Inhibition of staphylococcal nuclease by benzimidazole-based Ligand: Implications in DNA-Mediated entrapment and uptake of MRSA by Macrophage-like cells.

The staphylococcal nuclease also referred as micrococcal nuclease (MNase) is a key drug target as the enzyme degrades the neutrophil extracellular trap (NET) and empowers the pathogen to subvert the host innate immune system. To this end, the current study presents a critical evaluation of MNase inhibition rendered by benzimidazole-based ligands (C1 and C2) and probes its therapeutic implications. A nuclease assay indicated that MNase inhibition rendered by C1 and C2 was âˆ¼ 55 % and âˆ¼ 72 %, respectively, at the highest tested concentration of 10 µM. Studies on enzyme kinetics revealed that C2 rendered non-competitive inhibition and significantly reduced MNase turnover number (Kcat) and catalytic efficiency (Kcat/Km) with an IC50 value of âˆ¼ 1122 nM. In CD spectroscopy, a notable perturbation in the ß-sheet content of MNase was observed in presence of C2. Fluorescence-microscope analysis indicated that MNase inhibition by C2 could restore entrapment of methicillin-resistant Staphylococcus aureus (MRSA) in calf-thymus DNA (CT-DNA). Flow cytometry and confocal microscope analysis revealed that uptake of DNA-entrapped MRSA by activated THP-1 cells was reinstated by MNase inhibition rendered by C2. Inhibition of nuclease by the non-toxic ligand C2 holds therapeutic prospect as it has the potential to bolster the DNA-mediated entrapment machinery and mitigate MRSA infections.

Detection and Quantification of Secreted Nuclease Activity in Staphylococcus aureus Culture Supernatants.

Staphylococcal secreted nuclease contributes to S. aureus virulence by degrading neutrophil extracellular traps (NETs), which allows the bacterium to evade the host immune system and has also been shown to promote biofilm dispersal. In this chapter, two methods for detecting nuclease activity are described, both of which have increased sensitivity compared to the traditional nuclease agar method.

Ultrasensitive detection of micrococcal nuclease activity and Staphylococcus aureus contamination using optical biosensor technology-A review.

One of the most common and important pathogenic bacteria is Staphylococcus aureus (S. aureus) which is known as a foodborne illness all over the world. The detection of micrococcal nuclease (MNase) can act as a unique diagnostic biomarker for the identification of S. aureus. So far, various complex methods have been introduced for the evaluation of S. aureus bacterium. However, they have different limitations such as labor-intensive, inaccurate results and time-consuming procedures. Thus, it is of particular attention to develop fast, easy, simple and more approachable detection methods based on nanotechnology and MNase detection. In this review, recent advances and modern techniques of ultrasensitive biosensors based on quantum dots (QDs), noble metal and magnetic nanoparticles (NPs), graphene oxide (GO) nanosheets, and also transfer energy strategy have been discussed for the identification of MNase activity and S. aureus contamination. Besides, advantages and disadvantages of different types of fluorescent, phosphorescent and colorimetric biosensors have been discussed.

Thermonuclease test accuracy is preserved in methicillin-resistant Staphylococcus aureus isolates.

Introduction. The nuc gene encodes a thermonuclease which is present in Staphylococcus aureus but not in coagulase-negative staphylococci (CoNS) and is the target of the rapid phenotypic thermonuclease test. The effect of nuc gene variation in methicillin-resistant S. aureus (MRSA) on the performance of PCR testing has been noted, although there are no reports about the effect of MRSA on the activity of the thermonuclease enzyme.Aim. Our goals were to examine the sensitivity and specificity of the thermonuclease test used to distinguish S. aureus from CoNS cultured from blood. In addition, we aimed to assess differences in the sensitivity, specificity and accuracy of the thermonuclease test between methicillin-sensitive S. aureus (MSSA) and MRSA isolates.Methodology. We performed a retrospective analysis of 1404 isolates. Each isolate from a positive blood culture was identified as a Gram-positive coccus by microscopy then analysed with the thermonuclease test (Southern Group Laboratory) prior to confirmatory identification using VITEK microbial identification platforms (bioMérieux) and cefoxitin disc diffusion testing.Results. Of 1331 samples included in the final analysis, 189 were thermonuclease-positive, of which 176 were identified as S. aureus. Of the 1142 thermonuclease-negative samples, 13 were finally identified as S. aureus, giving a sensitivity of 93.1 % (95 % confidence interval [CI] 88.5-96.3) and specificity of 98.9 % (95 % CI 98.1-99.4). Of the nine proven MRSA samples, eight were thermonuclease-positive, giving a sensitivity of 88.9 % (95 % CI 51.8-99.7). Thermonuclease test accuracy for MSSA and MRSA isolates was 98.1 % (95 % CI 97.2-98.8) versus 98.8 % (95 % CI 98.0-99.3), respectively.Conclusions. In the era of increasing use of molecular-based microbiology assays, the thermonuclease test remains a simple, inexpensive and robust test for the presumptive identification of S. aureus cultured from blood, irrespective of methicillin sensitivity.

