Development Of Antibiotics Research Articles

Climate warming promotes collateral antibiotic resistance development in cyanobacteria

Both cyanobacterial blooms and antibiotic resistance have aggravated worldwide and posed a great threat to public health in recent years. As a significant source and reservoir of water environmental resistome, cyanobacteria exhibit confusing discrepancy between their reduced susceptibility and their chronic exposure to antibiotic mixtures at sub-inhibitory concentrations. How the increasing temperature affects the adaptive evolution of cyanobacteria-associated antibiotic resistance in response to low-level antibiotic combinations under climate change remains unclear. Here we profiled the antibiotic interaction and collateral susceptibility networks among 33 commonly detected antibiotics in 600 cyanobacterial strains isolated from 50 sites across four eutrophicated lakes in China. Cyanobacteria-associated antibiotic resistance level was found positively correlated to antibiotic heterogeneity across all sites. Among 528 antibiotic combinations, antagonism was observed for 62 % interactions and highly conserved within cyanobacterial species. Collateral resistance was detected in 78.5 % of pairwise antibiotic interaction, leading to a widened or shifted upwards mutant selection window for increased opportunity of acquiring second-step mutations. We quantified the interactive promoting effect of collateral resistance and increasing temperature on the evolution of both phenotypic and genotypic cyanobacteria-associated resistance under chronic exposure to environmental level of antibiotic combinations. With temperature increasing from 16 °C to 36 °C, the evolvability index and genotypic resistance level increased by 1.25 – 2.5 folds and 3 – 295 folds in the collateral-resistance-informed lineages, respectively. Emergence of resistance mutation pioneered by tolerance, which was jointly driven by mutation rate and persister fraction, was found to be accelerated by increased temperature and antibiotic switching rate. Our findings provided mechanic insights into the boosting effect of climate warming on the emergence and development of cyanobacteria-associated resistance against collateral antibiotic phenotypes.

The Phytochemical Composition and Antibacterial Effects of Allium sativum Clove Extracts on Some Enteric Bacterial Pathogens

In this study, the phytochemicals present in the cloves of Allium sativum were determined, and their antibacterial activities against some enteric bacterial pathogens were assessed. The phytochemical constituents were determined after the extraction process was completed using water and ethanol as the solvents. Furthermore, the aqueous and ethanolic extracts of A. sativum were tested against Escherichia coli, Salmonella typhi and Shigella dysenteriae isolated from faeces of gastroenteritis patients. The agar diffusion technique (punch method) was used for this. Additionally, the extracts' minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The mean values of the zones of inhibition obtained were statistically analyzed using ANOVA. The least significant difference was determined according to the LSD test at P<0.05. The results of the phytochemical analysis revealed the presence of saponin, alkaloids, flavonoids, and tannins. Furthermore, the antibacterial susceptibility test showed that the aqueous and ethanolic extracts possess antibacterial properties against all the test organisms. The ethanolic extract at the concentration of 500mg/ml had zones of inhibition of 29.00b against E. coli, 24.00b against S. dysenteriae and the lowest 20.00b against S. typhi. On the other hand, at that same concentration, the aqueous extract had zones of inhibition of 20.00b against E. coli and 18.00b against S. dysenteriae and S. typhi. This study suggests that A. sativum extracts possess antibacterial properties. Furthermore, since the ethanolic extract was more effective than the aqueous extract, it could be that the antibacterial potency of A. sativum is solvent-dependent. In conclusion, the findings from this study suggest that further purification of the constituents of the plant might lead to the development of novel antibiotics.

Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis.

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

A folate inhibitor exploits metabolic differences in Pseudomonas aeruginosa for narrow-spectrum targeting.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and in vitro assays using purified folA confirm that fluorofolin inhibits P. aeruginosa DHFR. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN, but these mutants also decrease pathogenesis. Our findings demonstrate how understanding species-specific genetic differences can enable selective targeting of important pathogens while revealing trade-offs between resistance and pathogenesis.

Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli.

