Changchang Li

Bio: Changchang Li is an academic researcher from Chinese Academy of Sciences. The author has contributed to research in topics: Acinetobacter baumannii & Multiplex. The author has an hindex of 2, co-authored 5 publications receiving 18 citations.

A Novel Acinetobacter baumannii Bacteriophage Endolysin LysAB54 With High Antibacterial Activity Against Multiple Gram-Negative Microbes

Abstract: The rapid spread and emergence of multidrug-resistant Acinetobacter baumannii and other pathogenic Gram-negative bacteria spurred scientists and clinicians to look for alternative therapeutic agents to conventional antibiotics. In the present study, an A. baumannii bacteriophage p54 was isolated and characterized. Morphological and genome analysis revealed that bacteriophage p54 belongs to Myoviridae family with a genome size of 165,813 bps. A novel endolysin, namely LysAB54, showing low similarity with other well-known related endolysins, was cloned, expressed, and characterized from the bacteriophage p54. LysAB54 showed significant bactericidal activity against multidrug-resistant A. baumannii and other Gram-negative bacteria, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli, in the absence of outer membrane permeabilizers. Based on all those observations, LysAB54 could represent a potential agent for the treatment of multidrug-resistant Gram-negative superbugs.

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing.

Abstract: Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening.Methods: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system.Results: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug could not.Conclusion: Our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude-based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Abstract: Multiplexing has been highlighted to save on costs, increase sample throughput, and maximize on the number of targets that can be sensitively detected within a small sample. With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. In this study, we developed simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and fourplex (4 targets) assays based on a two color ddPCR system for SARS-CoV-2 detection. Results showed that the fourplex assay had the similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical isolates demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug couldn’t. Conclusively, our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.

Intrinsic Antimicrobial Peptide Facilitates a New Broad-Spectrum Lysin LysP53 to Kill Acinetobacter baumannii In Vitro and in a Mouse Burn Infection Model.

Abstract: Antimicrobial resistance-related infections of Gram-negative pathogens pose a huge threat to global public health. Lysins, peptidoglycan hydrolases from bacteriophages, are expected as an alternative weapon against drug-resistant bacteria. In the present study, we report a new lysin LysP53 from Acinetobacter baumannii phage 53. Bioinformatic analysis revealed that LysP53 contains a positively charged N-terminal region and a putative peptidase catalytic domain. In vitro biochemical experiments showed that LysP53 is active against multiple antibiotic-resistant Gram-negative bacteria, including A. baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli, with a reduction of 5 logs in viable A. baumannii number after exposure to 100 μg/mL LysP53 for 1 h. Further studies showed that LysP53 contains a functional antimicrobial peptide, i.e., N-terminal 33 aa, with a comparable spectrum of activity to LysP53. In an A. baumannii-associated mouse model of burn infection, a single dose of 14 μg/mouse LysP53 (57.6 μM) showed higher decolonization efficacy than 4 μg/mouse minocycline- (874 μM; p < 0.05) and buffer-treated groups (p <0.001), leading to a bacterial reduction of 3 logs. Our findings collectively establish that LysP53 could be a promising candidate in the treatment of topical infections caused by multiple Gram-negative pathogens.

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification.

Abstract: Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

Endolysin, a Promising Solution against Antimicrobial Resistance.

Abstract: Antimicrobial resistance (AMR) is a global crisis for human public health which threatens the effective prevention and control of ever-increasing infectious diseases. The advent of pandrug-resistant bacteria makes most, if not all, available antibiotics invalid. Meanwhile, the pipeline of novel antibiotics development stagnates, which prompts scientists and pharmacists to develop unconventional antimicrobials. Bacteriophage-derived endolysins are cell wall hydrolases which could hydrolyze the peptidoglycan layer from within and outside of bacterial pathogens. With high specificity, rapid action, high efficiency, and low risk of resistance development, endolysins are believed to be among the best alternative therapeutic agents to treat multidrug resistant (MDR) bacteria. As of now, endolysins have been applied to diverse aspects. In this review, we comprehensively introduce the structures and activities of endolysins and summarize the latest application progress of recombinant endolysins in the fields of medical treatment, pathogen diagnosis, food safety, and agriculture.

Pharmaceutical Approaches on Antimicrobial Resistance: Prospects and Challenges.

Abstract: The rapid increase in pathogenic microorganisms with antimicrobial resistant profiles has become a significant public health problem globally. The management of this issue using conventional antimicrobial preparations frequently results in an increase in pathogen resistance and a shortage of effective antimicrobials for future use against the same pathogens. In this review, we discuss the emergence of AMR and argue for the importance of addressing this issue by discovering novel synthetic or naturally occurring antibacterial compounds and providing insights into the application of various drug delivery approaches, delivered through numerous routes, in comparison with conventional delivery systems. In addition, we discuss the effectiveness of these delivery systems in different types of infectious diseases associated with antimicrobial resistance. Finally, future considerations in the development of highly effective antimicrobial delivery systems to combat antimicrobial resistance are presented.

Current Status of Endolysin-Based Treatments against Gram-Negative Bacteria

Abstract: The prevalence of multidrug-resistant Gram-negative bacteria is a public health concern. Bacteriophages and bacteriophage-derived lytic enzymes have been studied in response to the emergence of multidrug-resistant bacteria. The availability of tRNAs and endolysin toxicity during recombinant protein expression is circumvented by codon optimization and lower expression levels using inducible pET-type plasmids and controlled cultivation conditions, respectively. The use of polyhistidine tags facilitates endolysin purification and alters antimicrobial activity. Outer membrane permeabilizers, such as organic acids, act synergistically with endolysins, but some endolysins permeate the outer membrane of Gram-negative bacteria per se. However, the outer membrane permeation mechanisms of endolysins remain unclear. Other strategies, such as the co-administration of endolysins with polymyxins, silver nanoparticles, and liposomes confer additional outer membrane permeation. Engineered endolysins comprising domains for outer membrane permeation is also a strategy used to overcome the current challenges on the control of multidrug-resistant Gram-negative bacteria. Metagenomics is a new strategy for screening endolysins with interesting antimicrobial properties from uncultured phage genomes. Here, we review the current state of the art on the heterologous expression of endolysin, showing the potential of bacteriophage endolysins in controlling bacterial infections.