Metagenomic perspectives on antibiotic resistance genes in tap water: The environmental characteristic, potential mobility and health threat.

As an emerging environmental contaminant, antibiotic resistance genes (ARGs) in tap water have attracted great attention. Although studies have provided ARG profiles in tap water, research on their abundance levels, composition characteristics, and potential threat is still insufficient. Here, 9 household tap water samples were collected from the Guangdong-Hong Kong-Macao Greater Bay Area (GBA) in China. Additionally, 75 sets of environmental sample data (9 types) were downloaded from the public database. Metagenomics was then performed to explore the differences in the abundance and composition of ARGs. 221 ARG subtypes consisting of 17 types were detected in tap water. Although the ARG abundance in tap water was not significantly different from that found in drinking water plants and reservoirs, their composition varied. In tap water samples, the three most abundant classes of resistance genes were multidrug, fosfomycin and MLS (macrolide-lincosamide-streptogramin) ARGs, and their corresponding subtypes ompR, fosX and macB were also the most abundant ARG subtypes. Regarding the potential mobility, vanS had the highest abundance on plasmids and viruses, but the absence of key genes rendered resistance to vancomycin ineffective. Generally, the majority of ARGs present in tap water were those that have not been assessed and are currently not listed as high-threat level ARG families based on the World Health Organization Guideline. Although the current potential threat to human health posed by ARGs in tap water is limited, with persistent transfer and accumulation, especially in pathogens, the potential danger to human health posed by ARGs should not be ignored.

Tracking Antibiotic Resistance Trends in Central Iran Amidst the COVID-19 Pandemic From 2021 to 2023: A Comprehensive Epidemiological Study.

Background: The emergence of coronavirus disease in 2019 (COVID-19) appears to be having an impact on antibiotic resistance patterns. Specific circumstances during the COVID-19 era may have played a role in the spread of antimicrobial resistance (AMR). This study aimed to look at the changes in AMR patterns of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii at Al-Zahra Hospital. Materials and Methods: From March 2021 to January 2023, 3651 clinical samples were collected from patients hospitalized at Isfahan's Al-Zahra Hospital. The Clinical and Laboratory Standards Institute recommended procedures for detecting gram-negative bacteria and assessing antibiotic susceptibility were used. We divided the information into three years. Results: Highest resistance rates were seen in A. baumannii to Ciprofloxacin (98.0%) and Ampicillin-Sulbactam (97.0%). For P. aeruginosa the resistance rate for Ceftazidime (36.1), Levofloxacin (37.8), and Meropenem (47.1) dropped seriously in 2022. Conclusion: During the second year of the pandemic in central Iran, all three species studied showed rising rates of AMR. This can be attributable to two peaks within Iran on May 6, 2021 and August 27, 2021. The results of this study show that P. aeruginosa, K. pneumoniae, and A. baumannii bacteria in central Iran have a higher level of antibiotic resistance than previously studied strains before the pandemic.

An Antibiotic-Degrading Engineered Biofilm Platform to Combat Environmental Antibiotic Resistance.

The presence of antibiotics in natural water bodies is a growing problem regarding the occurrence of antibiotic resistance among various species. This is mainly caused by the excessive use of medical and veterinary antibiotics as well as the lack of effective treatment processes for eliminating residual antibiotics from wastewaters. In this study, we introduce a genetically engineered biomaterial as a solution for the effective degradation of one of the dominantly found antibiotics in natural water bodies. Our biomaterial harnesses laccase-type enzymes, which are known to attack specific types of antibiotics, i.e., fluoroquinolone-type synthetic antibiotics, and as a result degradation occurs. The engineered biomaterial is built using Escherichia coli biofilm protein CsgA as a scaffold, which is fused separately to two different laccase enzymes with the SpyTag-SpyCatcher peptide-protein duo. The designed biofilm materials were successful in degrading ciprofloxacin, as demonstrated with the data obtained from mass spectrometry analysis and cell viability assays.

Carbapenemase and extended spectrum beta-lactamase producing bacteria in waters originating from a single landfill in Slovenia.

