probiotics, a transcriptome analysis of the zebrafish spleen was conducted 48 hours after infection by A. hydrophila, as well as after treatment using Lactococcus lactis KUST48 4 hours after infection. A total of 36,499 genes were obtained. There were 3,337 genes found to have significant differential expression between treatment and control groups. According to further annotation and enrichment analysis, differentially expressed genes (DEGs) were involved in signal transduction, endocrine system cancer, and the immune system. Insulin resistance disappeared in the zebrafish after treatment. Quantitative real-time PCR was performed to confirm the significant regulation of immune defense DEGs, the results of which were consistent with the RNA-sequencing data. These results could serve as a basis for future studies on the immune response to A. hydrophila and provide suggestions for probiotic alternatives to antibiotics, which will be of great significance to aquaculture and environmental protection.IMPORTANCEIn recent years, the unreasonable use of antibiotics has led to the emergence of drug-resistant pathogenic bacteria, antibiotic residues, cross infection, toxic side effects, and so on, which has caused a serious threat to human food safety and life health. In recent years, many studies have demonstrated the potential of probiotics as a substitute for antibiotics, but there is still a lack of understanding of the molecular mechanisms underlying probiotic therapy. We conduct a research on the impact of Lactococcus lactis KUST48 on the transcription profile of Aeromonas hydrophila-infected zebrafish spleen. Mortality of zebrafish infected with A. hydrophila was significantly reduced after treatment with L. lactis KUST48. Our results can help to strengthen our understanding of the pathogenic mechanisms of zebrafish and provide a valuable reference for the molecular mechanisms of probiotic therapy.

The antibacterial mechanism of the novel bacteriocin LpH25 and the synergistic preservation effect of this bacteriocin and Nisin in fresh milk

Bacteriocin, a natural preservative in the food industry, has been found to have a strong inhibitory effect on foodborne pathogens. This study focuses on the structure, stability and antibacterial mechanism of the novel bacteriocin LpH25, as well as the synergistic antibacterial effect of LpH25 in combination with Nisin on E. coli and S. aureus in milk. It was shown that the growth of L. rhamnosus LS-8 was inhibited by acidic environments and pepsin. LpH25 was an unsaturated polypeptide containing an aromatic ring and had good amphipathic. Additionally, bacteriocin LpH25 displayed remarkable thermal stability (121 °C, 30 min) and broad pH stability (pH 2.5–11), remaining stable at −80 °C for over 270 days and exhibiting insensitivity to various enzymes within 2 h. Moreover, LpH25 showed strong inhibitory activity against Escherichia coli, Staphylococcus aureus, and Cronobacter sakazakii within 5 h. Further experiments revealed that LpH25 could effectively inhibit the formation of E. coli outer membranes and flagella to exert antibacterial activity, ultimately leading to cell death. LpH25 inhibited the growth of S. aureus by repressing peptidoglycan synthesis. In addition, the number of viable bacteria in contaminated milk treated with LpH25 mixed with Nisin remained stable (6.5log10 CFU/mL) for seven days. This study provides important theoretical support for promoting the development of antibacterial agents, addressing the challenge of drug resistance of pathogenic bacteria, and extending the shelf life of milk.

Surface-enhanced Raman spectroscopy for the characterization of bacterial pellets of Staphylococcus aureus infected by bacteriophage.

