Integrated Sample to Detection of Carbapenem-Resistant Bacteria Extracted from Water Samples Using a Portable Gold Nanoparticle-Based Biosensor. - Related Documents




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507201.0000Integrated Sample to Detection of Carbapenem-Resistant Bacteria Extracted from Water Samples Using a Portable Gold Nanoparticle-Based Biosensor. Antimicrobial resistance (AMR) is a significant global threat and is driven by the overuse of antibiotics in both clinical and agricultural settings. This issue is further complicated by the lack of rapid surveillance tools to detect resistant bacteria in clinical, environmental, and food systems. Of particular concern is the rise in resistance to carbapenems, a critical class of beta-lactam antibiotics. Rapid detection methods are necessary for prevention and surveillance effort. This study utilized a gold nanoparticle-based plasmonic biosensor to detect three CR genes: bla(KPC-3), bla(NDM-1), and bla(OXA-1). Optical signals were analyzed using both a spectrophotometer and a smartphone app that quantified visual color changes using RGB values. This app, combined with a simple boiling method for DNA extraction and a portable thermal cycler, was used to evaluate the biosensor's potential for POC use. Advantages of the portable bacterial detection device include real time monitoring for immediate decision-making in critical situations, field and on-site testing in resource-limited settings without needing to transport samples to a centralized lab, minimal training required, automatic data analysis, storage and sharing, and reduced operational cost. Bacteria were inoculated into sterile water, river water, and turkey rinse water samples to determine the biosensor's success in detecting target genes from sample matrices. Magnetic nanoparticles were used to capture and concentrate bacteria to avoid time-consuming cultivation and separation steps. The biosensor successfully detected the target CR genes in all tested samples using three gene-specific DNA probes. Target genes were detected with a limit of detection of 2.5 ng/L or less, corresponding to ~10(3) CFU/mL of bacteria.202540942723
507310.9999Parallel Detection of the Unamplified Carbapenem Resistance Genes bla(NDM-1) and bla(OXA-1) Using a Plasmonic Nano-Biosensor with a Field-Portable DNA Extraction Method. Antimicrobial resistance (AMR) is a rapidly growing global concern resulting from the overuse of antibiotics in agricultural and clinical settings. The challenge is exacerbated by the lack of rapid surveillance for resistant bacteria in clinical, environmental, and food supply settings. The increasing resistance to carbapenems, an important sub-class of beta-lactam antibiotics, is a major concern in the healthcare community. Carbapenem resistance (CR) has been found in the environment and food supply chain, where it has the potential to spread to pathogens, animals, and humans through direct or indirect contact. Rapid detection for preventative and control measures should be developed. This study utilized a gold nanoparticle-based plasmonic biosensor for the parallel detection of the CR genes bla(NDM-1) and bla(OXA-1). To explore the field portability, DNA was extracted using two methods: a commercial extraction kit and a boiling method. The results were compared between the two methods using a spectrophotometer and a cellphone application for RGB values to quantify the visual results. The results showed that the boiling method of extraction was more effective than extraction with a commercial kit for this analysis. The parallel detection of unamplified genes extracted via the boiling method is novel. When combined with other portable testing equipment, the approach has the potential to be an inexpensive, rapid, and simple on-site CR gene detection protocol.202539997014
328020.9997Optimization of five qPCR protocols toward the detection and the quantification of antimicrobial resistance genes in environmental samples. Here, we describe the optimization and validation of five quantitative PCR (qPCR) assays by employing the SYBRGreen chemistry paired with melting curve analysis to detect and quantify clinically relevant antimicrobial resistance genes (ARGs) (i.e. ermB, bla(CTXM1-like), bla(CMY-2), qnrA and qnrS) from environmental samples (i.e. soil and manure). These five protocols accurately detected and quantified the aforementioned ARGs in complex environmental matrices and represent useful tools for both diagnostic and monitoring activities of resistant bacteria and ARGs into the environment.202134754761
568930.9997A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales. Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-β-lactamase (NDM) are particularly concerning due to their resistance to most β-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (bla(NDM)) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry bla(NDM) and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of bla(NDM)-(1) carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 10(0) CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.202438667187