Antibiotic resistance is an alarming public health concern that affects millions of individuals across the globe each year. A major challenge in the development of effective antibiotics lies in their limited ability to permeate cells, noting that numerous susceptible antibiotic targets reside within the bacterial cytosol. Consequently, improving the cellular permeability is often a key consideration during antibiotic development, underscoring the need for reliable methods to assess the permeability of molecules across cellular membranes. Currently, methods used to measure permeability often fail to discriminate between the arrival within the cytoplasm and the overall association of molecules with the cell. Additionally, these techniques typically possess throughput limitations. In this work, we describe a luciferase-based assay designed for assessing the permeability of molecules in the cytosolic compartment of Gram-negative bacteria. Our findings demonstrate a robust system that can elucidate the kinetics of intracellular antibiotic accumulation in live bacterial cells in real time.

Antibiotics influence the risk of anti-drug antibody formation during anti-TNF therapy in Chinese inflammatory bowel disease patients.

Aims: The formation of anti-drug antibodies (ADAs) during anti-tumor necrosis factor (anti-TNF) therapy is reported to lead to reducing serum drug levels, which may bring about a loss of response to treatment. Previous research has suggested an association between specific antibiotic classes and ADA formation during anti-TNF therapy. However, there are few studies specifically examining this association in Chinese inflammatory bowel disease (IBD) patients. Therefore, our study aimed to evaluate the possible effect of antibiotic use on ADA formation to anti-TNF therapy in Chinese patients with IBD. Methods: A total of 166 patients with IBD, including 149 with Crohn's disease (CD) and 17 with ulcerative colitis (UC), were included in this retrospective analysis. These patients were initially treated with anti-TNF therapy (infliximab or adalimumab) after January 2018 and reviewed with available ADA levels before October 2023. After univariable analysis of all the variables, a multivariate Cox proportional hazards model was used to assess the association between antibiotic use and ADA development. Results: Among 166 IBD patients treated with infliximab (108/166, 65.1%) or adalimumab (58/166, 34.9%), 31 patients (18.7%) were measured as positive ADA levels. Cox proportional hazard model demonstrated an increased risk of ADA formation in IBD patients who used β-lactam-β-lactamase inhibitor combinations (BL-BLIs) (HR = 5.143, 95%CI 1.136-23.270, p = 0.033), or nitroimidazoles (HR = 4.635, 95%CI 1.641-13.089, p = 0.004) during 12months before the ADA test. On the contrary, a reduced risk was noted in patients treated with fluoroquinolones (HR = 0.258, 95% CI 0.072-0.924, p = 0.037). Moreover, the median serum infliximab or adalimumab concentration in patients with positive ADA levels was significantly lower than that in patients with negative ADA levels (infliximab: 0.30 vs. 1.85μg/mL, p < 0.0001; adalimumab: 0.45 vs. 7.55μg/mL, p = 0.0121). Conclusion: ADA development is associated with various antibiotic classes. BL-BLIs and nitroimidazoles might increase the risk of ADA formation during anti-TNF therapy in Chinese IBD patients, while the treatment with fluoroquinolones could probably reduce such risk. There were certain limitations in the retrospective analysis of the study, therefore, the results are just for reference, and other studies are needed to further confirm our findings.

Electrochemical and Infrared Studies of a Model Bilayer of the Outer Membrane of Gram-Negative Bacteria and its Interaction with polymyxin─the Last-Resort Antibiotic.

A model bilayer of the outer membrane (OM) of Gram-negative bacteria, composed of lipid A and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), was assembled on the β-Tg modified gold (111) single crystal surface using a combination of Langmuir-Blodgett and Langmuir-Schaefer transfer. Electrochemical and spectroscopic methods were employed to study the properties of the model bilayer and its interaction with polymyxin. The model bilayer is stable on the gold surface in the transmembrane potential region between 0.0 and -0.7 V. The presence of Mg2+ coordinates with the phosphate and carboxylate groups in the leaflet of lipid A and stabilizes the structure of the model bilayer. Polymyxin causes the model bilayer leakage and damage in the transmembrane potential region between 0.2 and -0.4 V. At transmembrane potentials lower than -0.5 V, polymyxin does not affect the membrane integrity. Polymyxin binds to the phosphate and carboxylate groups in lipid A molecules and causes the increase of the tilt angle of acyl chains and the decrease of the tilt of the C═O bond. The results in this paper indicate that the antimicrobial activity of polymyxin depends on the transmembrane potential at the model bilayer and provides useful information for the development of new antibiotics.

American crocodiles (Crocodylus acutus: Reptilia: Crocodilidae) visiting the facilities of a freshwater aquaculture of the Northern Pacific region, Costa Rica, carry tetracycline-resistant Escherichia coli.

