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503300.9850A novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes. Imipenem-resistant Escherichia coli strains represent a growing public health concern, posing a threat due to their resistance to last-resort antibiotics. Here, we present the discovery of the Imipenem-Linked Resistance Gene VIN (ILR-VIN) within E. coli isolates from Vietnam, revealing its absence in non-resistant E. coli and local bacterial species. ILR-VIN constitutes a previously unrecognized genetic element potentially linked to Imipenem resistance, with notable prevalence in Vietnamese E. coli strains.We conducted an in-depth examination of the genetic basis of Carbapenem resistance in E. coli strains causing urinary tract infections. In a set of 47 UTI strains, we identified five displaying Imipenem resistance, with four of them carrying known resistance genes. Interestingly, ECV219, despite exhibiting Imipenem resistance, lacked known resistance genes, suggesting an unreported resistance mechanism. Comparative genetic analysis revealed distinct genes in ECV219, indicating a novel Imipenem resistance gene. To assess its function, we conducted transformation experiments in E. coli Rosetta™(DE3)pLysS and performed bioinformatics analyses using BLASTp, InterProScan, and Pfam to characterize the gene's structure and potential functions.Our study identifies ILR-VIN as a novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes. Experimental evidence confirmed that ILR-VIN expression enhances bacterial survival under Imipenem stress, providing direct evidence of its role in resistance. This discovery highlights the importance of ongoing research into antibiotic resistance genes to develop effective treatment strategies against antibiotic-resistant bacterial infections.202540097570
303410.9842The Integrative and Conjugative Element ICECspPOL2 Contributes to the Outbreak of Multi-Antibiotic-Resistant Bacteria for Chryseobacterium Spp. and Elizabethkingia Spp. Antibiotic resistance genes (ARGs) and horizontal transfer of ARGs among bacterial species in the environment can have serious clinical implications as such transfers can lead to disease outbreaks from multidrug-resistant (MDR) bacteria. Infections due to antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the multi-antibiotic-resistant strain Chryseobacterium sp. POL2 was isolated from the wastewater of a livestock farm. Whole-genome sequencing and annotation revealed that the POL2 genome encodes dozens of ARGs. The integrative and conjugative element (ICE) ICECspPOL2, which encodes ARGs associated with four types of antibiotics, including carbapenem, was identified in the POL2 genome, and phylogenetic affiliation analysis suggested that ICECspPOL2 evolved from related ICEEas of Elizabethkingia spp. Conjugation assays verified that ICECspPOL2 can horizontally transfer to Elizabethkingia species, suggesting that ICECspPOL2 contributes to the dissemination of multiple ARGs among Chryseobacterium spp. and Elizabethkingia spp. Because Elizabethkingia spp. is associated with clinically significant infections and high mortality, there would be challenges to clinical treatment if these bacteria acquire ICECspPOL2 with its multiple ARGs, especially the carbapenem resistance gene. Therefore, the results of this study support the need for monitoring the dissemination of this type of ICE in Chryseobacterium and Elizabethkingia strains to prevent further outbreaks of MDR bacteria. IMPORTANCE Infections with multiple antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the mobile integrative and conjugative element ICECspPOL2, which was associated with the transmission of a carbapenem resistance gene, was identified in the genome of the multi-antibiotic-resistant strain Chryseobacterium sp. POL2. ICECspPOL2 is closely related to the ICEEas from Elizabethkingia species, and ICECspPOL2 can horizontally transfer to Elizabethkingia species with the tRNA-Glu-TTC gene as the insertion site. Because Elizabethkingia species are associated with clinically significant infections and high mortality, the ability of ICECspPOL2 to transfer carbapenem resistance from environmental strains of Chryseobacterium to Elizabethkingia is of clinical concern.202134937181
153320.9842A Transferable IncC-IncX3 Hybrid Plasmid Cocarrying bla(NDM-4), tet(X), and tmexCD3-toprJ3 Confers Resistance to Carbapenem and Tigecycline. Tigecycline is a last-resort antimicrobial against carbapenemase-producing Enterobacterales (CPE). However, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, have emerged in China and have spread possibly worldwide. Tet(X) family proteins function as tigecycline-inactivating enzymes, and TMexCD-TOprJ complexes function as efflux pumps for tigecycline. Here, to the best of our knowledge we report a CPE isolate harboring both emerging tigecycline resistance factors for the first time. A carbapenem- and tigecycline-resistant Klebsiella aerogenes strain, NUITM-VK5, was isolated from an urban drainage in Vietnam in 2021, and a plasmid, pNUITM-VK5_mdr, cocarrying tet(X) and tmexCD3-toprJ3 along with the carbapenemase gene bla(NDM-4) was identified in NUITM-VK5. pNUITM-VK5_mdr was transferred to Escherichia