345740.9997Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR. The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, bla(TEM), bla(SHV), and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs' biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R(2) > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments.202438748252
507150.9995Versatile and Portable Cas12a-mediated Detection of Antibiotic Resistance Markers. Antibiotic-resistant bacteria are spreading in clinical, industrial, and environmental ecosystems. The spreading dynamics to and from the environment are unknown, largely due to the lack of appropriate (robust, fast, low-cost) analytical assays. In this study, we developed C12a, a versatile molecular toolbox to detect genetic markers of antibiotic resistance using CRISPR/Cas12a. Biochemical characterization show that the C12a toolbox can detect less than 100 attoMolar of pure DNA fragments from the blaCTX-M15 and floR genes, conferring resistance to b-lactams and amphenicols, respectively important for human and veterinary uses. In microbiological assays, C12a detected less than 10(2) CFU/mL and high concordance was observed if compared to antibiotic susceptibility tests, PCR, or to whole genome sequencing. Additionally, C12a confirmed a high prevalence of the integrase/integron system in E. coli isolates containing multiple antibiotic resistance genes (ARGs). The C12a toolbox shows equivalent detection performance in diverse laboratory settings, results redout (Fluorescence vs FLA) or input sample. Altogether, this work presents a comprehensive proof-of-concept, development description, and biochemical characterization of a collection of molecular tools to detect antibiotic resistance markers in a one health setup.202539605319
345560.9995Quantifying nonspecific TEM beta-lactamase (blaTEM) genes in a wastewater stream. To control the antibiotic resistance epidemic, it is necessary to understand the distribution of genetic material encoding antibiotic resistance in the environment and how anthropogenic inputs, such as wastewater, affect this distribution. Approximately two-thirds of antibiotics administered to humans are beta-lactams, for which the predominant bacterial resistance mechanism is hydrolysis by beta-lactamases. Of the beta-lactamases, the TEM family is of overriding significance with regard to diversity, prevalence, and distribution. This paper describes the design of DNA probes universal for all known TEM beta-lactamase genes and the application of a quantitative PCR assay (also known as Taqman) to quantify these genes in environmental samples. The primer set was used to study whether sewage, both treated and untreated, contributes to the spread of these genes in receiving waters. It was found that while modern sewage treatment technologies reduce the concentrations of these antibiotic resistance genes, the ratio of bla(TEM) genes to 16S rRNA genes increases with treatment, suggesting that bacteria harboring bla(TEM) are more likely to survive the treatment process. Thus, beta-lactamase genes are being introduced into the environment in significantly higher concentrations than occur naturally, creating reservoirs of increased resistance potential.200918997031
327870.9995Prevalence of Antibiotic-Resistant Bacteria and Antibiotic-Resistant Genes and the Quantification of Antibiotics in Drinking Water Treatment Plants of Malaysia: Protocol for a Cross-sectional Study. BACKGROUND: Antimicrobial resistance is a known global public health threat. In addition, it brings serious economic consequences to agriculture. Antibiotic resistance in humans, animals, and environment is interconnected, as proposed in the tricycle surveillance by the World Health Organization. In Malaysia, research and surveillance of antimicrobial resistance are mainly performed in clinical samples, agricultural settings, and surface waters, but no surveillance of the drinking water systems has been performed yet. Hence, this policy-driven study is a combined effort of microbiologists and engineers to provide baseline data on the magnitude of antimicrobial resistance in the drinking water systems of Malaysia. OBJECTIVE: The aim of this study was to study the baseline level of antibiotic-resistant bacteria in the drinking water distribution systems of Malaysia by collecting samples from the pretreatment and posttreatment outlets of water treatment plants in a selected state of Malaysia. We aimed to determine the prevalence of antibiotic-resistant bacteria, the occurrence of antibiotic-resistant genes, and the level of antibiotics present in the drinking water systems. METHODS: This is a laboratory-based, cross-sectional study in a selected state of Malaysia. Water samples from 6 drinking water treatment plants were collected. Samples were collected at 3 sampling