Rapid Detection Of Antibiotic Resistance Research Articles

Graphene-Assisted Sensor for Rapid Detection of Antibiotic Resistance in Escherichia coli.

In recent years, antibiotic-resistant bacteria caused by antibiotic abuse in the medical industry have become a new environmental pollutant that endangers public health. Therefore, it is necessary to establish a detection method for evaluating drug-resistant bacteria. In this work, we used Escherichia coli as a target model and proposed a method to evaluate its drug resistance for three antibiotics. Graphene dispersion was used to co-mix with E. coli cells for the purpose of increasing the current signal. This electrochemical-based sensor allows the evaluation of the activity of E. coli on the electrode surface. When antibiotics were present, the electrocatalytic reduction signal was diminished because of the reduced activity of E. coli. Based on the difference in the electrochemical reduction signal, we can evaluate the antibiotic resistance of different E. coli strains.

Rapid detection of antibiotic resistance in Salmonella with screen printed carbon electrodes

Salmonella, one of the most common foodborne pathogens, poses a serious threat to human health. In recent years, the antibiotic resistance of Salmonella has become serious as well, which has strengthened the harm of Salmonella to human health. In this paper, a simple and effective electrochemical approach was developed to obtain profile of the antimicrobial susceptibility. Screen-printed carbon electrodes (SPCEs) were used to detect various concentrations of target bacteria at the same time, which have good stability, low cost, and easy mass production. The electroactive redox, resazurin, was used to monitor levels of metabolically active bacteria. As a demonstration, the antibacterial effect of ofloxacin and penicillin on Salmonella gallinarum (S. gallinarum) isolates was evaluated, and the minimum inhibitory concentration (MIC) was measured as well. In the real sample measurement, the MIC obtained was similar to that obtained by conventional antimicrobial susceptibility testing (AST). In this assay, bacterial activity was quantified sensitively and accurately, and the detection time was greatly reduced compared with conventional AST (16–20 h), which means that metabolic capacity of live bacteria could be observed after 1 h of incubation. We were able to clearly detect bacteria above 102 CFU/m. This method aims to nonspecific and can be widely applied to the detection of a variety of drug-resistant bacteria, providing an experimental basis for the rational use of antibiotics and bacterial resistance mechanisms.

Rapid detection of antibiotic resistance in positive blood cultures by MALDI-TOF MS and an automated and optimized MBT-ASTRA protocol for Escherichia coli and Klebsiella pneumoniae

Introduction: For fast and effective antibiotic therapy of serious infections like sepsis, it is crucial with rapid information about antibiotic susceptibility, especially in a time when the number of infections caused by multi resistant bacteria has escalated in the world.Methods: Here, we have used a semi-quantitative MALDI-TOF-MS based method for antibiotic resistance detection, MBT-ASTRA™, which is based on the comparison of growth rate of the bacteria cultivated with and without antibiotics. We demonstrate a new protocol where several parameters have been optimized and automated leading to reduced hands-on time and improved capacity to simultaneously analyse multiple clinical samples and antibiotics.Results: Ninety minutes of incubation at 37 °C with agitation was sufficient to differentiate the susceptible and resistant strains of E. coli and K. pneumoniae, for the antibiotics cefotaxime, meropenem and ciprofloxacin. In total, 841 positive blood culture analyses of 14 reference strains were performed. The overall sensitivity was 99%, specificity 99% and the accuracy 97%. The assay gave no errors for cefotaxime (n = 263) or meropenem (n = 289) for sensitive and resistant strains, whilst ciprofloxacin (n = 289) gave six (0.7%) major errors (false resistance) and four (0.5%) very major errors (false susceptibility). The intermediate strains showed a larger variety compared to the E-test MIC values.Conclusions: The hands-on time and the analysis time to detect antibiotic resistance of clinical blood samples can be substantially reduced and the sample capacity can be increased by using automation and this improved protocol.

