A TaqMan real-time PCR assay for detection of qacEΔ1 gene in Gram-negative bacteria. - Related Documents




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509001.0000A TaqMan real-time PCR assay for detection of qacEΔ1 gene in Gram-negative bacteria. The transfer of biocide and antibiotic resistance genes by mobile genetic elements is the most common mechanism for rapidly acquiring and spreading resistance among bacteria. The qacEΔ1 gene confers the resistance to quaternary ammonium compounds (QACs). It has also been considered a genetic marker for the presence of class 1 integrons associated with multidrug-resistant (MDR) phenotypes in Gram-negative bacteria. In this study, a TaqMan real-time PCR assay was developed to detect the qacEΔ1 gene in Gram-negative bacteria. The assay has a detection limit of 80 copies of the qacEΔ1 gene per reaction. No false-positive or false-negative results have been observed. Simultaneous amplification and detection of the 16S rRNA gene is performed as an endogenous internal amplification control (IAC). The TaqMan real-time PCR assay developed is a rapid, sensitive, and specific method that could be used to monitor resistance to QACs, the spread of class 1 integrons, and the prediction of associated MDR phenotypes in Gram-negative bacteria.202439395725
509110.9999Quantitative 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
508820.9999A 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
597330.9998DNA microarray detection of antimicrobial resistance genes in diverse bacteria. High throughput genotyping is essential for studying the spread of multiple antimicrobial resistance. A test oligonucleotide microarray designed to detect 94 antimicrobial resistance genes was constructed and successfully used to identify antimicrobial resistance genes in control strains. The microarray was then used to assay 51 distantly related bacteria, including Gram-negative and Gram-positive isolates, resulting in the identification of 61 different antimicrobial resistance genes in these bacteria. These results were consistent with their known gene content and resistance phenotypes. Microarray results were confirmed by polymerase chain reaction and Southern blot analysis. These results demonstrate that this approach could be used to construct a microarray to detect all sequenced antimicrobial resistance genes in nearly all bacteria.200616427254
508940.9998A TaqMan-based multiplex real-time PCR assay for the rapid detection of tigecycline resistance genes from bacteria, faeces and environmental samples. BACKGROUND: Tigecycline is a last-resort antibiotic used to treat severe infections caused by extensively drug-resistant bacteria. Recently, novel tigecycline resistance genes tet(X3) and tet(X4) have been reported, which pose a great challenge to human health and food security. The current study aimed to establish a TaqMan-based real-time PCR assay for the rapid detection of the tigecycline-resistant genes tet(X3) and tet(X4). RESULTS: No false-positive result was found, and the results of the TaqMan-based real-time PCR assay showed 100% concordance with the results of the sequencing analyses. This proposed method can detect the two genes at the level of 1 × 10(2) copies/μL, and the whole process is completed within an hour, allowing rapid screening of tet(X3) and tet(X4) genes in cultured bacteria, faeces, and soil samples. CONCLUSION: Taken together, the TaqMan-based real-time PCR method established in this study is rapid, sensitive, specific, and is capable of detecting the two genes not only in bacteria, but also in environmental samples.202032571294
569350.9998Evaluation of an expanded microarray for detecting antibiotic resistance genes in a broad range of gram-negative bacterial pathogens. A microarray capable of detecting genes for resistance to 75 clinically relevant antibiotics encompassing 19 different antimicrobial classes was tested on 132 Gram-negative bacteria. Microarray-positive results correlated >91% with antimicrobial resistance phenotypes, assessed using British Society for Antimicrobial Chemotherapy clinical breakpoints; the overall test specificity was >83%. Microarray-positive results without a corresponding resistance phenotype matched 94% with PCR results, indicating accurate detection of genes present in the respective bacteria by microarray when expression was low or absent and, hence, undetectable by susceptibility testing. The low sensitivity and negative predictive values of the microarray results for identifying resistance to some antimicrobial resistance classes are likely due to the limited number of resistance genes present on the current microarray for those antimicrobial agents or to mutation-based resistance mechanisms. With regular updates, this microarray can be used for clinical diagnostics to help accurate therapeutic options to be taken following infection with multiple-antibiotic-resistant Gram-negative bacteria and prevent treatment failure.201323129055