Apex predators are exposed to antimicrobial compounds and resistant microbes, which accumulate at different trophic levels of the related ecosystems. The study aimed to characterize the presence and the antimicrobial resistance patterns of fecal Escherichia coli isolated from cloacal swab samples obtained from wild-living American crocodiles (Crocodylus acutus) (n = 53). Sampling was conducted within the distinctive context of a freshwater-intensive aquaculture farm in Costa Rica, where incoming crocodiles are temporarily held in captivity before release. Phenotypic antimicrobial susceptibility profiles were determined in all isolates, while resistant isolates were subjected to whole-genome sequencing and bioinformatics analyses. In total, 24 samples contained tetracycline-resistant E. coli (45.3%). Isolates carried either tet(A), tet(B), or tet(C) genes. Furthermore, genes conferring resistance to ß-lactams, aminoglycosides, fosfomycin, sulfonamides, phenicol, quinolones, trimethoprim, and colistin were detected in single isolates, with seven of them carrying these genes on plasmids. Genome sequencing further revealed that sequence types, prevalence of antibiotic resistance carriage, and antibiotic resistance profiles differed between the individuals liberated within the next 24 h after their capture in the ponds and those liberated from enclosures after longer abodes. The overall presence of tetracycline-resistant E. coli, coupled with potential interactions with various anthropogenic factors before arriving at the facilities, hinders clear conclusions on the sources of antimicrobial resistance for the studied individuals. These aspects hold significant implications for both the aquaculture farm's biosecurity and the planning of environmental monitoring programs using such specimens. Considering human-crocodile conflicts from the One Health perspective, the occurrence of antimicrobial resistance underscores the importance of systematical surveillance of antibiotic resistance development in American crocodiles.

Antibiotic Development: Lessons from the Past and Future Opportunities.

The challenge of antimicrobial resistance is broadly appreciated by the clinical and scientific communities. To assess progress in the development of medical countermeasures to combat bacterial infections, we deployed information gleaned from clinical trials conducted from 2000 to 2021. Whereas private sector interest in cancer grew dramatically over this period, activity to combat bacterial infections remained stagnant. The comparative ambivalence to antimicrobial resistance is reflected in the number of investigative drugs under clinical investigation, their stage of development and most troublingly, a declining number of organizations that are actively involved in the development of new products to treat bacterial infections. This drop reflects the exits of many companies that had previously developed antibacterial agents.

Combatting Antibiotic Resistance: Regulatory Exclusivities for New Antibiotic Development in the United States and Europe

Abstract Antimicrobial resistance is a global concern that has led to millions of deaths worldwide. Genetic changes occur naturally in pathogens such as viruses, bacteria, parasites, and fungi. To overcome these concerns, there is a need to develop a new generation of antimicrobials. In an effort to encourage innovation, both the United States (US) and the European Union have introduced exclusivity programs. The US offered 5 years of additional nonpatent exclusivity for Qualified Infectious Disease Products under the Generating Antibiotic Incentives Now Act. In parallel, the European Commission offers a patent extension of 12 months across Europe (EU) countries or the option to sell the vouchers to other pharmaceutical companies through transferable exclusivity extension vouchers. This review focuses on the most important innovative strategies of antibiotic development the drug approvals, their mode of action, spectra of activity, and targeting of microorganisms, in past 5 years were mentioned, and discusses how to overcome the challenges of drug approval under antibiotic exclusivity in the US and EU, which will benefit the companies to develop novel drugs in several nations.

Structural Insights into the Lipopolysaccharide Transport (Lpt) System as a Novel Antibiotic Target.

Lipopolysaccharide (LPS) is a critical component of the extracellular leaflet within the bacterial outer membrane, forming an effective physical barrier against environmental threats in Gram-negative bacteria. After LPS is synthesized and matured in the bacterial cytoplasm and the inner membrane (IM), LPS is inserted into the outer membrane (OM) through the ATP-driven LPS transport (Lpt) pathway, which is an energy-intensive process. A trans-envelope complex that contains seven Lpt proteins (LptA-LptG) is crucial for extracting LPS from the IM and transporting it across the periplasm to the OM. The last step in LPS transport involves the mediation of the LptDE complex, facilitating the insertion of LPS into the outer leaflet of the OM. As the Lpt system plays an essential role in maintaining the impermeability of the OM via LPS decoration, the interactions between these interconnected subunits, which are meticulously regulated, may be potential targets for the development of new antibiotics to combat multidrug-resistant Gram-negative bacteria. In this review, we aimed to provide an overview of current research concerning the structural interactions within the Lpt system and their implications to clarify the function and regulation of LPS transport in the overall process of OM biogenesis. Additionally, we explored studies on the development of therapeutic inhibitors of LPS transport, the factors that limit success, and future prospects.