Drug-resistant pathogenic bacteria are a major cause of infectious diseases in the world and they have become a major threat through the reduced efficacy of developed antibiotics. This issue can be addressed by using bacteriophages, which can kill lethal bacteria and prevent them from causing infections. Surface-enhanced Raman spectroscopy (SERS) is a promising technique for studying the degradation of infectious bacteria by the interaction of bacteriophages to break the vicious cycle of drug-resistant bacteria and help to develop chemotherapy-independent remedial strategies. The phage (viruses)-sensitive Staphylococcus aureus (S. aureus) bacteria are exposed to bacteriophages (Siphoviridae family) in the time frame from 0 min (control) to 50 minutes with intervals of 5 minutes and characterized by SERS using silver nanoparticles as SERS substrate. This allows us to explore the effects of the bacteriophages against lethal bacteria (S. aureus) at different time intervals. The differentiating SERS bands are observed at 575 (C-C skeletal mode), 620 (phenylalanine), 649 (tyrosine, guanine (ring breathing)), 657 (guanine (COO deformation)), 728-735 (adenine, glycosidic ring mode), 796 (tyrosine (C-N stretching)), 957 (C-N stretching (amide lipopolysaccharides)), 1096 (PO2 (nucleic acid)), 1113 (phenylalanine), 1249 (CH2 of amide III, N-H bending and C-O stretching (amide III)), 1273 (CH2, N-H, C-N, amide III), 1331 (C-N stretching mode of adenine), 1373 (in nucleic acids (ring breathing modes of the DNA/RNA bases)) and 1454 cm-1 (CH2 deformation of saturated lipids), indicating the degradation of bacteria and replication of bacteriophages. Multivariate data analysis was performed by employing principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) to study the biochemical differences in the S. aureus bacteria infected by the bacteriophage. The SERS spectral data sets were successfully differentiated by PLS-DA with 94.47% sensitivity, 98.61% specificity, 94.44% precision, 98.88% accuracy and 81.06% area under the curve (AUC), which shows that at 50 min interval S. aureus bacteria is degraded by the replicating bacteriophages.

In-vitro Antimicrobial Effects and Phytochemical Contents of Stingless Bee Meliponula beccarii Honey and Pollen from Baringo County, Kenya

Background: Afro-tropical stingless bees produce several products including honey, propolis, cerumen and pollen, which are widely used as traditional medicine and food. In Baringo County, Kenya stingless bee honey in particular is widely used as a traditional remedy for respiratory disorders, stomach disorders and oral thrush, commonly associated with bacterial and fungal infections. However, scientific data on the antimicrobial activities and phytochemical content of stingless bee products from Baringo is scarce. Objectives: The objective of this study was to investigate the in vitro antimicrobial activities and phytochemical content of Meliponula beccarii stingless bee honey and pollen from Baringo County. Materials and methods: Eleven honey and pollen samples were conveniently sampled from eleven wild occurring stingless bee nests in three ecologically distinct areas. Increasing concentrations of honey and pollen samples were then prepared and tested against H. influenzae, E. coli, MRSA and C. albicans using agar well diffusion assay. The broth microdilution test was further performed to determine the Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs). Standard qualitative methods were used to analyse the phytochemical contents of the honey and pollen samples. Data was analysed by two-way ANOVA and Tukey’s post-hoc tests. Results: in comparison to distilled water (negative control), the honey samples had a significantly higher mean zones of inhibition at concentration of 100% v/v against E. coli (9.0±4.7 mm vs 0 mm, p<0.0001, N=11), H. influenzae (11.1±5.0 mm vs 0 mm, p<0.0001, N=11), and MRSA (10.8±5.9 mm vs 0 mm, p>0.0001, N=11). However, compared to ciprofloxacin standard (30ug/ml), all the concentrations of honey samples had significantly lower mean zones of inhibition against H. influenzae (8.8±2.4 mm vs 25±0.58 mm, p<0.0001, N=11), E. coli (5.7±3.4 mm vs 31.7±1.5 mm, p<0.0001, N=11) and MRSA (8.1±2.8 mm vs 27±0 mm). All the honey samples did not exhibit activity against C. albicans even at 100% (v/v). The mean MICs against E. coli, H. influenzae and MRSA were 9.38% (v/v), 18.75% (v/v) and 18.75% (v/v), respectively. Notably, the honey samples exhibited bactericidal activity, only against MRSA with a mean MBC of 60.94% (v/v). All the pollen samples showed no antibacterial and antifungal activities against the tested micro-organisms. Qualitative analyses revealed that the honey and pollen samples of Meliponula beccarii contain alkaloids, phenolics, triterpenoids, flavonoids, saponins, tannins, glycosides and steroids, but not terpenoids. Conclusion: Some but not all Meliponula beccarii honey samples from Baringo County has antibacterial activities. The honey and pollen are rich in various phytochemical compounds. Our findings validate the use of Meliponula beccarii honey in traditional treatment of bacterial infections and its further investigation as a potential source of novel agents against drug resistant pathogenic bacteria.