points, that is, the intake sampling station, service reservoir outlet station, and the distribution system sampling station. These were tested against 7 types of antibiotics in triplicates. Samples were screened for antibiotic-resistant bacteria and antibiotic-resistant genes and quantified for the level of antibiotics present in the drinking water treatment plants. RESULTS: We will show the descriptive statistics of the number of bacterial colonies harvested from water samples grown on Reasoner's 2A agar with or without antibiotics, the occurrence of antibiotic-resistant genes, and the level of antibiotics detected in the water samples. The sampling frame was scheduled to start from November 2021 and continue until December 2022. Data analysis is expected to be completed by early 2023, and the results are expected to be published in mid-2023. CONCLUSIONS: This study provides baseline information on the status of the antimicrobial-resistant bacteria, the presence of resistance genes as contaminants, and the level of antibiotics present in the drinking water systems of Malaysia, with the aim of demonstrating to policymakers the need to consider antimicrobial resistance as a parameter in drinking water surveillance. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/37663.202236409546
498080.9995Co-selection of antibiotic and disinfectant resistance in environmental bacteria: Health implications and mitigation strategies. BACKGROUND: The rapid emergence of co-selection between antimicrobials, including antibiotics and disinfectants, presents a significant challenge to healthcare systems. This phenomenon exacerbates contamination risks and limits the effectiveness of strategies to combat antibiotic resistance in clinical settings. This study aimed to investigate the prevalence and characteristics of bacteria in hospital environments that exhibit co-selection mechanisms and their potential implications for patient health, framed within the One Health perspective. METHODS: Air and surface samples were collected from seven large hospitals and analyzed to detect antibiotic-resistant bacteria (ARB). The resistance profiles of isolated ARB to various disinfectants were determined. Bacterial species were identified using 16S rRNA gene sequencing, and the presence of antibiotic resistance genes (ARGs) and class 1 integrons (intI1) was investigated. RESULTS: A high percentage (85%) of samples contained ARB, with β-lactam resistance being the most frequently observed. Alarmingly, 94% of isolated ARB exhibited resistance to at least one disinfectant, and 91% demonstrated resistance to three or more disinfectants. Staphylococcus and Bacillus emerged as the dominant genera displaying co-selection. The presence of ARGs, including mecA (associated with methicillin resistance) and qacB (associated with disinfectant resistance), along with intI1, provided further evidence supporting co-selection mechanisms. CONCLUSION: These findings underscore the critical need for robust antimicrobial resistance surveillance and the prudent use of disinfectants in healthcare settings. Further research into co-selection mechanisms is essential to inform the development of effective infection control strategies and minimize the spread of resistant bacteria.202539732420
465390.9995Modelling the effectiveness of surveillance based on metagenomics in detecting, monitoring, and forecasting antimicrobial resistance in livestock production under economic constraints. Current surveillance of antimicrobial resistance (AMR) is mostly based on testing indicator bacteria using minimum inhibitory concentration (MIC) panels. Metagenomics has the potential to identify all known antimicrobial resistant genes (ARGs) in complex samples and thereby detect changes in the occurrence earlier. Here, we simulate the results of an AMR surveillance program based on metagenomics in the Danish pig population. We modelled both an increase in the occurrence of ARGs and an introduction of a new ARG in a few farms and the subsequent spread to the entire population. To make the simulation realistic, the total cost of the surveillance was constrained, and the sampling schedule was set at one pool per month with 5, 20, 50, or 100 samples. Our simulations demonstrate that a pool of 20-50 samples and a sequencing depth of 250 million fragments resulted in the shortest time to detection in both scenarios, with a time delay to detection of change of [Formula: see text]15 months in all scenarios. Compared with culture-based surveillance, our simulation indicates that there are neither significant reductions nor increases in time to detect a change using metagenomics. The benefit of metagenomics is that it is possible to monitor all known resistance in one sampling and laboratory procedure in contrast to the current monitoring that is based on the phenotypic characterisation of selected indicator bacterial species. Therefore, overall changes in AMR in a population will be detected earlier using metagenomics due to the fact that the resistance gene does not have to be transferred to and expressed by an indicator bacteria before it is possible to detect.202337990114
