Application of microarray technology for the detection of intracranial bacterial infection. - Related Documents




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579401.0000Application of microarray technology for the detection of intracranial bacterial infection. The aim of this study was to assess the value of microarray technology for the detection of intracranial bacterial infection. A small gene chip was prepared based on the four pathogens commonly known to cause intracranial infection and the corresponding six types of common resistance genes in The Affiliated Hospital of Nantong University and The Affiliated Haian People's Hospital of Nantong University. Cerebrospinal fluid samples were then collected from 30 patients with clinically diagnosed intracranial infection for the detection of the bacteria and resistance genes. The results were compared with the bacterial culture and sensitivity test results from the Department of Clinical Laboratories. The laboratory bacterial culture took 4-5 days, and revealed that 12 cases were positive and 18 cases were negative for bacteria. The microarray analysis took 1 day, and bacteria and resistance genes were detected in 15 cases. The 16S gene and drug resistance genes were detected in 8 cases; however, the bacterial strain was not identified. Seven cases appeared negative for bacteria and resistance genes. Microarray technology is rapid, sensitive and suitable for use in the detection of intracranial infections and other diseases for which conventional bacterial culture has a low positive rate.201424396433
597310.9996DNA 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
597120.9996Detection 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
509230.9996Rapid 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
509140.9995Quantitative 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
579650.9995Antibiotic treatment algorithm development based on a microarray nucleic acid assay for rapid bacterial identification and resistance determination from positive blood cultures. Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management.201626712265
509360.9995Evaluation 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
508870.9995A 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
563780.9995Preparation 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
509090.9995A 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
5640100.9995Antibiotic consumption and faecal bacterial susceptibility in surgical in-patients. A one-day prevalence study of resistance of faecal bacteria to 19 antibacterial agents was performed in 144 surgical inpatients. Most of the drug-resistant isolates were of aerobic and anaerobic species commonly seen in infections, which indicates that surveys of faecal flora can yield rapid information on local patterns of drug resistance in pathogens relevant to abdominal infection. In faecal bacteria the drug resistance pattern only weakly reflected the local antibiotic consumption. The amount of administered aminoglycosides was relatively small, and no gentamicin-resistant aerobes were found. Absence of resistance was found also for some of the newer agents not yet in clinical use (aztreonam, latamoxef, norfloxacin), but not for others (ceftazidime, ceftriaxone). Despite heavy use of fosfomycin and metronidazole, resistance had not emerged among aerobic and anaerobic bacteria, respectively. Imipenem was unique in inhibiting growth of all aerobic and anaerobic faecal bacteria, in the studied patients with the single exception of a strain of Enterobacter.19873673450
5974110.9995Use 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
2247120.9995Metagenomic identification of pathogens and antimicrobial-resistant genes in bacterial positive blood cultures by nanopore sequencing. Nanopore sequencing workflows have attracted increasing attention owing to their fast, real-time, and convenient portability. Positive blood culture samples were collected from patients with bacterial bloodstream infection and tested by nanopore sequencing. This study compared the sequencing results for pathogen taxonomic profiling and antimicrobial resistance genes to those of species identification and phenotypic drug susceptibility using traditional microbiology testing. A total of 37 bacterial positive blood culture results of strain genotyping by nanopore sequencing were consistent with those of mass spectrometry. Among them, one mixed infection of bacteria and fungi was identified using nanopore sequencing and confirmatory quantitative polymerase chain reaction. The amount of sequencing data was 21.89 ± 8.46 MB for species identification, and 1.0 MB microbial strain data enabled accurate determination. Data volumes greater than or equal to 94.6 MB nearly covered all the antimicrobial resistance genes of the bacteria in our study. In addition, the results of the antimicrobial resistance genes were compared with those of phenotypic drug susceptibility testing for Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Therefore, the nanopore sequencing platform for rapid identification of causing pathogens and relevant antimicrobial resistance genes complementary to conventional blood culture outcomes may optimize antimicrobial stewardship management for patients with bacterial bloodstream infection.202338192400
5641130.9995A 7-year survey of drug resistance in aerobic and anaerobic fecal bacteria of surgical inpatients: clinical relevance and relation to local antibiotic consumption. One-day studies of bacteriological cultures of fecal specimens obtained from 409 surgical inpatients at 5 occasions enabled rapid assessment of antibiotic resistance in aerobic and anaerobic bacteria, relevant to abdominal infection. This novel approach to surveillance of drug resistance was tested in a 7-year survey at a surgical department. A distinct correlation between local drug consumption and prevalence of resistant fecal bacteria was recorded for ampicillin and doxycycline. 17 other agents studied showed no such obvious correlations. Huge increases of cefuroxime and metronidazole consumption caused no emergence of drug resistant aerobic and anaerobic fecal bacteria. Imipenem was the only agent tested, which inhibited both the aerobic and anaerobic fecal bacteria of nearly all patients.19892617202