563860.9998PCR monitoring for tetracycline resistance genes in subgingival plaque following site-specific periodontal therapy. A preliminary report. BACKGROUND: The selection of antibiotic resistance genes during antibiotic therapy is a critical problem complicated by the transmission of resistance genes to previously sensitive strains via conjugative plasmids and transposons and by the transfer of resistance genes between gram-positive and gram-negative bacteria. The purpose of this investigation was to monitor the presence of selected tetracycline resistance genes in subgingival plaque during site specific tetracycline fiber therapy in 10 patients with adult periodontitis. METHOD: The polymerase chain reaction (PCR) was used in separate tests for the presence of 3 tetracycline resistance genes (tetM, tetO and tetQ) in DNA purified from subgingival plaque samples. Samples were collected at baseline, i.e., immediately prior to treatment, and at 2 weeks, and 1, 3, and 6 months post-fiber placement. The baseline and 6-month samples were also subjected to DNA hybridization tests for the presence of 8 putative periodontal pathogenic bacteria. RESULTS: PCR analysis for the tetM resistance gene showed little or no change in 5 patients and a decrease in detectability in the remaining 5 patients over the 6 months following tetracycline fiber placement. The results for tetO and tetQ were variable showing either no change in detectability from baseline through the 6-month sampling interval or a slight increase in detectability over time in 4 of the 10 patients. DNA hybridization analysis showed reductions to unmeasurable levels of the putative periodontal pathogenic bacteria in all but 2 of the 10 patients. CONCLUSIONS: These results complement earlier studies of tet resistance and demonstrate the efficacy of PCR monitoring for the appearance of specific resistance genes during and after antibiotic therapy.200010883874
569470.9998Multiplex characterization of human pathogens including species and antibiotic-resistance gene identification. The efficient medical treatment of infections requires detailed information about the pathogens involved and potential antibiotic-resistance mechanisms. The dramatically increasing incidence of multidrug-resistant bacteria especially highlights the importance of sophisticated diagnostic tests enabling a fast patient-customized therapy. However, the current molecular detection methods are limited to either the detection of species or only a few antibiotic-resistance genes.In this work, we present a human pathogen characterization assay using a rRNA gene microarray identifying 75 species comprising bacteria and fungi. A statistical classifier was developed to facilitate the automated species identification. Additionally, the clinically most important β-lactamases were identified simultaneously in a 100-plex reaction using padlock probes and the same microarray. The specificity and sensitivity of the combined assay was determined using clinical isolates. The detection limit was 10(5) c.f.u. ml(-1), recovering 89 % of the detectable β-lactamase-encoding genes specifically. The total assay time was less than 7 hand the modular character of the antibiotic-resistance detection allows the easy integration of further genetic targets. In summary, we present a fast, highly specific and sensitive multiplex pathogen characterization assay.201626489938
584080.9998Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa. Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. To reduce the selection pressure for resistance, it is important to determine the antibiotic susceptibility pattern of bacteria so that hospital patients can be treated with more narrow-spectrum and target-specific antibiotics. This study describes the development of a technique for detecting point muations in the fluoroquinolone resistance-determining region of the gyrA and parC genes as well as the efflux regulatory genes mexR, mexZ and mexOZ that are associated with fluoroquinolone and aminoglycoside resistance. The assay is based on a short DNA sequencing method using multiplex-fast polymerase chain reaction (PCR) and Pyrosequencing for amplification and sequencing of the selected genes. Fifty-nine clinical isolates of P. aeruginosa were examined for mutations in the abovementioned genes. Mutations related to antibiotic resistance were detected in codons 83 and 87 of gyrA and codon 126 of the mexR regulatory gene. Results of this study suggest Pyrosequencing as a substitute for traditional methods as it provides a rapid and reliable technique for determining the antibiotic resistance pattern of a given bacterial strain in <1 h.200919656662
504190.9998Development 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
5773100.9998LBJMR medium: a new polyvalent culture medium for isolating and selecting vancomycin and colistin-resistant bacteria. BACKGROUND: Multi-drug resistant bacteria are a phenomenon which is on the increase around the world, particularly with the emergence of colistin-resistant Enterobacteriaceae and vancomycin-resistant enterococci strains. The recent discovery of a plasmid-mediated colistin resistance with the description of the transferable mcr-1 gene raised concerns about the need for an efficient detection method for these pathogens, to isolate infected patients as early as possible. The LBJMR medium was developed to screen for all polymyxin-resistant Gram-negative bacteria, including mcr-1 positive isolates, and vancomycin-resistant Gram-positive bacteria. RESULTS: The LBJMR medium was developed by adding colistin sulfate salt at a low concentration (4 μg/mL) and vancomycin (50 μg/mL), with glucose (7.5 g/L) as a fermentative substrate, to a Purple Agar Base (31 g/L). A total of 143 bacterial strains were used to evaluate this universal culture medium, and the sensitivity and specificity of detection were 100% for the growth of resistant strains. 68 stool samples were cultured on LBJMR, and both colistin-resistant Gram-negative and vancomycin-resistant Gram-positive strains were specifically detected. CONCLUSIONS: The LBJMR medium is a multipurpose selective medium which makes it possible to identify bacteria of interest from clinical samples and to isolate contaminated patients in hospital settings. This is a simple medium that could be easily used for screening in clinical microbiology laboratories.201729169321
