The Isolation of specifically lytic phages along with their extracted endolysins as antibacterial agents to MDR Enterococcus faecalis

Abstract

Background: Multidrug-resistant (MDR) enterococci have become a major problem in recent times and have been reported increasingly around the world. Lytic phages infect bacteria leading to rapid host death with limited risk of phage transduction, underlining the increasing interest in potential phage therapy in the future. Objective (s): The aim of this study is to use phage therapy as alternative approach for treatment of Enterococcus faecalis infections that recorded as MDR in Iraq to tackle this problem. Materials and Methods: Thirty E. faecalis isolates were collected from patients with different infectious diseases such as urinary tract infection (UTI), diabetic foot, septicemia, and wound infections. The isolation of specific lytic phages was from different environmental sources such as (sewage, and wastewater). The biokinetic assays were carried out to measure the characteristics of the isolated phage. The study of the bacteriophage and the formed phage cocktail infectivity against isolates E.faecalis was tested by the top layer assay. The phage endolysin was extracted from the best bacteriophage that gave best results. Results: All the isolated E.faecalis was reported as MDR in this study. About 75 E.faecalis specific phages were isolated and purified. All the isolated bacteria were 100% sensitive to the lytic phages. The formed phage cocktail was capable to create inhibition zones on the most bacterial isolates' lawns. The molecular weight and the concentration of the extracted endolysin was evaluated in this study and found to be as (48 kDa) and (0.5mg/ml), respectively. The antibacterial activity of the extracted endolysin was evaluated by the turbidity reduction assay. A clear decline in the bacterial growth was manifested (5x107 CFU/ml) to (1x104 CFU/ml), in which the bacterial growth was reduced by (3.63 log). The endolysin found to be effective against 90% of E.faeclais isolates. Conclusion: The activity of the isolated specific phage together with the activity of the formed phage cocktail, were efficient as successful and inexpensive method of therapy against MDR E.faecalis. The potential of the extracted endolysin over the phage therapy was verified in this study. The coverage rate and the absence of resistant E.faecalis to the phage and its endolysin had emphasized on the importance of this alternative therapy to commonly used antibiotics. List of abbreviation: MDR = multiple drug-resistant, E. faecalis = Enterococcus faecalis, BT = burst time, BS = burst size, IP = infective percentage, Phage =bacteriophage, CFU = Colony forming unit, kDa = Kilodalton, OD = Optical Densities.

Estimation of the In-Vitro Synergistic Effect of Vancomycin with Bacteriophage and its Endolysin on Iraqi Local Isolates of Enterococcus faecalis

“…Unlikely, the susceptibility was found to be with Tigecyclin and Inducible-clindamycin. The susceptibility to Tigecyclin among the isolates from urine samples was similar to a previous study done in our laboratory 11 . The antibiotic resistance genes in E. faecalis isolated from urine samples are alarming the researchers, since these genes have the ability to transmit among enterococci 25 .…”

“…The next day, the susceptibility to the phage/ phage cocktail was determined by the formation of the lyses zones. Moreover, the coverage rate of the phage cocktail was determined against another 12 new samples of E.faecalis using the same method mentioned above 11 .…”

“…The dialyzed solution was added with 20 ml of 0.05 M phosphate-buffered saline at pH 7.5 as eluent. Every five minutes the elute 1 ml was collected in 50 Eppendorf tubes 11,12 . A spectrophotometer was used to measure the absorbance of each fraction at 280 nm.…”

“…The Bacterial concentration (CFU/ml) was measured according to the below formulae 11 . ΔOD = OD (before treatment) -OD (after treatment) Δ Bacterial concentration (CFU/ml) = CFU/ml (before treatment) -CFU/ml (after treatment)…”

“…Bacteriophages or phages, with their related cellwall lysing enzymes (endolysins), have the ability to be useful tools to tackle MDR bacteria 10 . Many studies indicate the potential of phages and their endolysins as suitable alternatives to treat E. faecalis 11,12 . A recent study has come to the fact that a combination of a phage and an antibiotic has better killing efficiency against E. faecalis 13 .…”