5833140.9995Rapid identification, virulence analysis and resistance profiling of Staphylococcus aureus by gene segment-based DNA microarrays: application to blood culture post-processing. Up to now, blood culturing systems are the method of choice to diagnose bacteremia. However, definitive pathogen identification from positive blood cultures is a time-consuming procedure, requiring subculture and biochemical analysis. We developed a microarray for the identification of Staphylococcus aureus comprising PCR generated gene-segments, which can reduce the blood culture post-processing time to a single day. Moreover, it allows concomitant identification of virulence factors and antibiotic resistance determinants directly from positive blood cultures without previous amplification by PCR. The assay unambiguously identifies most of the important virulence genes such as tsst-1, sea, seb, eta and antibiotic resistance genes such as mecA, aacA-aphD, blaZ and ermA. To obtain positive signals, 20 ng of purified genomic S. aureus DNA or 2 microg of total DNA extracted from blood culture was required. The microarray specifically distinguished S. aureus from gram-negative bacteria as well as from closely related coagulase negative staphylococci (CoNS). The microarray-based identification of S. aureus can be accomplished on the same day blood cultures become positive in the Bactec. The results of our study demonstrate the feasibility of microarray-based systems for the direct identification and characterization of bacteria from cultured clinical specimens.200717141897
5972150.9995Method 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
5089160.9995A 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
2240170.9995Evaluation of multiplex tandem PCR (MT-PCR) assays for the detection of bacterial resistance genes among Enterobacteriaceae in clinical urines. BACKGROUND: Increasing resistance drives empirical use of less potent and previously reserved antibiotics, including for urinary tract infections (UTIs). Molecular profiling, without culture, might better guide early therapy. OBJECTIVES: To explore the potential of AusDiagnostics multiplex tandem (MT) PCR UTI assays. METHODS: Two MT-PCR assays were developed successively, seeking 8 or 16 resistance genes. Amplification was tracked in real time, with melting temperatures used to confirm product identity. Assays were variously performed on: (i) extracted DNA; (ii) cultured bacteria; (iii) urine spiked with reference strains; and (iv) bacteria harvested from clinical urines. Results were compared with those from sequencing, real-time SybrGreen PCR or phenotypic susceptibility. RESULTS: Performance was similar irrespective of whether DNA, cultures or urines were used, with >90% sensitivity and specificity with respect to common β-lactamases, dfr genes and aminoglycoside resistance determinants except aadA1/A2/A3, for which carriage correlated poorly with streptomycin resistance. Fluoroquinolone-susceptible and -resistant Escherichia coli (but not other species) were distinguished by the melting temperatures of their gyrA PCR products. The time from urine to results was <3 h. CONCLUSIONS: The MT-PCR assays rapidly identified resistance genes from Gram-negative bacteria in urines as well as from cultivated bacteria. Used directly on urines, this assay has the potential to guide early therapy.201930476137
5638180.9995PCR 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
5795190.9995Direct identification of Gram-positive bacteria and resistance determinants from blood cultures using a microarray-based nucleic acid assay: in-depth analysis of microarray data for undetermined results. BACKGROUND: The Verigene Gram-Positive Blood Culture (BC-GP) nucleic acid assay (Nanosphere, Inc., Northbrook, IL, USA) is a newly developed microarray-based test with which 12 Gram-positive bacterial genes and three resistance determinants can be detected using blood culture broths. We evaluated the performance of this assay and investigated the signal characteristics of the microarray images. METHODS: At the evaluation stage, we tested 80 blood cultures that were positive for various bacteria (68 bacteria covered and 12 not covered by the BC-GP panel) collected from the blood of 36 patients and 44 spiked samples. In instances where the automated system failed and errors were called, we manually inspected microarray images, measured the signal intensities of target spots, and reclassified the results. RESULTS: With the manual analysis of the microarray images of 14 samples for which error calls were reported, we could obtain correct identification results for 12 samples without the need for retesting, because strong signals in the target spots were clearly discriminable from background noise. With our interpretation strategy, we could obtain 97.1% sensitivity and 100% specificity for bacterial identification by using the BC-GP assay. The two unidentified bacteria were viridans group streptococci, which produced weaker target signals. During the application stage, among 25 consecutive samples positive for Gram-positive bacteria, we identified two specimens with error calls as Streptococcus spp. by using manual analysis. CONCLUSIONS: With help of the manual review of the microarray images, the BC-GP assay could successfully identify species and resistance markers for many clinically important Gram-positive bacteria.201525536666