Nano-biosensors for rapid detection of antibiotic resistance genes blaCTX-M in Escherichia coli and blaKPC in Klebsiella pneumoniae. - Related Documents




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247401.0000Nano-biosensors for rapid detection of antibiotic resistance genes blaCTX-M in Escherichia coli and blaKPC in Klebsiella pneumoniae. The wide spread of antibiotic resistance genes in clinical isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) has become a worrying issue worldwide. Thus, electrochemical DNA-based biosensors were developed and applied for the rapid detection of antibiotic resistance genes (blaCTX-M extended-spectrum β-lactamase (ESBL) in E. coli and blaKPC carbapenemase in Klebsiella pneumoniae). Selective ssDNA probes specific to the targeting complementary genes were designed and individually immobilized onto disposable sensor chips that were functionalized with nanocomposite (ssDNA-probe@ATP@AuNPs-PPY-V(2)O(5)). In this regard, the sensor surfaces were first modified with vanadium oxide (V(2)O(5)) and polypyrrole (PPy) then gold nanoparticles (AuNPs) to effectively promote the chemical immobilization of each of the designed bio-receptor (ssDNA probe) onto the sensor matrix using the 4-aminothiophenol (ATP) as the chemical cross-linker. Different parameters that affect the biosensing performance were studied and optimized. Accordingly, high sensitivity to both E. coli blaCTX-M and K. pneumoniae blaKPC genes were obtained with a dynamic linear range from 10(-6) to 0.1 ng/μL with correlation coefficient (R(2)) of 0.991 and 0.988, limit of detection (LODs) of 0.5 × 10(-7) ng/μL and 1 × 10(-7) ng/μL, and limit of quantification (LOQs) of 1.2 × 10(-7) and 3 × 10(-7) ng/μL, respectively. High selectivity was provided since no electrochemical signals were obtained from various competitors of common bacteria. Finally, different types of clinical samples were analyzed and validated.202540250654
222410.9992Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes. The global prevalence of antibiotic-resistant bacteria has increased the risk of dangerous infections, requiring rapid diagnosis and treatment. The standard method for diagnosis of bacterial infections remains dependent on slow culture-based methods, carried out in central laboratories, not easily extensible to rapid identification of organisms, and thus not optimal for timely treatments at the point-of-care (POC). Here, we demonstrate rapid detection of bacteria by combining electrochemical immunoassays (EC-IA) for pathogen identification with confirmatory quantitative mass spectral immunoassays (MS-IA) based on signal ion emission reactive release amplification (SIERRA) nanoparticles with unique mass labels. This diagnostic method uses compatible reagents for all involved assays and standard fluidics for automatic sample preparation at POC. EC-IA, based on alkaline phosphatase-conjugated pathogen-specific antibodies, quantified down to 10(4) bacteria per sample when testing Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa lysates. EC-IA quantitation was also obtained for wound samples. The MS-IA using nanoparticles against S. aureus, E. coli, Klebsiella pneumoniae, and P. aeruginosa allowed selective quantitation of ∼10(5) bacteria per sample. This method preserves bacterial cells allowing extraction and amplification of 16S ribosomal RNA genes and antibiotic resistance genes, as was demonstrated through identification and quantitation of two strains of E. coli, resistant and nonresistant due to β-lactamase cefotaximase genes. Finally, the combined immunoassays were compared against culture using remnant deidentified patient urine samples. The sensitivities for these immunoassays were 83, 95, and 92% for the prediction of S. aureus, P. aeruginosa, and E. coli or K. pneumoniae positive culture, respectively, while specificities were 85, 92, and 97%. The diagnostic platform presented here with fluidics and combined immunoassays allows for pathogen isolation within 5 min and identification in as little as 15 min to 1 h, to help guide the decision for additional testing, optimally only on positive samples, such as multiplexed or resistance gene assays (6 h).202133819029