Elongation Factor P is Required for Processes Associated with Acinetobacter Pathogenesis

Antibiotic resistance is one of the world’s fastest-growing and most prevalent problems today. The influx of antibiotics within our environment from inadequate antibiotic stewardship has led to a surge of drug-resistant microorganisms. The CDC has classified Carbapenem-resistant Acinetobacter (CRA) as an urgent threat within this crisis. New drug development is imperative to combat infections caused by drug-resistant pathogens such as CRA. Bacterial translation, the process of protein synthesis by the ribosome, is a common target for new antibiotic development. Elongation factor P (EF-P) is a universally conserved translation factor required for antibiotic resistance in many bacteria. In this study, we assessed the importance of EF-P in processes associated with Acinetobacter pathogenesis. In the absence of EF-P, Acinetobacter baylyi displays decreased biofilm formation, surface-associated motility, and resistance to beta-lactams and carbapenems. This data holds hope for future drug development targeting EF-P in pathogens closely related to A. baylyi. KEYWORDS: Acinetobacter baylyi; Translation; Ribosome; Elongation Factor P; Polyproline; Biofilm; Surface Associated Motility; Antibiotic Resistance

Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

Antibiotic resistome in landfill leachate and impact on groundwater

Landfill leachate is a hotspot in antibiotic resistance development. However, little is known about antibiotic resistome and host pathogens in leachate and their effects on surrounding groundwater. Here, metagenomic sequencing was used to explore profiles, host bacteria, environmental risks and influencing factors of antibiotic resistome in raw and treated leachate and surrounding groundwater of three landfills. Results showed detection of a total of 324 antibiotic resistance genes (ARGs). The ARGs conferring resistance to multidrug (8.8 %–25.7 %), aminoglycoside (13.1 %–39.2 %), sulfonamide (10.0 %–20.9 %), tetracycline (5.7 %–34.4 %) and macrolide-lincosamide-streptogramin (MLS, 5.3 %–29.5 %) were dominant in raw leachate, while multidrug resistance genes were the major ARGs in treated leachate (64.1 %–83.0 %) and groundwater (28.7 %–76.6 %). Source tracking analysis suggests non-negligible influence of leachate on the ARGs in groundwater. The pathogens including Acinetobacter pittii, Pseudomonas stutzeri and P. alcaligenes were the major ARG-carrying hosts. Variance partitioning analysis indicates that the microbial community, abiotic variables and their interaction contributed most to the antibiotic resistance development. Our results shed light on the dissemination and driving mechanisms of ARGs from leachate to the groundwater, indicating that a comprehensive risk assessment and efficient treatment approaches are needed to deal with ARGs in landfill leachate and nearby groundwater. Environmental implicationsAntibiotic resistance genes are found abundant in the landfill sites, and these genes could be disseminated into groundwater via leaching of wastewater and infiltration of leachate. This results in deterioration of groundwater quality and human health risks posed by these ARGs and related pathogens. Thus measures should be taken to minimize potential negative impacts of landfills on the surrounding environment.

Metabolic Profiling Reveals the Importance of Arginine Metabolism via the Arginine Deiminase Pathway in Vancomycin-intermediate S. aureus

Gene mutations located in VraSR and GraSR, two-component regulatory systems have been reported to cause vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). Subsequent comparative proteomic profiling between vancomycin-susceptible S. aureus (VSSA) and VISAs of the Mu50 genetic lineage revealed up-regulated level of catabolic ornithine carbamoyltransferase (ArcB) in the latter, suggesting a role of arginine catabolism in VISA development. This study aimed to further investigate metabolic pathways associated with VISA development using liquid chromatography mass spectrometry (LCMS)-based untargeted metabolomic profiling. Metabolite profiles were compared among 3 isogenic strains of the Mu50 lineage: Mu50Ω (VSSA), Mu50Ω-vraSm (VISA) and Mu50Ω-vraSm-graRm (VISA). Intracellular levels of α-hydroxyglutaric acid, choline, lysine, malic acid, N-acetylornithine, nicotinamide, and cystathionine were found to be significantly different between VISAs compared to VSSA. VISA cells were found to have lower levels of intracellular citrulline and extracellular arginine, with higher extracellular level of ornithine compared to VSSA. These differences in metabolite levels between VISA and VSSA suggested the importance of arginine metabolism. Activation of the arginine deiminase pathway (ADI) in VISAs of the Mu50 lineage could be further explored as a target for antibiotic development.