Smartphone detection of antibiotic resistance using convective PCR and a lateral flow assay

Rapid and simple nucleic acid tests are critical for the emerging field of personalised medicine. In particular, rapid detection of antibiotic resistance in Staphylococcus aureus is crucial in initiating appropriate antibiotic treatment that can be used in routine clinical situations. Our aim was to develop a rapid detection method based on convective PCR (cPCR) in combination with a nucleic acid lateral flow assay. cPCR offers an alternative rapid amplification of nucleic acids in less than 30 min without the need for complex equipment. A low-cost cPCR device was developed with off-the shelf electronic components that allows amplification of nucleic acids without the need for electrical power. The multiplex cPCR was performed using fluorescein- and digoxigenin-modified primers targeting the femA and mecA genes to differentiate methicillin-sensitive and -resistant S. aureus in a single tube. The mecA gene is responsible for methicillin-resistance while the femA gene allows identification of the causative organism. The modified amplicons were analysed with a duplex lateral flow assay using quantum dot-labelled reporter probes and the fluorescent signal was acquired via a smartphone camera. The utility of our diagnostic system was demonstrated by detecting clinical samples of MRSA with a detection limit of 4.7 × 103 copies of DNA. The smartphone system developed may find facile applications outside centralised laboratories, such as in doctor’s offices and at the bed-side of patients.

Rapid detection of antibiotic resistance by MALDI-TOF mass spectrometry using a novel direct-on-target microdroplet growth assay

ObjectivesWe aimed to develop a universal phenotypic method, which allows easy and rapid antimicrobial susceptibility testing independently of underlying resistance mechanisms. MethodsWe established a novel direct-on-target microdroplet growth assay for the detection of antibiotic resistance within a few hours, which is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microorganisms were incubated with and without meropenem in nutrient broth as microdroplets directly on MALDI-TOF MS target. Subsequently, broth was separated from microbial cells by contacting the microdroplets with an absorptive material. The microorganisms grown in the presence of antibiotic were detected by MALDI-TOF MS. A total of 24 Klebsiella pneumoniae and 24 Pseudomonas aeruginosa isolates were used to assess performance for detection of meropenem resistance. The microdroplet volumes investigated were 2, 4, 6, 8 and 10 μL. ResultsThe best performance was achieved using 6-μL microdroplets. Applying this volume, all growth controls were successfully detected (definition of valid test), and all isolates were correctly categorized as susceptible or non-susceptible after an 18-h incubation. For K. pneumoniae, rate of valid tests, sensitivity and specificity all reached 100% after a 4-h incubation of 6-μL microdroplets. Using the same microdroplet volume for P. aeruginosa, incubation for 5 h resulted in 83.3% of valid tests with 100% sensitivity and 100% specificity. ConclusionsWe demonstrated easy, rapid and accurate resistance detection using carbapenem-resistant Gram-negative bacteria as an example. Our technology is suitable for automatization and expandable to further applications, e.g. simultaneous testing of multiple antibiotics as well as resistance determination directly from clinical samples.

Simple and Fast Detection of Resistance to Antibiotic Inhibitors of Protein Synthesis in Gram-Negative Pathogens Through Evaluation of Mitomycin C-Induced Cell Elongation.

Increasing the resistance of Gram-negative pathogens to antibiotics that inhibit protein synthesis is of great concern. In life-threatening situations, an early detection of antibiotic resistance may improve patient outcome. A rapid assay for the identification of antibiotic resistance to gentamicin, tobramycin, and tigecycline has been designed and tested in clinical strains of Acinetobacter baumannii, Pseudomonas aeruginosa, and the Enterobacteriaceae Escherichia coli and Klebsiella pneumoniae. Exponentially growing cultures were incubated with 0.5 mg/L mitomycin C (MMC) for 2 hr (10 mg/L for A. baumannii), which induced significant cell enlargement as visualized under the microscope. Addition of the appropriate antibiotic dose 15 min before the addition of MMC prevented elongation when the strain was susceptible to the antibiotic, thereby inhibiting protein synthesis. Cell enlargement was not precluded in the antibiotic resistant strains, where protein synthesis had not been successfully inhibited. In comparison with the standard dilution-based antibiogram, the sensitivity of the assay was 100% and the specificity ranged between 96.0% and 100%. Results were obtained after 2 hr and 45 min from exponentially growing cultures. The procedure is easy, reliable, and demonstrates the suitability of the evaluation of simple morphological changes, which are protein synthesis dependent, for the rapid detection of antibiotic resistance.