223120.9992Detection of the common resistance genes in Gram-negative bacteria using gene chip technology. OBJECTIVE: To design a resistance gene detection chip that could, in parallel, detect common clinical drug resistance genes of Gram-negative bacteria. MATERIALS AND METHODS: Seventy clinically significant Gram-negative bacilli (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii) were collected. According to the known resistance gene sequences, we designed and synthesized primers and probes, which were used to prepare resistance gene detection chips, and finally we hybridized and scanned the gene detection chips. RESULTS: The results between the gene chip and polymerase chain reaction (PCR) were compared. The rate was consistently 100% in the eight kinds of resistance genes tested (TEM, SHV, CTX-M, DHA, CIT, VIM, KPC, OXA-23). One strain of Pseudomonas aeruginosa had the IMP, but it was not found by gene chip. CONCLUSION: The design of Gram-negative bacteria-resistant gene detection chip had better application value.201323867670
504130.9992Development and Validation of a Clinical Laboratory Improvement Amendments-Compliant Multiplex Real-Time PCR Assay for Detection of mcr Genes. Increased use of colistin in both human and veterinary medicine has led to the emergence of plasmid-mediated colistin resistance (mcr genes). In this study, we report the development of a real-time PCR assay using TaqMan probe-based chemistry for detection of mcr genes from bacterial isolates. Positive control isolates harboring mcr-1 and mcr-2 yielded exponential amplification curves with the assay, and the amplification efficiency was 98% and 96% for mcr-1 and mcr-2, respectively. Each target gene could be reproducibly detected from a sample containing 10(3) cfu/mL of mcr-harboring bacteria, and there was no cross-reactivity with DNA extracted from several multidrug-resistant bacteria harboring other resistance genes, but lacking mcr genes. Both sensitivity and specificity of the mcr real-time PCR assay were 100% in a method validation performed with a set of 25 previously well-characterized bacterial isolates containing mcr-positive and -negative bacteria. This newly developed assay is a rapid and sensitive tool for detecting emerging mcr genes in cultured bacterial isolates. The assay was successfully validated according to quality standards of the Clinical Laboratory Improvement Amendments (CLIA).201930942652
222540.9991Evaluation of the DNA microarray "AMR Direct Flow Chip Kit" for detection of antimicrobial resistance genes from Gram-positive and Gram-negative bacterial isolated colonies. INTRODUCTION: The AMR Direct Flow Chip assay allows the simultaneous detection of a large variety of antibiotic resistance genetic markers. To assess this kit's performance, we use isolated colonies as starting material. The assay has been approved by the European Economic Area as a suitable device for in vitro diagnosis (CE IVD) using clinical specimens. METHODS: A total of 210 bacterial isolates harbouring either one or more antimicrobial resistance genes including plasmid-encoded extended-spectrum β-lactamases (SHV, CTX-M) and carbapenemases (GES, SME, KPC, NMC/IMI, SIM, GIM, SPM, NDM, VIM, IMP, and OXA), mecA, vanA and vanB, and 30 controls were included. RESULTS: The assay displayed a sensitivity and specificity of 100% for all target genes included in the array. CONCLUSION: The AMR Direct Flow Chip Kit is an accurate assay for detecting genes which commonly confer resistance to β-lactams and vancomycin from isolated colonies in culture of Gram-positive and Gram-negative bacteria.201930857832
222850.9991Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry. BACKGROUND: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. METHODS: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. RESULTS: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. CONCLUSION: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.201931849899
276360.9991Faecal indicator bacteria and antibiotic-resistant β-lactamase producing Escherichia coli in blackwater: a pilot study. The aim of this study was to identify and quantify faecal indicator bacteria in blackwater collected from a source separation unit and determine the amount of E. coli isolates resistant to antimicrobials and their potential to produce extended spectrum β-lactamases (ESβLs) and metallo-β-lactamases (MβLs), which hydrolyse the most important antibiotics used in clinical practice. Most of the isolates were resistant to amoxicillin with clavulanic acid (36.4 %), followed by ticarcillin with clavulanic acid (22.7 %) and tetracycline (18.2 %). ESβL-producing genes blaCTX-M and blaTEM were found in three (13.6 %) and four (18.2 %) E. coli strains, respectively, while MβL genes were found in two (9.1 %). By separating at source, this pilot study clearly shows that gastrointestinal bacteria of healthy people can be an important source of antibiotic resistance released into the environment through wastewaters. One way to prevent that is to treat wastewater with a combination of TiO2, UV light, or ozone, as successful methods to remove resistant bacteria and prevent their spread in the environment.201931246572