An "OFF-ON" quantum dot-graphene oxide bioprobe for sensitive detection of micrococcal nuclease of Staphylococcus aureus.

Micrococcal nuclease (MNase) is an extracellular endonuclease of Staphylococcus aureus (S. aureus). It digests single stranded nucleic acid. The presence of MNase is the gold standard to identify S. aureus and its content. The present study reports the ultrahigh sensitive and selective fluorescence platform for MNase detection, designed and developed based on the surface energy transfer mechanism. A "proof of concept" is being developed based on monoclonal antibody-conjugated quantum dots (mAb-QDs), wherein mAb-QDs act as donors and graphene oxide (GO) acts as an acceptor. mAb-QDs in close proximity to GO undergo adsorption due to weak affinity between them and this results in fluorescence quenching by the transfer of surface energy from mAb-QDs to GO. During sensing, a much stronger affinity of mAb-QDs towards MNase inhibits the energy transfer to GO and this allows the regaining of fluorescence. Immobilized mAb-QDs on nitrocellulose membrane strips were fabricated and tested for "ON-OFF-ON" sensing of MNase. The limit of detection for fluorescence based assay and strips is found to be 0.3 ng mL-1 and 0.5 ng mL-1, respectively. The developed strips were applied on real samples for the detection of S. aureus.

Detection of micrococcal nuclease for identifying Staphylococcus aureus based on DNA templated fluorescent copper nanoclusters.

Micrococcal nuclease (MNase) is a naturally-secreted nucleic acid degrading enzyme with important role in the spread of the bacteria in an infected host. The content of MNase can be used to estimate the pathogenicity of Staphylococcus aureus (S. aureus). A fluorometric method is described here for determination of the activity of MNase and for identification of S. aureus using DNA templated fluorescent copper nanoclusters (CuNC). A double-stranded DNA (dsDNA) with AT-rich regions and protruding 3'-termini was identified as a high-selectivity substrate for MNase and as a template for CuNC. In the absence of MNase, the long AT-rich dsDNA templates the formation of CuNC that display bright yellow fluorescence, with excitation/emission peaks at 340/570 nm. However, the substrates are enzymatically digested to mononucleotides or short-oligonucleotide fragments, which fail to synthesize fluorescent CuNC. The method works in the 1.0 × 10-3 - 5.0 × 10-2 U mL-1 MNase activity range, has a 1.0 mU mL-1 detection limit, and is highly selective over other exonucleases. The assay was successfully applied to the detection of MNase secreted by S. aureus and to the identification of S. aureus. Graphical abstract A smart dsDNA, with AT-rich regions and 3'-protruding termini, is screened as the high-selectivity substrate for MNase and template for the formation of copper nanoclusters (CuNC). A method is described for determination of the activity of MNase and for identification of S. aureus via smart DNA templated formation of fluorescent CuNCs.

Short communication: Characterization of enterotoxin-producing Staphylococcus aureus isolated from mastitic cows.

Staphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. In an attempt to determine whether staphylococci causing bovine mastitis could also cause food poisoning, 60 isolates of presumed S. aureus were isolated in the period between March and August 2017 from 3,384 routine, composite, quarter milk samples of individual cows raised on 12 dairy farms in central Italy. Seventeen out of 60 isolates were confirmed as S. aureus after coagulase, thermonuclease, and biochemical tests. These isolates were analyzed by PCR for the presence of the nuc, sea, seb, sec, sed, and see genes. The positive isolates were nuc, 100% (17); sea, 35.29% (6); seb, 5.88% (1); sec, 5.88% (1); sed, 29.41% (5); and see, 47.06% (8). The isolates were also tested with 2 enzyme immunoassay diagnostic kits, one for the screening detection of the production of staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE) and one for the detection of specific enterotoxin produced by each isolate. Seven out of 17 (41.18%) were enterotoxin producers: 7 produced SEA (41.18%), 1 SEB (5.88%), 1 SEC (5.88%), 5 SED (29.41%), and 6 SEE (35.29%). To further characterize the isolates, they were analyzed by the Kirby Bauer test for susceptibility to 13 antimicrobials (ampicillin, ciprofloxacin, kanamycin, tetracycline, gentamicin, methicillin, nalidixic acid, erythromycin, amoxicillin/clavulanic acid, streptomycin, vancomycin, neomycin, and enrofloxacin), and we detected resistance to ampicillin (52.94%), nalidixic acid (70.59%), erythromycin (5.88%), and amoxicillin/clavulanic acid (17.65%). The isolates were sensitive to the main classes of antimicrobials used for the treatment of bovine subclinical mastitis. The presence of enterotoxin-producing isolates of S. aureus in bovine milk means that a temperature abuse or a breakdown in the thermal treatment of the milk could present a food safety risk, particularly if all enterotoxigenic isolates could potentially produce SEA in milk.

High Occurrence of Staphylococcus aureus Isolated from Fitness Equipment from Selected Gymnasiums.

Introduction: Staphylococcus aureus is a leading cause of cutaneous bacterial infection involving community. Methods: In this study, a total of 42 swab samples were collected from the surface of various fitness equipment such as back machines, exercise mats, dip stations, dumbbells, and treadmills. Identification of the bacterial isolates was conducted using biochemical tests and further analysed molecularly using the PCR method targeting nuc gene (270 bp). The nuc gene encodes for the thermonuclease enzyme, a virulent factor of S. aureus. Results: The findings showed 31 out of 42 swab samples (73.81%) were positive with S. aureus. Conclusion: This study showed that gymnasium equipment is a potential reservoir for S. aureus and might play an important role in transmitting the pathogen to humans. Objective: This study was undertaken to assess the presence of S. aureus on the surface of fitness equipment from selected gymnasiums in Kuching and Kota Samarahan, Sarawak (Malaysia).

Electrochemical DNA biosensor with chitosan-Co(3)O(4) nanorod-graphene composite for the sensitive detection of Staphylococcus aureus nuc gene sequence.

In this paper a novel nanocomposite material prepared by Co(3)O(4) nanorods (nano-Co(3)O(4)), graphene (GR) and chitosan (CTS) was fabricated and further modified on carbon ionic liquid electrode (CILE), which was used as the substrate electrode to construct a new electrochemical DNA biosensor. The single-stranded DNA (ssDNA) probe was immobilized on the CTS-Co(3)O(4)-GR/CILE surface by electrostatic attraction, which could hybridize with the target ssDNA sequence under the selected conditions. By using methylene blue (MB) as the electrochemical indicator, the hybridization reactions were monitored with the reduction peak current. By combining the biocompatibility of Co(3)O(4) nanorods, excellent electron transfer ability and big surface of GR, good film-forming ability of CTS and the high conductivity of CILE, the amount of ssDNA adsorbed on the electrode surface was increased and the electrochemical response of MB was accelerated. Under the optimal conditions differential pulse voltammetric responses of MB were in linear with the specific target ssDNA sequence in the concentration range from 1.0×10(-12) to 1.0×10(-6)M with the detection limit as 4.3×10(-13)M (3σ). Good discrimination ability to the one-base and three-base mismatched ssDNA sequences could be achieved and the polymerase chain reaction (PCR) amplification products of Staphylococcus aureus nuc gene sequence were detected with satisfactory results.

Micrococcal nuclease detection based on peptide-bridged energy transfer between quantum dots and dye-labeled DNA.