coli by conjugation and simultaneously conferred high-level resistance against multiple antimicrobials, including carbapenems and tigecycline. An efflux pump inhibitor reduced TMexCD3-TOprJ3-mediated tigecycline resistance, suggesting that both tigecycline resistance factors independently and additively contribute to the high-level resistance. The plasmid had the IncX3 and IncC replicons and was estimated to be a hybrid of plasmids with different backbones. Unlike IncX3 plasmids, IncC plasmids are stably maintained in an extremely broad range of bacterial hosts in humans, animals, and the environment. Thus, the future global spread of multidrug resistance plasmids such as pNUITM-VK5_mdr poses a public health crisis. IMPORTANCE Tigecycline is important as a last-resort antimicrobial and effective against antimicrobial-resistant bacteria, such as carbapenem-producing Enterobacterales (CPE), whose infections are difficult to treat with antimicrobials. Since 2019, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, and their variants have been reported mainly from China, and it has become important to understand their epidemiological situation and detailed genetic mechanisms. In this study, we identified a bacterial isolate coharboring tet(X) and tmexCD-toprJ on the same plasmid. A Klebsiella aerogenes isolate in Vietnam carried both these tigecycline resistance genes on a transferable plasmid leading to high-level resistance to multiple clinically important antimicrobials, including carbapenem and tigecycline, and could actually transfer the plasmid to other bacteria. The spread of such a multidrug resistance plasmid among bacterial pathogens should be of great concern because there are few antimicrobials to combat bacteria that have acquired the plasmid.202134346701
902830.9841Efflux Pumps in Chromobacterium Species Increase Antibiotic Resistance and Promote Survival in a Coculture Competition Model. Members of the Chromobacterium genus include opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics, including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis We also demonstrate that adding sublethal bactobolin concentrations to the coculture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show that in cells exposed to sublethal bactobolin concentrations, the cseAB-oprN genes are transcriptionally induced, and this corresponds to an increase in bactobolin resistance. Induction of this pump is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of antibiotic recognition. We examine the distribution of cseAB-oprN and cdeAB-oprM gene clusters in members of the Chromobacterium genus and find the cseAB-oprN genes are limited to the nonpathogenic C. subtsugae strains, whereas the cdeAB-oprM genes are more widely distributed among members of the Chromobacterium genus. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps for saprophytic bacteria existing in multispecies communities.IMPORTANCE Antibiotic efflux pumps are best known for increasing antibiotic resistance of pathogens; however, the role of these pumps in saprophytes is much less well defined. This study describes two predicted efflux pump gene clusters in the Chromobacterium genus, which is comprised of both nonpathogenic saprophytes and species that cause highly fatal human infections. One of the predicted efflux pump clusters is present in every member of the Chromobacterium genus and increases resistance to a broad range of antibiotics. The other gene cluster has more narrow antibiotic specificity and is found only in Chromobacterium subtsugae, a subset of entirely nonpathogenic species. We demonstrate the role of both pumps in increasing antibiotic resistance and demonstrate the importance of efflux-dependent resistance induction for C. subtsugae survival in a dual-species competition model. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside the host.201931324628
334740.9840Freshwater viral metagenome reveals novel and functional phage-borne antibiotic resistance genes. BACKGROUND: Antibiotic resistance developed by bacteria is a significant threat to global health. Antibiotic resistance genes (ARGs) spread across different bacterial populations through multiple dissemination routes, including horizontal gene transfer mediated by bacteriophages. ARGs carried by bacteriophages are considered especially threatening due to their prolonged persistence in the environment, fast replication rates, and ability to infect diverse bacterial hosts. Several studies employing qPCR and viral metagenomics have shown that viral fraction and viral sequence reads in clinical and environmental samples carry many ARGs. However, only a few ARGs have been found in viral contigs assembled from metagenome reads, with most of these genes lacking effective antibiotic resistance phenotypes. Owing to the wide application of viral metagenomics, nevertheless, different classes of ARGs are being continuously found in viral metagenomes acquired from diverse environments. As such, the presence and functionality of ARGs encoded by bacteriophages remain up for debate. RESULTS: We evaluated ARGs excavated from viral contigs recovered from urban surface water viral metagenome data. In virome reads and contigs, diverse