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Multidrug efflux pumps excrete a range of small molecules from bacterial cells. In this study, we show that bacterial efflux pumps have affinity for a range of SYTO™ dyes that are commonly used to label bacteria. Efflux pump activity will there lead to false negative results from bacterial staining and SYTO™ dyes should be used with caution on live samples.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objectives: We developed a rat model of prosthetic vascular graft infection to assess, whether the fibrinolytic tissue plasminogen activator (tPA) could increase the efficacy of antibiotic therapy., Materials and Methods: Rats were implanted a polyethylene graft in the common carotid artery, pre-inoculated with approx. 6 log10 colony forming units (CFU) of methicillin resistant Staphylococcus aureus. Ten days after surgery, rats were randomized to either: 0.9% NaCl (n = 8), vancomycin (n = 8), vancomycin + tPA (n = 8), vancomycin + rifampicin (n = 18) or vancomycin + rifampicin + tPA (n = 18). Treatment duration was seven days. Approximately 36 hours after the end of treatment, the rats were euthanized, and grafts and organs were harvested for CFU enumeration., Results: All animals in the control group had significantly higher CFU at the time of euthanization compared to bacterial load found on the grafts prior to inoculation (6.45 vs. 4.36 mean log10 CFU/mL, p = 0.0011), and both the procedure and infection were well tolerated. Vancomycin and rifampicin treatment were superior to monotherapy with vancomycin, as it lead to a marked decrease in median bacterial load on the grafts (3.50 vs. 6.56 log10 CFU/mL, p = 0.0016). The addition of tPA to vancomycin and rifampicin combination treatment did not show a further decrease in bacterial load (4.078 vs. 3.50 log10 CFU/mL, p = 0.26). The cure rate was 16% in the vancomycin + rifampicin group vs. 37.5% cure rate in the vancomycin + rifampicin + tPA group. Whilst interesting, this trend was not significant at our sample size (p = 0.24)., Conclusion: We developed the first functional model of an arterial prosthetic vascular graft infection in rats. Antibiotic combination therapy with vancomycin and rifampicin was superior to vancomycin monotherapy, and the addition of tPA did not significantly reduce bacterial load, nor significantly increase cure rate., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Johansen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Applications of conventional linear ligation-rolling circle amplification (RCA) are restricted by the sophisticated operation steps and unsatisfactory picomolar-level detection limits. We herein demonstrate an RCA-based cascade amplification reaction that converts a side-reaction to secondary amplification, which improves the detection limit and simplifies the operation compared to linear ligation-RCA assays. The proposed nicking-assisted enzymatic cascade amplification (NECA) comprises an on-loop amplification reaction using circular templates to generate intermediate amplicons, and an off-loop amplification reaction using intermediate amplicons as primers for end amplicons. The whole NECA reaction is homogeneous and isothermal. Amplicons anneal to detection probes that are grafted onto magnetic nanoparticles (MNPs), such that MNP clusters form and can be detected in real-time using optomagnetic measurements. The optomagnetic sensor detects the presence and size increase of MNP clusters by optical transmission measurements in an oscillating magnetic field. A detection limit of 2 fM was achieved with a total assay time of ca. 70 min. By combining optomagnetic readouts of signal phase lag and hydrodynamic size increase of MNPs, NECA-based target quantification provided a wide dynamic detection range of ca. 4.5 orders of magnitude. Moreover, the specificity and the serum detection capability of the proposed method were investigated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Detection of a single base mutation in Mycobacterium tuberculosis DNA can provide fast and highly specific diagnosis of antibiotic-resistant tuberculosis. Mutation-specific ligation of padlock probes (PLPs) on the target followed by rolling circle amplification (RCA) is highly specific, but challenging to integrate in a simple microfluidic device due to the low temperature stability of the phi29 polymerase and the interference of phi29 with the PLP annealing and ligation. Here, we utilized the higher operation temperature and temperature stability of Equiphi29 polymerase to simplify the integration of the PLP ligation and RCA steps of an RCA assay in two different strategies performed at uniform temperature. In strategy I, PLP annealing took place off-chip and the PLP ligation and RCA were performed in one pot and the two reactions were clocked by a change of the temperature. For a total assay time of about 1.5 h, we obtained a limit of detection of 2 pM. In strategy II, the DNA ligation mixture and the RCA mixture were separated into two chambers on a microfluidic disc. After on-disc PLP annealing and ligation, the disc was spun to mix reagents and initiate RCA. For a total assay time of about 2 h, we obtained a limit of detection of 5 pM. Graphical abstract.