Groundwater, rainwater, and leachate associated with a single landfill were analysed to detect extended spectrum beta-lactamase (ESBL) producing and carbapenemase (CP) producing bacteria. After cultivation on three commercial selective-differential media, 240 bacterial isolates were obtained and identified by MALDI-TOF MS. Isolates from clinically relevant species were further genotyped by Enterobacterial Repetitive Intergenic Consensus (ERIC) polymerase chain reaction (PCR), tested for antibiotic susceptibility and presence of carbapenemases and ESBL enzymes. Two ESBL producing isolates and two isolates producing carbapenemases were detected in rainwater, groundwater and in leachate: Klebsiella oxytoca complex with the gene for the ESBL enzyme CTX-M-1 and the gene for the carbapenemase OXA-48, Serratia fonticola with the gene for the ESBL enzyme FONA-2 and Pseudomonas aeruginosa with gene coding VIM metallo-beta lactamase. Our study indicates that bacteria with ESBL and CP genes can be present in landfill-associated waters.

Sulfonamide inhibitors of bacterial carbonic anhydrases.

The increasing prevalence of antibiotic-resistant bacteria necessitates the exploration of novel therapeutic targets. Bacterial carbonic anhydrases (CAs) have been known for decades, but only in the past ten years they have garnered significant interest as drug targets to develop antibiotics having a diverse mechanism of action compared to the clinically used drugs. Significant progress has been made in the field in the past three years, with the validation in vivo of CAs from Neisseria gonorrhoeae, and vancomycin-resistant enterococci as antibiotic targets. This chapter compiles the state-of-the-art research on sulfonamide derivatives described as inhibitors of all known bacterial CAs. A section delves into the mechanisms of action of sulfonamide compounds with the CA classes identified in pathogenic bacteria, specifically α, ß, and γ classes. Therefore, the inhibitory profiling of the bacterial CAs with classical and clinically used sulfonamide compounds is reported and analyzed. Another section covers various other series of sulfonamide CA inhibitors studied for the development of new antibiotics. By synthesizing current research findings, this chapter highlights the potential of sulfonamide inhibitors as a novel class of antibacterial agents and paves the way for future drug design strategies.

Bacterial α-CAs: a biochemical and structural overview.

Carbonic anhydrases belonging to the α-class are widely distributed in bacterial species. These enzymes have been isolated from bacteria with completely different characteristics including both Gram-negative and Gram-positive strains. α-CAs show a considerable similarity when comparing the biochemical, kinetic and structural features, with only small differences which reflect the diverse role these enzymes play in Nature. In this chapter, we provide a comprehensive overview on bacterial α-CA data, with a highlight to their potential biomedical and biotechnological applications.

Pathogenicity, phylogenomic, and comparative genomic study of Pseudomonas syringae sensu lato affecting sweet cherry in California.

To gain insights into the diversity of Pseudomonas syringae sensu lato affecting sweet cherry in California, we sequenced and analyzed the phylogenomic and genomic architecture of 86 fluorescent pseudomonads isolated from symptomatic and asymptomatic cherry tissues. Fifty-eight isolates were phylogenetically placed within the P. syringae species complex and taxonomically classified into five genomospecies: P. syringae pv. syringae, P. syringae, Pseudomonas cerasi, Pseudomonas viridiflava, and A. We annotated components of the type III secretion system and phytotoxin-encoding genes and correlated the data with pathogenicity phenotypes. Intact probable regulatory protein HrpR was annotated in the genomic sequences of all isolates of P. syringae pv. syringae, P. syringae, P. cerasi, and A. Isolates of P. viridiflava had atypical probable regulatory protein HrpR. Syringomycin and syringopeptin-encoding genes were annotated in isolates of all genomospecies except for A and P. viridiflava. All isolates of P. syringae pv. syringae caused cankers, leaf spots, and fruit lesions in the field. In contrast, all isolates of P. syringae and P. cerasi and some isolates of P. viridiflava caused only cankers. Isolates of genomospecies A could not cause any symptoms suggesting phytotoxins are essential for pathogenicity. On detached immature cherry fruit pathogenicity assays, isolates of all five genomospecies produced symptoms (black-dark brown lesions). However, symptoms of isolates of genomospecies A were significantly (P < 0.01) less severe than those of other genomospecies. We also mined for genes conferring resistance to copper and kasugamycin and correlated these data with in vitro antibiotic sensitivity tests. IMPORTANCE: Comprehensive identification of phytopathogens and an in-depth understanding of their genomic architecture, particularly virulence determinants and antibiotic-resistant genes, are critical for several practical reasons. These include disease diagnosis, improved knowledge of disease epidemiology, pathogen diversity, and determination of the best possible management strategies. In this study, we provide the first report of the presence and pathogenicity of genomospecies Pseudomonas cerasi and Pseudomonas viridiflava in California sweet cherry. More importantly, we report a relatively high level of resistance to copper among the population of Pseudomonas syringae pv. syringae (47.5%). This implies copper cannot be effectively used to control bacterial blast and bacterial canker of sweet cherries. On the other hand, no isolates were resistant to kasugamycin, an indication that kasugamycin could be effectively used for the control of bacterial blast and bacterial canker. Our findings are important to improve the management of bacterial blast and bacterial canker of sweet cherries in California.