ARGs, including polymyxin resistance genes, multidrug efflux proteins, and β-lactamases, were identified. In particular, when a lenient threshold of e value of ≤ 1 × e(-5) and query coverage of ≥ 60% were employed in the Resfams database, the novel β-lactamases bla(HRV-1) and bla(HRVM-1) were found. These genes had unique sequences, forming distinct clades of class A and subclass B3 β-lactamases, respectively. Minimum inhibitory concentration analyses for E. coli strains harboring bla(HRV-1) and bla(HRVM-1) and catalytic kinetics of purified HRV-1 and HRVM-1 showed reduced susceptibility to penicillin, narrow- and extended-spectrum cephalosporins, and carbapenems. These genes were also found in bacterial metagenomes, indicating that they were harbored by actively infecting phages. CONCLUSION: Our results showed that viruses in the environment carry as-yet-unreported functional ARGs, albeit in small quantities. We thereby suggest that environmental bacteriophages could be reservoirs of widely variable, unknown ARGs that could be disseminated via virus-host interactions. Video abstract.202032482165
509750.9839Comparing Graph Sample and Aggregation (SAGE) and Graph Attention Networks in the Prediction of Drug-Gene Associations of Extended-Spectrum Beta-Lactamases in Periodontal Infections and Resistance. INTRODUCTION: Gram-negative bacteria exhibit more antibiotic resistance than gram-positive bacteria due to their cell wall structure and composition differences. Porins, or protein channels in these bacteria, can allow small, hydrophilic antibiotics to diffuse, affecting their susceptibility. Mutations in porin protein genes can also impair antibiotic entry. Predicting drug-gene associations of extended-spectrum beta-lactamases (ESBLs) is crucial as they confer resistance to beta-lactam antibiotics, challenging the treatment of infections. This aids clinicians in selecting suitable treatments, optimizing drug usage, enhancing patient outcomes, and controlling antibiotic resistance in healthcare settings. Graph-based neural networks can predict drug-gene associations in periodontal infections and resistance. The aim of the study was to predict drug-gene associations of ESBLs in periodontal infections and resistance. METHODS: The study focuses on analyzing drug-gene associations using probes and drugs. The data was converted into graph language, assigning nodes and edges for drugs and genes. Graph neural networks (GNNs) and similar algorithms were implemented using Google Colab and Python. Cytoscape and CytoHubba are open-source software platforms used for network analysis and visualization. GNNs were used for tasks like node classification, link prediction, and graph-level prediction. Three graph-based models were used: graph convolutional network (GCN), Graph SAGE, and graph attention network (GAT). Each model was trained for 200 epochs using the Adam optimizer with a learning rate of 0.01 and a weight decay of 5e-4. RESULTS: The drug-gene association network has 57 nodes, 79 edges, and a 2.730 characteristic path length. Its structure, organization, and connectivity are analyzed using the GCN and Graph SAGE, which show high accuracy, precision, recall, and an F1-score of 0.94. GAT's performance metrics are lower, with an accuracy of 0.68, precision of 0.47, recall of 0.68, and F1-score of 0.56, suggesting that it may not be as effective in capturing drug-gene relationships. CONCLUSION: Compared to ESBLs, both GCN and Graph SAGE demonstrate excellent performance with accuracy, precision, recall, and an F1-score of 0.94. These results indicate that GCN and Graph SAGE are highly effective in predicting drug-gene associations related to ESBLs. GCN and Graph SAGE outperform GAT in predicting drug-gene associations for ESBLs. Improvements include data augmentation, regularization, and cross-validation. Ethical considerations, fairness, and open-source implementations are crucial for future research in precision periodontal treatment.202439347119
512660.9838Blanket antimicrobial resistance gene database with structural information, BOARDS, provides insights on historical landscape of resistance prevalence and effects of mutations in enzyme structure. Antimicrobial resistance (AMR) in pathogenic bacteria poses a significant threat to public health, yet there is still a need for development in the tools to deeply understand AMR genes based on genetic or structural information. In this study, we present an interactive web database named Blanket Overarching Antimicrobial-Resistance gene Database with Structural information (BOARDS, sbml.unist.ac.kr), a database that comprehensively includes 3,943 reported AMR gene information for 1,997 extended spectrum beta-lactamase (ESBL) and 1,946 other genes as well as a total of 27,395 predicted protein structures. These structures, which include both wild-type AMR genes and their mutants, were derived from 80,094 publicly available whole-genome sequences. In addition, we developed the rapid analysis and detection tool of antimicrobial-resistance (RADAR), a one-stop analysis pipeline to detect AMR genes across whole-genome sequencing (WGSs). By integrating BOARDS and RADAR, the AMR prevalence landscape for eight multi-drug resistant pathogens was