Cajaninstilbene acid derivatives conjugated with siderophores of 3-hydroxypyridin-4(1H)-ones as novel antibacterial agents against Gram-negative bacteria based on the Trojan horse strategy

The low permeability of the outer membrane of Gram-negative bacteria is a serious obstacle to the development of new antibiotics against them. Conjugation of antibiotic with siderophore based on the “Trojan horse strategy” is a promising strategy to overcome the outer membrane obstacle. In this study, series of antibacterial agents were designed and synthesized by conjugating the 3-hydroxypyridin-4(1H)-one based siderophores with cajaninstilbene acid (CSA) derivative 4 which shows good activity against Gram-positive bacteria by targeting their cell membranes but is ineffective against Gram-negative bacteria. Compared to the inactive parent compound 4, the conjugates 45c or 45d exhibits significant improvement in activity against Gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae and especially P. aeruginosa (minimum inhibitory concentrations, MICs = 7.8–31.25 μM). The antibacterial activity of the conjugates is attributed to the CSA derivative moiety, and the action mechanism is by disruption of bacterial cell membranes. Further studies on the uptake mechanisms showed that the bacterial siderophore-dependent iron transport system was involved in the uptake of the conjugates. In addition, the conjugates 45c and 45d showed a lower cytotoxic effects in vivo and in vitro and a positive therapeutic effect in the treatment of C. elegans infected by P. aeruginosa. Overall, our work describes a new class and a promising 3-hydroxypyridin-4(1H)-one-CSA derivative conjugates for further development as antibacterial agents against Gram-negative bacteria.

Identification of phytoconstituents from Dicliptera paniculata and study of antibacterial activity guided by molecular docking.

According to WHO, antibiotic resistance is one of the biggest healthcare challenges to the global community. Therefore, it is absolutely essential to discover new antibiotics to address the challenge. Dicliptera paniculata (ForssK.) I. Darbysh, a rare medicinal herb of Acanthaceae, is known for its noteworthy uses as a flavoring, spicing, and antibacterial agent. The primary goal of the study is to identify novel antibacterials from D. paniculata. The petroleum ether fraction of the methanol extract of D. paniculata was subjected to GC-MS and identified 14 compounds. Several bacterial target proteins were used for molecular docking. The antibacterial activity of petroleum-ether fraction was evaluated on bacteria whose target protein interacts most strongly with identified molecules. The molecules DP_02, DP_06, and DP_14 exhibited the highest docking scores with Staphylococcus aureus dihydrofolate reductase, which were - 6.283, - 7.705, and - 6.364kcal/mol, respectively. The MM-GBSA binding energy of compounds DP_02, DP_06, and DP_14 were - 46.736, - 42.366, and - 35.734kcal/mol, respectively. The MM-GBSA binding energy and decent docking score of the compounds DP_02 and DP_06 were both encouraging, and both of the compounds are drug-like. The finding was validated through studies on antibacterial effectiveness against S. aureus and showed encouraging results. These two molecules might serve as the building blocks for the future development of potent antibiotics.

The Combination of Membrane Disruption and FtsZ Targeting by a Chemotherapeutic Hydrogel Synergistically Combats Pathogens Infections.

The emergence of multidrug-resistant (MDR) bacteria poses a significant challenge to global health. Due to a shortage of antibiotics, alternative therapeutic strategies are urgently needed. Unfortunately, colistin, the last-resort antibiotic, has unavoidable nephrotoxicity and hepatotoxicity, and its single killing mechanism is prone to drug resistance. To address this challenge, a promising combinatorial approach that includes colistin, a membrane-disrupting antimicrobial agent, and chelerythrine (CHE), a FtsZ protein inhibitor is proposed. This approach significantly reduces antibiotic dose and development of resistance, leading to almost complete inactivation of MDR pathogens in vitro. To address solubility issues and ensure transport, the antimicrobial hydrogel system LNP-CHE-CST@hydrogel, which induced reactive oxygen species (ROS) and apoptosis-like cell death by targeting the FtsZ protein, is used. In an in vivo mouse skin infection model, the combination therapy effectively eliminated MDR bacteria within 24 h, as monitored by fluorescence tracking. The findings demonstrate a promising approach for developing multifunctional hydrogels to combat MDR bacterial infections.

