Development of an enzyme-linked phage receptor-binding protein assay (ELPRA) based on a novel biorecognition molecule- receptor-binding protein Gp130 of Pseudomonas aeruginosa bacteriophage Henu5.

Pseudomonas aeruginosa is a Gram-negative bacterium associated with life-threatening healthcare-associated infections (HAIs), including burn wound infections, pneumonia and sepsis. Moreover, P. aeruginosa has been considered a pathogen of global concern due to its rising antibiotic resistance. Efficient identification of P. aeruginosa would significantly benefit the containment of bacterial infections, prevent pathogen transmission, and provide orientated treatment options. The accuracy and specificity of bacterial detection are primarily dictated by the biorecognition molecules employed. Lytic bacteriophages (or phages) could specifically attach to and lyse host bacterial cells. Phages' host specificity is typically determined by their receptor-binding proteins (RBPs), which recognize and adsorb phages to particular bacterial host receptors. This makes RBPs promising biorecognition molecules in bacterial detection. This study identified a novel RBP (Gp130) from the P. aeruginosa phage Henu5. A modified enzyme-linked phage receptor-binding protein assay (ELPRA) was developed for P. aeruginosa detection employing Gp130 as biorecognition molecules. Optimized conditions provided a calibration curve for P. aeruginosa with a range from 1.0 × 103 to 1.0 × 107 CFU/mL, with a limit of detection as low as 10 CFU/mL in phosphate-buffered saline (PBS). With VITEKⓇ 2 Compact system identification (40 positives and 21 negatives) as the gold standard, the sensitivity of ELPRA was 0.950 (0.818-0.991), and the specificity was 0.905 (0.682-0.983) within a 95 %confidence interval. Moreover, the recovery test in spiked mouse serum showed recovery rates ranging from 82.79 %to 98.17%, demonstrating the prospect of the proposed ELPRA for detecting P. aeruginosa in biological samples.

Electrochemical biosensors for pathogenic microorganisms detection based on recognition elements.

The worldwide spread of pathogenic microorganisms poses a significant risk to human health. Electrochemical biosensors have emerged as dependable analytical tools for the point-of-care detection of pathogens and can effectively compensate for the limitations of conventional techniques. Real-time analysis, high throughput, portability, and rapidity make them pioneering tools for on-site detection of pathogens. Herein, this work comprehensively reviews the recent advances in electrochemical biosensors for pathogen detection, focusing on those based on the classification of recognition elements, and summarizes their principles, current challenges, and prospects. This review was conducted by a systematic search of PubMed and Web of Science databases to obtain relevant literature and construct a basic framework. A total of 171 publications were included after online screening and data extraction to obtain information of the research advances in electrochemical biosensors for pathogen detection. According to the findings, the research of electrochemical biosensors in pathogen detection has been increasing yearly in the past 3 years, which has a broad development prospect, but most of the biosensors have performance or economic limitations and are still in the primary stage. Therefore, significant research and funding are required to fuel the rapid development of electrochemical biosensors. The overview comprehensively evaluates the recent advances in different types of electrochemical biosensors utilized in pathogen detection, with a view to providing insights into future research directions in biosensors.

Bacteriophages and their derived enzymes as promising alternatives for the treatment of Acinetobacter baumannii infections.

Nosocomial infections with the opportunistic bacterium Acinetobacter baumannii pose a severe challenge to clinical treatment, which is aggravated by the increasing occurrence of multi-drug resistance, especially resistance to carbapenems. The use of phage therapy as an alternative and supplement to the current antibiotics has become an important research topic in the post-antibiotic era. This review summarizes in vivo and in vitro studies on phage therapy against multi-drug-resistant A. baumannii infection that have used different approaches, including treatment with a single phage, combination with other phages or non-phage agents, and administration of phage-derived enzymes. We also briefly discuss the current challenges of phage-based therapy as well as promising approaches for the treatment of A. baumannii infection in the future.