Detection of Carbapenemase‐Producing Organisms with a Carbapenem‐Based Fluorogenic Probe

AbstractAntibiotic resistance has become a major challenge to public health worldwide. This crisis is further aggravated by the increasing emergence of bacterial resistance to carbapenems, typically considered as the antibiotics of last resort, which is mainly due to the production of carbapenem‐hydrolyzing carbapenemases in bacteria. Herein, the carbapenem‐based fluorogenic probe CB‐1 with an unprecedented enamine–BODIPY switch is developed for the detection of carbapenemase activity. This reagent is remarkably specific towards carbapenemases over other prevalent β‐lactamases. Furthermore, the efficient imaging of live clinically important carbapenemase‐producing organisms (CPOs) with CB‐1 demonstrates its potential for the rapid detection of antibiotic resistance and timely diagnosis of CPO infections.

Detection of Carbapenemase-Producing Organisms with a Carbapenem-Based Fluorogenic Probe.

Antibiotic resistance has become a major challenge to public health worldwide. This crisis is further aggravated by the increasing emergence of bacterial resistance to carbapenems, typically considered as the antibiotics of last resort, which is mainly due to the production of carbapenem-hydrolyzing carbapenemases in bacteria. Herein, the carbapenem-based fluorogenic probe CB-1 with an unprecedented enamine-BODIPY switch is developed for the detection of carbapenemase activity. This reagent is remarkably specific towards carbapenemases over other prevalent β-lactamases. Furthermore, the efficient imaging of live clinically important carbapenemase-producing organisms (CPOs) with CB-1 demonstrates its potential for the rapid detection of antibiotic resistance and timely diagnosis of CPO infections.

Automation Highlights from the Literature

Automation Highlights from the Literature

Rapid Detection of Antibiotic Resistance in Gram-Negative Bacteria Through Assessment of Changes in Cellular Morphology.

Rapid antimicrobial susceptibility testing has the potential to improve patient outcomes and reduce healthcare-associated costs. In this study, a novel assay based on bacterial cell elongation after exposure to an antibiotic (ceftazidime) was evaluated for its ability to rapidly detect resistance in Gram-negative bacteria. The assay was used to detect resistance in a large collection of strains containing 320 clinical isolates of Acinetobacter baumannii, 171 clinical isolates of Klebsiella pneumoniae, and 212 clinical isolates of Pseudomonas aeruginosa, and the results were compared to those obtained using standard antimicrobial susceptibility testing methods. The assay identified ceftazidime-resistant strains with 100% sensitivity and 100% specificity for A. baumannii, 100% sensitivity and 97.2% specificity for K. pneumoniae, and with 82.3% sensitivity and 100% specificity for P. aeruginosa. Importantly, results were obtained in 1 hour 15 minutes from exponentially growing cultures. This study demonstrates that changes in cell length are highly correlated with phenotypic antibiotic susceptibility determined using standard susceptibility testing methods. This study therefore provides proof-of-concept that changes in cell morphology can be used as the basis for rapid detection of antibiotic resistance and provides the basis for the development of novel rapid diagnostics for the detection of antibiotic resistance.

An integrated microfluidic system for diagnosis of the resistance of Helicobacter pylori to quinolone-based antibiotics

Helicobacter pylori (H. pylori) is a species of bacteria that can colonize the human stomach mucosa. It is closely associated with gastric diseases such as ulcer and inflammation. Recently, some H. pylori strains were found to express resistance to a family of antibiotics known as quinolones due to single-point mutations. Although traditional polymerase chain reaction (PCR) and molecular diagnostic-based approaches can be used to determine the presence and abundance of antibiotic-resistant H. pylori strains, such processes are relatively expensive, labor-intensive, and require bulky and costly equipment. This study therefore reports an advanced diagnostic assay performed on an integrated microfluidic system for rapid detection of antibiotic resistance in H. pylori. The assay features three components: (1) nucleic acid extraction by specific probe-conjugated magnetic beads, (2) amplification of the target deoxyribonucleic acid (DNA) fragments by using single-nucleotide-polymorphism polymerase chain reaction (SNP-PCR), and (3) optical detection of the PCR products. The device integrates several microfluidic components including micro-pumps, normally-closed micro-valves, and reaction chambers such that the entire diagnostic assay can be automatically executed on a single microfluidic system within one hour with detection limits of 100, 102, and 102 bacterial cells for H. pylori detection and two different SNP sites strains. Three PCR-based assays for determining presence of H. pylori infection and two DNA single-point mutation assays aimed at determining whether the infected strains were resistant to quinolone can be performed simultaneously on a single chip, suggesting that this microfluidic system could be a promising tool for rapid diagnosis of the presence of antibiotic-resistant H. pylori strains.