5092110.9998Rapid detection of antibiotic resistance genes in lactic acid bacteria using PMMA-based microreactor arrays. The emergence of lactic acid bacteria (LABs) resistant to existing antimicrobial drugs is a growing health crisis. To decrease the overuse of antibiotics, molecular diagnostic systems that can rapidly determine the presence of antibiotic resistance (AR) genes in LABs from yogurt samples are needed. This paper describes a fully integrated, miniaturized plastic chip and closed-tube detection chemistry that performs multiplex nucleic acid amplification. High-throughput identification of AR genes was achieved through this approach, and six AR genes were analyzed simultaneously in < 2 h. This time-to-result included the time required for the extraction of DNA. The detection limit of the chip was 10(3) CFU mL(-1), which was consistent with that of tube LAMP. We detected and identified multiple DNAs, including streptomycin, tetracycline, and vancomycin resistance-associated genes, with complete concordance to the Kirby-Bauer disk diffusion method.Key Points• A miniaturized chip was presented, and multiplex nucleic acid amplification was performed.• The device can be integrated with LAMP for rapid detection of antibiotic resistance genes.• The approach had a high throughput of AR gene analysis in lactic acid bacteria.202032488313
5974120.9998Use of a bacterial antimicrobial resistance gene microarray for the identification of resistant Staphylococcus aureus. As diagnostic and surveillance activities are vital to determine measures needed to control antimicrobial resistance (AMR), new and rapid laboratory methods are necessary to facilitate this important effort. DNA microarray technology allows the detection of a large number of genes in a single reaction. This technology is simple, specific and high-throughput. We have developed a bacterial antimicrobial resistance gene DNA microarray that will allow rapid antimicrobial resistance gene screening for all Gram-positive and Gram-negative bacteria. A prototype microarray was designed using a 70-mer based oligonucleotide set targeting AMR genes of Gram-negative and Gram-positive bacteria. In the present version, the microarray consists of 182 oligonucleotides corresponding to 166 different acquired AMR gene targets, covering most of the resistance genes found in both Gram-negative and -positive bacteria. A test study was performed on a collection of Staphylococcus aureus isolates from milk samples from dairy farms in Québec, Canada. The reproducibility of the hybridizations was determined, and the microarray results were compared with those obtained by phenotypic resistance tests (either MIC or Kirby-Bauer). The microarray genotyping demonstrated a correlation between penicillin, tetracycline and erythromycin resistance phenotypes with the corresponding acquired resistance genes. The hybridizations showed that the 38 antimicrobial resistant S. aureus isolates possessed at least one AMR gene.201021083822
5692130.9998Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria. We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations.200818243668
5977140.9998Methods to determine antibiotic resistance gene silencing. The occurrence of antibiotic-resistant bacteria is an increasingly serious problem world-wide. In addition, to phenotypically resistant bacteria, a threat may also be posed by isolates with silent, but intact, antibiotic resistance genes. Such isolates, which have recently been described, possess wild-type genes that are not expressed, but may convert to resistance by activating expression of the silent genes. They may therefore compromise the efficacy of antimicrobial treatment, particularly if their presence has not been diagnosed. This chapter describes the detection of silent resistance genes by PCR and DNA sequencing. A method to detect five potentially silent acquired resistance genes; aadA, bla (OXA-2), strAB, sul1, and tet(A) is described. First, the susceptibility of the isolates to the relevant antibiotics is determined by an appropriate susceptibility testing method, such as E-test. Then the presence of the genes is investigated by PCR followed by agarose gel electrophoresis of the amplification products. If a resistance gene is detected in a susceptible isolate, the entire open-reading frame and promoter sequence of the gene is amplified by PCR and their DNA sequences obtained. The DNA sequences are then compared to those of known resistant isolates, to detect mutations that may account for susceptibility. If no mutations are detected the expression of the gene is investigated by RT-PCR following RNA extraction. The methods described here can be applied to all acquired resistance genes for which sequence and normal expression data are available.201020401584