223270.9991Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.201020356716
276580.9991Quantification of β-lactamase producing bacteria in German surface waters with subsequent MALDI-TOF MS-based identification and β-lactamase activity assay. Environmental oligotrophic bacteria are suspected to be highly relevant carriers of antimicrobial resistance (AMR). However, there is a lack of validated methods for monitoring in the aquatic environment. Since extended-spectrum β-lactamases (ESBLs) play a particularly important role in the clinical sector, a culturing method based on R2A-medium spiked with different combinations of β-lactams was applied to quantify β-lactamase-producing environmental bacteria from surface waters. In German surface water samples (n = 28), oligotrophic bacteria ranging from 4.0 × 10(3) to 1.7 × 10(4) CFU per 100 mL were detected on the nutrient-poor medium spiked with 3(rd) generation cephalosporins and carbapenems. These numbers were 3 log(10) higher compared to ESBL-producing Enterobacteriales of clinical relevance from the same water samples. A MALDI-TOF MS identification of the isolates demonstrated, that the method leads to the isolation of environmentally relevant strains with Pseudomonas, Flavobacterium, and Janthinobacterium being predominant β-lactam resistant genera. Subsequent micro-dilution antibiotic susceptibility tests (Micronaut-S test) confirmed the expression of β-lactamases. The qPCR analysis of surface waters DNA extracts showed the presence of β-lactamase genes (bla(TEM), bla(CMY-2), bla(OXA-48), bla(VIM-2), bla(SHV), and bla(NDM-1)) at concentrations of 3.7 (±1.2) to 1.0 (±1.9) log(10) gene copies per 100 mL. Overall, the results demonstrate a widespread distribution of cephalosporinase and carbapenemase enzymes in oligotrophic environmental bacteria that have to be considered as a reservoir of ARGs and contribute to the spread of antibiotic resistance.202438486766
225590.9991Diversity and metallo-β-lactamase-producing genes in Pseudomonas aeruginosa strains isolated from filters of household water treatment systems. The microbiological quality of drinking water has long been a critical element in public health. Considering the high clinical relevance of Pseudomonas aeruginosa, we examined the filters of household water treatment systems for its presence and characteristics to determine the systems' efficiency in eliminating the bacteria. In total, filters of 50 household water treatment systems were examined. Microbiological and molecular methods were used for the detection and confirmation of P. aeruginosa isolates. Random Amplification of Polymorphic DNA-polymerase chain reaction (RAPD-PCR) was performed to detect similarities and differences among P. aeruginosa isolates. Combined disk (CD) method and double disk synergy test (DDST) were performed to detect metallo-beta-lactamase (MBL)-producing P. aeruginosa isolates. Finally, PCR was performed to detect MBL genes in MBL-producing strains. From the 50 analyzed systems, 76 colonies of P. aeruginosa were identified. In some systems, isolated bacteria from different filters harbored similar genetic profiles, indicating that these isolates may be able to pass through the filter and reach higher filters of the system. Phenotypic tests revealed 7 (9.2%) MBL-producing strains. Two isolates were positive for bla(VIM-1), whereas one isolate was positive for bla(NDM) and bla(IMP-1). The wide distribution of resistant phenotypes and genetic plasticity of these bacteria in household water treatment systems indicate that resistance mechanisms circulate among P. aeruginosa isolates in the environment of the filtration systems. The presence of MBL-producing genes in these systems and P. aeruginosa as a potential reservoir of these resistance genes can be a major concern for public health.201930368151