Tannic acid and carboxymethyl chitosan-based multi-functional double-layered hydrogel with pH-stimulated response behavior for smart real-time infection monitoring and wound treatment

The dramatic increase of drug-resistant pathogenic bacteria has seriously effect on human health, appealing the needs of developing theranostic platforms with stimuli-responsive materials to realize the accurate bacterial diagnostics and therapeutics. Herein, a tannic acid and carboxymethyl chitosan-based multifunctional ZIF-90@i-PPOPs-phenol red double-layered hydrogel with stimuli-responsiveness and antibacterial activity was fabricated. The inner layer hydrogel (ZIF-90@i-PPOPs-based TFC hydrogels) was fabricated based on ZIF-90@i-PPOPs, integrate tannic acid and carboxymethyl chitosan linked by formylphenylboronic acid (FPBA), which exhibited outstanding injectable, biodegradability and antibacterial activity. The outer layer hydrogel (PR@PAM hydrogels) were constructed from polyacrylamide (PAM) and pH indicator phenol red, owning porous structure and excellent tissue adhesion. Due to the weakly acidic microenvironment within wound, the inner-layer hydrogel was stimulus-responsively decomposed, resulting in the accurate delivery of the positively charged ZIF-90@i-PPOPs to the lesion site to capture and kill bacteria by enhanced Zn2+ and ROS release. Meantime, the outer-layer hydrogel could real-timely monitor the pH changes to evaluate the wound recovery status. These double-layered hydrogels possessed precisely pH monitoring capacity, excellent antibacterial ability and negligible side effect to normal tissue in vivo, implying the high potential of the suggested hydrogels as theranostic platform for antibacterial treatment.

Prevalence of Extended Spectrum β-Lactamase Producers (ESBLs) with antibiotic resistance pattern of Gram negative pathogenic bacteria isolated from door handles in hospitals of Pokhara, Western Nepal

BackgroundThe presence of drug-resistant Gram-negative pathogenic bacteria and Extended Spectrum β-Lactamase Producers (ESBLs) in hospital associated fomites like door handles can serve as vehicles in transmission and may be the key factor in epidemiology of ESBL producing bacterial infection not only in a hospital setting but also in the community. The aim of this study was to determine the prevalence of ESBLs and antibiotic resistance of Gram-Negative pathogenic Bacteria isolated from door-handles in two selected hospitals in Pokhara Metropolitan City, Nepal. The study was conducted in selected hospitals in Pokhara Metropolitan City, Western Nepal. A cross-sectional study design was used. The hospitals were selected randomly. A total of 100 swab samples were taken from door-handles. Isolation and identification of bacteria were done using standard microbiological procedures. An antibiotic susceptibility test, screening and confirmation of ESBLs were performed using the Clinical Laboratory Standard Institute’s guidelines. ResultsOut of the 100 swab samples cultured, 96 (96%) showed bacterial growth. A total of one hundred and forty isolates were isolated in this study which were further identified based on cultural, morphological and biochemical characteristics. The study also found that door handles/knobs had higher level of contamination in Outpatient Departments (OPDs), Emergency, Laboratory, General wards and Toilets, in that order as compared to Radiology Room, Staff rooms, Intensive Care Unit and Operation Theatre which were lower. The level of contamination varies depending on the traffic exposure and the environment. The most prevalent Gram-negative bacteria identified was Escherichia coli 28.85%, followed by Klebsiella spp 21.15%, Pseudomonas aeruginosa 15.38%, Proteus spp 11.54%, Enterobacter spp 9.62%, Acenetobacter spp 7.69%, Citrobacter spp 5.77%. The most effective drug of choice was Amikacin, Nitrofurantoin, Norfloxacin, Ciprofloxacin, Tetracycline and Imipenem for many Gram-negative isolates. The overall prevalence of ESBLs in this study was 27.14%. Out of total 15 Escherichia coli isolated, 11(73.3%), Klebsiella spp 9/11 (81.8%); Pseudomonas spp 7/8 (87.5%), Proteus spp 4/6 (66.6%); Enterobacter spp 3/5 (60%), Acenetobacter spp 3/4 (75%) and Citrobacter spp 1/3 (33.3%) were found to be Extended β-Lactamase Producers (ESBLs). ConclusionThe isolation of of pathogenic Gram-negative bacteria and ESBLs in hospital environments and subsequent detection of high drug resistance patterns indicates a potentially serious public health challenge that strengthens the need for the effective and routine cleaning of door-handles in hospitals.