A novel and simple method was presented for micrococcal nuclease (MNase) detection based on fluorescence resonance energy transfer (FRET) realized by electrostatic interaction. In this study, mercaptoacetic acid capped quantum dots (MAA-QDs) and ROX-modified single-stranded DNA (ROX-ssDNA) were chosen as energy donor and acceptor, respectively. At slightly basic pH, the positively charged peptide served as a bridge to bring negatively charged QDs and negatively charged ROX-ssDNA into close contact to energy transfer. When the ROX-ssDNA was cleaved into small fragments by MNase, the relatively weak electrostatic interaction between the fragmented ssDNA chains and the QDs/peptide complex should make the ROX away from the QDs/peptide complex, and thus the FRET efficiency decreased. Consequently, the fluorescence intensity of acceptor decreased and a quantification of the MNase was enabled. Under the optimal conditions, experimental results showed that the fluorescence intensity of acceptor was proportional to the logarithm of MNase concentration in a range of 4.0×10(-3)-8.0×10(-2) U mL(-1). The proposed approach offered adequate sensitivity for the detection of the MNase at 2.9×10(-3) U mL(-1).

Bispecific antibody-mediated detection of the Staphylococcus aureus thermonuclease.

We report a novel fluorescence-based immunoassay which enables qualitative detection of the Staphylococcus aureus Thermonuclease (TNase) enzyme, thus providing confirmation of the presence of the S. aureus bacterium in vitro. The biomedical problem of chronic wound healing and the continuing emergence of antibiotic-resistant species is addressed in the development of a detection system capable of the rapid, real-time assessment of bacterial load and diversity. The use of bispecific antibodies (BsAb) provides integration of the molecular detection and signal response components of a standard immunoassay due to steric hindrance-mediated release of prebound fluorescent reporter molecules upon specific binding of TNase to adjacent sites. Rhodamine and fluorescein-labeled hemocyanin from Megathura crenulata (KLH) were prepared as effective immunoconjugates containing a sensitive fluorescent reporter moiety. BsAb that both specifically quenched the fluorescence of the reporter conjugate and bound the TNase target antigen were produced using cell fusion techniques. Assays were then performed to analyze the properties attributable to the steric hindrance-mediated release of the fluorescent reporter molecules upon adjacent TNase binding. This was performed by monitoring the intensity of fluorescence emission of the immunogenic reporter conjugate released into an aqueous environment at 578 and 520 nm, respectively.

Molecular characterization of Staphylococcus pseudintermedius strains isolated from clinical samples of animal origin.

The aim of this study was to determine the species distribution among 44 randomly selected clinical isolates (30 mecA-positive and 14 mecA-negative) of animal origin previously identified as Staphylococcus intermedius by phenotypic tests and species-specific PCR amplification of the 16S rRNA gene. For this purpose, we used a multiplex PCR for the detection of the nuc gene and restriction fragment length polymorphism analysis of pta gene amplified by PCR. Both methods allow discrimination of Staphylococcus pseudintermedius from the other closely related members of the S. intermedius group and other coagulase-positive staphylococci isolated from animals. Genetic diversity of S. pseudintermedius strains was analyzed by staphylococcal protein A-encoding gene (spa) typing. Multiplex PCR method was used to identify staphylococcal cassette chromosome mec (SCCmec) type in mecA-positive strains. All isolates previously identified as S. intermedius were shown to belong to S. pseudintermedius. According to PCR-based SCCmec typing, SCCmecIII was the most prevalent type (n = 23), and solely seven isolates were designated as non-typeable. Furthermore, the assessment of spa-typing results revealed that the majority of all strains (n = 27) harbored spa type t02, and 17 strains were classified as non-typeable.

A coagulase-negative variant of Staphylococcus aureus from bovine mastitis milk.

Bacteriological analysis of milk samples from quarters of a dairy cow suffering from subclinical mastitis yielded two isolates of Staphylococcus aureus which gave a negative reaction in the standard coagulase test. Both isolates were also clumping factor and thermonuclease negative, and gave a negative reaction in the Staphaurex® test. The isolates were identified by using commercial biochemical systems, and by PCR analysis of different staphylococcal cell surface protein and exoprotein genes. Further molecular identification of the isolates, which included sequencing of the 16S rRNA gene and RT-PCR of coagulase (coa), clumping-factor (clfA) and thermonuclease (nuc) genes, was consistent with the diagnosis phenotypically 'coagulase-negative variant of Staph. aureus'. The fact that coagulase-negative Staph. aureus variants can occur in the context of intramammary infections in cattle may result in the incorrect diagnosis 'coagulase-negative staphylococci (CNS)' in routine mastitis diagnostic, at least in rare cases. To fully ensure correct species diagnosis, sequencing of the 16S rRNA gene and amplification of specific genes such as coa is necessary in these cases.