1717100.9991Integrated detection of extended-spectrum-beta-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes. Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in gram-negative bacteria by simultaneously genotyping bla(TEM), bla(SHV), and bla(CTX-M). The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla(CTX-M) (76%), bla(SHV) (22%), or both (2%), whereas no ESBL variant of bla(TEM) was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).201020007393
2745110.9991Dissemination of multi-resistant Gram-negative bacteria into German wastewater and surface waters. Carbapenem antibiotics constitute the mainstay therapy of nosocomial infections with extended spectrum beta-lactamase producing Gram-negative bacteria; however, resistance against these compounds is increasing. This study was designed to demonstrate that carbapenemase-producing bacteria are disseminated from hospitals into the environment. To this end, resistant bacteria were isolated from a clinical/urban and from a rural catchment system in Germany in 2016/17. The study followed the dissemination of resistant bacteria from the wastewater through the wastewater treatment plant (WWTP) into the receiving surface waters. The bacteria were cultivated on selective agar and characterized by antibiotic testing, real-time PCR targeting carbapenemase genes and typing. Bacteria with resistance to third generation cephalosporins were isolated from all sample sites. 134 isolates harboring carbapenemase genes encoding VIM, NDM and OXA-48 and 26 XDR (extensively drug-resistant) strains with susceptibility to only one or two antibiotics were isolated from the clinical/urban system. The rural system yielded eight carbapenemase producers and no XDR strains. In conclusion, clinical wastewaters were charged with a high proportion of multidrug resistant bacteria. Although most of these bacteria were eliminated during wastewater treatment, dissemination into surface waters is possible as single carbapenemase producers were still present in the effluent of the WWTP.201829659796
2226120.9991Evaluation of the Microbiological Performance and Potential Clinical Impact of New Rapid Molecular Assays for the Diagnosis of Bloodstream Infections. Bloodstream infection (BSI) is a critical medical emergency associated with a high mortality rate. Rapid and accurate identification of the causative pathogen and the results of antimicrobial susceptibility testing are crucial for initiating appropriate antimicrobial therapy. The aim of this study was to evaluate the performance of a new rapid PCR Molecular Mouse System (MMS) for the identification of Gram-negative bacteria (GNB) and GNB resistance genes directly from a positive blood culture (BC). The validation of these rapid multiplex assays was carried out in a real hospital setting. A total of 80 BSI episodes were included in our study and the results were compared with culture-based methods. BC samples in which GNB had previously been detected microscopically and which originated from different hospital wards were analysed. The MMS GNB identification assay achieved a sensitivity of 98.7% and a specificity of 100% for the covered pathogens. In one BC sample, Klebsiella aerogenes was identified at the family level (Enterobacteriaceae) with MMS. However, in three polymicrobial samples, MMS identified bacteria that were not detected by culture-based methods (Klebsiella pneumoniae, K. aerogenes and Stenotrophomonas maltophilia). MMS also showed excellent overall performance in the detection of GNB resistance markers (100% sensitivity and 100% specificity). The type of extended-spectrum beta-lactamase (ESBL) resistance gene identified correctly with MMS was CTX-M-1/9 (n = 17/20), alone or in combination with SHV-type β-lactamase or with the different types of carbapenemase genes. MMS detected one carbapenemase gene of each type (KPC, NDM and OXA-23) and six OXA-48 genes. In addition, the colistin resistance gene mcr-1 was detected in one positive BC with Escherichia coli (E. coli). The time to result was significantly shorter for MMS than for routine culture methods. A retrospective analysis of the patients' medical records revealed that a change in empirical antimicrobial therapy would have been made in around half of the patients following the MMS results. These results support the use of MMS as a valuable complement to conventional culture methods for more rapid BSI diagnosis and adjustment of empirical therapy.202540142509