Nano-selenium foliar intervention-induced resistance of cucumber to Botrytis cinerea by activating jasmonic acid biosynthesis and regulating phenolic acid and cucurbitacin.

Botrytis cinerea is the primary disease affecting cucumber production. It can be managed by applying pesticides and cultivating disease-resistant cucumber strains. However, challenges, such as drug resistance in pathogenic bacteria and changes in physiological strains, are obstacles in the effective management of B. cinerea. Nano-selenium (Nano-Se) has potential in enhancing crop resistance to biological stress, but the exact mechanism for boosting disease resistance remains unclear. Here, we used metabolomics and transcriptomics to examine how Nano-Se, as an immune activator, induces plant resistance. Compared with the control group, the application of 10.0 mg/L Nano-Se on the cucumber plant's leaf surface resulted in increased levels of chlorophyll, catalase (10.2%), glutathione (326.6%), glutathione peroxidase (52.2%), cucurbitacin (41.40%), and metabolites associated with the phenylpropane synthesis pathway, as well as the total antioxidant capacity (21.3%). Additionally, the expression levels of jasmonic acid (14.8 times) and related synthetic genes, namely LOX (264.1%), LOX4 (224.1%), and AOC2 (309.2%), were up-regulated. A transcription analysis revealed that the CsaV3_4G002860 gene was up-regulated in the KEGG enrichment pathway in response to B. cinerea infection following the 10.0 mg/L Nano-Se treatment. In conclusion, the activation of the phenylpropane biosynthesis and branched-chain fatty acid pathways by Nano-Se promotes the accumulation of jasmonic acid and cucurbitacin in cucumber plants. This enhancement enables the plants to exhibit resistance against B. cinerea infections. Additionally, this study identified a potential candidate gene for cucumber resistance to B. cinerea induced by Nano-Se, thereby laying a theoretical foundation for further research in this area. © 2023 Society of Chemical Industry.

Isolation and identification of bioactive compounds from Antrodia camphorata against ESKAPE pathogens.

Antimicrobial resistance is a major threat to human health globally. Antrodia camphorata was grown in a malt/yeast extract broth liquid medium for 15 days. Then, 4-L fermentation broth was harvested, yielding 7.13 g of the ethyl acetate extract. By tracing the antimicrobial activity, 12.22 mg of the antimicrobial compound was isolated. The structure of 5-methyl-benzo [1,3]-dioxole-4,7-diol (MBBD) was elucidated using NMR and MS data analyses. The antibacterial activity of MBBD was detected through the microbroth dilution method. MBBD exhibited broad-spectrum antibacterial activity. The minimum inhibitory concentration (MIC) range of MBBD for drug-resistant pathogenic bacteria was 64-256 μg/mL, with the lowest MIC observed for Acinetobacter baumannii (64 μg/mL), followed by Pseudomonas aeruginosa (MIC = 128 μg/mL). Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli were also sensitive, with an MIC of 256 μg/mL. The MIC range of MBBD against 10 foodborne pathogens was 12.5-100 μg/mL. Based on the results of this study, MBBD exhibits broad-spectrum antibacterial activity, particularly demonstrating excellent inhibitory effects against A. baumannii. MBBD will be good candidates for new antimicrobial drugs.

Acquisition of Type I methyltransferase via horizontal gene transfer increases the drug resistance of Aeromonas veronii.