Spotlight on the epidemiology and antimicrobial susceptibility profiles of Vibrio species in the MENA region, 2000-2023.

Recent cholera outbreaks in many countries in the Middle East and North Africa (MENA) region have raised public health concerns and focused attention on the genus Vibrio. However, the epidemiology of Vibrio species in humans, water, and seafood is often anecdotal in this region. In this review, we screened the literature and provided a comprehensive assessment of the distribution and antibiotic resistance properties of Vibrio species in different clinical and environmental samples in the region. This review will contribute to understanding closely the real burden of Vibrio species and the spread of antibiotic-resistant strains in the MENA region. The overall objective is to engage epidemiologists, sanitarians and public health stakeholders to address this problem under the One-health ethos.

The Vibrio genus contains many bacterial species normally found in freshwater, estuaries and marine environments. Some of these species can be transmitted by water and food and can make people severely ill. For instance, some groups of the bacterium Vibrio cholerae (serogroups O1 and O139) can cause serious watery diarrhea called cholera. Other pathogenic Vibrio bacteria can cause other types of infections such as gastroenteritis and wound infections. Some of these bacteria are becoming increasingly resistant to antibiotics, which will threaten and complicate therapy. This review discusses the occurrence and antibiotic resistance of different important Vibrio species in the Middle East and North Africa (MENA) region.

Antibiotic heteroresistance and persistence: an additional aid in hospital acquired infections by Enterococcus spp.?

Enterococcus, particularly E. faecium and E. faecalis, are responsible for many hospital-acquired infections. With their intrinsic antibiotic resistance and ability to form biofilms, enterococcal infections are already challenging to manage. However, when heterogenous populations are present, such as those exhibiting heteroresistance and persistence, the complexity of these infections increases exponentially not only due to their treatment but also due to their difficult diagnosis. In this study, we provide a summary of the current understanding of both heteroresistance and persistence in terms of mechanisms, diagnosis and treatment and subsequently review recent literature pertaining to these susceptibility types specifically in enterococci.

Some bacteria are common causes of illness among hospital patients. Some of these infections are very difficult to treat, as the bacteria can respond differently to antibiotics. This review looks at how a type of bacteria called Enterococcus can respond differently to antibiotics, and how we can diagnose or kill them more easily.

Metagenomic Profiling of Internationally Sourced Sewage Influents and Effluents Yields Insight into Selecting Targets for Antibiotic Resistance Monitoring.

It has been debated whether wastewater treatment plants (WWTPs) primarily act to attenuate or amplify antibiotic resistance genes (ARGs). However, ARGs are highly diverse with respect to their resistance mechanisms, mobilities, and taxonomic hosts and therefore their behavior in WWTPs should not be expected to be universally conserved. We applied metagenomic sequencing to wastewater influent and effluent samples from 12 international WWTPs to classify the behavior of specific ARGs entering and exiting WWTPs. In total, 1079 different ARGs originating from a variety of bacteria were detected. This included ARGs that could be mapped to assembled scaffolds corresponding to nine human pathogens. While the relative abundance (per 16S rRNA gene) of ARGs decreased during treatment at 11 of the 12 WWTPs sampled and absolute abundance (per mL) decreased at all 12 WWTPs, increases in relative abundance were observed for 40% of the ARGs detected at the 12th WWTP. Also, the relative abundance of mobile genetic elements (MGE) increased during treatment, but the fraction of ARGs known to be transmissible between species decreased, thus demonstrating that increased MGE prevalence may not be generally indicative of an increase in ARGs. A distinct conserved resistome was documented in both influent and effluent across samples, suggesting that well-functioning WWTPs generally attenuate influent antibiotic resistance loads. This work helps inform strategies for wastewater surveillance of antibiotic resistance, highlighting the utility of tracking ARGs as indicators of treatment performance and relative risk reduction.

Metapopulation model of phage therapy of an acute Pseudomonas aeruginosa lung infection.

