Isolation, screening and characterization of phage.

Bacterial resistance threatens public health due to a lack of novel antibacterial classes since the 21st century. Bacteriophages, the most ubiquitous microorganism on Earth and natural predators of bacteria, have the potential to save the world from the post-antibiotic era. Therefore, phage isolation and characterization are in high demand to find suitable phages for therapeutic and bacterial control applications. The chapter presents brief guidance supported by recommendations on the isolation of phages, and initial screening of phage antimicrobial efficacy, in addition to, conducting comprehensive characterization addressing morphological, biological, genomic, and taxonomic features.

Phages for treatment of Klebsiella pneumoniae infections.

Klebsiella pneumoniae is an opportunistic pathogen involved in both hospital- and community-acquired infections. K. pneumoniae is associated with various infections, including pneumonia, septicemia, meningitis, urinary tract infection, and surgical wound infection. K. pneumoniae possesses serious virulence, biofilm formation ability, and severe resistance to many antibiotics especially hospital-acquired strains, due to excessive use in healthcare systems. This limits the available effective antibiotics that can be used for patients suffering from K. pneumoniae infections; therefore, alternative treatments are urgently needed. Bacteriophages (for short, phages) are prokaryotic viruses capable of infecting, replicating, and then lysing (lytic phages) the bacterial host. Phage therapy exhibited great potential for treating multidrug-resistant bacterial infections comprising K. pneumoniae. Hence, this chapter emphasizes and summarizes the research articles in the PubMed database from 1948 until the 15th of December 2022, addressing phage therapy against K. pneumoniae. The chapter provides an overview of K. pneumoniae phages covering different aspects, including phage isolation, different morphotypes of isolated phages, in vitro characterization, anti-biofilm activity, various therapeutic forms, in vivo research and clinical studies.

Morphological, biological, and genomic characterization of Klebsiella pneumoniae phage vB_Kpn_ZC2.

BACKGROUND: Bacteriophages (phages) are one of the most promising alternatives to traditional antibiotic therapies, especially against multidrug-resistant bacteria. Klebsiella pneumoniae is considered to be an opportunistic pathogen that can cause life-threatening infections. Thus, this study aims at the characterization of a novel isolated phage vB_Kpn_ZC2 (ZCKP2, for short). METHODS: The phage ZCKP2 was isolated from sewage water by using the clinical isolate KP/08 as a host strain. The isolated bacteriophage was purified and amplified, followed by testing of its molecular weight using Pulse-Field Gel Electrophoresis (PFGE), transmission electron microscopy, antibacterial activity against a panel of other Klebsiella pneumoniae hosts, stability studies, and whole genome sequencing. RESULTS: Phage ZCKP2 belongs morphologically to siphoviruses as indicated from the Transmission Electron Microscopy microgram. The Pulsed Field Gel Electrophoresis and the phage sequencing estimated the phage genome size of 48.2 kbp. Moreover, the absence of lysogeny-related genes, antibiotic resistance genes, and virulence genes in the annotated genome suggests that phage ZCKP2 is safe for therapeutic use. Genome-based taxonomic analysis indicates that phage ZCKP2 represents a new family that has not been formally rated yet. In addition, phage ZCKP2 preserved high stability at different temperatures and pH values (-20 - 70 °C and pH 4 - 9). For the antibacterial activity, phage ZCKP2 maintained consistent clear zones on KP/08 bacteria along with other hosts, in addition to effective bacterial killing over time at different MOIs (0.1, 1, and 10). Also, the genome annotation predicted antibacterial lytic enzymes. Furthermore, the topology of class II holins was predicted in some putative proteins with dual transmembrane domains that contribute significantly to antibacterial activity. Phage ZCKP2 characterization demonstrates safety and efficiency against multidrug-resistant K. pneumoniae, hence ZCKP2 is a good candidate for further in vivo and phage therapy clinical applications.

Occurrence and Molecular Study of Hypermucoviscous/Hypervirulence Trait in Gut Commensal K. pneumoniae from Healthy Subjects.