5663100.9995Development of multiplex Luminex assays for the surveillance of antimicrobial resistance genes in nasal samples. Bovine respiratory disease (BRD) is the major cause of morbidity and mortality in feedlot cattle. It is the major driver for the therapeutic use of antimicrobials in feedlot cattle with their continued use and effectiveness being underpinned through the implementation of stewardship programs that include monitoring of resistance levels. To enable these programs, rapid and user-friendly assays are needed to detect antimicrobial resistance genes (ARG) for efficient monitoring. This study developed multiplex Luminex assays targeting 34 ARGs and validated them using reference strains of Pasteurellaceae and other bacteria, as well as field samples from nasal swabs of cattle (n = 94) undergoing BRD treatment at an Australian feedlot. One swab was collected from each nostril of every animal, with one being used for bacterial culture and conventional PCR analyses for ARGs, while the DNA extracted from the second swab was analyzed using the novel Luminex assays for the presence or absence of the ARGs of interest. The pathogens isolated by culture were tested for macrolide resistance genes erm(42), mph(E) and msr(E); sulfonamide resistance genes, sul1 and sul2; florfenicol resistance gene floR; β-lactam resistance gene bla(Rob-1) and tetracycline resistance genes tet(Q) and tet(Y), by conventional PCR. Kappa statistics suggested a moderate agreement between the tests in detecting the macrolide resistance genes. Luminex based analyses identified more resistance genes than PCR on cultured organisms, revealing the presence of a broader array of these genes than previously reported. In addition to detecting more genes, Luminex assays could process a higher number of samples in a single day, making them well-suited for ongoing surveillance of antimicrobial resistance in BRD affected cattle. This capability is essential for optimising therapeutic use and detecting emerging resistance patterns.202540848749
3934110.9995Prevalence of antimicrobial resistance genes and its association with restricted antimicrobial use in food-producing animals: a systematic review and meta-analysis. BACKGROUND: There is ongoing debate regarding potential associations between restrictions of antimicrobial use and prevalence of antimicrobial resistance (AMR) in bacteria. OBJECTIVES: To summarize the effects of interventions reducing antimicrobial use in food-producing animals on the prevalence of AMR genes (ARGs) in bacteria from animals and humans. METHODS: We published a full systematic review of restrictions of antimicrobials in food-producing animals and their associations with AMR in bacteria. Herein, we focus on studies reporting on the association between restricted antimicrobial use and prevalence of ARGs. We used multilevel mixed-effects models and a semi-quantitative approach based on forest plots to summarize findings from studies. RESULTS: A positive effect of intervention [reduction in prevalence or number of ARGs in group(s) with restricted antimicrobial use] was reported from 29 studies for at least one ARG. We detected significant associations between a ban on avoparcin and diminished presence of the vanA gene in samples from animals and humans, whereas for the mecA gene, studies agreed on a positive effect of intervention in samples only from animals. Comparisons involving mcr-1, blaCTX-M, aadA2, vat(E), sul2, dfrA5, dfrA13, tet(E) and tet(P) indicated a reduced prevalence of genes in intervention groups. Conversely, no effects were detected for β-lactamases other than blaCTX-M and the remaining tet genes. CONCLUSIONS: The available body of scientific evidence supported that restricted use of antimicrobials in food animals was associated with an either lower or equal presence of ARGs in bacteria, with effects dependent on ARG, host species and restricted drug.202133146719
2574120.9995Detection of Antibiotic Resistance Genes in Source and Drinking Water Samples from a First Nations Community in Canada. Access to safe drinking water is now recognized as a human right by the United Nations. In developed countries like Canada, access to clean water is generally not a matter of concern. However, one in every five First Nations reserves is under a drinking water advisory, often due to unacceptable microbiological quality. In this study, we analyzed source and potable water from a First Nations community for the presence of coliform bacteria as well as various antibiotic resistance genes. Samples, including those from drinking water sources, were found to be positive for various antibiotic resistance genes, namely, ampC, tet(A), mecA, β-lactamase genes (SHV-type, TEM-type, CTX-M-type, OXA-1, and CMY-2-type), and carbapenemase genes (KPC, IMP, VIM, NDM, GES, and OXA-48 genes). Not surprisingly, substantial numbers of total coliforms, including Escherichia coli, were recovered from these samples, and this result was also confirmed using Illumina sequencing of the 16S rRNA gene. These findings deserve further attention, as the presence of coliforms and antibiotic resistance genes potentially puts the health of the community members at risk. IMPORTANCE: In this study, we highlight the poor microbiological quality of drinking water in a First Nations community in Canada. We examined the coliform load as well as the presence of antibiotic resistance genes in these samples. This study examined the presence of antibiotic-resistant genes in drinking water samples from a First Nations Community in Canada. We believe that our findings are of considerable significance, since the issue of poor water quality in First Nations communities in Canada is often ignored, and our findings will help shed some light on this important issue.201627235436
3286130.9995Impact of Vegetative Treatment Systems on Multiple Measures of Antibiotic Resistance in Agricultural Wastewater. Wastewater is an important vector of antibiotic resistant bacteria and antibiotic resistance genes (ARB/G). While there is broad agreement that ARB/G from agricultural (ag) wastewaters can be transported through the environment and may contribute to untreatable infectious disease in humans and animals, there remain large knowledge gaps surrounding applied details on the types and amounts of ARB/G associated with different agricultural wastewater treatment options and different ag production systems. This study evaluates a vegetative treatment system (VTS) built to treat the wastewater from a beef cattle feedlot. Samples were collected for three years, and plated on multiple media types to enumerate tetracycline and cefotaxime-resistant bacteria. Enterobacteriaceae isolates (n = 822) were characterized for carriage of tetracycline resistance genes, and E. coli isolates (n = 673) were phenotyped to determine multi-drug resistance (MDR) profiles. Tetracycline resistance in feedlot runoff wastewater was 2-to-3 orders of magnitude higher compared to rainfall runoff from the VTS fields, indicating efficacy of the VTA for reducing ARB over time following wastewater application. Clear differences in MDR profiles were observed based on the specific media on which a sample was plated. This result highlights the importance of method, especially in the context of isolate-based surveillance and monitoring of ARB in agricultural wastewaters.201829933547
4942140.9995Nanopore-based enrichment of antimicrobial resistance genes - a case-based study. Rapid screening of hospital admissions to detect asymptomatic carriers of resistant bacteria can prevent pathogen outbreaks. However, the resulting isolates rarely have their genome sequenced due to cost constraints and long turn-around times to get and process the data, limiting their usefulness to the practitioner. Here we used real-time, on-device target enrichment ("adaptive") sequencing as a highly multiplexed assay covering 1,147 antimicrobial resistance genes. We compared its utility against standard and metagenomic sequencing, focusing on an isolate of Raoultella ornithinolytica harbouring three carbapenemases (NDM, KPC, VIM). Based on this experimental data, we then modelled the influence of several variables on the enrichment results and predicted the large effect of nucleotide identity (higher is better) and read length (shorter is better). Lastly, we showed how all relevant resistance genes are detected using adaptive sequencing on a miniature ("Flongle") flow cell, motivating its use in a clinical setting to monitor similar cases and their surroundings.202336949817
5093150.9995Evaluation of filter paper to transport inactivated bacteria to detect carbapenem resistance genes by multiplex real-time PCR using high-resolution melting. Infections caused by resistant microorganisms are a complex global public health challenge, and the way to combat the increase of resistance is the development of more modern and faster techniques for resistance detection. This study aimed to evaluate the transport of inactivated bacteria impregnated in a filter paper disk to detect carbapenem resistance genes by multiplex real-time PCR (qPCR) using high-resolution melting (HRM). A total of 88 isolates of 10 different species of Enterobacterales harboring well-characterized carbapenem resistance genes were evaluated. A full 10-µL loop of fresh growth of bacteria were impregnated in a filter paper disk, which was left at room temperature for 2 days in order to simulate the time spent in transportation. Bacterial inactivation was performed with 70% ethanol at 15 min. Afterwards, the DNA was extracted from the paper disks for further analysis by qPCR HRM. The time of 15 min in 70% ethanol was enough to inactivate all the isolates tested. It was possible to correctly identify the presence of the carbapenem resistance gene by HRM qPCR in 87 isolates (98.87%) that were transported in the filter paper disks. Our results indicated that it is possible to use filter paper to transport inactivated bacteria and to identify carbapenem resistance genes by qPCR HRM. This alternative tends to facilitate the access to this technology by many laboratories which do not have the qPCR equipment.202134213734