Infections caused by multidrug resistant (MDR) pathogenic bacteria are a global health threat. Bacteriophages ("phage") are increasingly used as alternative or last-resort therapeutics to treat patients infected by MDR bacteria. However, the therapeutic outcomes of phage therapy may be limited by the emergence of phage resistance during treatment and/or by physical constraints that impede phage-bacteria interactions in vivo. In this work, we evaluate the role of lung spatial structure on the efficacy of phage therapy for Pseudomonas aeruginosa infections. To do so, we developed a spatially structured metapopulation network model based on the geometry of the bronchial tree, including host innate immune responses and the emergence of phage-resistant bacterial mutants. We model the ecological interactions between bacteria, phage, and the host innate immune system at the airway (node) level. The model predicts the synergistic elimination of a P. aeruginosa infection due to the combined effects of phage and neutrophils, given the sufficient innate immune activity and efficient phage-induced lysis. The metapopulation model simulations also predict that MDR bacteria are cleared faster at distal nodes of the bronchial tree. Notably, image analysis of lung tissue time series from wild-type and lymphocyte-depleted mice revealed a concordant, statistically significant pattern: infection intensity cleared in the bottom before the top of the lungs. Overall, the combined use of simulations and image analysis of in vivo experiments further supports the use of phage therapy for treating acute lung infections caused by P. aeruginosa, while highlighting potential limits to therapy in a spatially structured environment given impaired innate immune responses and/or inefficient phage-induced lysis. IMPORTANCE: Phage therapy is increasingly employed as a compassionate treatment for severe infections caused by multidrug-resistant (MDR) bacteria. However, the mixed outcomes observed in larger clinical studies highlight a gap in understanding when phage therapy succeeds or fails. Previous research from our team, using in vivo experiments and single-compartment mathematical models, demonstrated the synergistic clearance of acute P. aeruginosa pneumonia by phage and neutrophils despite the emergence of phage-resistant bacteria. In fact, the lung environment is highly structured, prompting the question of whether immunophage synergy explains the curative treatment of P. aeruginosa when incorporating realistic physical connectivity. To address this, we developed a metapopulation network model mimicking the lung branching structure to assess phage therapy efficacy for MDR P. aeruginosa pneumonia. The model predicts the synergistic elimination of P. aeruginosa by phage and neutrophils but emphasizes potential challenges in spatially structured environments, suggesting that higher innate immune levels may be required for successful bacterial clearance. Model simulations reveal a spatial pattern in pathogen clearance where P. aeruginosa are cleared faster at distal nodes of the bronchial tree than in primary nodes. Interestingly, image analysis of infected mice reveals a concordant and statistically significant pattern: infection intensity clears in the bottom before the top of the lungs. The combined use of modeling and image analysis supports the application of phage therapy for acute P. aeruginosa pneumonia while emphasizing potential challenges to curative success in spatially structured in vivo environments, including impaired innate immune responses and reduced phage efficacy.

Simultaneous removal of ammonia nitrogen, sulfamethoxazole, and antibiotic resistance genes in self-corrosion microelectrolysis-enhanced counter-diffusion biofilm system.

A self-corrosion microelectrolysis (SME)-enhanced membrane-aerated biofilm reactor (eMABR) was developed for the removal of pollutants and reduction of antibiotic resistance genes (ARGs). Fe2+ and Fe3+ formed iron oxides on the biofilm, which enhanced the adsorption and redox process. SME can induce microorganisms to secrete more extracellular proteins and up-regulate the expression of ammonia monooxygenase (AMO) (0.92 log2). AMO exposed extra binding sites (ASP-69) for antibiotics, weakening the competition between NH4+-N and sulfamethoxazole (SMX). The NH4+-N removal efficiency in the S-eMABR (adding SMX and IC) increased by 44.87 % compared to the S-MABR (adding SMX). SME increased the removal performance of SMX by approximately 1.45 times, down-regulated the expressions of sul1 (-1.69 log2) and sul2 (-1.30 log2) genes, and controlled their transfer within the genus. This study provides a novel strategy for synergistic reduction of antibiotics and ARGs, and elucidates the corresponding mechanism based on metatranscriptomic and molecular docking analyses.

Neglected contributors to the transmission of bacterial antibiotic resistance in drinking water: Extracellular antibiotic resistance genes and the natural transformation.