Aeromonas veronii is an opportunistic pathogen that affects both fish and mammals, including humans, leading to bacteraemia, sepsis, meningitis and even death. The increasing virulence and drug resistance of A. veronii are of significant concern and pose a severe risk to public safety. The Type I restriction-modification (RM) system, which functions as a bacterial defence mechanism, can influence gene expression through DNA methylation. However, little research has been conducted to explore its origin, evolutionary path, and relationship to virulence and drug resistance in A. veronii. In this study, we analysed the pan-genome of 233 A. veronii strains, and the results indicated that it was 'open', meaning that A. veronii has acquired additional genes from other species. This suggested that A. veronii had the potential to adapt and evolve rapidly, which might have contributed to its drug resistance. One Type I methyltransferase (MTase) and two complete Type I RM systems were identified, namely AveC4I, AveC4II and AveC4III in A. veronii strain C4, respectively. Notably, AveC4I was exclusive to A. veronii C4. Phylogenetic analysis revealed that AveC4I was derived from horizontal gene transfer from Thiocystis violascens and exchanged genes with the human pathogen Comamonas kerstersii. Single molecule real-time sequencing was applied to identify the motif methylated by AveC4I, which was unique and not recognized by any reported MTases in the REBASE database. We also annotated the functions and pathways of the genes containing the motif, revealing that AveC4I may control drug resistance in A. veronii C4. Our findings provide new insight on the mechanisms underlying drug resistance in pathogenic bacteria. By identifying the specific genes and pathways affected by AveC4I, this study may aid in the development of new therapeutic approaches to combat A. veronii infections.

Broad-spectrum antibacterial and antibiofilm activity of biogenic silver nanoparticles synthesized from leaf extract of Phyllanthus niruri

Multidrug resistance (MDR) among pathogenic bacteria is a global health concern as it has rendered the current antibiotic therapy ineffective. A recent and rapidly developing research area in the biomedical sector is the use of biogenic nanoparticles as antibacterial agents. In the current investigation, aqueous leaf extract of Phyllanthus niruri (Pn-AgNPs) were employed for the biogenic synthesis of silver nanoparticles, further characterized by SEM, EDX, FTIR, TEM, UV–Vis and XRD techniques. Electron microscopy images of the green-synthesized Pn-AgNPs showed them as evenly dispersed spherical particles having an average size of about ∼ 20 nm. The Pn-AgNPs were further tested for potential antibacterial activity against isolated drug-resistant pathogenic bacteria such as Burkholderia cepacia complex, carbapenem-resistant Enterobacterales (CRE), Citrobacter freundii, Enterococcus faecalis, Escherichia coli, Enterobacter clocae, MRSA, Salmonella typhi, Streptococcus spp, and vancomycin-resistant Enterococci (VRE).The Pn-AgNPs significantly inhibited growth all of the studied bacterial isolates, including antibiotic-resistant isolates such as MRSA, VRE, and CRE. Electron micrographs clearly showed the enhanced penetration of AgNPs leading to disruption of cell membrane causing death of the cell. Further, Pn-AgNPs dramatically reduced the ability of P. aeruginosa, Bacillus cereus, E. coli, and S. aureus to form biofilms. Leakage of protein from bacterial cells could be main reason for the effective bactericidal action of synthesized AgNPs. This antibacterial and antibiofilm potential of Phyllanthus niruri biosynthesized AgNPs against clinically significant drug-resistant bacteria is impressive and can be developed as a novel antimicrobial agent to combat the threat of drug-resistant bacterial infections.

Directed evolution of TetR for constructing sensitive and broad-spectrum tetracycline antibiotics whole-cell biosensor

Antibiotic abuse is the main reason for the drug resistance of pathogenic bacteria, posing a potential health risk. Antibiotic surveillance is critical for preventing antibiotic contamination. This study aimed to develop a sensitive and broad-spectrum whole-cell biosensor for tetracycline antibiotics (TCs) detection. Wild-type TCs-responsive biosensor was constructed by introducing a tetracycline operon into a sfGFP reporter plasmid. Using error-prone PCR, mutation libraries containing approximately 107 variants of the tetracycline repressor (TetR) gene were generated. The tigecycline-senstive mutants were isolated using high-throughput flow cytometric sorting. After 2 rounds of directed evolution, a mutant epS2–22 of TerR was isolated and assembled as a TCs biosensor. The epS2–22 biosensor was more sensitive and broad-spectrum than the wild-type biosensors. The detection limits of the epS2–22 biosensor for seven TCs were 4- to 62-fold lower than the wild-type biosensor (no response to tigecycline). Meanwhile, the epS2–22 biosensor had a shorter detection time and a stronger signal output than the wild type. In addition, the evolved epS2–22 biosensor showed excellent performance in detecting low traces of TCs in environmental water. These results suggest that directed evolution is a powerful tool for developing high-performance whole-cell biosensors, and the evolved epS2–22 biosensors have the potential for wider applications in real-world TCs detection.

