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.

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.

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.

Peroral endoscopic myotomy (POEM) for the treatment of achalasia: A multicenter Middle Eastern experience.

BACKGROUND: Peroral endoscopic myotomy (POEM) was proposed in 2010 as a minimally invasive procedure for the treatment of achalasia. In this article, we describe the Middle Eastern experience with the procedure in terms of efficacy, length of admission, and short- and long-term complications. METHODS: A retrospective analysis of our prospectively collected data on patients who underwent a POEM procedure was conducted between March 2019 and May 2020. The primary outcome was clinical success rate, defined as a postprocedure Eckardt score ≤3 at ≥3 months. Secondary outcomes included the length of hospital stay, presence of reflux symptoms or need for proton pump inhibitors (PPIs) ≥3 months, and adverse events. RESULTS: During the study period, 67 patients (35 females) underwent the procedure for achalasia. The participants' ages ranged from 11 to 80 years (mean 41 ± 18 years). Eckardt scores before the treatment ranged between 4 and 12 (mean 8.85 ± 1.75). Sixty-four patients (95.5%) achieved Eckardt scores of ≤3 at ≥3 months after the procedure (95% confidence interval [CI]: 91%-100%). The difference between pre- and post-procedural Eckardt scores averaged around -8 points (95% CI: -7.5 to -8.5 P < 0.0001). Adverse events were reported in 24 patients (35.8%) and included pneumoperitoneum (32.8%), reflux symptoms at 3 months (29.9%), and surgical emphysema (3%). Six patients had adverse events that led to prolongation of admission; 3% of whom had aspiration pneumonia, 3% had pneumoperitoneum, 1.5% had both, and 1.5% had an esophageal tear. CONCLUSIONS: POEM is a promising procedure for the treatment of achalasia with a high clinical success rate, short hospital admission, and a reassuring safety profile.

Peroral endoscopic myotomy as treatment for Zenker's diverticulum (Z-POEM): a multi-center international study.

BACKGROUND: Peroral endoscopic myotomy for the treatment of Zenker's diverticulum (Z-POEM) is a novel technique that has been described in several recent reports. This method utilizes the third space (submucosal layer) to create a tunnel to facilitate complete visualization of the septum and hence cutting it entirely. Conventional endoscopic septotomy carries the risk of recurrence due to incomplete visualization of the septum. While surgical correction is a risky and lengthy procedure in old comorbid patients with Zenker's diverticulum. The aim of this study is to assess the efficacy and safety of Z-POEM. METHODS: The study enrolled 24 patients diagnosed with Zenker's diverticulum (ZD) who underwent Z-POEM at seven independent endoscopy centers in five different countries. RESULTS: Mean patient age ± standard deviation (SD) was 74.3 ± 11 years. Most of the patients were males (n = 20, 83.3%); four (16.7%) were females. More than 50% of the patients (n = 14, 58.3%) had associated comorbidities. The mean size of the diverticula was 4 cm (range 2-7 cm). The Kothari-Haber Score was used to assess clinical symptoms; values ranged from 6 to 14 (median = 9). We achieved 100% technical success with a median procedure time of 61 min and no adverse events. Median hospital stay was 1 day (range 1-5 days). There is a significant reduction in the Kothari-Haber Score after Z-POEM (P < 0.0001). Technical success was achieved in 100% of the patients. Clinical success was achieved in 23/24 (95.8%) of the patients with a median follow-up of 10 months (range 6-24 months). CONCLUSION: Z-POEM is a safe and effective modality for managing ZD.

Tissue Inhibitors of Metalloproteinase-1 and 2 and Obesity Related Non-Alcoholic Fatty Liver Disease: Is There a Relationship.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease is a spectrum of clinical conditions, including simple steatosis and non-alcoholic steatohepatitis (NASH). The aim of the study is to evaluate the tissue inhibitors of metalloproteinase-1 and 2 (TIMPs) as noninvasive predictors of NASH. METHODS: Three groups were included in the study. Obese patients (n = 30) with normal liver enzymes were included in group I and obese patients (n = 30) with elevated liver enzymes with liver biopsy-based diagnosis of NASH were included in group II. Age-matched subjects (n = 30) formed the control as group III. The lipid profile, liver enzyme levels and levels of TIMPs were compared among all the patients and subjects. RESULTS: Comparison of groups I and II showed significantly elevated levels of TIMP-1 and TIMP-2 in group II as compared to group I (p < 0.05). Similarly, comparison between groups II and III showed significantly increased levels of TIMP-1 and TIMP-2 in group II as compared to group III (p < 0.05). TIMP-1 (sensitivity 96.7%, specificity 100%) and TIMP-2 (sensitivity 93.3%, specificity 100%) showed high accuracy in NASH diagnosis. CONCLUSION: TIMP-1 and TIMP-2 may be considered noninvasive markers for the diagnosis of NASH.