Hypervirulent Klebsiella pneumoniae (hvKp) is emerging worldwide. Hypermucoviscousity is the characteristic trait that distinguishes it from classic K. pneumoniae (cKp), which enables Kp to cause severe invasive infections. This research aimed to investigate the hypermucoviscous Kp (hmvKp) phenotype among gut commensal Kp isolated from healthy individuals and attempted to characterize the genes encoding virulence factors that may regulate the hypermucoviscosity trait. Using the string test, 50 identified Kp isolates from healthy individuals' stool samples were examined for hypermucoviscosity and investigated by transmission electron microscopy (TEM). Antimicrobial susceptibility profiles of Kp isolates were determined using the Kirby Bauer disc method. Kp isolates were tested for genes encoding different virulence factors by PCR. Biofilm formation was assayed by the microtiter plate method. All Kp isolates were multidrug-resistant (MDR). Phenotypically, 42% of isolates were hmvKp. PCR-based genotypic testing revealed the hmvKp isolates belonged to capsular serotype K2. All study Kp isolates harbored more than one virulence gene. The genes magA and rmpA were not detected, while the terW gene was present in all isolates. The siderophores encoding genes entB and irp2 were most prevalent in hmvKp isolates (90.5%) and non-hmvKp (96.6%), respectively. hmvKp isolates harbored the genes wabG and uge with rates of 90.5% and 85.7%, respectively. The outcomes of this research highlight the potential health risk of commensal Kp to cause severe invasive diseases, owing to being hmvKp and MDR, and harboring multiple virulence genes. The absence of essential genes related to hypermucoviscosity such as magA and rmpA in hmvKp phenotypes suggests the multifactorial complexity of the hypermucoviscosity or hypervirulence traits. Thus, further studies are warranted to verify the hypermucoviscosity-related virulence factors among pathogenic and commensal Kp in different colonization niches.

Characterization and comprehensive genome analysis of novel bacteriophage, vB_Kpn_ZCKp20p, with lytic and anti-biofilm potential against clinical multidrug-resistant Klebsiella pneumoniae.

Introduction: The rise of infections by antibiotic-resistant bacterial pathogens is alarming. Among these, Klebsiella pneumoniae is a leading cause of death by hospital-acquired infections, and its multidrug-resistant strains are flagged as a global threat to human health, which necessitates finding novel antibiotics or alternative therapies. One promising therapeutic alternative is the use of virulent bacteriophages, which specifically target bacteria and coevolve with them to overcome potential resistance. Here, we aimed to discover specific bacteriophages with therapeutic potential against multiresistant K. pneumoniae clinical isolates. Methods and Results: Out of six bacteriophages that we isolated from urban and medical sewage, phage vB_Kpn_ZCKp20p had the broadest host range and was thus characterized in detail. Transmission electron microscopy suggests vB_Kpn_ZCKp20p to be a tailed phage of the siphoviral morphotype. In vitro evaluation indicated a high lytic efficiency (30 min latent period and burst size of ∼100 PFU/cell), and extended stability at temperatures up to 70°C and a wide range of (2-12) pH. Additionally, phage vB_Kpn_ZCKp20p possesses antibiofilm activity that was evaluated by the crystal violet assay and was not cytotoxic to human skin fibroblasts. The whole genome was sequenced and annotated, uncovering one tRNA gene and 33 genes encoding proteins with assigned functions out of 85 predicted genes. Furthermore, comparative genomics and phylogenetic analysis suggest that vB_Kpn_ZCKp20p most likely represents a new species, but belongs to the same genus as Klebsiella phages ZCKP8 and 6691. Comprehensive genomic and bioinformatics analyses substantiate the safety of the phage and its strictly lytic lifestyle. Conclusion: Phage vB_Kpn_ZCKp20p is a novel phage with potential to be used against biofilm-forming K. pneumoniae and could be a promising source for antibacterial and antibiofilm products, which will be individually studied experimentally in future studies.

Relation Between Familial Mediterranean Fever and QT Markers (QTc, QTd, and QTcd): A Systematic Review and Meta-Analysis.