2577160.9995Molecular Detection of bla(TEM) and bla(SHV) Genes in ESBL-Producing Acinetobacter baumannii Isolated from Antarctic Soil. The phenomenon of antimicrobial resistance (AMR) in cold environments, exemplified by the Antarctic, calls into question the assumption that pristine ecosystems lack clinically significant resistance genes. This study examines the molecular basis of AMR in Acinetobacter spp. Isolated from Antarctic soil, focusing on the bla(TEM) and bla(SHV) genes associated with extended-spectrum beta-lactamase (ESBL) production; Soil samples were collected and processed to isolate Antarctic soil bacteria. Molecular detection was then conducted using polymerase chain reaction (PCR) to identify the bacteria species by 16S rRNA/rpoB and 10 different beta-lactamase-producing genes. PCR amplicons were sequenced to confirm gene identity and analyze genetic variability. Acinetobacter baumannii were identified by both microbiological and molecular tests. Notably, both the bla(TEM) and bla(SHV) genes encoding the enzymes responsible for resistance to penicillins and cephalosporins were identified, indicating the presence of resistance determinants in bacteria from extreme cold ecosystems. The nucleotide sequence analysis indicated the presence of conserved ARGs, which suggest stability and the potential for horizontal gene transfer within microbial communities. These findings emphasize that AMR is not confined to human-impacted environments but can emerge and persist in remote, cold habitats, potentially facilitated by natural reservoirs and global microbial dispersal. Understanding the presence and role of AMR in extreme environments provides insights into its global dissemination and supports the development of strategies to mitigate the spread of resistance genes in both environmental and clinical contexts.202540142377
4943170.9995Targeted sequencing of Enterobacterales bacteria using CRISPR-Cas9 enrichment and Oxford Nanopore Technologies. Sequencing DNA directly from patient samples enables faster pathogen characterization compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed to improve the yield of low abundance sequences but has not been thoroughly explored with Oxford Nanopore Technologies (ONT) for use in clinical bacterial epidemiology. We designed CRISPR-Cas9 guide RNAs to enrich the human pathogen Klebsiella pneumoniae, by targeting multi-locus sequence type (MLST) and transfer RNA (tRNA) genes, as well as common antimicrobial resistance (AMR) genes and the resistance-associated integron gene intI1. We validated enrichment performance in 20 K. pneumoniae isolates, finding that guides generated successful enrichment across all conserved sites except for one AMR gene in two isolates. Enrichment of MLST genes led to a correct allele call in all seven loci for 8 out of 10 isolates that had depth of 30× or more in these regions. We then compared enriched and unenriched sequencing of three human fecal samples spiked with K. pneumoniae at varying abundance. Enriched sequencing generated 56× and 11.3× the number of AMR and MLST reads, respectively, compared to unenriched sequencing, and required approximately one-third of the computational storage space. Targeting the intI1 gene often led to detection of 10-20 proximal resistance genes due to the long reads produced by ONT sequencing. We demonstrated that CRISPR-Cas9 enrichment combined with ONT sequencing enabled improved genomic characterization outcomes over unenriched sequencing of patient samples. This method could be used to inform infection control strategies by identifying patients colonized with high-risk strains. IMPORTANCE: Understanding bacteria in complex samples can be challenging due to their low abundance, which often results in insufficient data for analysis. To improve the detection of harmful bacteria, we implemented a technique aimed at increasing the amount of data from target pathogens when combined with modern DNA sequencing technologies. Our technique uses CRISPR-Cas9 to target specific gene sequences in the bacterial pathogen Klebsiella pneumoniae and improve recovery from human stool samples. We found our enrichment method to significantly outperform traditional methods, generating far more data originating from our target genes. Additionally, we developed new computational techniques to further enhance the analysis, providing a thorough method for characterizing pathogens from complex biological samples.202539772804