reconstructed, leading to unexpected findings such as the pre-existence of the MCR genes before their official reports. Enzymatic structure prediction-based analysis revealed that the occurrence of mutations found in some ESBL genes was found to be closely related to the binding affinities with their antibiotic substrates. Overall, BOARDS can play a significant role in performing in-depth analysis on AMR.IMPORTANCEWhile the increasing antibiotic resistance (AMR) in pathogen has been a burden on public health, effective tools for deep understanding of AMR based on genetic or structural information remain limited. In this study, a blanket overarching antimicrobial-resistance gene database with structure information (BOARDS)-a web-based database that comprehensively collected AMR gene data with predictive protein structural information was constructed. Additionally, we report the development of a RADAR pipeline that can analyze whole-genome sequences as well. BOARDS, which includes sequence and structural information, has shown the historical landscape and prevalence of the AMR genes and can provide insight into single-nucleotide polymorphism effects on antibiotic degrading enzymes within protein structures.202438085058
975870.9837Study on collateral sensitivity of tigecycline to colistin-resistant Enterobacter cloacae complex. The past decade has witnessed the emergence and spread of carbapenem-resistant Enterobacter cloacae complex (CRECC), presenting a significant clinical challenge and urgently demanding new treatment strategies against antimicrobial resistance (AMR). This study focused on the impact of tigecycline on the susceptibility of CRECC isolates to colistin and the collateral sensitivity in CRECC. Under tigecycline pressure, the resistance of five clinically isolated CRECC strains to colistin was converted from resistance to sensitivity. These mutants exhibited significantly higher expression of acrA, acrB, and ramA genes, with mutations in the ramR gene. Overexpression of ramA in certain mutants did not alter ramR expression. No mutations were identified in lipid A synthesis genes; however, phoQ was consistently downregulated, and arnA expression varied among CRECC405-resistant mutants. Low-dose colistin and tigecycline combination therapy outperformed monotherapy in antimicrobial efficacy. Overall, collateral susceptibility to tigecycline was observed in CRECC isolates with colistin resistance. The overexpression of acrA, acrB, and ramA, due to ramR mutations, led to tigecycline resistance. Inconsistent expression levels of lipid A synthesis genes affected lipid A modification, which in turn upregulated arnA expression in CRECC405-resistant mutants. Increased sensitivity to colistin was associated with the downregulation of phoQ and arnA expression. IMPORTANCE: Antimicrobial resistance (AMR) is escalating faster than our ability to manage bacterial infections, with antibiotic-resistant bacteria emerging as a significant public health risk. Innovative strategies are urgently needed to curb AMR dissemination. Our research identified collateral sensitivity in Enterobacter cloacae complex following tigecycline (TGC) resistance, resulting in newfound sensitivity to colistin (COL), a drug to which it was once resistant. Synergistic tigecycline and colistin therapy significantly suppress bacterial growth, offering a promising approach to combat infections and curb AMR progression through the strategic pairing of antibiotics with complementary sensitivities.202540407373
334680.9835Genes encoding antibiotic modifying enzymes conferring resistance against aminoglycosides in bacteria: Their identification and detection from wastewater. Global reporting of antibiotic resistant bacteria (ARB) bearing antibiotic resistance genes (ARGs) have increased in the past decade. Sewage systems act as breeding grounds for these pathogens. Dumping of untreated sewage effluent in river water systems have aided in their dissemination and spread. The molecular pathways circumventing antibiotics through ARGs is rising owing to overuse of these drugs. Use of aminoglycoside spectrum drugs has been increased exponentially. The genes providing resistance to these antibiotics are transferred through extra-chromosomal circular DNA elements. Polluted water bodies are ground zero for exchange of these genetic factors. Through literature survey, we shortlisted some clinically relevant genes which provide resistance against aminoglycosides and hold immense importance in present scenario. Initial screening for these genes was done on water samples collected from Swarna Rekha River channel in Gwalior District of Madhya Pradesh, India. A total of five identified genes were sequence verified using conventional PCR followed by targeted sequencing. Further, diagnostic platforms were designed for two reoccurring genes npmA & sat4(A) and their presence evaluated from wastewater samples collected from urban establishments of the district. Prevalence of these genes in sewage samples validated the broad impact of urban waste burden in polluting local water bodies. We were able to identify some indispensable and high risk aminoglycoside resistance providing genes, unreported in Indian context. This approach towards ARG screening could support risk assessment of future antibiotic resistance associated public health hazards.202539708933