False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Rolling circle amplification (RCA) of a synthetic nucleic acid target is detected using magnetic nanoparticles (MNPs) combined with an optomagnetic (OM) readout. Two RCA assays are developed with on-chip detection of rolling circle products (RCPs) either at end-point where MNPs are mixed with the sample after completion of RCA or in real time where MNPs are mixed with the sample during RCA. The plastic chip acts as a cuvette, which is positioned in a setup integrated with temperature control and simultaneous detection of four parallel DNA hybridization reactions between functionalized MNPs and products of DNA amplification. The OM technique probes the small-angle rotation of MNPs bearing oligonucleotide probes complementary to the repeated nucleotide sequence of the RCPs. This rotation is restricted when MNPs bind to RCPs, which can be observed as a turn-off of the signal from MNPs that are free to rotate. The amount of MNPs bound to RCPs is found to increase in response to the amplification time as well as in response to the synthetic DNA target concentration (2-40 pM dynamic range). We report OM real-time results obtained with MNPs present during RCA and compare to relevant end-point OM results for RCPs generated for different RCA times. The real-time approach avoids opening of tubes post-RCA and thus reduces risk of lab contamination with amplification products without compromising the sensitivity and dynamic range of the assay.

Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Padlock probe ligation-based rolling circle amplification (RCA) can distinguish single-nucleotide variants, which is promising for the detection of drug-resistance mutations in, e.g., Mycobacterium tuberculosis ( Mtb ). However, the clinical application of conventional linear RCA is restricted by its unsatisfactory picomolar-level limit of detection (LOD). Herein, we demonstrate the mechanism of a nicking-enhanced RCA (NickRCA) strategy that allows several polymerases to act simultaneously on the same looped template, generating single-stranded amplicon monomers. Limiting factors of NickRCA are investigated and controlled for higher amplification efficiency. Thereafter, we describe a NickRCA-based magnetic nanoparticle (MNP) dimer formation strategy combined with a real-time optomagnetic sensor monitoring MNP dimers. The proposed methodology is applied for the detection of a common Mtb rifampicin-resistance mutation, rpoB 531 (TCG/TTG). Without additional operation steps, an LOD of 15 fM target DNA is achieved with a total assay time of ca . 100 min. Moreover, the proposed biosensor holds the advantages of single-nucleotide mutation discrimination and the robustness to quantify targets in 10% serum samples. NickRCA produces short single-stranded monomers instead of the DNA coils produced in conventional RCA, which makes it more convenient for downstream operation, immobilization or detection, thus being applicable with different molecular tools and biosensors.

Rolling circle amplification (RCA) is a linear isothermal amplification technique that is widely applied in biomolecular assays due to its high specificity. Handling of a target sample using magnetic microbeads (MMBs) in a multi-step assay is appealing as the MMBs enable separation and transportation using an external magnet. Detection of amplicons using optomagnetic measurements of the rotational diffusion properties of magnetic nanoparticles (MNPs) is also appealing as it can be performed on any transparent sample container. Two strategies are described for integration of MMB sample handling in an RCA assay with on-chip optomagnetic detection of the amplification products. The first strategy relies on selective and irreversible release of the amplicons from the MMBs so that the binding of functionalized MNPs to the amplicons can be detected optomagnetically. The second strategy relies on the incorporation of MNPs into RCA products during RCA, followed by their separation on MMBs and subsequent optomagnetic detection upon release from the RCA products. Using MMB handling of RCA steps, the limits of detection (LODs) for a synthetic DNA target representative of Victoria Influenza type B were found to be between 4 and 20 pM with total assay times between 2 and 2.5 h. Without magnetic microbead sample handling, the LOD was 200 fM. The findings provide deeper insight into the use of magnetic microbeads as solid substrates to handle a DNA target for integration of RCA as well as other DNA-based assays. Graphical Abstract Schematic illustration of magnetic microbeads transporting a DNA target through the steps in a rolling circle amplification assay. Optomagnetic measurements detect the binding of magnetic nanoparticles to amplicons released from microbeads (top) or the pH-induced release of magnetic nanoparticles trapped in amplicons (bottom).

We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background.

We report on optomagnetic dose-dependent detection of DNA triplex-mediated and pH-switchable clusters of functionalised magnetic nanoparticles.