Antibiotic resistance genes (ARGs) have increasingly gained recognition as an "emerging contaminant" that poses a threat to the biosafety of drinking water. However, previous researches have primarily focused on the intracellular state of ARGs and rarely investigated the ecological characteristics (e.g., distribution and origin), environmental behavior (spread), and risks of extracellular form (eARGs) within drinking water systems. Therefore, this review evaluated isolation strategies and extraction methods for recovering eARGs from drinking water, elucidated the distribution characteristics of eARGs, and examined their impact on the antibiotic resistome from source water to tap water. We emphasized that chlorination and biological treatments significantly contribute to the prevalence and persistence of eARGs in drinking water. Moreover, we highlighted the role of biological reactors (e.g., biofilter, biological activated carbon) and drinking water distribution systems in facilitating the natural transformation of eARGs while significantly contributing to bacterial antibiotic resistance (BAR) propagation. Finally, we summarized the current risk assessment systems for ARGs and critically address remaining challenging questions necessary for better forecasting health risks associated with eARGs in drinking water environments. Collectively, this review enhances the understanding of ecological characteristics and environmental behavior of eARGs in drinking water while providing important implications for controlling and reducing BAR contamination not only in drinking water but also in other aquatic environments.

Disentangling abiotic and biotic effects of treated wastewater on stream biofilm resistomes enables the discovery of a new planctomycete beta-lactamase.

BACKGROUND: Environmental reservoirs of antibiotic resistance pose a threat to human and animal health. Aquatic biofilms impacted by wastewater effluent (WW) are known environmental reservoirs for antibiotic resistance; however, the relative importance of biotic factors and abiotic factors from WW on the abundance of antibiotic resistance genes (ARGs) within aquatic biofilms remains unclear. Additionally, experimental evidence is limited within complex aquatic microbial communities as to whether genes bearing low sequence similarity to validated reference ARGs are functional as ARGs. RESULTS: To disentangle the effects of abiotic and biotic factors on ARG abundances, natural biofilms were previously grown in flume systems with different proportions of stream water and either ultrafiltered or non-ultrafiltered WW. In this study, we conducted deep shotgun metagenomic sequencing of 75 biofilm, stream, and WW samples from these flume systems and compared the taxonomic and functional microbiome and resistome composition. Statistical analysis revealed an alignment of the resistome and microbiome composition and a significant association with experimental treatment. Several ARG classes exhibited an increase in normalized metagenomic abundances in biofilms grown with increasing percentages of non-ultrafiltered WW. In contrast, sulfonamide and extended-spectrum beta-lactamase ARGs showed greater abundances in biofilms grown in ultrafiltered WW compared to non-ultrafiltered WW. Overall, our results pointed toward the dominance of biotic factors over abiotic factors in determining ARG abundances in WW-impacted stream biofilms and suggested gene family-specific mechanisms for ARGs that exhibited divergent abundance patterns. To investigate one of these specific ARG families experimentally, we biochemically characterized a new beta-lactamase from the Planctomycetota (Phycisphaeraceae). This beta-lactamase displayed activity in the cleavage of cephalosporin analog despite sharing a low sequence identity with known ARGs. CONCLUSIONS: This discovery of a functional planctomycete beta-lactamase ARG is noteworthy, not only because it was the first beta-lactamase to be biochemically characterized from this phylum, but also because it was not detected by standard homology-based ARG tools. In summary, this study conducted a metagenomic analysis of the relative importance of biotic and abiotic factors in the context of WW discharge and their impact on both known and new ARGs in aquatic biofilms. Video Abstract.

Polyvinyl chloride microplastics disseminate antibiotic resistance genes in Chinese soil: A metagenomic analysis.

The widespread prevalence of microplastics (MPs) in the environment poses concerns as they are vectors of antibiotic resistance genes (ARGs). The relationships between antibiotic resistomes and MPs remain unexplored in soil which was considered as the reservoirs of MPs and ARGs. This study investigated the effects of polyvinyl chloride (PVC) MPs on soil bacterial communities and ARG abundance which soil samples sourced from 20 provinces across China. We found that PVC significantly influences soil bacterial community structure and ARG abundance. Structural equation modeling revealed that PVC alters soil characteristics, ultimately affecting soil bacterial communities, including ARG-containing bacterial hosts, and the relative abundance of ARGs. This study enhances our understanding of how MPs influence the proliferation and hosts of ARGs within diverse soil environments, offering crucial insights for future strategies in plastic management and disposal.

Antibiotic Resistance of Helicobacter pylori in Mainland China: A Focus on Geographic Differences through Systematic Review and Meta-analysis.

