グエン ミンフオン

Phage therapy, the use of bacteriophages (phages) to treat bacterial infections, is regaining momentum as a promising weapon against the rising threat of multidrug-resistant (MDR) bacteria. This comprehensive review explores the historical context, the modern resurgence of phage therapy, and phage-facilitated advancements in medical and technological fields. It details the mechanisms of action and applications of phages in treating MDR bacterial infections, particularly those associated with biofilms and intracellular pathogens. The review further highlights innovative uses of phages in vaccine development, cancer therapy, and as gene delivery vectors. Despite its targeted and efficient approach, phage therapy faces challenges related to phage stability, immune response, and regulatory approval. By examining these areas in detail, this review underscores the immense potential and remaining hurdles in integrating phage-based therapies into modern medical practices.

Abstract In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.

ABSTRACT The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to β-lactam antibiotics, these strains can easily acquire reduced β-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC , which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced β-lactam susceptibility, as it does not directly associate with the expression of mecA . This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoB ^H929P , RpoB ^Q645H , RpoC ^G950R , RpoC ^G498D , RpiA ^A64E , and FruB ^A211E , using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to β-lactam in OS-MRSA. IMPORTANCE The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to β-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC , leading to treatment failure. This study investigated the mechanisms underlying reduced β-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to β-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Abstract Transforming growth factor-β-activated kinase 1 (TAK1) plays a pivotal role in innate and adaptive immunity. TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways downstream of diverse immune receptors, including toll-like receptors (TLRs). Upon stimulation with TLR ligands, TAK1 is activated via recruitment to the lysine 63-linked polyubiquitin chain through TAK1-binding protein 2 (TAB2) and TAB3. However, the physiological importance of TAB2 and TAB3 in macrophages is still controversial. A previous study has shown that mouse bone marrow-derived macrophages (BMDMs) isolated from mice double deficient for TAB2 and TAB3 produced tumor necrosis factor (TNF)-α and interleukin (IL)-6 to the similar levels as control wild-type BMDMs in response to TLR ligands such as lipopolysaccharide (LPS) or Pam3CSK4, indicating that TAB2 and TAB3 are dispensable for TLR signaling. In this study, we revisited the role of TAB2 and TAB3 using an improved mouse model. We observed a significant impairment in the production of pro-inflammatory cytokines and chemokine in LPS- or Pam3CSK4-treated BMDMs deficient for both TAB2 and TAB3. Double deficiency of TAB2 and TAB3 resulted in the decreased activation of NF-κB and MAPK pathways as well as the slight decrease in TAK1 activation in response to LPS or Pam3CSK4. Notably, the TLR-mediated expression of inhibitor of NF-κB (IκB)ζ was severely compromised at the protein and messenger RNA (mRNA) levels in the TAB2/TAB3 double-deficient BMDMs, thereby impeding IL-6 production. Our results suggest that TAB2 and TAB3 play a redundant and indispensable role in the TLR signaling pathway.

RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.

Salmonella enterica is one of the most dangerous foodborne pathogens listed by the World Health Organization. In this study, whole-duck samples were collected at wet markets in five districts in Hanoi, Vietnam, in October 2019 to assess their Salmonella infection rates and evaluate the susceptibility of the isolated strains to antibiotics currently used in the prophylaxis and treatment of Salmonella infection. Based on the antibiotic resistance profiles, eight multidrug resistance strains were whole-genome-sequenced, and their antibiotic resistance genes, genotypes, multi-locus sequence-based typing (MLST), virulence factors, and plasmids were analyzed. The results of the antibiotic susceptibility test indicate that phenotypic resistance to tetracycline and cefazolin was the most common (82.4%, 28/34 samples). However, all isolates were susceptible to cefoxitin and meropenem. Among the eight sequenced strains, we identified 43 genes associated with resistance to multiple classes of antibiotics such as aminoglycoside, beta-lactam, chloramphenicol, lincosamide, quinolone, and tetracycline. Notably, all strains carried the blaCTX-M-55 gene, which confers resistance to third-generation antibiotics including cefotaxime, cefoperazone, ceftizoxime, and ceftazidime, as well as resistance genes of other broad-spectrum antibiotics used in clinical treatment such as gentamicin, tetracycline, chloramphenicol, and ampicillin. Forty-three different antibiotic resistance genes were predicted to be present in the isolated Salmonella strains’ genomes. In addition, three plasmids were predicted in two strains, 43_S11 and 60_S17. The sequenced genomes also indicated that all strains carried SPI-1, SPI-2, and SPI-3. These SPIs are composed of antimicrobial resistance gene clusters and thus represent a potential threat to public health management. Taken together, this study highlights the extent of multidrug-resistant Salmonella contamination in duck meat in Vietnam.