The aim of this study is to perform a meta-analysis to evaluate the possible association betweenQT markers and familial Mediterranean fever (FMF). PUBMED, Web of Science, OVID, and SCOPUS databases were searched. Inclusion criteria were randomized control trials or observational studies that compared measurement of the QT markers in FMF patients and healthy controls in both males and females without any age restriction or other comorbidities. RevMan software (5.4) was used to perform the analysis. A total of 14 studies with 1,154 individuals were included in the study. The pooled effect estimate showed a statistically significant association between FMF group and prolonged corrected QT (QTc) and QT dispersion (QTd) (MD= 7.06, 95% CI = 2.68 to 11.43, p-value = 0.002) and (MD= 6.08, 95% CI = 0.84 to 11.32, p-value= 0.02), respectively. No statistically significant difference between FMF group and QT interval and corrected QT dispersion (QTcd) (MD= 2.34, 95% CI = -1.21 to 5.89, p-value = 0.20) and (MD= 4.82, 95% CI = -0.57 to 10.20, p-value = 0.08), respectively. Our findings revealed a statistically significant relationship between FMF and extended QTc and QTd. More randomized multicenter trials are required to confirm our findings.

Bacteriophage as a potential therapy to control antibiotic-resistant Pseudomonas aeruginosa infection through topical application onto a full-thickness wound in a rat model.

BACKGROUND: Antibiotic-resistant Pseudomonas aeruginosa (P. aeruginosa) is one of the most critical pathogens in wound infections, causing high mortality and morbidity in severe cases. However, bacteriophage therapy is a potential alternative to antibiotics against P. aeruginosa. Therefore, this study aimed to isolate a novel phage targeting P. aeruginosa and examine its efficacy in vitro and in vivo. RESULTS: The morphometric and genomic analyses revealed that ZCPA1 belongs to the Siphoviridae family and could infect 58% of the tested antibiotic-resistant P. aeruginosa clinical isolates. The phage ZCPA1 exhibited thermal stability at 37 °C, and then, it decreased gradually at 50 °C and 60 °C. At the same time, it dropped significantly at 70 °C, and the phage was undetectable at 80 °C. Moreover, the phage ZCPA1 exhibited no significant titer reduction at a wide range of pH values (4-10) with maximum activity at pH 7. In addition, it was stable for 45 min under UV light with one log reduction after 1 h. Also, it displayed significant lytic activity and biofilm elimination against P. aeruginosa by inhibiting bacterial growth in vitro in a dose-dependent pattern with a complete reduction of the bacterial growth at a multiplicity of infection (MOI) of 100. In addition, P. aeruginosa-infected wounds treated with phages displayed 100% wound closure with a high quality of regenerated skin compared to the untreated and gentamicin-treated groups due to the complete elimination of bacterial infection. CONCLUSION: The phage ZCPA1 exhibited high lytic activity against MDR P. aeruginosa planktonic and biofilms. In addition, phage ZCPA1 showed complete wound healing in the rat model. Hence, this research demonstrates the potential of phage therapy as a promising alternative in treating MDR P. aeruginosa.

In trust we trust: The impact of trust in government on excess mortality during the COVID-19 pandemic.

The COVID-19 pandemic has brought forward myriad challenges to public policy, central of which is understanding the different contextual factors that can influence the effectiveness of policy responses across different systems. In this article, we explore how trust in government can influence the ability of COVID-19 policy responses to curb excess mortality during the pandemic. Our findings indicate that stringent policy responses play a central role in curbing excess mortality. They also indicate that such relationship is not only influenced by systematic and structural factors, but also by citizens' trust in government. We leverage our findings to propose a set of recommendations for policymakers on how to enhance crisis policymaking and strengthen the designs of the widely used underlying policy learning processes.

A Guide to Benchmarking COVID-19 Performance Data.

If the COVID-19 pandemic has taught us anything, it is that policy makers, experts, and public managers need to be capable of interpreting comparative data on their government's performance in a meaningful way. Simultaneously, they are confronted with different data sources (and measurements) on COVID-19 without necessarily having the tools to assess these sources strategically. Because of the speed with which decisions are required and the different data sources, it can be challenging for any policy maker, expert, or public manager to make sense of how COVID-19 has an impact, especially from a comparative perspective. Starting from the question "How can we benchmark COVID-19 performance data across countries?," this article presents important indicators, measurements, and their strengths and weaknesses, and concludes with practical recommendations. These include a focus on measurement equivalence, systems thinking, spatial and temporal thinking, multilevel governance, and multimethod designs.