Investigation and isolation of lytic bacteriophages effective on Klebsiella resistant tocolistin

One of the most important global health problems in the last decade is the increasing prevalenceof antibiotic-resistant infections, including bacteria from the Enterobacteriaceae family,especially the Klebsiella pneumoniae strain. Excessive use of antibacterial drugs leads toincreased resistance to several drugs and changes in the bacterial population in patients. Gramnegativebacteria such as Klebsiella pneumoniae by expressing extended-spectrum betalactamases(ESBL) have become resistant to antibiotics such as colistin, which are among thelast lines of treatment, and these antibiotics cannot be used to control the infection.Due to the increasing prevalence of multi-drug resistant infections, treatment problems andlong-term treatment lead to the production of new high-efficiency antibacterial drugs with lowside effects and cost-effective treatment of multi-drug resistant infections. One of the treatmentoptions as an alternative to antibiotics are bacterial viruses or bacteriophages. The aim of thepresent study is to screen and isolate lytic bacteriophage against clinical samples isolated fromKlebsiella pneumoniae strains resistant to the antibiotic colistin from wastewater. Materialsand methods: Clinical samples of Klebsiella pneumoniae were isolated and identified fromJanuary ۲۰۱۸ to September ۲۰۱۹ from hospitals and medical centers in Isfahan. Using theantibiotic sensitivity test method (antibiogram) and the E-TEST test method, the level ofresistance to colistin was used to determine the resistance pattern of the isolates to differentantibiotics. Strains with multi-drug resistance and colistin-resistant strains were tested andselected to investigate the effect of isolated phage. To isolate the bacteriophage, sampling wasdone from the water and sewage treatment plant in the south of Isfahan. After primaryenrichment, the bacteriophage was isolated and then purified, secondary enrichment andstaining, the morphology of the bacteriophage was determined by transmission electronmicroscopy (TEM). Phage identification tests including host range and growth stages,temperature sensitivity test and pH sensitivity test were performed. Findings: Examination ofthe images (TEM) showed that the phage has a ۲-sided head and a non-contractible and longtail and belongs to the Siphoviridae family. Among the ۱۰ Klebsiella pneumoniae strains thatwere resistant to colistin, the isolated phage was able to lyse ۶ strains. Also, the isolatedbacteriophage maintained its antibacterial activity at a pH between ۳ and ۹ and a temperatureof -۲۰ to ۴۵ degrees Celsius. Conclusion: The formation of transparent plaques shows the highlysing power of the phage, so it has a good potential for further testing and analysis for clinicaluse as a therapeutic agent in the future.

Keywords:

bacteriophages , Klebsiella , antibiotic colistin