506990.9835MC-PRPA-HLFIA Cascade Detection System for Point-of-Care Testing Pan-Drug-Resistant Genes in Urinary Tract Infection Samples. Recently, urinary tract infection (UTI) triggered by bacteria carrying pan-drug-resistant genes, including carbapenem resistance gene bla(NDM) and bla(KPC), colistin resistance gene mcr-1, and tet(X) for tigecycline resistance, have been reported, posing a serious challenge to the treatment of clinical UTI. Therefore, point-of-care (POC) detection of these genes in UTI samples without the need for pre-culturing is urgently needed. Based on PEG 200-enhanced recombinase polymerase amplification (RPA) and a refined Chelex-100 lysis method with HRP-catalyzed lateral flow immunoassay (LFIA), we developed an MCL-PRPA-HLFIA cascade assay system for detecting these genes in UTI samples. The refined Chelex-100 lysis method extracts target DNA from UTI samples in 20 min without high-speed centrifugation or pre-incubation of urine samples. Following optimization, the cascade detection system achieved an LOD of 10(2) CFU/mL with satisfactory specificity and could detect these genes in both simulated and actual UTI samples. It takes less than an hour to complete the process without the use of high-speed centrifuges or other specialized equipment, such as PCR amplifiers. The MCL-PRPA-HLFIA cascade assay system provides new ideas for the construction of rapid detection methods for pan-drug-resistant genes in clinical UTI samples and provides the necessary medication guidance for UTI treatment.202337047757
3842100.9834Mutation-driven resistance development in wastewater E. coli upon low-level cephalosporins: Pharmacophore contribution and novel mechanism. Cephalosporins have been widely applied in clinical and veterinary settings and detected at increasing concentrations in water environments. They potentially induce high-level antibiotic resistance at environmental concentrations. This study characterized how typical wastewater bacteria developed heritable antibiotic resistance under exposure to different cephalosporins, including pharmacophore-resistance correlation, resistance mechanism, and occurrence of resistance-relevant mutations in different water environments. Wastewater-isolated E. coli JX1 was exposed to eight cephalosporins individually at 25 µg/L for 60 days. Multidrug resistance developed and diverse mutations arose in selected mutants, where a single mutation in ATP phosphoribosyltransferase encoding gene (hisG) resulted in up to 128-fold increase in resistance to meropenem. Molprint2D pharma RQSAR analysis revealed that hydrogen-bond acceptors and hydrophobic groups in the R1 and R2 substituents of cephalosporins contributed positively to antibiotic resistance. Some of these pharmacophores may persist during bio- or photo-degradation in the environment. hisG mutation confers a novel resistance mechanism by inhibiting fatty acid degradation, and its variants were more abundant in water-related E. coli (especially in the effluent of wastewater treatment plants) compared with those in non-water environments. These results suggest that specific degradation of particular pharmacophores in cephalosporins could be useful for controlling resistance development, and mutations in previously unreported resistance genes (e.g., hisG) can lead to overlooked antibiotic resistance risks in water environments.202438310801
9878110.9834Two novel trimethoprim resistance genes, dfra50 and dfra51, identified in phage-plasmids. Phage-plasmids carry a significant burden of clinically relevant antibiotic resistance genes (ARGs). Intriguingly, the majority of these ARGs are found within plasmids with phage features, with a single exception residing in a phage genome with plasmid features. Therefore, we speculate that phage genomes with plasmid features, whose sequences are highly homologous to bacterial plasmids, may carry novel ARGs. We subsequently identified 46 such phage genomes by employing Hidden Markov models (HMMs) based on plasmid-specific protein profiles andbasic local alignment search tool (BLASTn) searches against the National Center for Biotechnology Information (NCBI) RefSeq Plasmid Database. Among them, six phages harbored seven ARGs identified through a lenient-threshold search strategy, of which only two had been previously reported. The remaining five ARGs were categorized as novel ARGs since their encoded proteins differed from known ARGs. Notably, half of the phages carried trimethoprim-resistant dfrA-like genes. Functional studies characterized these genes and demonstrated that the expression of two of these dfrA genes (dfrA50 and dfrA51) can confer resistance to trimethoprim in Escherichia coli. Through genome analysis, we found that these phages with plasmid features likely contributed to the natural dissemination of these dfrA genes, as evidenced by their widespread presence in plasmids across various pathogenic bacteria. These findings underscore the importance of identifying and monitoring ARGs encoded by phage genomes with plasmid features that also function as plasmids in bacteria, aiming to proactively address the antibiotic resistance challenges posed by these phage-mediated dissemination events.202540503927