4999180.9995Dissemination Routes of Carbapenem and Pan-Aminoglycoside Resistance Mechanisms in Hospital and Urban Wastewater Canalizations of Ghana. Wastewater has a major role in antimicrobial resistance (AMR) dynamics and public health. The impact on AMR of wastewater flux at the community-hospital interface in low- and middle-income countries (LMICs) is poorly understood. Therefore, the present study analyzed the epidemiological scenario of resistance genes, mobile genetic elements (MGEs), and bacterial populations in wastewater around the Tamale metropolitan area (Ghana). Wastewater samples were collected from the drainage and canalizations before and after three hospitals and one urban waste treatment plant (UWTP). From all carbapenem/pan-aminoglycoside-resistant bacteria, 36 isolates were selected to determine bacterial species and phenotypical resistance profiles. Nanopore sequencing was used to screen resistance genes and plasmids, whereas, sequence types, resistome and plasmidome contents, pan-genome structures, and resistance gene variants were analyzed with Illumina sequencing. The combination of these sequencing data allowed for the resolution of the resistance gene-carrying platforms. Hospitals and the UWTP collected genetic and bacterial elements from community wastewater and amplified successful resistance gene-bacterium associations, which reached the community canalizations. Uncommon carbapenemase/β-lactamase gene variants, like bla(DIM-1), and novel variants, including bla(VIM-71), bla(CARB-53), and bla(CMY-172), were identified and seem to spread via clonal expansion of environmental Pseudomonas spp. However, bla(NDM-1), bla(CTX-M-15), and armA genes, among others, were associated with MGEs that allowed for their dissemination between environmental and clinical bacterial hosts. In conclusion, untreated hospital wastewater in Ghana is a hot spot for the emergence and spread of genes and gene-plasmid-bacterium associations that accelerate AMR, including to last-resort antibiotics. Urgent actions must be taken in wastewater management in LMICs in order to delay AMR expansion. IMPORTANCE Antimicrobial resistance (AMR) is one the major threats to public health today, especially resistance to last-resort compounds for the treatment of critical infections, such as carbapenems and aminoglycosides. Innumerable works have focused on the clinical ambit of AMR, but studies addressing the impact of wastewater cycles on the emergence and dissemination of resistant bacteria are still limited. The lack of knowledge is even greater when referring to low- and middle-income countries, where there is an absence of accurate sanitary systems. Furthermore, the combination of short- and long-read sequencing has surpassed former technical limitations, allowing the complete characterization of resistance genes, mobile genetic platforms, plasmids, and bacteria. The present study deciphered the multiple elements and routes involved in AMR dynamics in wastewater canalizations and, therefore, in the local population of Tamale, providing the basis to adopt accurate control measures to preserve and promote public health.202235103490
2578190.9994Bacteria tolerant to colistin in coastal marine environment: Detection, microbiome diversity and antibiotic resistance genes' repertoire. The global spread of mobilized colistin resistance (mcr) genes in clinical and natural environments dangerously diminishes the effectiveness of this last-resort antibiotic, becoming an urgent health threat. We used a multidisciplinary approach to detect mcr-1 gene and colistin (CL)-resistant bacteria in seawater from two Croatian public beaches. Illumina-based sequencing of metagenomic 16S rRNA was used to assess the taxonomic, functional, and antibiotic resistance genes (ARGs) profiling of the bacterial community tolerant to CL regarding different culture-based isolation methodologies. Data revealed that the choice of methodology alters the diversity and abundance of taxa accounting for the CL-resistance phenotype. The mcr-1 gene was identified by cloning and sequencing in one sample, representing the first report of mcr-1 gene in Croatia. Culturing of CL-resistant strains revealed their resistance phenotypes and concurrent production of clinically significant β-lactamases, such as CTX-M-15, CTX-M-3 and SHV-12. We also report the first identification of bla(CTX-M-15) gene in Klebsiella huaxiensis and K. michiganensis, as well as the bla(TEM-1+CTX-M-3) in Serratia fonticola. ARGs profiles derived from metagenomic data and predicted by PICRUSt2, showed the highest abundance of genes encoding for multidrug efflux pumps, followed by the transporter genes accounting for the tetracycline, macrolide and phenicol resistance. Our study evidenced the multidrug resistance features of CL-tolerant bacterial communities thriving in surface beach waters. We also showed that combined application of the metagenomic approaches and culture-based techniques enabled successful detection of mcr-1 gene, which could be underreported in natural environment.202134289613