Biofilm Production and Porin Permeability Activity in Clinical Isolates of Ciprofloxacin-Resistant Klebsiella pneumoniae in the Tertiary Hospital in Purwokerto, Indonesia

World Health Organization reports that the current use of ciprofloxacin as a broad-spectrum antibiotic shows a pattern of high bacterial resistance in many countries. Klebsiella pneumoniae is known as a Gram-negative bacterium that most often causes infection and is resistant to antibiotics. This study aimed to find patterns of resistance mechanisms based on biofilm production and porin permeability activity in K. pneumoniae clinical specimens from patients at RSUD Prof. Dr Margono Soekardjo Purwokerto, Indonesia. Several isolates of ciprofloxacin-resistant K. pneumoniae were isolated from clinical specimens of blood, sputum, urine, pus, stool and pleural fluid from August to October 2022. Identification and sensitivity testing of K. pneumoniae to ciprofloxacin were performed using Vitek® 2 Compact. A test for biofilm production is carried out by measuring optical density (OD) with a microplate at a wavelength of 630 nm. The porin permeability activity was determined using the value of the minimum inhibitory concentration (MIC) by the LC-MS spectrophotometry method at a wavelength of 630 nm. The results showed that 72 (47 %) isolates were resistant to ciprofloxacin. Among them, 41.3 % (24/58) of ciprofloxacin-resistant K. pneumoniae were able to produce strong biofilm. Porin permeability is indicated by MIC values of 1, 2 and 4 mg/L with bacterial cell counts of 32×106, 19×106 and 34×106 CFU/mL, respectively. This number decreased from the initial control with a bacterial cell count of 10.15×107 CFU/mL. Analysis of the correlation between the significance of biofilm production and the number of bacterial cells indicated that biofilm formation was related to the number of bacterial cells (R = 0.628, p ≤ 0.05). In conclusion, the resistance mechanism of ciprofloxacin-resistant K. pneumoniae involves biofilm production and decreased porin permeability activity. HIGHLIGHTS Major resistance mechanisms in pneumoniae decreased cell permeability from porin activity and biofilm formation Bacterial outer membrane permeability to antimicrobial substances influences inherent resistance. Bacteria produce biofilm to increase their resistance to antibiotics. Porins are non-specific diffusion transport proteins that play a part in the antibiotic resistance process The evolutionary development in the case of antibiotic resistance has an impact on the emergence of understanding in new perspective patterns on how to overcome health problems related to antibiotic resistance GRAPHICAL ABSTRACT

Transposon sequencing reveals metabolic pathways essential for Mycobacterium tuberculosis infection.

New drugs are needed to shorten and simplify treatment of tuberculosis caused by Mycobacterium tuberculosis. Metabolic pathways that M. tuberculosis requires for growth or survival during infection represent potential targets for anti-tubercular drug development. Genes and metabolic pathways essential for M. tuberculosis growth in standard laboratory culture conditions have been defined by genome-wide genetic screens. However, whether M. tuberculosis requires these essential genes during infection has not been comprehensively explored because mutant strains cannot be generated using standard methods. Here we show that M. tuberculosis requires the phenylalanine (Phe) and de novo purine and thiamine biosynthetic pathways for mammalian infection. We used a defined collection of M. tuberculosis transposon (Tn) mutants in essential genes, which we generated using a custom nutrient-rich medium, and transposon sequencing (Tn-seq) to identify multiple central metabolic pathways required for fitness in a mouse infection model. We confirmed by individual retesting and complementation that mutations in pheA (Phe biosynthesis) or purF (purine and thiamine biosynthesis) cause death of M. tuberculosis in the absence of nutrient supplementation in vitro and strong attenuation in infected mice. Our findings show that Tn-seq with defined Tn mutant pools can be used to identify M. tuberculosis genes required during mouse lung infection. Our results also demonstrate that M. tuberculosis requires Phe and purine/thiamine biosynthesis for survival in the host, implicating these metabolic pathways as prime targets for the development of new antibiotics to combat tuberculosis.