3033120.9834Glabridin inhibited the spread of polymyxin-resistant Enterobacterium carrying ICEMmoMP63. INTRODUCTION: The role of integrative and conjugative elements (ICEs) in antibiotic resistance in Morganella morganii is unknown. This study aimed to determine whether an ICE identified in the M. morganii genome contributed to the polymyxin resistance. METHODS: Whole-genome sequencing was performed followed by bioinformatics analyses to identify ICEs and antibiotic resistance genes. Conjugation assays were performed to analyze the transferability of a discovered ICE. A drug transporter encoded on the ICE was heterogeneously expressed in Escherichia coli, minimum inhibitory concentrations of antibiotics were determined, and a traditional Chinese medicine library was screened for potential efflux pump inhibitors. RESULTS: An antibiotic resistance-conferring ICE, named ICEMmoMP63, was identified. ICEMmoMP63 was verified to be horizontally transferred among Enterobacteriaceae bacteria. G3577_03020 in ICEMmoMP63 was found to mediate multiple antibiotic resistances, especially polymyxin resistance. However, natural compound glabridin was demonstrated to inhibit polymyxin resistance. DISCUSSION: Our findings support the need for monitoring dissemination of ICEMmoMP63 in Enterobacteriaceae bacteria. Combined glabridin and polymyxin may have therapeutic potential for treating infections from multi-drug resistant bacteria carrying ICEMmoMP63.202337283918
7316130.9834Nanopore-based long-read metagenomics uncover the resistome intrusion by antibiotic resistant bacteria from treated wastewater in receiving water body. Wastewater treatment plant (WWTP) effluent discharge could induce the resistome enrichment in the receiving water environments. However, because of the general lack of a robust antibiotic-resistant bacteria (ARB) identification method, the driving mechanism for resistome accumulation in receiving environment is unclear. Here, we took advantage of the enhanced ARBs recognition by nanopore long reads to distinguish the indigenous ARBs and the accumulation of WWTP-borne ARBs in the receiving water body of a domestic WWTP. A bioinformatic framework (named ARGpore2: https://github.com/sustc-xylab/ARGpore2) was constructed and evaluate to facilitate antibiotic resistance genes (ARGs) and ARBs identification in nanopore reads. ARGs identification by ARGpore2 showed comparable precision and recall to that of the commonly adopt BLASTP-based method, whereas the spectrum of ARBs doubled that of the assembled Illumina dataset. Totally, we identified 33 ARBs genera carrying 65 ARG subtypes in the receiving seawater, whose concentration was in general 10 times higher than clean seawater's. Notably we report a primary resistome intrusion caused by the revival of residual microbes survived from disinfection treatment. These WWTP-borne ARBs, including several animal/human enteric pathogens, contributed up to 85% of the receiving water resistome. Plasmids and class 1 integrons were reckoned as major vehicles facilitating the persistence and dissemination of ARGs. Moreover, our work demonstrated the importance of extensive carrier identification in determining the driving force of multifactor coupled resistome booming in complicated environmental conditions, thereby paving the way for establishing priority for effective ARGs mitigation strategies.202236332295
5170140.9834Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori. BACKGROUND: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism. RESULTS: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs. CONCLUSION: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.200919594901
4697150.9834Biocide-Induced Emergence of Antibiotic Resistance in Escherichia coli. Biocide use is essential and ubiquitous, exposing microbes to sub-inhibitory concentrations of antiseptics, disinfectants, and preservatives. This can lead to the emergence of biocide resistance, and more importantly, potential cross-resistance to antibiotics, although the degree, frequency, and mechanisms that give rise to this phenomenon are still unclear. Here, we systematically performed adaptive laboratory evolution of the gut bacteria Escherichia coli in the presence of sub-inhibitory, constant concentrations of ten widespread biocides. Our results show that 17 out of 40 evolved strains (43%) also decreased the susceptibility to medically relevant antibiotics. Through whole-genome sequencing, we identified mutations related to multidrug efflux proteins (mdfA and acrR), porins (envZ and ompR), and RNA polymerase (rpoA and rpoBC), as mechanisms behind the resulting (cross)resistance. We also report an association of several genes (yeaW, pyrE, yqhC, aes, pgpA, and yeeP-isrC) and specific mutations that induce cross-resistance, verified through mutation repairs. A greater capacity for biofilm formation with respect to the parent strain was also a common feature in 11 out of 17 (65%) cross-resistant strains. Evolution in the biocides chlorophene, benzalkonium chloride, glutaraldehyde, and chlorhexidine had the most impact in antibiotic susceptibility, while