Isolation and identification of specific lytic bacteriophages as a biocontrol agent against Serratia odorifera PBA-IAUF-1 and Kluyvera intermedia PBA-IAUF-6 causing bacterial canker in the grape and Siberian pear.

Bacterial canker, a prevalent disease among fruit trees, is a significant concern. The use of phage therapy is presently seen as a dependable biological strategy to control bacterial diseases in fruits. The objective of this research was to use various biochemical and molecular techniques to determine the types of bacteria responsible for causing cankers in various fruits. Additionally, their ability to cause disease in the fruit tissues was assessed, the specific bacteriophages targeting these bacteria were isolated and identified. The bacteria were separated from different parts of the infected fruits like grapes and Siberian pears. The selection of fruit tissues showing signs of canker disease was performed, and the validation of the isolates' pathogenicity was confirmed following Koch's principles. Subsequently, in order to establish a conclusive identification of the bacterial species, molecular identification was conducted through the sequencing of a specific fragment within the 16S rRNA following amplification by PCR by using universal primers, RW01 and DG74. Isolation and titration of phages specific to fruit spoilage bacteria was done by spot and double-layer agar method, and the growth curve of the isolated bacteriophage was drawn. The phages were detected by transmission electron microscopy (TEM). The results of the study proved the presence of canker causing agents, Kluyvera intermedia PBA-IAUF-6 with the code Sh6 in the Siberian pears, and Serratia odorifera PBA-IAUF-1 with the code Rz3 in the grape fruits, which were deposited in GenBank with the accession numbers of KU878579 and KU168605, respectively. Isolation of the specific bacteriophages to the S. odorifera PBA-IAUF-1 and K. intermedia PBA-IAUF-6 bacterial strains were done from the effluent of South Isfahan wastewater treatment plant and Caspian Sea water, respectively. The titer of the specific phage to S. odorifera PBA-IAUF-1 and K. intermedia PBA-IAUF-6 was detected in the samples as 2.2 × 10-5 and 5 × 10-11 PFU/ml, respectively. An electron micrograph of a bacteriophage that targets two different bacterial strains revealed phages with a geometrically shaped head and a flexible tail, which resembled viruses from the Siphoviridae family.

Bacteriophages PɸEn-CL and PɸEn-HO can eliminate MDR Enterobacter cloacae and Enterobacter hormaechei isolated from burn wound infections without toxicity for human skin cells.

The prevalence of multidrug-resistant (MDR) strains has caused serious problems in the treatment of burn infections. MDR Enterobactercloacae and Enterobacterhormaechei have been defined as the causative agents of nosocomial infections in burn patients. In this situation, examination of phages side effects on human cell lines before any investigation on human or animal that can provide beneficial information about the safety of isolated phages. The aim of this study was to isolate and identify the specific bacteriophages on MDR E. cloacae and E. hormaechei isolated from burn wounds and to analyze the efficacy, cell viability and cell cytotoxicity of phages on A-375 and HFSF-PI cell lines by MTT (3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) colorimetric assay and lactate dehydrogenase (LDH) release assay. Phages were isolated from urban sewage Isfahan, Iran. Enterobactercloacae strain Iau-EC100 (GenBank accession number: MZ314381) and E. hormaechei strain Iau-EHO100 (GenBank accession number: MZ348826) were sensitive to the isolated phages. Transmission electron microscopy (TEM) results revealed that PɸEn-CL and PɸEn-HO that were described had the morphologies of Myovirus and Inovirus, respectively. Overall, MTT and LDH assays showed moderate to excellent correlation in the evaluation of cytotoxicity of isolated phages. The results of MTT and LDH assays showed that, phages PɸEn-CL and PɸEn-HO had no significant toxicity effect on A375 and HFSF-PI 3 cells. Phage PɸEn-HO had a better efficacy on the two tested cell lines than other phage. Our results indicated that, there were significant differences between the two cytotoxicity assays in phage treatment compared to control.

Isolation, characterization, and effectiveness of bacteriophage Pɸ-Bw-Ab against XDR Acinetobacter baumannii isolated from nosocomial burn wound infection.

OBJECTIVES: With emergence of drug resistance, novel approaches such as phage therapy for treatment of bacterial infections have received significant attention. The purpose of this study was to isolate and identify effective bacteriophages on extremely drug-resistant (XDR) bacteria isolated from burn wounds. MATERIALS AND METHODS: Pathogenic bacteria were isolated from hospitalized patient wounds in specialized burn hospitals in Iran, and their identification was performed based on biochemical testing and sequencing of the gene encoding 16S rRNA. Bacteriophages were isolated from municipal sewage, Isfahan, Iran. The phage morphology was observed by TEM. After detection of the host range, adsorption rate, and one-step growth curve, the phage proteomics pattern and restriction enzyme digestion pattern were analyzed. RESULTS: All isolates of bacteria were highly resistant to antibiotics. Among isolates, Acinetobacter baumannii strain IAU_FAL101 (GenBank accession number: MW845680), which was an XDR bacterium, showed significant sensitivity to phage Pɸ-Bw-Ab. TEM determined the phage belongs to Siphoviridae. They had double-stranded DNA. This phage showed the highest antibacterial effect at 15 °C and pH 7. Analysis of the restriction enzyme digestion pattern showed Pɸ-Bw-Ab phage was sensitive to most of the used enzymes and based on SDS-PAGE, protein profiles were revealed 43 to 90 kDa. CONCLUSION: Considering the potential ability of the isolated phage, it had an antibacterial impact on other used bacterial spp and also strong antibacterial effects on XDR A. baumannii. Also, it had long latency and low burst size. This phage can be a suitable candidate for phage therapy.