[A multi-stage and multi-epitope vaccine against Mycobacterium tuberculosis based on an immunoinformatics approach].

Objectives To develop a multi-stage and multi-epitope vaccine, which consists of epitopes from the early secretory and latency-associated antigens of Mycobacterium tuberculosis (MTB). Methods The B-cell, cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes of 12 proteins were predicted using an immunoinformatics. The epitopes with antigenicity, without cytotoxicity and sensitization, were further screened to construct the multi-epitope vaccine. Furthermore, the proposed vaccine underwent physicochemical properties analysis and secondary structure prediction as well as 3D structure modeling, refinement and validation. Then the refined model was docked with TLR4. Finally, an immune simulation of the vaccine was carried out. Results The proposed vaccine, which consists of 12 B-cell, 11 CTL and 12 HTL epitopes, had a flexible and stable globular conformation as well as a thermostable and hydrophilic structure. A stable interaction of the vaccine with TLR4 was confirmed by molecular docking. The efficiency of the candidate vaccine to trigger effective cellular and humoral immune responses was assessed by immune simulation. Conclusion A multi-stage multi-epitope MTB vaccine construction strategy based on immunoinformatics is proposed, which is expected to prevent both active and latent MTB infection.

Antimicrobial Peptides: A Promising Strategy for Anti-tuberculosis Therapeutics.

The high global burden of tuberculosis (TB) and the increasing emergence of the drugresistant (DR) strain of Mycobacterium tuberculosis (Mtb) emphasize the urgent need for novel antimycobacterial agents. Antimicrobial peptides (AMPs) are small peptides widely existing in a variety of organisms and usually have amphiphilic cationic structures, which have a selective affinity to the negatively charged bacterial cell wall. Besides direct bactericidal mechanisms, including interacting with the bacterial cell membrane and interfering with the biosynthesis of the cell wall, DNA, or protein, some AMPs are involved in the host's innate immunity. AMPs are promising alternative or complementary agents for the treatment of DR-TB, given their various antibacterial mechanisms and low cytotoxicity. A large number of AMPs, synthetic or natural, from human to bacteriophage sources, have displayed potent anti-mycobacterial activity in vitro and in vivo. In this review, we summarized the features, antimycobacterial activity, and mechanisms of action of the AMPs according to their sources. Although AMPs have not yet met the expectations for clinical application due to their low bioavailabilities, high cost, and difficulties in large-scale production, their potent antimycobacterial activity and action mechanisms, which are different from conventional antibiotics, make them promising antibacterial agents against DR-Mtb in the future.

Diagnostic efficiency of RPA/RAA integrated CRISPR-Cas technique for COVID-19: A systematic review and meta-analysis.

OBJECTIVE: To evaluate the diagnostic value of recombinase polymerase/ aided amplification (RPA/RAA) integrated clustered regularly interspaced short palindromic repeats (CRISPR) in the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We searched relevant literature on CRISPR technology for COVID-19 diagnosis using "novel coronavirus", "clustered regularly interspaced short palindromic repeats" and "RPA/RAA" as subject terms in PubMed, Cochrane, Web of Science, and Embase databases. Further, we performed a meta-analysis after screening the literature, quality assessment, and data extraction. RESULTS: The pooled sensitivity, specificity and a rea under the summary receiver operator characteristic curve (AUC) were 0.98 [95% confidence interval (CI):0.97-0.99], 0.99 (95% CI: 0.97-1.00) and 1.00 (95% CI: 0.98-1.00), respectively. For CRISPR-associated (Cas) proteins-12, the sensitivity, specificity was 0.98 (95% CI: 0.96-1.00), 1.00 (95% CI: 0.99-1.00), respectively. For Cas13, the sensitivity and specificity were 0.99 (95% CI: 0.97-1.00) and 0.95 (95% CI: 0.91-1.00). The positive likelihood ratio (PLR) was 183.2 (95% CI: 28.8, 1166.8); the negative likelihood ratio (NLR) was 0.02 (95% CI: 0.01, 0.03). CONCLUSION: RPA/RAA integrated with CRISPR technology is used to diagnose coronavirus disease-19 (COVID-19) with high accuracy and can be used for large-scale population screening.