Covalent Conjugates of Allylbenzenes and Terpenoids as Antibiotics Enhancers with the Function of Prolonged Action.

The drug resistance of pathogenic bacteria is often due efflux pumps-specific proteins that remove foreign compounds from bacterial cells. To overcome drug resistance, adjuvants are often used that can inhibit efflux pumps or other systems that ensure the resistance of bacteria to the action of antibiotics. We assumed that a new level of effectiveness with the use of an antibiotic + an adjuvant pair could be achieved by their joint delivery into the pathogen. To test this hypothesis, we constructed a series of molecular carriers based on poly-(olygo-, dendry)mers based on cyclodextrin-grafted PEI or mannan, as well as glycol chitosan, covalently bound to antibiotic, adjuvant, and the oligosaccharide ligand to the macrophage mannose receptor (CD206), which we studied earlier and showed high efficiency and selectivity of delivery of a therapeutic "cargo" to macrophages. Moxifloxacin was used as an antibiotic, and terpenoid and allylbenzene compounds were used as adjuvants, for which we previously discovered the ability to inhibit bacterial efflux pumps. We show that: (a) the resulting structures were stable in vitro for a long time (up to 10 days); (b) they were adsorbed on bacterial cells, providing a local increase in the concentration of the antibiotic and adjuvant in pathogen cells; (c) they were internalized by bacterial cells, ensuring the accumulation of both antibiotic and adjuvant inside bacterial cells; (d) the adjuvant, after entering the bacterial cell, provided inhibition of the efflux pumps; (e) due to this action of the adjuvant, combined with the targeted delivery by the carrier, the antibiotic's half-life in rats increased by more than 2 times, the effective concentration of the drug in the blood plasma (AUC) increased up to 8-10 times; (f) a significant increase in the effectiveness of the antibacterial action against Gram+ and Gram- cells was achieved (up to 3 times). Potentially, such an approach would significantly increase the effectiveness of therapies for a number of infectious and other diseases, reduce the dosage of antibiotics, shorten the duration of treatment, and reduce the risk of developing bacterial resistance. Moreover, the use of a polymer carrier with covalently bound organic molecules of different structures will avoid problems linked to different (suboptimal) solubility and bio-distribution of the administered molecules, which would be almost inevitable when using the same compounds separately. It would be very difficult to find antibiotic/adjuvant pairs that simultaneously achieve optimal concentrations in the same target cells. In our case, terpenoids and alkylbenzenes used as adjuvants are practically insoluble as individual compounds, and their unacceptable pharmacological properties would not allow them to be used as efflux pump inhibitors.

ИССЛЕДОВАНИЕ ФАРМАКОКИНЕТИКИ ТВЕРДОДИСПЕРСНОЙ И КРИСТАЛЛИЧЕСКОЙ ФОРМ АНТИВИРУЛЕНТНОГО СРЕДСТВА ФТОРТИАЗИНОН У КРЫС

Prevalent drug resistance of pathogenic bacteria is a major concern for healthcare. Decreased bacterial susceptibility to antibiotics reduces treatment efficiency, putting lives of patients at risk. To overcome this issue, a novel anti-virulence drug Fluorothiazinon (FT) was developed. The drug inhibits type three secretion system and flagella motility of many Gram-negative bacteria. It showed therapeutic activity against a number of pathogenic microorganisms, such as Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis and Salmonella enterica Typhimurium among others. To find appropriate form for subsequent drug development solid dispersion and crystalline forms of FT were manufactured. In this study we aimed to investigate pharmacokinetics of FT in two forms to find the formulation with higher bioavailability. For comparative pharmacokinetics study rats were allocated to receive a single oral dose 50 mg/kg of the drug in either solid dispersion or crystalline forms. Determination of FT and its metabolite was carried out by high-performance liquid chromatography – tandem mass-spectrometry analysis. Our results show that the solid dispersion form of Fluorothiazinon has a higher oral bioavailability of 235.4% compared to the crystalline form in rats. Therefore, solid dispersion form was demonstrated to be more suitable for dug development to have enhanced bioavailability, leading to increased absorption and possible improved therapeutic potential of the developed drug based on pharmacokinetics data.

A deep learning method for predicting the minimum inhibitory concentration of antimicrobial peptides against Escherichia coli using Multi-Branch-CNN and Attention.