Autoinducers enable bacteria to sense cell density and to coordinate collective behavior. NTD/kanosamine is an autoinducer produced by B. subtilis and several close relatives, although its physiological function remains unknown.

<jats:p>In Vietnam and around the world, Staphylococcus aureus remains a major hazard of food safety and food poisoning. S. aureus is present in many places and easily contaminates food production during processing chains. In this study, we successfully isolated S. aureus strains from suspected samples of two food borne poisoning outbreaks in Ha Giang and Vinh Phuc in 2017 and 2018, respectively. The collected samples were examined for presence of staphylococcal enterotoxins (SEs) by using 3MTMTECRATM Staph Enterotoxin kit, from there all the samples were positive with SEs. Different strains of S. aureus were isolated and then confirmed by MALDI-TOF technique. Those strains then were stored in Brain heart solution with 15% glycerol until further analysis. Our results identified three STs, ST96, ST88 (spa type t7558), and ST72 (spa type t3092), were responsible for two outbreaks. Two virulence genes detected from the above strains were sea and sec. Furthermore, these strains are test for antibiotic resistance susceptibility with commonly antibiotics. Penicillin are found to be resisted by all three STs, in particularly, ST96 and ST88 are both resistant to erythromycin while ST72 is resistant to gentamicin. Taken together, our study highlights the usefulness of molecular characterization to study and monitor bacterial pathogens associated with food poisoning outbreaks in Vietnam.</jats:p>

Background Neisseria meningitidis remains the main cause of sporadic meningitis and sepsis in military camps in Vietnam. Yet, very limited molecular data of their genotypic and epidemiological characteristics are available from Vietnam, and particularly the military environment. Whole genome sequencing (WGS) has proven useful for meningococcal disease surveillance and guiding preventative vaccination programs. Previously, we characterized key genetic and epidemiological features of an invasive N. meningitidis B isolate from a military unit in Vietnam. Here, we extend these findings by sequencing two additional invasive N. meningitidis B isolated from cerebrospinal fluid (CSF) of two meningitis cases at another military unit and compared their genomic sequences and features. We also report the sequence types and antigenic profiles of 25 historical and more recently emerged N. meningitidis isolates from these units and other units in proximity. Methods Strains were sequenced using the Illumina HiSeq platform, de novo assembled and annotated. Genomes were compared within and between military units, as well as against the global N. meningitidis collection and other isolates from the Southeast Asia region using PubMLST. Variations at the nucleotide level were determined, and phylogenetic relationships were estimated. Antigenic genotypes and vaccine coverage were analyzed using gMATS and PubMLST. Susceptibility of isolates against commonly used antibiotic agents was examined using E-test. Results Genome comparison revealed a high level of similarity among isolates both within and between units. All isolates showed resistance to chloramphenicol and carried identical catP gene with other Southeast Asian isolates, suggesting a common lineage. Their antigenic genotypes predicted no coverage by either Bexsero^®or Trumenba^®, and nucleotide variation analysis revealed diverse new, unassigned alleles at multiple virulence loci of all strains. Groups of singleton and unique novel sequence types extending beyond individual camps were found from epidemiological data of 25 other isolates. Our results add to the sparse published molecular data of N. meningitidis in the military units in Vietnam, highlight their diversity, distinct genetic features and antibiotic resistance pattern, and emphasize the need for further studies on the molecular characteristics of N. meningitidis in Vietnam.