hydrogen peroxide and povidone-iodine the least. No cross-resistance to antibiotics was observed for isopropanol, ethanol, sodium hypochlorite, and peracetic acid. This work reinforces the link between exposure to biocides and the potential for cross-resistance to antibiotics, presents evidence on the underlying mechanisms of action, and provides a prioritized list of biocides that are of greater concern for public safety from the perspective of antibiotic resistance. SIGNIFICANCE STATEMENT: Bacterial resistance and decreased susceptibility to antimicrobials is of utmost concern. There is evidence that improper biocide (antiseptic and disinfectant) use and discard may select for bacteria cross-resistant to antibiotics. Understanding the cross-resistance emergence and the risks associated with each of those chemicals is relevant for proper applications and recommendations. Our work establishes that not all biocides are equal when it comes to their risk of inducing antibiotic resistance; it provides evidence on the mechanisms of cross-resistance and a risk assessment of the biocides concerning antibiotic resistance under residual sub-inhibitory concentrations.202133717036
5119160.9833ROCker models for reliable detection and typing of short-read sequences carrying mcr, erm, mph, and lnu antibiotic resistance genes. Quantitative monitoring of emerging antimicrobial resistance genes (ARGs) using short-read sequences remains challenging due to the high frequency of amino acid functional domains and motifs shared with related but functionally distinct (non-target) proteins. To facilitate ARG monitoring efforts using unassembled short reads, we present novel ROCker models for mcr, mph, erm, and lnu ARG families, as well as models for variants of special public health concern within these families, including mcr-1, mphA, ermB, lnuF, lnuB, and lnuG genes. For this, we curated target gene sequence sets for model training and built these models using the recently updated ROCker V2 pipeline (Gerhardt et al., in review). To validate our models, we simulated reads from the whole genome of ARG-carrying isolates spanning a range of common read lengths and used them to challenge the filtering efficacy of ROCker versus common static filtering approaches, such as similarity searches using BLASTx with various e-value thresholds or hidden Markov models. ROCker models consistently showed F1 scores up to 10× higher (31% higher on average) and lower false-positive (by 30%, on average) and false-negative (by 16%, on average) rates based on 250 bp reads compared to alternative methods. The ROCker models and all related reference materials and data are freely available through http://enve-omics.ce.gatech.edu/rocker/models, further expanding the available model collection previously developed for other genes. Their application to short-read metagenomes, metatranscriptomes, and PCR amplicon data should facilitate more accurate classification and quantification of unassembled short-read sequences for these ARG families and specific genes.IMPORTANCEAntimicrobial resistance gene families encoding erm and mph genes confer resistance to the macrolide class of antimicrobials, which are used to treat a wide range of infections. Similarly, the mcr gene family confers resistance to polymyxin E (colistin), a drug of last resort for many serious drug-resistant bacterial infections, and the lnu gene family confers resistance to lincomycin, which is reserved for patients allergic to penicillin or where bacteria have developed resistance to other antimicrobials. Assessing the prevalence of these genes in clinical or environmental samples and monitoring their spread to new pathogens are thus important for quantifying the associated public health risk. However, detecting these and other resistance genes in short-read sequence data is technically challenging. Our ROCker bioinformatic pipeline achieves reliable detection and typing of broad-range target gene sequences in complex data sets, thus contributing toward solving an important problem in ongoing surveillance efforts of antimicrobial resistance.202541143534
5096170.9833A comprehensive computer-based assessment of Deacetylnomilin as an inhibitor for antibiotic-resistant genes identified from the whole genome sequence of the multidrug-resistant Enterobacter cloacae isolate 1382. The twenty-first century presents a serious threat to public health due to the growth in antibiotic resistance among opportunistic bacteria, particularly within the ESKAPE group, which includes Enterobacter species with high morbidity, mortality, virulence, and nosocomial dissemination rates. Enterobacter species, especially Enterobacter cloacae, bacteria have developed resistance to multiple antibiotics through mechanisms, such as continuous production of AmpC beta-lactamase. In this study, a comprehensive bioinformatics approach was employed to analyze the genome of Enterobacter cloacae, utilizing sequence data from GenBank (ID: OW968328.1). The AbritAMR and ResFinder tools were utilized to identify antibiotic-resistant genes, which included the presence of blaOXA-48, blaCMH, FosA, OqxA, and OqxB each conferring resistance to specific antibiotics such as β-lactams and fluoroquinolones. These