5088130.9990A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples. The aim of the study was to develop a multiplex assay for rapid detection of mcr-1, mcr-2, and mcr-3, a group of genes of conferring resistance to colistin mediated by plasmid in Enterobacteriaceae. A SYBR Green based real-time PCR assay has been designed to detect the mcr genes, and applied to cultured bacteria, feces and soil samples. All three mcr genes could be detected with a lower limit of 10(2) cultured bacteria. This test was highly specific and sensitive, and generated no false-positive results. The assay was also conclusive when applied to feces and soil samples containing mcr-1-positive Escherichia coli, which could facilitate the screening of mcr genes not only in the bacteria, but also directly from the environment. This simple, rapid, sensitive, and specific multiplex assay will be useful for rapid screening of the colistin resistance in both clinical medicine and animal husbandry.201729163387
5664140.9990Array based detection of antibiotic resistance genes in Gram negative bacteria isolated from retail poultry meat in the UK and Ireland. The use of antibiotics in birds and animals intended for human consumption within the European Union (EU) and elsewhere has been subject to regulation prohibiting the use of antimicrobials as growth promoters and the use of last resort antibiotics in an attempt to reduce the spread of multi-resistant Gram negative bacteria. Given the inexorable spread of antibiotic resistance there is an increasing need for improved monitoring of our food. Using selective media, Gram negative bacteria were isolated from retail chicken of UK-Intensively reared (n=27), Irish-Intensively reared (n=19) and UK-Free range (n=30) origin and subjected to an oligonucleotide based array system for the detection of 47 clinically relevant antibiotic resistance genes (ARGs) and two integrase genes. High incidences of β-lactamase genes were noted in all sample types, acc (67%), cmy (80%), fox (55%) and tem (40%) while chloramphenicol resistant determinants were detected in bacteria from the UK poultry portions and were absent in bacteria from the Irish samples. Denaturing Gradient Gel Electrophoresis (DGGE) was used to qualitatively analyse the Gram negative population in the samples and showed the expected diversity based on band stabbing and DNA sequencing. The array system proved to be a quick method for the detection of antibiotic resistance gene (ARG) burden within a mixed Gram negative bacterial population.201424713169
2251150.9990Direct-PCR from rectal swabs and environmental reservoirs: A fast and efficient alternative to detect bla(OXA-48) carbapenemase genes in an Enterobacter cloacae outbreak setting. Carbapenemase-producing bacteria are a risk factor in clinical settings worldwide. The aim of the study was to accelerate the time to results during an outbreak situation with bla(OXA-48)-positive Enterobacter cloacae by using a real-time multiplex quantitative PCR (qPCR) directly on rectal swab specimens and on wastewater samples to detect carbapenemase-producing bacteria. Thus, we analyzed 681 rectal swabs and 947 environmental samples during a five-month period by qPCR and compared the results to culture screening. The qPCR showed a sensitivity of 100% by testing directly from rectal swabs and was in ten cases more sensitive than the culture-based methods. Environmental screening for bla(OXA-48)-carbapenemase genes by qPCR revealed reservoirs of different carbapenemase genes that are potential sources of transmission and might lead to new outbreaks. The rapid identification of patients colonized with those isolates and screening of the hospital environment is essential for earlier patient treatment and eliminating potential sources of nosocomial infections.202234343553