<jats:p>Meningococcal bacterium Neisseria meningitidis is one of the major causes of meningitis worldwide. Currently in Vietnam, N. meningitidis B and C are the most prevalent invasive serogroups. N. meningitidis expresses two major transmembrane proteins, PorA and PorB, of which PorA showed high levels of polymorphism among strains and has been studied as one component of vaccines against N. meningitidis B. Polymorphisms in porA gene clustered into three variable regions VR1, VR2 and VR3. In this study, we sequenced the entired porA gene of 19 N. meningitidis strains isolated from military units in northern Vietnam during the period 2008 – 2017 in order to: (1) evaluate the polymorphism of porA gene at nucleotide level and predict its immuno-reactivity from annotated amino acide sequences; and (2) compare porA variants of Vietnamese strains with known strains worldwide. Our results showed a high level of polymorphisms among Vietnamese strains, with the highest polymorphism found in VR1, followed by VR2 and VR3. Notably, 5/19 strains in this study showed novel variants at VR2 with unknown immunological reactivity. This result suggested potential novel immunological characteristics of meningococcal strains from Vietnam.</jats:p>

<jats:p>The discovery of antibiotics is considered to be one of the greatest medical achievements in the early part of 20th. Over the past six decades, these ‘wonder drugs’ have played a critical role in reducing the global burden of communicable diseases. As a country in the tropical zone, Vietnam faces numerous infectious disease outbreaks every year. In addition, the overuse of antibiotics in healthcare and the misuse of antibiotics as growth promoter in agriculture these past decades have led to serious antibiotic-resistance in bacterial pathogens of human, crops and livestock in Vietnam. This poses an urgent need for alternative strategies to fight against these pathogens. Lysins are phage-encoded peptidoglycan hydrolases which were recently demonstrated the strong potential in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. These enzymes when applied exogenously to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell and therefore promise an effective alternative therapy against antibiotic-resistant bacterial pathogens. In this study, we applied the DNA technology to produce a recombinant S. aureus lysin (LysK). LysK was PCR amplified from phage S. aureus and cloned into a pET32a(+) expression vector. The recombinant fusion protein (LysK-Trx) was successfully expressed in E. coli, purified through nickel column chromatography, and further digested with Thrombin protease. The cleaved protein (intact LysK) was purified by nickel column again. The recombinant LysK was tested for its ability to kill S. aureus by a spot inoculation assay. The results showed that recombinant LysK induced the lysis of host bacteria, indicating that the protein was expressed and functionally active. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains in Vietnam.</jats:p>

<jats:p>The green alga Chlamydomonas reinhardtii is a model organism for studying mechanism of the body in response to changes in environmental conditions, photosynthesis as well as chloroplast biogenesis and can be modified to produce high value proteins. In this study, we present results on the selection of cultural conditions for the growth of cell wall - deficient mutant C. reinhardtii (503 strain- control) and recombinant 503-2 strain expressing VP28 protein of White Spot Syndrome Virus (WSSV) in 250 ml flasks. Suitable conditions for the growth of both control and recombinant strains were TAP medium, temperature of 25 C, pH = 7, light intensity of 2 klux, C source - Trisbase, N source - nitrate and salinity of 50 - 100 mM. In these conditions, maximum cell density of the control and recombinant strains was 3.20 ± 0.08 x 106 and 2.96 ± 0.17 x 106 cells/ mL after 3 and 7 days of cultivation, respectively. The presence of vp28 gene in the recombinant microalgal strain was stably maintained at all levels of culture. The results of this paper will be a scientific basis for culturing the recombinant C. reinhardtii to obtain algal biomass which had high expression of surface VP28 protein of WSSV’s envelope for edible vaccine production vaccine by feeding in order to prevent white spot disease of shrimp.</jats:p>