A positively charged QDs-based FRET probe for micrococcal nuclease detection.

A new strategy for quantitatively detecting micrococcal nuclease (MNase) is proposed using electrostatic interaction-based fluorescence resonance energy transfer (FRET) between positively charged QDs and negatively charged dye-labeled single-stranded DNA (dye-ssDNA). Herein, we have made our attempt to develop a strategy where the variation of FRET efficiency is due to the change of the electrostatic interaction between QDs and the ssDNA that result from the cleavage of dye-ssDNA by a single-strand-specific nuclease. To demonstrate the feasibility of this design, positively charged QDs (lysozyme modified QDs, Lyz-QDs) are prepared as the energy donor, with the fluorescent dye 6-carboxy-X-rhodamine (ROX) that is labeled to ssDNA serving as the energy acceptor. The ROX-labeled probe ssDNA (ROX-ssDNA) is absorbed to the surface of the QDs through electrostatic interaction, which results in resonance energy transfer between the QDs and the dye. In the presence of MNase which cleaves the ROX-ssDNA into small fragments, the weakened interaction between QDs and the shortened ssDNA causes the decrease of FRET efficiency. At given amounts of donor and acceptor, the ratio of fluorescence intensity of QDs to ROX changes in a MNase concentration-dependent manner. Under optimized conditions, the ratio is linear with MNase concentration over the range of 8 x 10(-3) to 9.0 x 10(-2) unit mL(-1), with a limit of detection of 1.6 x 10(-3) unit mL(-1). This new detection strategy features straightforward design and easy operation, which is capable of expanding the application of the positively charged QDs-based FRET in DNA-related bioassays.

Failure of cloxacillin in treatment of a patient with borderline oxacillin-resistant Staphylococcus aureus endocarditis.

Clinical evidence for failure with beta-lactam therapy has been lacking for patients with borderline oxacillin-resistant Staphylococcus aureus (BORSA) infections. We describe a failure of cloxacillin for a patient with endocarditis due to BORSA. The isolate also had false-negative thermonuclease and coagulase test results.

A high sensitive and specific QDs FRET bioprobe for MNase.

We used CdSe/ZnS quantum dots-ssDNA-fluorescent dye conjugates as bioprobes to detect micrococcal nuclease with high specificity and sensitivity, and further utilized the bioprobe to monitor the micrococcal nuclease activity in the culture medium of Staphylococcus aureus by fluorescence microscopy.

Effect of N-terminal deletions on the foldability, stability, and activity of staphylococcal nuclease.

The effect of N-terminally successive deletions on the foldability, stability, and activity of staphylococcal nuclease was examined. The structural changes in the nuclease caused by the deletions follow a hierarchical pattern: N-terminal truncation of the nuclease by up to nine residues clearly perturbs the conformation of the N-terminal beta-subdomain but does not affect the C-terminal alpha-subdomain; deletion of 11 or 12 residues perturbs the C-terminal alpha-subdomain, resulting in formation of a molten globule state; deletion of 13 residues causes the nuclease to become highly unfolded. N-terminally deleted nuclease delta11 retains the ability to fold but delta12 is not able to fold into an enzymatically active conformation, suggesting that 11 residues is the maximum length that can be deleted from the N-terminus while still retaining the folding competence of the nuclease. Further, the results suggest that proper folding of the C-terminal alpha-subdomain probably relies on the integrity of the N-terminal beta-subdomain.

[Expression and nuclease activity analysis of staphylococcus nuclease in E.coli].

AIM: To express staphylococcus nuclease (SN) in E.coli and prepare rabbit antisera against SN. METHODS: The SN gene was amplified by high-fidelity PCR from plasmid pPLC-SN and then subcloned into expression vector pLEX to obtain the recombinant plasmid pLEX-SN. The expression of recombinant protein was induced by tryptophan. The expressed SN was used to immunize a rabbit to prepare specific antibody. RESULTS: SDS-PAGE analysis showed that the relative molecular mass (M(r)) of the expressed SN was about 17,000 and the expressed SN accounted for about 37% of total bacterial proteins. The prepared antisera were specific to react with recombinant SN. CONCLUSION: Expression vector of SN has been successfully constructed and rabbit antibody against SN was prepared. These results lay the foundation for developing SN as antiviral protein.