3412160.9990Bacterial Resistance to β-Lactam Antibiotics in Municipal Wastewater: Insights from a Full-Scale Treatment Plant in Poland. This study investigated enzymatic and genetic determinants of bacterial resistance to β-lactam antibiotics in the biocenosis involved in the process of biological treatment of wastewater by activated sludge. The frequency of bacteria resistant to selected antibiotics and the activity of enzymes responsible for resistance to β-lactam antibiotics were estimated. The phenomenon of selection and spread of a number of genes determining antibiotic resistance was traced using PCR and gene sequencing. An increase in the percentage of bacteria showing resistance to β-lactam antibiotics in the microflora of wastewater during the treatment process was found. The highest number of resistant microorganisms, including multi-resistant strains, was recorded in the aeration chamber. Significant amounts of these bacteria were also present in treated wastewater, where the percentage of penicillin-resistant bacteria exceeded 50%, while those resistant to the new generation β-lactam antibiotics meropenem and imipenem were found at 8.8% and 6.4%, respectively. Antibiotic resistance was repeatedly accompanied by the activity of enzymes such as carbapenemases, metallo-β-lactamases, cephalosporinases and β-lactamases with an extended substrate spectrum. The activity of carbapenemases was shown in up to 97% of the multi-resistant bacteria. Studies using molecular biology techniques showed a high frequency of genes determining resistance to β-lactam antibiotics, especially the blaTEM1 gene. The analysis of the nucleotide sequences of blaTEM1 gene variants present in bacteria at different stages of wastewater treatment showed 50-100% mutual similarity of.202236557576
5689170.9990A 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
5091180.9990Quantitative multiplex real-time PCR for detecting class 1, 2 and 3 integrons. OBJECTIVES: Integrons are bacterial genetic elements that can capture and express genes contained in mobile cassettes. Integrons have been described worldwide in Gram-negative bacteria and are a marker of antibiotic resistance. We developed a specific and sensitive Taqman probe-based real-time PCR method with three different primer-probe pairs for simultaneous detection of the three main classes of integron. METHODS: Sensitivity was assessed by testing mixtures of the three targets (intI integrase genes of each integron class) ranging from 10 to 10(8) copies. Specificity was determined with a panel of integron-containing and integron-free control strains. The method was then applied to clinical samples. RESULTS: The PCR method was specific and had a sensitivity of 10(2) copies for all three genes, regardless of their respective quantities. The method was quantitative from 10(3) to 10(7) copies, and was able to detect integrons directly in biological samples. CONCLUSIONS: We have developed a rapid, quantitative, specific and sensitive method that could prove useful for initial screening of Gram-negative isolates, or clinical samples, for likely multidrug resistance.201020542899
3312190.9990Monitoring of antimicrobial resistance in hospital, municipal, and treated wastewater in Mbarara, Uganda. OBJECTIVE: The aim of this study was to estimate the prevalence of antimicrobial resistance in the population of Mbarara through analysis of wastewater and determine the effectiveness of wastewater treatment in reducing discharge of antibiotic-resistant bacteria and antibiotic resistance genes into the environment. METHODS: Hospital, municipal, and treated wastewater (collected on 10 different dates) from Mbarara, Uganda, were analysed for extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli using a culture-based method and selected clinically relevant antibiotic resistance genes using quantitative PCR. RESULTS: The finding of this study demonstrated that 30.6% of the total E. coli were ESBL producers, constituting a high proportion compared to studies in other countries. Furthermore, the investigation revealed the widespread distribution of the carbapenemase gene bla(CMY-2) within the population. The comparative study of the inflow and outflow of the waste stabilisation pond system, which is used for wastewater treatment, demonstrated a log reduction of 1.9-2.4 for coliform bacteria and total as well as ESBL-producing E. coli. Conversely, the wastewater treatment was associated with an increase of the antibiotic resistance genes sul1 and tetC. CONCLUSIONS: The study shows that the waste stabilisation pond system is releasing significant amounts of coliform bacteria, E. coli, ESBL-producing E. coli, somatic bacteriophages, and antibiotic resistance genes into the Rwizi River. We also demonstrated that wastewater-based surveillance is a cost-effective method of obtaining information on the prevalence of AMR in the population, especially in countries where clinical surveillance is limited due to a lack of resources and infrastructure.202540962206