BACKGROUND: Empirical treatment needs to be supported by regional data, but knowledge of interregional differences is currently lacking in China. This study aimed to summarize and map the primary and secondary antibiotic resistance of H. pylori in different regions of mainland China. METHODS: PubMed, EMBASE, Web of Science, China National Knowledge Infrastructure and Wanfang databases were systematically reviewed for studies published between January 1st, 2000 and July 15th, 2023. Data related to primary and secondary H. pylori antibiotic resistance rates were included. Random-effects models were used to synthesize the pooled resistance rates. RESULTS: Ultimately, 74 studies were included in the final analysis. Sixteen provinces reported resistance data. The overall resistance rates of H. pylori in mainland China were 30.72% (95% CI 27.53%-33.99%) to clarithromycin, 70.14% (95% CI 29.53%-37.46%) to metronidazole, and 32.98% (95% CI 28.73%-37.37%) to levofloxacin; for amoxicillin, tetracycline, and furazolidone, the rates were 2.41% (95% CI 1.43%-3.60%), 2.53% (95% CI 1.19%-4.28%) and 1.54% (95% CI 0.28%-3.62%), respectively. Spatial and temporal differences were observed. The resistance rates increased after treatment failure, however, secondary resistance to amoxicillin, tetracycline, and furazolidone were still low across the vast majority of study regions. CONCLUSION: Surveillance of the updated prevalence of antibiotic resistance of H. pylori for different regions is warranted, which should factor into clinical decision making and guideline recommendations.

Conjugative transfer of the IncN plasmid pKM101 is mediated by dynamic interactions between the TraK accessory factor and TraI relaxase.

Conjugative dissemination of mobile genetic elements (MGEs) among bacteria is initiated by assembly of the relaxosome at the MGE's origin-of-transfer (oriT) sequence. A critical but poorly defined step of relaxosome assembly involves recruitment of the catalytic relaxase to its DNA strand-specific nicking site within oriT. Here, we present evidence by AlphaFold modeling, affinity pulldowns, and in vivo site-directed photocrosslinking that the TraK Ribbon-Helix-Helix DNA-binding protein recruits TraI to oriT through a dynamic interaction in which TraI's C-terminal unstructured domain (TraICTD) wraps around TraK's C-proximal tetramerization domain. Upon relaxosome assembly, conformational changes disrupt this contact, and TraICTD instead self-associates as a prerequisite for relaxase catalytic functions or substrate engagement with the transfer channel. These findings delineate key early-stage processing reactions required for conjugative dissemination of a model MGE.

Phytochemical profiling and antibacterial activities of Ziziphora tenuior root extracts: a molecular docking against VanA of vancomycin-resistant enterococci.

Medicinal plants, renowned for their antibacterial phytocompounds and secondary metabolites, hold significant promise in addressing antibiotic-resistant bacterial strains. This study aimed to conduct phytochemical profiling of the methanolic and dichloromethane extracts of Ziziphora tenuior root using the GC-MS technique. These extracts' antioxidant potential was assessed via DPPH assay and their antibacterial activity was evaluated against S. aureus, E. coli, and VRE bacterial strains. Furthermore, the drug-ligand interactions between the extracts' biocompounds and d-alanyl-d-lactate ligase (VanA) protein of vancomycin-resistant enterococci strains (VRE) were analyzed using molecular docking. Based on the results, 74% of methanolic extract consisted of (3methyl, 24S)-stigmast-5-en-3-ol (which is a ß-sitosterol), followed by Tetrasiloxane, decamethyl (15.5%), and 1-methyl-4-phenyl-5-thioxo-1,2,4-triazolidin-3-one (10.5%). Also, the only predominant compound identified in the dichloromethane extract was Benzo[h]quinoline, 2,4-dimethyl-. Both extracts showed antioxidant activity, while the antioxidant activity of the methanolic extract (IC50 = 95.33 µg/ml) was significantly higher than that of the dichloromethane extract (IC50 = 934.23 µg/ml). Also, both extracts displayed substantial antibacterial efficacy against the tested pathogens, particularly against VRE. Moreover, the in silico analysis revealed that (3methyl, 24S)-stigmast-5-en-3-ol and Benzo[h]quinoline,2,4-dimethyl- exhibited notable interactions with VanA through docking energy values of - 9.0 and - 9.1 kcal/mol, respectively. Furthermore, these compounds formed 2 and 1 hydrogen bonds with VanA, respectively, highlighting their potential as effective interactants. These findings provide valuable visions into the therapeutic potentials of these plant-derived biocompounds in combating antibiotic-resistant bacterial infections.

Efflux pumps and membrane permeability contribute to intrinsic antibiotic resistance in Mycobacterium abscessus.

Mycobacterium abscessus is a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized, M. abscessus-specific protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.