Rapid detection of antibiotic resistance based on mass spectrometry and stable isotopes

With the emergence and growing complexity of bacterial drug resistance, rapid and reliable susceptibility testing has become a topical issue. Therefore, new technologies that assist in predicting the effectiveness of empiric antibiotic therapy are of great interest. Although the use of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid detection of antibiotic resistance is an attractive option, the current methods for MALDI-TOF MS susceptibility testing are restricted to very limited conditions. Here, we describe a technique that may allow for rapid susceptibility testing to an extent that is comparable to phenotypic methods. The test was based on a stable isotope labelling by amino acids in cell culture (SILAC)-like approach. This technique was used to visualise the growth of bacteria in the presence of an antibiotic. Pseudomonas aeruginosa was chosen as the model organism, and strains were incubated in normal medium, medium supplemented with (13)C6-(15) N2-labelled lysine and medium supplemented with labelled lysine and antibiotic. Peak shifts occurring due to the incorporation of the labelled amino acids were detected by MALDI-TOF MS. Three antibiotics with different mechanisms of action, meropenem, tobramycin and ciprofloxacin, were tested. A semi-automated algorithm was created to enable rapid and unbiased data evaluation. With the proposed test, a clear distinction between resistant and susceptible isolates was possible for all three antibiotics. The application of SILAC technology for the detection of antibiotic resistance may contribute to accelerated and reliable susceptibility testing.

MALDI‐TOF MS: an upcoming tool for rapid detection of antibiotic resistance in microorganisms

MALDI-TOF MS profiling for microorganism detection has already been demonstrated in the 1990s, but has evolved to the first-line identification method in many laboratories just during the past five years. While this application of MALDI-TOF MS has proven its broad applicability, accuracy, robustness, and cost-effectiveness it is of particular interest to expand the capabilities of the mass spectrometric platform. Resistance detection is the most desirable further application of MALDI-TOF MS in microbiology, but maybe also the most challenging. Different approaches have been published regarding diverse antibiotic drugs and distinct microorganism classes. The current review shall give an overview about the developments of the recent years and their potential to get transformed in clinical useful assays in the future.

Rapid detection of antibiotic resistance in Acinetobacter baumannii using quantitative real-time PCR

The rapid detection of antibiotic resistance in clinical isolates of Acinetobacter baumannii would shorten the period during which patients receive empirical therapy and facilitate the early initiation of directed antibiotic therapy. The objective of this study was to evaluate the ability of a real-time PCR assay to detect antibiotic resistance to four clinically relevant antibiotics from different antibiotic classes in clinical isolates of A. baumannii. The growth of 48 clinical isolates of A. baumannii with a broad range of MICs of imipenem, ciprofloxacin, colistin and amikacin was evaluated using a real-time PCR assay targeting a highly conserved region of the ompA gene. Fold changes in the number of copies of genomic DNA after 6 h of growth were used to determine resistance and the results were compared with those obtained using broth microdilution. The results obtained using the real-time PCR assay were concordant with broth microdilution for 184 of 192 determinations (95.8%). The global values for specificity (97.5%), sensitivity (92.9%), positive predictive value (95.6%) and negative predictive value (96.0%) indicated that the real-time PCR assay was able to reliably differentiate between resistant and non-resistant strains. The use of real-time PCR to monitor bacterial growth in the presence of antibiotics is effective for rapidly identifying antibiotic resistance in A. baumannii.

Determination of Bacterial Antibiotic Resistance Based on Osmotic Shock Response

We investigate the buoyant mass of bacterial cells in real time with the suspended microchannel resonator (SMR) as the population recovers from an osmotic shock. The density of the culture medium is chosen such that the bacteria initially have a positive buoyant mass which becomes negative as they recover from the hyperosmotic stress. This behavior can be used to differentiate between an antibiotic-resistant and an antibiotic-susceptible strain of the pathogenic bacteria Citrobacter rodentium, and we propose a general approach for exploiting the high precision of the SMR for rapid detection of antibiotic resistance.