Simple and Sensitive Multi-components Detection Using Synthetic Nitrogen-doped Carbon Dots Based on Soluble Starch.

Although carbon dots (CDs) as fluorescent sensors have been widely exploited, multi-component detection using CDs without tedious surface modification is always a challenging task. Here, two kinds of nitrogen-doped CDs (NCD-m and NCD-o) based on soluble starch (SS) as carbon source were prepared through one-pot hydrothermal process using m-phenylenediamine and o-phenylenediamine as nitrogenous dopant respectively. Through fluorescence "on-off" mechanism of CDs, NCD-m and NCD-o could be used as a fluorescence sensor for detection of Fe 3+ and Ag + with LOD of 0.25 and 0.51 µM, respectively. Additionally, NCD-m could be used for indirect detection of ascorbic acid (AA) with LOD of 5.02 µM. Moreover, fluorescence intensity of NCD-m also exhibited the sensitivity to pH change from 2 to 13. More importantly, Both NCD-m and NCD-o had potential application for analysis of complicated real samples such as tap water, Vitamin C tablets and orange juice. Ultimately, the small size of NCD-m could contribute to reinforcing intracellular endocytosis, which allowed them to be used for bacteria imaging. Obviously, these easily obtainable nitrogen-doped CDs were able to be used for multi-components detection. Strategy for synthesis of nitrogen-doped carbon dots (NCDs) and a schematic for fabrication of as-prepared NCDs for detection of Fe 3+, Ag + and ascorbic acid (AA).

Toll-Like Receptor-Mediated Activation of CD39 Internalization in BMDCs Leads to Extracellular ATP Accumulation and Facilitates P2X7 Receptor Activation.

Toll-like receptors (TLRs) trigger innate immune responses through their recognition of conserved molecular ligands of either endogenous or microbial origin. Although activation, function, and signaling pathways of TLRs were already well-studied, their precise function in specific cell types, especially innate immune cells, needs to be further clarified. In this study, we showed that when significantly decreased amounts of membrane CD39, an adenosine triphosphate (ATP)-degrading enzyme, were detected in lipopolysaccharide (LPS)-treated bone marrow-derived dendritic cells (BMDCs), Cd39 mRNA expression, and whole-cell CD39 expression were at the same levels as those in untreated BMDCs. Further experiments demonstrated that the downregulation of membrane CD39 expression in LPS-treated BMDCs was mediated by endocytosis, leading to membrane-exposed CD39 downregulation, which was positively associated with decreased enzymatic activity in ATP metabolism and increased extracellular ATP accumulation. The accumulated ATP promoted intracellular calcium accumulation and IL-1ß production in BMDCs through P2X7 signaling activation. Further research revealed that not only LPS but also other TLR ligands, excluding polyI:C, induced CD39 internalization in BMDCs and that the MyD88 pathway was critical in this process. The results suggested that the activation of CD39 internalization in DCs induced by a TLR ligand caused increased ATP accumulation, leading to P2X7 receptor activation that mediated a proinflammatory effect. Considering the strong modulatory effect of extracellular ATP accumulation on the immune response and inflammation, the manipulation of membrane CD39 expression on DCs may have implications on the regulation and treatment of inflammatory responses.

Lysin cell-binding domain-functionalized magnetic beads for detection of Staphylococcus aureus via inhibition of fluorescence of Amplex Red/hydrogen peroxide assay by intracellular catalase.

