DNA microarray detection of antimicrobial resistance genes in diverse bacteria. - Related Documents




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597301.0000DNA 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
597410.9999Use 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
597120.9999Detection of antibiotic resistance genes in different Salmonella serovars by oligonucleotide microarray analysis. In this study the feasibility of 50- and 60-mer oligonucleotides in microarray analysis for the detection and identification of antibiotic resistance genes in various Salmonella strains was assessed. The specificity of the designed oligonucleotides was evaluated, furthermore the optimal spotting concentration was determined. The oligonucleotide microarray was used to screen two sets of Salmonella strains for the presence of several antibiotic resistance genes. Set 1 consisted of strains with variant Salmonella Genomic Island 1 (SGI1) multidrug resistance (MDR) regions of which the antibiotic resistance profiles and genotypes were known. The second set contained strains of which initially only phenotypic data were available. The microarray results of the first set of Salmonella strains perfectly matched with the phenotypic and genotypic information. The microarray data of the second set were almost completely in concordance with the available phenotypic data. It was concluded that the microarray technique in combination with random primed genomic labeling and 50- or 60-mer oligonucleotides is a powerful tool for the detection of antibiotic resistance genes in bacteria.200515823391
597230.9999Method 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
569340.9999Evaluation 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
569250.9999Development 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
509060.9998A 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
597770.9998Methods 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
508880.9998A 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
577390.9998LBJMR 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
5978100.9998Evidences 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
5694110.9998Multiplex 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
5091120.9998Quantitative 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
5507130.9998Putative Protein Biomarkers of Escherichia coli Antibiotic Multiresistance Identified by MALDI Mass Spectrometry. The commensal bacteria Escherichia coli causes several intestinal and extra-intestinal diseases, since it has virulence factors that interfere in important cellular processes. These bacteria also have a great capacity to spread the resistance genes, sometimes to phylogenetically distant bacteria, which poses an additional threat to public health worldwide. Here, we aimed to use the analytical potential of MALDI-TOF mass spectrometry (MS) to characterize E. coli isolates and identify proteins associated closely with antibiotic resistance. Thirty strains of extended-spectrum beta-lactamase producing E. coli were sampled from various animals. The phenotypes of antibiotic resistance were determined according to Clinical and Laboratory Standards Institute (CLSI) methods, and they showed that all bacterial isolates were multi-resistant to trimethoprim-sulfamethoxazole, tetracycline, and ampicillin. To identify peptides characteristic of resistance to particular antibiotics, each strain was grown in the presence or absence of the different antibiotics, and then proteins were extracted from the cells. The protein fingerprints of the samples were determined by MALDI-TOF MS in linear mode over a mass range of 2 to 20 kDa. The spectra obtained were compared by using the ClinProTools bioinformatics software, using three machine learning classification algorithms. A putative species biomarker was also detected at a peak m/z of 4528.00.202032204308
5969140.9998Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.200515872258
5637150.9998Preparation and application of microarrays for the detection of antibiotic resistance genes in samples isolated from Changchun, China. The emergence of antibiotic-resistant bacteria, especially tetracycline- and beta-lactam-resistant bacteria, poses a great threat to human health. The purpose of this study was to develop and apply a suitable gene microarray for the detection of antibiotic resistance genes. We isolated 463 strains of bacteria from a hospital, a veterinary station, an animal nursery, and living environment of Changchun, China. After screening, it was found that 93.9% of these bacteria were resistant to tetracycline, 74.9% to ampicillin, 55.6% to deoxycycline, and 41.7% to ciprofloxacin. For amplification of antibiotic genes, we designed 28 pairs of primers. In addition, 28 hybridization probes for these genes were developed. The DNA microarray analysis was performed at 42 degrees C for 5 h. We were successful in detecting 12 resistance genes by microarray analysis. After detection, we also evaluated the sensitivity of the microarray analysis. The LDL (Lowest Detection Level) of the microarray was 1 x 10(6) copies/ml of template DNA. It is believed that such microarray-based determination of tetracycline and beta-lactam resistance genes can have a potential application in clinical studies in the future.201019642018
2082160.9998Rapid 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
5638170.9998PCR 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
5840180.9998Detection 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
5968190.9998A PCR assay for rapid detection of vancomycin-resistant enterococci. Since the first report of a vancomycin-resistant enterococcal clinical isolate, these Gram-positive bacteria have emerged as important nosocomial pathogens. Several glycopeptide resistance phenotypes can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin. In the present study, we developed a multiplex PCR, which allows the simultaneous identification of enterococci at the genus level and detection of the most frequent glycopeptide resistance genotypes. Five primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 and tuf were used in one reaction tube with bacterial DNA extracted from three to five colonies. This PCR method is suitable for the rapid detection of vancomycin-resistant enterococci.200212007446