Antimicrobial peptides (AMPs) are a promising alternative to antibiotics to combat drug resistance in pathogenic bacteria. However, the development of AMPs with high potency and specificity remains a challenge, and new tools to evaluate antimicrobial activity are needed to accelerate the discovery process. Therefore, we proposed MBC-Attention, a combination of a multi-branch convolution neural network architecture and attention mechanisms to predict the experimental minimum inhibitory concentration of peptides against Escherichia coli. The optimal MBC-Attention model achieved an average Pearson correlation coefficient (PCC) of 0.775 and a root mean squared error (RMSE) of 0.533 (log μM) in three independent tests of randomly drawn sequences from the data set. This results in a 5-12% improvement in PCC and a 6-13% improvement in RMSE compared to 17 traditional machine learning models and 2 optimally tuned models using random forest and support vector machine. Ablation studies confirmed that the two proposed attention mechanisms, global attention and local attention, contributed largely to performance improvement. IMPORTANCE Antimicrobial peptides (AMPs) are potential candidates for replacing conventional antibiotics to combat drug resistance in pathogenic bacteria. Therefore, it is necessary to evaluate the antimicrobial activity of AMPs quantitatively. However, wet-lab experiments are labor-intensive and time-consuming. To accelerate the evaluation process, we develop a deep learning method called MBC-Attention to regress the experimental minimum inhibitory concentration of AMPs against Escherichia coli. The proposed model outperforms traditional machine learning methods. Data, scripts to reproduce experiments, and the final production models are available on GitHub.

Antimicrobial activity of essential oils of Curcuma longa and Syzygium aromaticum against multiple drug-resistant pathogenic bacteria.

The present study was conducted to investigate the antimicrobial potential of essential oils of Curcuma longa and Syzygium aromaticum against multidrug-resistant pathogenic bacteria. Four identified bacterial isolates including Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii were selected and their antibiotic sensitivity was checked by disc diffusion assay. C. longa and S. aromaticum were subjected to steam distillation to obtain their essential oils. The crude essential oils were fractioned by employing column chromatography. Crude essential oils and their fractions were evaluated for their antibacterial activity by agar well diffusion assay and minimum inhibitory concentrations were calculated. All the selected bacterial isolates showed resistance to three or more than three antibiotic groups and were declared as multidrugresistant (MDRs). Crude essential oils of C. longa and S. aromaticum exhibited antimicrobial activity against all selected isolates but S. aromaticum activity was better than the C. longa with a maximum 19.3±1.50 mm zone of inhibition against A. baumannii at 1.04 µL/mL MIC. GC/MS analysis revealed the abundance of components including eugenol, eugenyl acetate, b- caryophyllene, and a- Humulene in both crude oil and fractions of S. aromaticum. While the main components of C. longa essential oil were Ar-tumerone, a-tumerone, b- Tumerone, I-Phellandrene, a-zingibirene, b- sesquiphellandrene, and p- Cymene. This study highlights that plant-based essential oils could be a promising alternative to antibiotics for which pathogens have developed resistance. C. longa and S. aromaticum carry compounds that have antimicrobial potential against multiple drug-resistant bacteria including MRSA. E. coli, K. pneumoniae and A. baumannii.

Actinomycetes as Producers of Biologically Active Terpenoids: Current Trends and Patents.

Terpenes and their derivatives (terpenoids and meroterpenoids, in particular) constitute the largest class of natural compounds, which have valuable biological activities and are promising therapeutic agents. The present review assesses the biosynthetic capabilities of actinomycetes to produce various terpene derivatives; reports the main methodological approaches to searching for new terpenes and their derivatives; identifies the most active terpene producers among actinomycetes; and describes the chemical diversity and biological properties of the obtained compounds. Among terpene derivatives isolated from actinomycetes, compounds with pronounced antifungal, antiviral, antitumor, anti-inflammatory, and other effects were determined. Actinomycete-produced terpenoids and meroterpenoids with high antimicrobial activity are of interest as a source of novel antibiotics effective against drug-resistant pathogenic bacteria. Most of the discovered terpene derivatives are produced by the genus Streptomyces; however, recent publications have reported terpene biosynthesis by members of the genera Actinomadura, Allokutzneria, Amycolatopsis, Kitasatosporia, Micromonospora, Nocardiopsis, Salinispora, Verrucosispora, etc. It should be noted that the use of genetically modified actinomycetes is an effective tool for studying and regulating terpenes, as well as increasing productivity of terpene biosynthesis in comparison with native producers. The review includes research articles on terpene biosynthesis by Actinomycetes between 2000 and 2022, and a patent analysis in this area shows current trends and actual research directions in this field.