Accurate and rapid identification of Staphylococcus aureus (S. aureus) is of great significance for controlling the food poisoning and infectious diseases caused by S. aureus. In this study, a novel strategy that combines lysin cell-binding domain (CBD)-based magnetic separation with fluorescence detection was developed for the specific and sensitive quantification of S. aureus in authentic samples. The S. aureus cells were separated from the sample matrix by lysin CBD-functionalized magnetic beads. Following lysis by lysostaphin, intracellular catalase was released from S. aureus cells and detected by a fluorometric system composed of horseradish peroxidase (HRP), hydrogen peroxide (H2O2), and Amplex Red. S. aureus was quantified via the inhibitory effect of the released intracellular catalase on the fluorometric system since the catalase could decompose the H2O2. Optimized conditions afforded a calibration curve for S. aureus ranging from 1.0 × 102 to 1.0 × 107 CFU mL-1. The detection limit was as low as 78 CFU mL-1 in phosphate-buffered saline (PBS), and the total detection process could be completed in less than 50 min. Other bacteria associated with common food-borne and nosocomial infections negligibly interfered with S. aureus detection, except for Staphylococcus epidermidis, which may have slightly interfered. Moreover, the potential of this proposed method for practical applications has been demonstrated by detection assays of sterilized milk and human serum. Graphical abstract.

A novel colorimetric sensing platform for the detection of S. aureus with high sensitivity and specificity.

In this study, a novel colorimetric sensing platform was developed for the detection of S. aureus using dog immunoglobulin G (IgG) as the capture antibody and chicken anti-protein A immunoglobulin Y labeled with horseradish peroxidase (HRP-IgY) as the detection antibody. Dog IgG labeled with magnetic beads was used to capture S. aureus through the interaction between the Fc region of dog IgG and Staphylococcal protein A (SPA). HRP-IgY was introduced to recognize the residual SPA on the surface of S. aureus and to create a sandwich format, after which a soluble 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added. A stop solution was utilized to cease the enzymatic chromogenic reaction, and then optical density was read at 450 nm. Under optimal conditions, the proposed method displayed a low detection limit of 1.0 × 103 CFU mL-1 and a wide linear range of 3.1 × 103 to 2.0 × 105 CFU mL-1. This detection method exhibited high specificity against other foodborne bacteria. The recovery rates ranged from 95.2% to 129.2%. To our knowledge, this is the first report to employ dog IgG and chicken IgY as an antibody pair to detect S. aureus. This technique exhibits high application potential for S. aureus monitoring in various kinds of samples.

Signature of circular RNAs in peripheral blood mononuclear cells from patients with active tuberculosis.

The study was to characterize the expression profiles of circular RNAs (circRNAs) in peripheral blood mononuclear cells (PBMCs) from active tuberculosis (TB) patients and to investigate their function. Microarray was applied to detect circRNA expression and reverse transcription-quantitative polymerase chain reaction was conducted to validate the microarray results. Meanwhile, receiver operating characteristic curve (ROC) curve was calculated to evaluate the predictive power of the selected circRNAs for TB diagnosis. Additionally, circRNA/miRNA interaction was predicted based on miRNA target prediction software, and gene ontology as well as Kyoto Encyclopedia of Genes and Genomes pathway analysis were used to predict their biological function. In total, 171 circRNAs were found to be dysregulated in TB samples. Specifically, circRNA_103017, circRNA_059914 and circRNA_101128 were confirmed to be increased, while circRNA_062400 was decreased in TB samples. ROC analysis revealed that circRNA_103017 had potential value for TB diagnosis, followed by circRNA_059914 and circRNA_101128. Moreover, circRNA_101128 expression in TB samples was negatively correlated with the level of its possible target let-7a and bioinformatics analysis showed that circRNA_101128 was potentially involved in MAPK and P13K-Akt pathway possibly via modulation of let-7a. Taken together, our results indicated that some dysregulated circRNAs were potential biomarkers for the diagnosis of TB and circRNA_101128-let-7a interplay may play considerable role in PBMCs response to Mtb infection.

Aptamer-based fluorometric assay for direct identification of methicillin-resistant Staphylococcus aureus from clinical samples.

Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is of important clinical significance. In this study, a novel aptamer-based fluorometric assay was developed for detection of MRSA in clinical samples by coupling with immunomagnetic separation. The S. aureus cells in clinical specimens were enriched by magnetic separation. Following lysis by staphylococcal lysin, the PBP2a proteins were released from S. aureus cells and detected by the aptamer-based fluorometric assay. Without lengthy period of bacteria cultivation in the traditional susceptibility testing, this test has an overall testing time of only 2 h with the detection limit of 2.63 × 103 and 1.38 × 103 CFU/mL in PBS and spiked nasal swab, respectively. Since it is simple, rapid and sensitive, this method could be used for the detection of MRSA in various clinical samples.

Rapid detection of viral antibodies based on multifunctional Staphylococcus aureus nanobioprobes.

Biosynthesis of nanoparticles inside S. aureus cells has enhanced the sensitivity of immunoassays based on the S. aureus nanoparticles. However, the current methods are limited to antigen detection by conjugating IgG antibodies on S. aureus nanoparticles. In this study, a simple way to conjugate antigens to the S. aureus nanobioparticles was developed by utilizing a cell wall binding domain (CBD) from a bacteriophage lysin PlyV12. Based on this novel design, simple agglutination tests of the IgG antibodies of Ebola virus (EBOV) nucleoprotein (NP) and Middle East Respiratory Virus (MERS) NP in rabbit sera were successfully developed by conjugating the S. aureus nanobioparticles with two fusion proteins EBOV NP- CBD and MERS NP-CBD, respectively. The conjugation was done easily by just mixing the fusion proteins with the S. aureus nanoparticles. The detection time was within 20 min without any special equipment or expertise. As far as we know, this is the first time to realize the detection of viral antibodies based on S. aureus nanoparticles.

Rapid detection of New Delhi metallo-ß-lactamase gene and variants coding for carbapenemases with different activities by use of a PCR-based in vitro protein expression method.

New Delhi metallo-ß-lactamase (NDM)-producing bacteria are considered potential global health threats. It is necessary to monitor NDM-1 and its variants in clinical isolates in order to understand the NDM-1 epidemic and the impact of its variants on ß-lactam resistance. To reduce the lengthy time needed for cloning and expression of NDM-1 variants, a novel PCR-based in vitro protein expression (PCR-P) method was used to detect blaNDM-1 and its variants coding for carbapenemases with different activities (functional variants). The PCR-P method combined a long-fragment real-time quantitative PCR (LF-qPCR) with in vitro cell-free expression to convert the blaNDM-1 amplicons into NDM for carbapenemase assay. The method could screen for blaNDM-1 within 3 h with a detection limit of 5 copies and identify functional variants within 1 day. Using the PCR-P to analyze 5 recent blaNDM-1 variants, 2 functional variants, blaNDM-4 and blaNDM-5, were revealed. In the initial testing of 23 clinical isolates, the PCR-P assay correctly found 8 isolates containing blaNDM-1. This novel method provides the first integrated approach for rapidly detecting the full-length blaNDM-1 and revealing its functional variants in clinical isolates.

PCR-based in vitro synthesis of hepatitis C virus NS3 protease for rapid phenotypic resistance testing of protease inhibitors.

Protease inhibitors (PIs) targeting the hepatitis C virus (HCV) NS3 protease, such as telaprevir, have significantly improved the sustained virologic response (SVR) rates of HCV genotype 1 antiviral therapy. Given the expanding antiviral therapy regimen, fast HCV PI resistance assays are urgently needed. In this view, we have developed a novel phenotypic resistance test for HCV PIs based on in vitro synthesis of patient-derived HCV NS3 protease and subsequent enzymatic testing in a fluorescent readout. The enzymatically active HCV NS3 proteases were synthesized from PCR-derived templates by an Escherichia coli S30 extract system. Tests of the protease genes with known mutations for telaprevir resistance showed that the phenotypic resistance test was fast, with a total turnaround time of <10 h, and was fully in agreement with the previous resistance results. The initial tests with 38 treatment-naive serum samples showed that the method was significantly less laborious and faster than currently available phenotypic resistance assays of HCV NS3 PIs.