proteins were analyzed using bioinformatics tools such as ProtParam, SOPMA, Robetta, I-TASSER, AlphaFold, and PROCHECK to investigate different structural models and their properties. The models from AlphaFold had the best quality in terms of structural accuracy, providing valuable insights into the 3D conformations of these resistant proteins. Based on the Molecular docking studies, these constructed targets were docked with 20 natural compounds known for their activity against Gram-negative bacteria. Among them, Deacetylnomilin showed the highest docking score and passed their ADMET properties. Molecular dynamic (MD) simulation was conducted for 100 ns for Deacetylnomilin with different resistant proteins. Deacetylnomilin exhibited more favorable binding free energies compared to the reference compounds across all five proteins, indicating higher stability and affinity. These results suggest that Deacetylnomilin could be an effective inhibitor against the resistant proteins of Enterobacter cloacae, making it a promising candidate for further drug development.202539702793
5183180.9833Development of phage resistance in multidrug-resistant Klebsiella pneumoniae is associated with reduced virulence: a case report of a personalised phage therapy. OBJECTIVES: Phage-resistant bacteria often emerge rapidly when performing phage therapy. However, the relationship between the emergence of phage-resistant bacteria and improvements in clinical symptoms is still poorly understood. METHODS: An inpatient developed a pulmonary infection caused by multidrug-resistant Klebsiella pneumoniae. He received a first course of treatment with a single nebulized phage (ΦKp_GWPB35) targeted at his bacterial isolate of Kp7450. After 14 days, he received a second course of treatment with a phage cocktail (ΦKp_GWPB35+ΦKp_GWPA139). Antibiotic treatment was continued throughout the course of phage therapy. Whole-genome analysis was used to identify mutations in phage-resistant strains. Mutated genes associated with resistance were further analysed by generating knockouts of Kp7450 and by measuring phage adsorption rates of bacteria treated with proteinase K and periodate. Bacterial virulence was evaluated in mouse and zebrafish infection models. RESULTS: Phage-resistant Klebsiella pneumoniae strains emerged after the second phage treatment. Comparative genomic analyses revealed that fabF was deleted in phage-resistant strains. The fabF knockout strain (Kp7450ΔfabF) resulted in an altered structure of lipopolysaccharide (LPS), which was identified as the host receptor for the therapeutic phages. Virulence evaluations in mice and zebrafish models showed that LPS was the main determinant of virulence in Kp7450 and alteration of LPS structure in Kp7450ΔfabF, and the bacteriophage-resistant strains reduced their virulence at cost. DISCUSSION: This study may shed light on the mechanism by which some patients experience clinical improvement in their symptoms post phage therapy, despite the incomplete elimination of pathogenic bacteria.202337652124
2494190.9833Dissemination of virulence and resistance genes among Klebsiella pneumoniae via outer membrane vesicle: An important plasmid transfer mechanism to promote the emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae. Klebsiella pneumoniae is well-known opportunistic enterobacteria involved in complex clinical infections in humans and animals. The domestic animals might be a source of the multidrug-resistant virulent K. pneumoniae to humans. K. pneumoniae infections in domestic animals are considered as an emergent global concern. The horizontal gene transfer plays essential roles in bacterial genome evolution by spread of virulence and resistance determinants. However, the virulence genes can be transferred horizontally via K. pneumoniae-derived outer membrane vesicles (OMVs) remains to be unreported. In this study, we performed complete genome sequencing of two K. pneumoniae HvK2115 and CRK3022 with hypervirulent or carbapenem-resistant traits. OMVs from K. pneumoniae HvK2115 and CRK3022 were purified and observed. The carriage of virulence or resistance genes in K. pneumoniae OMVs was identified. The influence of OMVs on the horizontal transfer of virulence-related or drug-resistant plasmids among K. pneumoniae strains was evaluated thoroughly. The plasmid transfer to recipient bacteria through OMVs was identified by polymerase chain reaction, pulsed field gel electrophoresis and Southern blot. This study revealed that OMVs could mediate the intraspecific and interspecific horizontal transfer of the virulence plasmid phvK2115. OMVs could simultaneously transfer two resistance plasmids into K. pneumoniae and Escherichia coli recipient strains. OMVs-mediated horizontal transfer of virulence plasmid phvK2115 could significantly enhance the pathogenicity of human carbapenem-resistant K. pneumoniae CRK3022. The CRK3022 acquired the virulence plasmid phvK2115 could become a CR-hvKp strain. It was critically important that OMVs-mediated horizontal transfer of phvK2115 lead to the coexistence of virulence and carbapenem-resistance genes in K. pneumoniae, resulting in the emerging of carbapenem-resistant hypervirulent K. pneumoniae.202235679514