5972150.9998Method of Selection of Bacteria Antibiotic Resistance Genes Based on Clustering of Similar Nucleotide Sequences. A new method for selection of bacterium antibiotic resistance genes is proposed and tested for solving the problems related to selection of primers for PCR assay. The method implies clustering of similar nucleotide sequences and selection of group primers for all genes of each cluster. Clustering of resistance genes for six groups of antibiotics (aminoglycosides, β-lactams, fluoroquinolones, glycopeptides, macrolides and lincosamides, and fusidic acid) was performed. The method was tested for 81 strains of bacteria of different genera isolated from patients (K. pneumoniae, Staphylococcus spp., S. agalactiae, E. faecalis, E. coli, and G. vaginalis). The results obtained by us are comparable to those in the selection of individual genes; this allows reducing the number of primers necessary for maximum coverage of the known antibiotic resistance genes during PCR analysis.201729063318
4956160.9998Rapid Identification of Plasmid Replicon Type and Coexisting Plasmid-Borne Antimicrobial Resistance Genes by S1-Pulsed-Field Gel Electrophoresis-Droplet Digital Polymerase Chain Reaction. Bacterial drug resistance is a significant food safety problem and public health threat. Plasmids carrying drug resistance genes may result in the rapid spread of resistance among different bacteria, hosts, and environments; therefore, antibiotic resistance monitoring and continuing research into the mechanisms of drug resistance are urgently needed. Southern blotting with probes for antibiotic resistance genes and even next-generation sequencing have been used previously to detect plasmid-borne resistance genes, but these approaches are complex and time-consuming. The next-generation sequencing requires strict laboratory conditions and bioinformatics analysis ability. In this study, we developed a simplified and sensitive method to detect plasmid-borne antimicrobial resistance genes and plasmid replicon types. Salmonella strains carrying plasmids of three different replicon types that contained mcr-1 and two ESBL-producing genes were used to verify the new method. The plasmids harbored by the Salmonella strains were separated by S1 nuclease treatment and pulsed-field gel electrophoresis (PFGE), then recovered and used as the templates for droplet digital polymerase chain reaction (ddPCR) to identify target genes. The target genes were present in significantly higher copy numbers on the plasmids than the background noise. These results were consistent with the plasmid sequencing results. This S1-PFGE-ddPCR method was less time-consuming to perform than Southern blot and complete plasmid sequencing. Therefore, this method represents a time-saving alternative for detecting plasmid-borne genes, and is likely to be a valuable tool for detecting coexisting plasmid-borne drug resistance genes.202133661029
5978170.9998Evidences of gentamicin resistance amplification in Klebsiella pneumoniae isolated from faeces of hospitalized newborns. The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenotypical gentamicin resistance amplification (frequencies of 10(-3) to 10(-5), compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromosomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.199910585658
2082180.9997Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology. A screening technique for integrons in members of the family Enterobacteriaceae and nonfermenting gram-negative bacteria by real-time PCR is reported. A total of 226 isolates of gram-negative bacteria obtained from a variety of clinical specimens were screened for class 1 integrons by real-time PCR performed on a LightCycler instrument. This technique used a primer pair specific for a 300-bp conserved region at the 5' ends of class 1 integrons. The screening assay was evaluated by comparison with results obtained by the conventional, thermal-block PCR (long PCR) by using established conditions and primers for the detection of class 1 integrons, and the real-time PCR technique was thus shown to be both sensitive and specific. DNA from 50 of 226 (22%) isolates screened was identified as containing an integron by the screening PCR, and sequence data were obtained across the integron for 34 of 50 (68%) of these isolates. In an attempt to study the molecular epidemiology of antimicrobial resistance genes carried within integrons, a comparison of the types of gene cassettes carried by isolates from different patients was made. Adenyltransferase genes conferring resistance to streptomycin and spectinomycin were the predominant gene cassettes amplified in the study. Resistance to trimethoprim was also frequently found to be encoded within integrons. Furthermore, multiple bacterial isolates obtained from one patient over a 5-month period were all shown to carry an integron containing the same single adenyltransferase gene cassette, suggesting that these elements were relatively stable in this case.200111257011
5093190.9997Evaluation 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