<jats:p>White spot syndrome virus (WSSV) is the leading cause of shrimp mortality in farms all over the world. In Vietnam, for the last five to ten years, WSSV has always been among the top causes of diseases and loss in our shrimp aquaculture. VP28 and VP26 are two capsid proteins of WSSV that commonly used as biomarkers for diagnosis and target antigens for vaccine against WSSV. Recombinant VP28 (rVP28) has been studied and expressed in various expression systems including E. coli, yeast and baculovirus. rVP28 expressed in these systems showed effective protection against WSSV in shrimps, though there remains limitation on expression efficiency and safety. Recently, green microalgae Chlamydomonas reinhardtii has been widely used to express pharmaceutical proteins and edible vaccines for aquaculture thanks to its advantages as a safe and efficient host. C. reinhardtii is also used as nutrious natural food for shrimps due to its benefits towards shrimp health and growth. In this study, the codons of vp28 gene was adapted and chemically synthesized, and transformed into the nucleus genome of C. reinhardtii using electroporation. The presence of a codon optimized vp28 gene in C. reinhardtii genome was confirmed by colony PCR and sequencing; and its expression level was examined by RT-PCR. These results proved our success in creating transgenic C. reinhardtiii expressing rVP28 and set the foundation for our future research on edible vaccine against WSSV for shrimp.</jats:p>

ABSTRACT Bacteriophage T7 terminator Tφ is a class I intrinsic terminator coding for an RNA hairpin structure immediately followed by oligo(U), which has been extensively studied in terms of its transcription termination mechanism, but little is known about its physiological or regulatory functions. In this study, using a T7 mutant phage, where a 31-bp segment of Tφ was deleted from the genome, we discovered that deletion of Tφ from T7 reduces the phage burst size but delays lysis timing, both of which are disadvantageous for the phage. The burst downsizing could directly result from Tφ deletion-caused upregulation of gene 17.5 , coding for holin, among other Tφ downstream genes, because infection of gp17.5-overproducing Escherichia coli by wild-type T7 phage showed similar burst downsizing. However, the lysis delay was not associated with cellular levels of holin or lysozyme or with rates of phage adsorption. Instead, when allowed to evolve spontaneously in five independent adaptation experiments, the Tφ-lacking mutant phage, after 27 or 29 passages, recovered both burst size and lysis time reproducibly by deleting early genes 0.5 , 0.6 , and 0.7 of class I, among other mutations. Deletion of genes 0.5 to 0.7 from the Tφ-lacking mutant phage decreased expression of several Tφ downstream genes to levels similar to that of the wild-type phage. Accordingly, phage T7 lysis timing is associated with cellular levels of Tφ downstream gene products. This suggests the involvement of unknown factor(s) besides the known lysis proteins, lysozyme and holin, and that Tφ plays a role of optimizing burst size and lysis time during T7 infection. IMPORTANCE Bacteriophages are bacterium-infecting viruses. After producing numerous progenies inside bacteria, phages lyse bacteria using their lysis protein(s) to get out and start a new infection cycle. Normally, lysis is tightly controlled to ensure phage progenies are maximally produced and released at an optimal time. Here, we have discovered that phage T7, besides employing its known lysis proteins, additionally uses its transcription terminator Tφ to guarantee the optimal lysis of the E. coli host. Tφ, positioned in the middle of the T7 genome, must be inactivated at least partially to allow for transcription-driven translocation of T7 DNA into hosts and expression of Tφ downstream but promoter-lacking genes. What role is played by Tφ before inactivation? Without Tφ, not only was lysis time delayed but also the number of progenies was reduced in this study. Furthermore, T7 can overcome Tφ deletion by further deleting some genes, highlighting that a phage has multiple strategies for optimizing lysis.