Synthesis of Green Copper Nanoparticles Using Medicinal Plant Krameria sp. Root Extract and Its Applications.

Nanotechnology is one of the most dynamic research areas and the fastest-growing market. Developing eco-friendly products using available resources to acquire maximum production, better yield, and stability is a great challenge for nanotechnology. In this study, copper nanoparticles (CuNP) were synthesized via the green method using root extract of the medical plant Rhatany (Krameria sp.) as a reducing and capping agent and used to investigate the influence of microorganisms. The maximum production of CuNP was noted at 70 °C after 3 h of reaction time. The formation of nanoparticles was confirmed through UV-spectrophotometer, and the product showed an absorbance peak in the 422-430 nm range. The functional groups were observed using the FTIR technique, such as isocyanic acid attached to stabilize the nanoparticles. The spherical nature and average crystal sizes of the particle (6.16 nm) were determined using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and X-ray diffractometer (XRD) analysis. In tests with a few drug-resistant pathogenic bacteria and fungus species, CuNP showed encouraging antimicrobial efficacy. CuNP had a significant antioxidant capacity of 83.81% at 200 g/m-1. Green synthesized CuNP are cost-effective and nontoxic and can be applied in agriculture, biomedical, and other fields.

Pathogenic bacteria recovered from Gene X-pert tuberculosis-negative adult patients in Gondar, Northwest Ethiopia

IntroductionLower respiratory tract infections (LRTIs) caused by drug-resistant pathogenic bacteria is a major problem in developing countries including Ethiopia. Therefore, this study aimed to determine the pathogenic bacteria and their antimicrobial susceptibility patterns among Gene X-pert tuberculosis-negative adult patients with clinically suspected LRTIs at the University of Gondar Comprehensive Specialized Referral Hospital, Gondar, Northwest Ethiopia.MethodsThis institutional-based cross-sectional study was conducted from February 01 to March 15, 2020. Socio-demographic data were collected by using a structured questionnaire. A total of 254 sputum specimens were collected from Gene X-pert tuberculosis-negative patients. Bacterial recovery was performed using blood, chocolate, and MacConkey agar plates. Bacterial isolates were identified based on Gram staining, colony characteristics, and biochemical reactions. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method. Methicillin resistance of S. aureus was confirmed using cefoxitin (30 µg). Descriptive statistics were calculated for each variable and results are shown in tables and figures.ResultsIn this study, the overall sputum culture positivity rate was 145/254 (57.1%). Gram-negative bacteria 111 (64.9%) were predominant compared to Gram-positive bacteria 60 (35.1%). Of the 145 culture-positive cases, 26 (14.8%) had poly-bacterial infections. S. aureus 40 (66.7%) was the predominant Gram-positive bacterium whereas K. pneumoniae 33 (29.7%), was the most isolated Gram-negative bacterium. Bacterial species, such as S. aureus were sensitive to ciprofloxacin 38/40 (95.0%), gentamicin 37/40 (92.5%), cefoxitin 36/40 (90.0%), and clindamycin 34/40 (85.0%). The proportion of Methicillin-resistant S. aureus was low, 4(10.0%). S. pneumoniae was sensitive to chloramphenicol 8/9 (88.9%) and resistant to ciprofloxacin 6/9 (66.7%). K pneumoniae, P. aeruginosa, E. coli, Serratia species, and H. influenzae also demonstrated high levels of resistance to ampicillin at rates of 21/33 (63.6%), 8/8 (100.0%), 15/17 (88.2%), 7/10 (70.0%), and 6/6 (100.0%), respectively.ConclusionThis study revealed a higher burden of Gram-negative and Gram-positive pathogenic bacterial agents, which is responsible for LRTs. Therefore, routine sputum culture identification and antibiotic susceptibility testing should be performed in Gene X-pert tuberculosis-negative patients.